anti-cd3 Search Results


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  • 99
    Thermo Fisher anti cd3
    IL-31R signaling did not influence the differentiation of CD4 + T cell sub-populations. Purified CD4 + T cells from WT and IL-31RA KO mice were stimulated with <t>anti-CD3/anti-CD28</t> under Th1-, Th2- or Th17- polarizing conditions. Cells were assayed for the transcription factors T-bet, GATA-3, ROR-γt and cytokines IFN-γ, IL-4, IL-17 by flow cytometry. No difference on the differentiation of Th1(A), Th2 (B) and Th17 (C) was found between WT ( n =4) and IL-31RA KO mice ( n =4). All values are plotted and lines indicate means. Statistical significance was determined by two-tailed Student's t - tests. The experiment was repeated three times. P, polarization; NP, non-polarization.
    Anti Cd3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12432 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti cd3
    Activated T cells up-regulate PCBP1. ( A ) Expression of mRNA encoding PCBP1-4 in human <t>CD3</t> + T cells before (Unstimulated) and after (Stimulated) stimulation for 24 hours with anti-CD3 and anti-CD28 (obtained from Gene Expression Omnibus accession code GSE13887). n = 4 biologically independent samples. ( B and C ) Immunoblotting for total PCBP1 and moesin expression in human CD4 + (B) and CD8 + (C) T cells left unstimulated (T0 and Tc-0) or stimulated (Th0 and Tc-1) with antibodies against CD3 and ICOS with IL-2 for 4 days. β-Actin was used as loading control. ( D ) Flow cytometry (left) and quantification (right) of PCBP1 expression in subsets of splenic lymphocytes from mice. FITC, fluorescein isothiocyanate. ( E and F ) Relative Pcbp1 mRNA expression (E) and fluorescence-activated cell sorting (FACS) analysis and PCBP1 mean fluorescence intensity (MFI) in subsets of in vitro polarized T cells. (E) n = 8; (F) n = 4. PE, phycoerythrin. ( G and H ) Immunoblotting of moesin, PCBP1, FoxP3, and β-actin (G) and quantification for PCBP1 and moesin (H) using splenic mouse CD4 + T cells activated with anti-CD3 and anti-CD28 for 3 days in the absence (Th0) or presence (iT reg ) of TGF-β in vitro. n = 5. ( I and J ) Mouse splenic CD8 + T cells from the same experiments as (G and H). n = 5. ( K and L ) Immunoblotting for phosphorylated and total PCBP1 (K) in Th0 and iT regs and quantification (L). (D) Error bars represent means ± SE and (E, F, H, J, and L) SD; * P
    Anti Cd3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 910 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti cd3
    Characterization of mice devoid of XBP1 in T cells. a , Deletion efficiency was analyzed by qRT-PCR using a primer set that specifically detects the exon 2 region of Xbp1 . Data were normalized to endogenous expression of Actb and presented as relative expression compared with WT ( n = 8). b , Absolute cell numbers in the thymus, spleen and lymph nodes. c , FACS-based phenotyping of double negative (CD4 - CD8 - ), double positive (CD4 + CD8 + ), or single positive (CD4 + or CD8 + ) thymocytes. d - g, Frequency of TCRβ + cells ( d , f ) and CD4 + or CD8 + cells (gated on TCRβ + cells) ( e , g ) in lymph nodes or spleen. h - i , Expression of CD44 and CD62L on both CD4 + ( h ) and CD8 + ( i ) TCRβ + subsets in the spleen. j , Frequency of splenic TCRβ + CD4 + FoxP3 + T cells. k - l , Frequency of non-T cell populations among total live cells in spleen ( k ) and lymph nodes ( l ). b - g , n = 5; h - j , n = 3; k - l , Xbp1 f/f ( n = 4), Xbp1 f/f Cd4 cre ( n = 5). m , Reconstitution efficiency of CD4 + and CD8 + T cells in bone marrow and spleen from mixed bone marrow chimeras ( n = 3 per chimera type). Chimeras were generated with a mixture of wild-type bone marrow (CD45.1 + ) plus either Xbp1 f/f or Xbp1 f/f Vav1 cre bone marrow (CD45.2 + ). n , Flow cytometry assessing cell proliferation of CD4 + T cells stained with the division-tracking dye (Cell Trace Violet). Cells were left unstimulated or stimulated for 72 and 96 h with plate-bound <t>anti-CD3</t> (5μg/ml) and soluble anti-CD28 (1μg/ml). Histograms (left) and proliferation index (right) are shown ( n = 4). o , Cell cycle analysis of CD4 + T cells activated for 72 h by staining with propidium iodide. Representative plots from two experiments. p , Transmission electron microscopy of in vitro activated WT versus XBP1-deficient CD4 + T cells. Naïve CD4 + T cells isolated from three biologically independent mice were activated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 48 h. White arrowheads indicate the endoplasmic reticulum (ER); M, Mitochondria; 12000× (left); 50000× (right). Average mitochondrial area of independent cells was estimated using image J software. Xbp1 f/f ( n = 19); Xbp1 f/f Cd4 cre ( n = 29). q , Histogram (left) and quantification (right) for mitochondrial staining (Mitotracker) in in vitro activated CD4 + T cells ( n = 2 from two independent experiments). r, Activated WT versus XBP1-deficient CD4 + T cells were incubated in glucose-containing, -depleted or 2-deoxyglucose (2-DG, 10mM)-treated media for 6 h and PGC1α expression was analyzed by immunoblot. β-ACTIN was used as loading control. Representative plots from two independent experiments. Data are shown as mean ± s.e.m. ( a - n, p ). n values represent biologically independent samples ( a - n, p-q ). Two-tailed Student’s t -tests ( b - l , n , p ); * P
    Anti Cd3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 15251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson plate bound anti cd3
    Variable ThPOK expression in γδ T cell subsets. A , ThPOK-GFP reporter expression in spleen γδ T cells and CD4 T cells WT and ThPOK GFP/WT . Numbers in quadrants indicate the percentage of γδ T cells that express GFP. B , GFP (ThPOK) and PLZF expression in spleen cells from ThPOK GFP/+ and ThPOK GFP/GFP mice. C , Expression of CD4 (APC-Cy7) and CD8 (Pacific Blue) in Vγ1.1 + Vδ6.3 + splenocytes from ThPOK GFP/WT and ThPOK GFP/KO reporter mice. D , INF-γ and IL-4 expression in spleen Vγ1.1 + Vδ6.3 + T cells from WT or ThPOK-deficient (ThPOK ko/ko ) mice following activation with PMA/ionomycin. γδ T cell subsets were identified with <t>anti-CD3</t> (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 3 × 10 6 events were acquired for these experiments. Doublet exclusion was done as indicated in the Materials and Methods. B–D , Numbers indicate the percentage of cells in each quadrant. Data are representative of at least two experiments. All flow cytometry plots are quantified in log10 fluorescence.
    Plate Bound Anti Cd3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies anti cd3
    Proliferation and apoptosis in lymphocytes from patients with PHTS. A, Gut tissue sections from patients with PHTS or control subjects were stained for Ki-67. Cell proliferation was analyzed within the T-cell areas. E , Epithelium. B, <t>CD3,</t> CD20, and CD10 costaining with terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) for apoptosis detection in control appendix sections. The graph demonstrates the percentage of TUNEL + cells that are CD3 + , CD20 + , or CD10 + . Differences were analyzed by using the Mann-Whitney test. Scale bars = 50 μm. Symbols represent individual patients. C and D, In vitro apoptosis assays. Apoptosis was induced in PHA-activated T-cell blasts by means of CD95L (FasL) stimulation (Fig E4, C ) or IL-2 deprivation (Fig E4, D ). Percentages of surviving cells are shown for 1 patient with PHTS (p.R130Q) and 2 healthy control subjects. Similar results have been obtained by using analysis of 6 further patients with PHTS with different mutations in an independent experiment (data not shown). Dapi , 4′, 6′-Diamidino-2-phenylindole.
    Anti Cd3, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 1812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd3  (Abcam)
    92
    Abcam cd3
    Proliferation and apoptosis in lymphocytes from patients with PHTS. A, Gut tissue sections from patients with PHTS or control subjects were stained for Ki-67. Cell proliferation was analyzed within the T-cell areas. E , Epithelium. B, <t>CD3,</t> CD20, and CD10 costaining with terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) for apoptosis detection in control appendix sections. The graph demonstrates the percentage of TUNEL + cells that are CD3 + , CD20 + , or CD10 + . Differences were analyzed by using the Mann-Whitney test. Scale bars = 50 μm. Symbols represent individual patients. C and D, In vitro apoptosis assays. Apoptosis was induced in PHA-activated T-cell blasts by means of CD95L (FasL) stimulation (Fig E4, C ) or IL-2 deprivation (Fig E4, D ). Percentages of surviving cells are shown for 1 patient with PHTS (p.R130Q) and 2 healthy control subjects. Similar results have been obtained by using analysis of 6 further patients with PHTS with different mutations in an independent experiment (data not shown). Dapi , 4′, 6′-Diamidino-2-phenylindole.
    Cd3, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 2421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher anti cd3 cd28 beads
    The ability to polarize Tfh cells is intrinsic for cDC2 but is tissue dependent for macrophages. (A–C) Purified human tonsil DC subsets and macrophages were cultured for 3 h with or without R848. After washing, preactivated DCs or macrophages were co-cultured with allogeneic naive CD4 + T cells for 6 d. T cells polarized with cDC1s, cDC2s, pDCs, or CD14 + macrophages are termed T cDC1 , T cDC2 , T pDC , and T MACRO , respectively. T Ø corresponds to T cells cultured without APCs. (A) T cell proliferation was assessed by dilution of a proliferation dye (CTV). Histogram representative of three independent experiments. Graphs show mean ± SEM ( n = 3). (B and C) Cytokine secretion was analyzed by cytometric bead array (CBA) (B) or ELISA (C) after restimulation with <t>anti-CD3/CD28</t> beads. Each symbol represents an individual donor ( n = 9). (D–G) Purified cDC1 and cDC2 from blood (D) or skin-draining lymph node (E), or DCs and macrophages from peritoneal tumor ascites (F) or synovial fluid of rheumatoid arthritis patients (G) were co-cultured with allogeneic naive CD4 + T cells for 6 d. IFN-γ and CXCL13 secretion was measured in the supernatant after restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor ( n = 6 for blood, 8 for lymph node, 5–7 for ascites, and 2 for synovial fluid). *, P
    Anti Cd3 Cd28 Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1691 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti cd3 fitc
    Structural and functional integrity of recombinant soluble NKT TCRs. (A) Purified bacterial NKT15 TCR was analyzed by SDS-PAGE under reducing (+DTT, dithiothreitol) and nonreducing (−DTT) conditions demonstrating αβ heterodimers. (B) Native gel electrophoresis of folded αβ heterodimeric NKT15 and control LC13 TCRs. (C) Recombinant soluble NKT12, NKT15, NKT18, and control LC13 TCRs were tested for their ability to block binding of mouse CD1d/α-GalCer tetramers to murine thymocytes. Phycoerythrin-conjugated mCD1d/α-GalCer tetramers were preincubated with the indicated soluble TCRs over a range of TCR concentrations before staining mouse thymocytes. Cells were analyzed by two-color flow cytometry showing mCD1d/α-GalCer tetramer staining on the vertical axis and <t>FITC-CD3</t> (mAb 145-2C11) staining on the horizontal axis. Cells staining positively with mCD1d/α-GalCer tetramer and FITC-CD3 are indicated with a circle.
    Anti Cd3 Fitc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2049 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen anti cd3
    Prominent expansion of erythroid cells in the spleen and of myeloid cells in the liver of irradiated mice with a liver shield. Two-colour staining for TER119 and <t>CD3</t> and that for Gr-1 and Mac-1 were conducted in control mice and irradiated mice with or without a liver shield (on day 14). Representative results of three experiments are depicted. Erythroid (TER119 + ) cells expanded prominently in the spleen of mice with a liver shield, whereas myeloid cells expanded in the liver of the same mice without a liver shield.
    Anti Cd3, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 1226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti cd3 percp
    Prominent expansion of erythroid cells in the spleen and of myeloid cells in the liver of irradiated mice with a liver shield. Two-colour staining for TER119 and <t>CD3</t> and that for Gr-1 and Mac-1 were conducted in control mice and irradiated mice with or without a liver shield (on day 14). Representative results of three experiments are depicted. Erythroid (TER119 + ) cells expanded prominently in the spleen of mice with a liver shield, whereas myeloid cells expanded in the liver of the same mice without a liver shield.
    Anti Cd3 Percp, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti cd3
    Prominent expansion of erythroid cells in the spleen and of myeloid cells in the liver of irradiated mice with a liver shield. Two-colour staining for TER119 and <t>CD3</t> and that for Gr-1 and Mac-1 were conducted in control mice and irradiated mice with or without a liver shield (on day 14). Representative results of three experiments are depicted. Erythroid (TER119 + ) cells expanded prominently in the spleen of mice with a liver shield, whereas myeloid cells expanded in the liver of the same mice without a liver shield.
    Anti Cd3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson anti cd3 apc
    Raltegravir intracellular accumulation analysis of <t>CD3+CD4+P-gp</t> high and P-gp low populations. (a) After Rho123 incubation (1 μM, 20 min, 37°C), PBMCs were put in culture (complete medium) during 2 h to let the dye be effluxed and then stained with <t>anti-CD3-APC</t> and anti-CD4-PerCP antibodies. CD3+CD4+ T cells were sorted based on their Rho123 staining: P-gp high (Rho123 very low) and P-gp low (Rho123 high). Cells were directly used in subsequent [ 3 H]raltegravir accumulation assays. (b) CD3+CD4+P-gp high and CD3+CD4+P-gp low cells were incubated in transport medium with 1 μM (1 μCi/mL) [ 3 H]raltegravir in the absence (mock) or presence of the P-gp-specific inhibitor XR9051 (1 μM). Unsorted (total) PBMCs were used as control of viability and transport, considering that they contain CD8+ T cells, NK and monocytes. The results represent the mean ± SEM of five independent experiments (each with a different blood donor) performed in duplicate. Statistical significance was assessed by paired t -test (* P
    Anti Cd3 Apc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti human cd3
    Dex treatment does not affect ERK and JNK activation. Cell extracts were prepared from Dex-treated and control naive CD4+ T cells stimulated with <t>anti-CD3,</t> anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28. The phosphorylation of ( a ) ERK and ( b ) JNK was assessed by Western blotting using specific Ab’s. Results from a representative experiment are shown.
    Anti Human Cd3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti mouse cd3
    IL-23 induces IL-17A production in CD4 + and particularly in CD4 − CD8 − T cells. Cells were extracted from the lymph nodes of 5-mo-old B6, B6/ lpr , 4-mo-old MRL/MPJ, and MRL/ lpr mice. The cells were incubated in DMEM with plate-bound <t>anti-CD3</t>
    Anti Mouse Cd3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1006 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    thermo fisher anti cd3 cd28 dynabeads
    Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by <t>anti-CD3/CD28</t> beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.
    Anti Cd3 Cd28 Dynabeads, supplied by thermo fisher, used in various techniques. Bioz Stars score: 93/100, based on 951 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd3 monoclonal antibody
    Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by <t>anti-CD3/CD28</t> beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.
    Anti Cd3 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti cd3 antibody
    Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by <t>anti-CD3/CD28</t> beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.
    Anti Cd3 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 402 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti cd3 pacific blue
    Gating strategy for menstrual and peripheral blood cells. A. PBMC and B. MBC. Initial gating is performed from the SSC-A vs. FSC-A to determine the total lymphocyte gate (small gate) and the larger gate is used to further gate for monocytes (CD14). From the lymphocytes, live cells are gated, followed by gating B cells (CD19), T cells <t>(CD3),</t> CD4+CD3+ and CD8+CD3+ cells. From the CD3- gate, NK cell phenotypes are selected. CD16-CD56+(immunoregulatory cells) and CD16+CD56+(Cytotoxic cells). Percentages of positive cells for a given marker are indicated above/near gate.
    Anti Cd3 Pacific Blue, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher plate bound anti cd3
    Effects of CSE on Treg differentiation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound <t>α-CD3</t> and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Cells were co-stained for CD25 and FOXP3 expression and measured by flow cytometry; representative pseudocolour dot plots gated on CD4 + T cells are shown. (B) Summary data of CD25 + FOXP3 + Tregs and CD25 + T cells in each condition, from (A) Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P
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    Thermo Fisher anti cd3 cd28 coated beads
    The Effect of Protocol A and Protocol B on the Expansion and Function of Treg Lines (A) Tregs were expanded by stimulation with <t>anti-CD3/CD28</t> beads and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. There was no difference in the fold expansion of Tregs between the different isolation strategies (protocol A and protocol B) or culture conditions. (B) The suppressive function of freshly isolated Tregs and expanded Treg lines. Suppressive ability of Tregs was measured as a decrease of proliferation of effector T cells in the presence of different concentrations of Tregs (Treg:Teff 1:1 and 1:10). Data represent the percentage Treg suppression at a Treg:Teff ratio of 1:1. Data represent mean ± SD of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA and revealed no significant difference.
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    Image Search Results


    IL-31R signaling did not influence the differentiation of CD4 + T cell sub-populations. Purified CD4 + T cells from WT and IL-31RA KO mice were stimulated with anti-CD3/anti-CD28 under Th1-, Th2- or Th17- polarizing conditions. Cells were assayed for the transcription factors T-bet, GATA-3, ROR-γt and cytokines IFN-γ, IL-4, IL-17 by flow cytometry. No difference on the differentiation of Th1(A), Th2 (B) and Th17 (C) was found between WT ( n =4) and IL-31RA KO mice ( n =4). All values are plotted and lines indicate means. Statistical significance was determined by two-tailed Student's t - tests. The experiment was repeated three times. P, polarization; NP, non-polarization.

    Journal: Biology Open

    Article Title: IL-31 plays dual roles in lung inflammation in an OVA-induced murine asthma model

    doi: 10.1242/bio.036244

    Figure Lengend Snippet: IL-31R signaling did not influence the differentiation of CD4 + T cell sub-populations. Purified CD4 + T cells from WT and IL-31RA KO mice were stimulated with anti-CD3/anti-CD28 under Th1-, Th2- or Th17- polarizing conditions. Cells were assayed for the transcription factors T-bet, GATA-3, ROR-γt and cytokines IFN-γ, IL-4, IL-17 by flow cytometry. No difference on the differentiation of Th1(A), Th2 (B) and Th17 (C) was found between WT ( n =4) and IL-31RA KO mice ( n =4). All values are plotted and lines indicate means. Statistical significance was determined by two-tailed Student's t - tests. The experiment was repeated three times. P, polarization; NP, non-polarization.

    Article Snippet: T cells at 2 million cells/ml were washed and resuspended in 2 μM CFSE (Invitrogen) staining solution for 10 min, and CFSE fluorescence was measured by flow cytometry at 96 h after culture with 1 μl/ml anti-CD3 (CAT#85-16-0032-81)/anti-CD28 (CAT#85-16-0281-81) (both eBioscience).

    Techniques: Purification, Mouse Assay, Flow Cytometry, Cytometry, Two Tailed Test

    IL-31R signaling suppressed the proliferation of CD4 + T cells. Purified CD4 + T cells from WT and IL-31RA KO mice were stimulated with anti-CD3/anti-CD28. The activation ( n =4) (A) and proliferation ( n =4) (B) of CD4 + T cells were assayed by flow cytometry. All values are plotted and lines indicate means. Statistical significance was determined by two-tailed Student's t - tests. The experiment was repeated twice. Ctrl, control; Act, activation.

    Journal: Biology Open

    Article Title: IL-31 plays dual roles in lung inflammation in an OVA-induced murine asthma model

    doi: 10.1242/bio.036244

    Figure Lengend Snippet: IL-31R signaling suppressed the proliferation of CD4 + T cells. Purified CD4 + T cells from WT and IL-31RA KO mice were stimulated with anti-CD3/anti-CD28. The activation ( n =4) (A) and proliferation ( n =4) (B) of CD4 + T cells were assayed by flow cytometry. All values are plotted and lines indicate means. Statistical significance was determined by two-tailed Student's t - tests. The experiment was repeated twice. Ctrl, control; Act, activation.

    Article Snippet: T cells at 2 million cells/ml were washed and resuspended in 2 μM CFSE (Invitrogen) staining solution for 10 min, and CFSE fluorescence was measured by flow cytometry at 96 h after culture with 1 μl/ml anti-CD3 (CAT#85-16-0032-81)/anti-CD28 (CAT#85-16-0281-81) (both eBioscience).

    Techniques: Purification, Mouse Assay, Activation Assay, Flow Cytometry, Cytometry, Two Tailed Test, Activated Clotting Time Assay

    Bcl2 transgenic mice can rescue cell apoptosis. ( A ) Cell surface staining of CD4 and CD8 on WT and cKO on Bcl2 − or Bcl2 + background thymocytes. Numbers in or adjacent to outlined areas (or in quadrants) indicate percent. Bar charts show the frequency and cell number of total, DN, DP, CD4 + , or CD8 + SP thymocyte subpopulations. ( B ) Cell surface staining of CD4 and CD8 on WT and cKO on Bcl2 − or Bcl2 + background splenocytes, and cell number of splenocyte subpopulations. ( C ) Cell staining of annexin V of WT, cKO, WT Bcl2 + , and cKO Bcl2 + DN and DP thymocytes with or without anti-CD3 stimulation, and quantification of annexin V cell percentage. Data are representative of five experiments. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Protein phosphatase 2A has an essential role in promoting thymocyte survival during selection

    doi: 10.1073/pnas.1821116116

    Figure Lengend Snippet: Bcl2 transgenic mice can rescue cell apoptosis. ( A ) Cell surface staining of CD4 and CD8 on WT and cKO on Bcl2 − or Bcl2 + background thymocytes. Numbers in or adjacent to outlined areas (or in quadrants) indicate percent. Bar charts show the frequency and cell number of total, DN, DP, CD4 + , or CD8 + SP thymocyte subpopulations. ( B ) Cell surface staining of CD4 and CD8 on WT and cKO on Bcl2 − or Bcl2 + background splenocytes, and cell number of splenocyte subpopulations. ( C ) Cell staining of annexin V of WT, cKO, WT Bcl2 + , and cKO Bcl2 + DN and DP thymocytes with or without anti-CD3 stimulation, and quantification of annexin V cell percentage. Data are representative of five experiments. * P

    Article Snippet: i.p. injection with anti-CD3 antibody (eBioscience, 1.0 mg/mL) was performed (10 µL per mouse).

    Techniques: Transgenic Assay, Mouse Assay, Staining

    Bone marrow-chimera experiments. ( A ) Flow cytometry of thymus cells from bone marrow chimeras deficient in recombination-activating gene 1 ( Rag1 −/− ) 8 wk after transfer of bone marrow cells from WT C57BL/6 (CD45.1 + ) mice and WT or PP2A cKO(CD45.2 + ) mice, assessed for expression of CD4 and CD8 after gating on CD45.1 or CD45.2. Data are representative of three experiments. ( B ) Apoptosis of C57BL/6 (CD45.1 + ) and WT (CD45.2 + ) total thymocytes in reconstituted Rag1 −/− mice. ( C ) The apoptosis of DN and DP cells by annexin V staining before and after anti-CD3 stimulation in WT or PP2A cKO(CD45.2 + ) derived cells. ( D ) BrdU incorporation in WT or PP2A cKO(CD45.2 + ) mice. Data are representative of three experiments. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Protein phosphatase 2A has an essential role in promoting thymocyte survival during selection

    doi: 10.1073/pnas.1821116116

    Figure Lengend Snippet: Bone marrow-chimera experiments. ( A ) Flow cytometry of thymus cells from bone marrow chimeras deficient in recombination-activating gene 1 ( Rag1 −/− ) 8 wk after transfer of bone marrow cells from WT C57BL/6 (CD45.1 + ) mice and WT or PP2A cKO(CD45.2 + ) mice, assessed for expression of CD4 and CD8 after gating on CD45.1 or CD45.2. Data are representative of three experiments. ( B ) Apoptosis of C57BL/6 (CD45.1 + ) and WT (CD45.2 + ) total thymocytes in reconstituted Rag1 −/− mice. ( C ) The apoptosis of DN and DP cells by annexin V staining before and after anti-CD3 stimulation in WT or PP2A cKO(CD45.2 + ) derived cells. ( D ) BrdU incorporation in WT or PP2A cKO(CD45.2 + ) mice. Data are representative of three experiments. * P

    Article Snippet: i.p. injection with anti-CD3 antibody (eBioscience, 1.0 mg/mL) was performed (10 µL per mouse).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Expressing, Staining, Derivative Assay, BrdU Incorporation Assay

    Ser/Thr phospho-peptide profiling of thymocytes and generation of PP2Ac conditional knockout mice. ( A ) Ser/Thr phosphorylation measured by MS-based iTRAQ phosphor-peptide analysis in C57BL/6 resting and activated thymocytes. About 350 proteins changed their phosphorylation (log 2 ). Proteins in which phosphorylation was notably changed are annotated such as Rab12 (green font) and Prpf4b (red font). ( B ) Ser/Thr phosphorylation measured by MS-based iTRAQ phosphor-peptide analysis in PP2Ac cKO resting and activated thymocytes. Proteins which cannot be found but which notably changed in wild-type cells ( A ) are annotated, for example Rab12 Prpf4b. The dephosphorylation of some proteins was reduced such as Smarca4. ( C ) Heatmap showing the 35 proteins showing the greatest change in phosphorylation as measured by mass spectrometry (mean change 1.75-fold). Six of them are related to cell survival (marked with red font). ( D ) Pathway analysis of the 35 proteins showed the greatest change in phosphorylation in CD3-stimulated PP2A-deficient thymocytes.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Protein phosphatase 2A has an essential role in promoting thymocyte survival during selection

    doi: 10.1073/pnas.1821116116

    Figure Lengend Snippet: Ser/Thr phospho-peptide profiling of thymocytes and generation of PP2Ac conditional knockout mice. ( A ) Ser/Thr phosphorylation measured by MS-based iTRAQ phosphor-peptide analysis in C57BL/6 resting and activated thymocytes. About 350 proteins changed their phosphorylation (log 2 ). Proteins in which phosphorylation was notably changed are annotated such as Rab12 (green font) and Prpf4b (red font). ( B ) Ser/Thr phosphorylation measured by MS-based iTRAQ phosphor-peptide analysis in PP2Ac cKO resting and activated thymocytes. Proteins which cannot be found but which notably changed in wild-type cells ( A ) are annotated, for example Rab12 Prpf4b. The dephosphorylation of some proteins was reduced such as Smarca4. ( C ) Heatmap showing the 35 proteins showing the greatest change in phosphorylation as measured by mass spectrometry (mean change 1.75-fold). Six of them are related to cell survival (marked with red font). ( D ) Pathway analysis of the 35 proteins showed the greatest change in phosphorylation in CD3-stimulated PP2A-deficient thymocytes.

    Article Snippet: i.p. injection with anti-CD3 antibody (eBioscience, 1.0 mg/mL) was performed (10 µL per mouse).

    Techniques: Knock-Out, Mouse Assay, Mass Spectrometry, De-Phosphorylation Assay

    Impaired cell survival of PP2Ac cKO thymocytes. ( A ) Cells were gated on a single cell by the exclusion of dead cells for analysis: Annexin V staining of DN and DP cells left unstimulated or stimulated for 12 h in vitro with different doses of anti-CD3; bar chart shows quantification of the results ( Lower ). Data are representative of five experiments. ( B ) Active Caspase-3 staining of DN and DP cells left unstimulated or stimulated for 12 h in vitro with anti-CD3 bar chart shows quantification of the results. ( C ) Percentages and cell numbers of annexin V staining of DN3, DN4, DN, and DP cells after coculture with OP9-DL1 at day 5. ( D ) Number of DP cells ( Left ) and percentage of WT and cKO DP cells ( Center ) after i.p. injected with PBS or anti-CD3 for 12 h. Percentages and quantification of annexin V + of DN and DP cells ( Right ). ( E ) Staining of total thymus with TUNEL (Green) in frozen sections from WT and cKO mice i.p. injected with PBS or anti-CD3 for 12 h and quantification of total TUNEL + thymocytes. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Protein phosphatase 2A has an essential role in promoting thymocyte survival during selection

    doi: 10.1073/pnas.1821116116

    Figure Lengend Snippet: Impaired cell survival of PP2Ac cKO thymocytes. ( A ) Cells were gated on a single cell by the exclusion of dead cells for analysis: Annexin V staining of DN and DP cells left unstimulated or stimulated for 12 h in vitro with different doses of anti-CD3; bar chart shows quantification of the results ( Lower ). Data are representative of five experiments. ( B ) Active Caspase-3 staining of DN and DP cells left unstimulated or stimulated for 12 h in vitro with anti-CD3 bar chart shows quantification of the results. ( C ) Percentages and cell numbers of annexin V staining of DN3, DN4, DN, and DP cells after coculture with OP9-DL1 at day 5. ( D ) Number of DP cells ( Left ) and percentage of WT and cKO DP cells ( Center ) after i.p. injected with PBS or anti-CD3 for 12 h. Percentages and quantification of annexin V + of DN and DP cells ( Right ). ( E ) Staining of total thymus with TUNEL (Green) in frozen sections from WT and cKO mice i.p. injected with PBS or anti-CD3 for 12 h and quantification of total TUNEL + thymocytes. * P

    Article Snippet: i.p. injection with anti-CD3 antibody (eBioscience, 1.0 mg/mL) was performed (10 µL per mouse).

    Techniques: Staining, In Vitro, Injection, TUNEL Assay, Mouse Assay

    Activated T cells up-regulate PCBP1. ( A ) Expression of mRNA encoding PCBP1-4 in human CD3 + T cells before (Unstimulated) and after (Stimulated) stimulation for 24 hours with anti-CD3 and anti-CD28 (obtained from Gene Expression Omnibus accession code GSE13887). n = 4 biologically independent samples. ( B and C ) Immunoblotting for total PCBP1 and moesin expression in human CD4 + (B) and CD8 + (C) T cells left unstimulated (T0 and Tc-0) or stimulated (Th0 and Tc-1) with antibodies against CD3 and ICOS with IL-2 for 4 days. β-Actin was used as loading control. ( D ) Flow cytometry (left) and quantification (right) of PCBP1 expression in subsets of splenic lymphocytes from mice. FITC, fluorescein isothiocyanate. ( E and F ) Relative Pcbp1 mRNA expression (E) and fluorescence-activated cell sorting (FACS) analysis and PCBP1 mean fluorescence intensity (MFI) in subsets of in vitro polarized T cells. (E) n = 8; (F) n = 4. PE, phycoerythrin. ( G and H ) Immunoblotting of moesin, PCBP1, FoxP3, and β-actin (G) and quantification for PCBP1 and moesin (H) using splenic mouse CD4 + T cells activated with anti-CD3 and anti-CD28 for 3 days in the absence (Th0) or presence (iT reg ) of TGF-β in vitro. n = 5. ( I and J ) Mouse splenic CD8 + T cells from the same experiments as (G and H). n = 5. ( K and L ) Immunoblotting for phosphorylated and total PCBP1 (K) in Th0 and iT regs and quantification (L). (D) Error bars represent means ± SE and (E, F, H, J, and L) SD; * P

    Journal: Science Advances

    Article Title: RNA binding protein PCBP1 is an intracellular immune checkpoint for shaping T cell responses in cancer immunity

    doi: 10.1126/sciadv.aaz3865

    Figure Lengend Snippet: Activated T cells up-regulate PCBP1. ( A ) Expression of mRNA encoding PCBP1-4 in human CD3 + T cells before (Unstimulated) and after (Stimulated) stimulation for 24 hours with anti-CD3 and anti-CD28 (obtained from Gene Expression Omnibus accession code GSE13887). n = 4 biologically independent samples. ( B and C ) Immunoblotting for total PCBP1 and moesin expression in human CD4 + (B) and CD8 + (C) T cells left unstimulated (T0 and Tc-0) or stimulated (Th0 and Tc-1) with antibodies against CD3 and ICOS with IL-2 for 4 days. β-Actin was used as loading control. ( D ) Flow cytometry (left) and quantification (right) of PCBP1 expression in subsets of splenic lymphocytes from mice. FITC, fluorescein isothiocyanate. ( E and F ) Relative Pcbp1 mRNA expression (E) and fluorescence-activated cell sorting (FACS) analysis and PCBP1 mean fluorescence intensity (MFI) in subsets of in vitro polarized T cells. (E) n = 8; (F) n = 4. PE, phycoerythrin. ( G and H ) Immunoblotting of moesin, PCBP1, FoxP3, and β-actin (G) and quantification for PCBP1 and moesin (H) using splenic mouse CD4 + T cells activated with anti-CD3 and anti-CD28 for 3 days in the absence (Th0) or presence (iT reg ) of TGF-β in vitro. n = 5. ( I and J ) Mouse splenic CD8 + T cells from the same experiments as (G and H). n = 5. ( K and L ) Immunoblotting for phosphorylated and total PCBP1 (K) in Th0 and iT regs and quantification (L). (D) Error bars represent means ± SE and (E, F, H, J, and L) SD; * P

    Article Snippet: Murine lentiviral DNA plasmid against Pcbp1 (hnRNP E1) (sequence: CCG GCC ATG ATC CAA CTG TGT AAT TCT CGA GAA TTA CAC AGT TGG ATC ATG GTT TTT G) (Sigma-Aldrich, TRC1-pLKO.1-puromycin) were transduced into CD4+ T cells activated for 48 hours with anti-CD3 and anti-CD28 with polybrene (8 μg/ml; Sigma-Aldrich).

    Techniques: Expressing, Flow Cytometry, Mouse Assay, Fluorescence, FACS, In Vitro

    Characterization of mice devoid of XBP1 in T cells. a , Deletion efficiency was analyzed by qRT-PCR using a primer set that specifically detects the exon 2 region of Xbp1 . Data were normalized to endogenous expression of Actb and presented as relative expression compared with WT ( n = 8). b , Absolute cell numbers in the thymus, spleen and lymph nodes. c , FACS-based phenotyping of double negative (CD4 - CD8 - ), double positive (CD4 + CD8 + ), or single positive (CD4 + or CD8 + ) thymocytes. d - g, Frequency of TCRβ + cells ( d , f ) and CD4 + or CD8 + cells (gated on TCRβ + cells) ( e , g ) in lymph nodes or spleen. h - i , Expression of CD44 and CD62L on both CD4 + ( h ) and CD8 + ( i ) TCRβ + subsets in the spleen. j , Frequency of splenic TCRβ + CD4 + FoxP3 + T cells. k - l , Frequency of non-T cell populations among total live cells in spleen ( k ) and lymph nodes ( l ). b - g , n = 5; h - j , n = 3; k - l , Xbp1 f/f ( n = 4), Xbp1 f/f Cd4 cre ( n = 5). m , Reconstitution efficiency of CD4 + and CD8 + T cells in bone marrow and spleen from mixed bone marrow chimeras ( n = 3 per chimera type). Chimeras were generated with a mixture of wild-type bone marrow (CD45.1 + ) plus either Xbp1 f/f or Xbp1 f/f Vav1 cre bone marrow (CD45.2 + ). n , Flow cytometry assessing cell proliferation of CD4 + T cells stained with the division-tracking dye (Cell Trace Violet). Cells were left unstimulated or stimulated for 72 and 96 h with plate-bound anti-CD3 (5μg/ml) and soluble anti-CD28 (1μg/ml). Histograms (left) and proliferation index (right) are shown ( n = 4). o , Cell cycle analysis of CD4 + T cells activated for 72 h by staining with propidium iodide. Representative plots from two experiments. p , Transmission electron microscopy of in vitro activated WT versus XBP1-deficient CD4 + T cells. Naïve CD4 + T cells isolated from three biologically independent mice were activated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 48 h. White arrowheads indicate the endoplasmic reticulum (ER); M, Mitochondria; 12000× (left); 50000× (right). Average mitochondrial area of independent cells was estimated using image J software. Xbp1 f/f ( n = 19); Xbp1 f/f Cd4 cre ( n = 29). q , Histogram (left) and quantification (right) for mitochondrial staining (Mitotracker) in in vitro activated CD4 + T cells ( n = 2 from two independent experiments). r, Activated WT versus XBP1-deficient CD4 + T cells were incubated in glucose-containing, -depleted or 2-deoxyglucose (2-DG, 10mM)-treated media for 6 h and PGC1α expression was analyzed by immunoblot. β-ACTIN was used as loading control. Representative plots from two independent experiments. Data are shown as mean ± s.e.m. ( a - n, p ). n values represent biologically independent samples ( a - n, p-q ). Two-tailed Student’s t -tests ( b - l , n , p ); * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: Characterization of mice devoid of XBP1 in T cells. a , Deletion efficiency was analyzed by qRT-PCR using a primer set that specifically detects the exon 2 region of Xbp1 . Data were normalized to endogenous expression of Actb and presented as relative expression compared with WT ( n = 8). b , Absolute cell numbers in the thymus, spleen and lymph nodes. c , FACS-based phenotyping of double negative (CD4 - CD8 - ), double positive (CD4 + CD8 + ), or single positive (CD4 + or CD8 + ) thymocytes. d - g, Frequency of TCRβ + cells ( d , f ) and CD4 + or CD8 + cells (gated on TCRβ + cells) ( e , g ) in lymph nodes or spleen. h - i , Expression of CD44 and CD62L on both CD4 + ( h ) and CD8 + ( i ) TCRβ + subsets in the spleen. j , Frequency of splenic TCRβ + CD4 + FoxP3 + T cells. k - l , Frequency of non-T cell populations among total live cells in spleen ( k ) and lymph nodes ( l ). b - g , n = 5; h - j , n = 3; k - l , Xbp1 f/f ( n = 4), Xbp1 f/f Cd4 cre ( n = 5). m , Reconstitution efficiency of CD4 + and CD8 + T cells in bone marrow and spleen from mixed bone marrow chimeras ( n = 3 per chimera type). Chimeras were generated with a mixture of wild-type bone marrow (CD45.1 + ) plus either Xbp1 f/f or Xbp1 f/f Vav1 cre bone marrow (CD45.2 + ). n , Flow cytometry assessing cell proliferation of CD4 + T cells stained with the division-tracking dye (Cell Trace Violet). Cells were left unstimulated or stimulated for 72 and 96 h with plate-bound anti-CD3 (5μg/ml) and soluble anti-CD28 (1μg/ml). Histograms (left) and proliferation index (right) are shown ( n = 4). o , Cell cycle analysis of CD4 + T cells activated for 72 h by staining with propidium iodide. Representative plots from two experiments. p , Transmission electron microscopy of in vitro activated WT versus XBP1-deficient CD4 + T cells. Naïve CD4 + T cells isolated from three biologically independent mice were activated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 48 h. White arrowheads indicate the endoplasmic reticulum (ER); M, Mitochondria; 12000× (left); 50000× (right). Average mitochondrial area of independent cells was estimated using image J software. Xbp1 f/f ( n = 19); Xbp1 f/f Cd4 cre ( n = 29). q , Histogram (left) and quantification (right) for mitochondrial staining (Mitotracker) in in vitro activated CD4 + T cells ( n = 2 from two independent experiments). r, Activated WT versus XBP1-deficient CD4 + T cells were incubated in glucose-containing, -depleted or 2-deoxyglucose (2-DG, 10mM)-treated media for 6 h and PGC1α expression was analyzed by immunoblot. β-ACTIN was used as loading control. Representative plots from two independent experiments. Data are shown as mean ± s.e.m. ( a - n, p ). n values represent biologically independent samples ( a - n, p-q ). Two-tailed Student’s t -tests ( b - l , n , p ); * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Mouse Assay, Quantitative RT-PCR, Expressing, FACS, Generated, Flow Cytometry, Cytometry, Staining, Cell Cycle Assay, Transmission Assay, Electron Microscopy, In Vitro, Isolation, Software, Incubation, Two Tailed Test

    XBP1 controls the abundance of glutamine transporters in glucose-deprived T cells. a - b , Pre-activated WT or XBP1-deficient CD4 + T cells were incubated in the indicated media for 6 h and then stained on poly-l-lysine coated discs using antibodies specific for ASCT2 (green) or SNAT2 (red). Nuclei are depicted in blue (DAPI staining). a , Representative confocal images of the indicated T cells from three experiments. b , The mean fluorescence intensity (MFI) of each glutamine transporter on ~50 individual cells from three independent slides ( n = 150) was computationally quantified using the Image J software by two independent investigators in a blinded manner. Individual dots depict the average MFI of each independent analysis ( n = 6). c , Naïve splenic CD4 + T cells isolated from WT or XBP1-deficient mice were activated via CD3/CD28 stimulation for 48 h and then incubated in media lacking glucose for 6 h. mRNA expression of genes encoding glutamine transporters was determined by qRT-PCR ( n = 6 from three experiments). Data were normalized to endogenous expression of Actb in each case. d - e , Pre-activated mouse CD4 + T cells were incubated in the indicated media for 6 h in the presence or absence of proteasome inhibitor MG132 (10 μM). d , Protein levels of the glutamine transporter SNAT1 were determined by immunoblot analysis where β-ACTIN was used as loading control. Representative image from five independent experiments. e , Densitometric quantification of SNAT1 ( n = 5). Results are presented as relative expression compared with untreated control T cells incubated in glucose-containing media. Data are shown as mean ± s.e.m ( b , c ). n values represent biologically independent samples ( c , e ). Two-tailed Student’s t -tests ( b ); Two-tailed paired Student’s t -tests ( e ); * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: XBP1 controls the abundance of glutamine transporters in glucose-deprived T cells. a - b , Pre-activated WT or XBP1-deficient CD4 + T cells were incubated in the indicated media for 6 h and then stained on poly-l-lysine coated discs using antibodies specific for ASCT2 (green) or SNAT2 (red). Nuclei are depicted in blue (DAPI staining). a , Representative confocal images of the indicated T cells from three experiments. b , The mean fluorescence intensity (MFI) of each glutamine transporter on ~50 individual cells from three independent slides ( n = 150) was computationally quantified using the Image J software by two independent investigators in a blinded manner. Individual dots depict the average MFI of each independent analysis ( n = 6). c , Naïve splenic CD4 + T cells isolated from WT or XBP1-deficient mice were activated via CD3/CD28 stimulation for 48 h and then incubated in media lacking glucose for 6 h. mRNA expression of genes encoding glutamine transporters was determined by qRT-PCR ( n = 6 from three experiments). Data were normalized to endogenous expression of Actb in each case. d - e , Pre-activated mouse CD4 + T cells were incubated in the indicated media for 6 h in the presence or absence of proteasome inhibitor MG132 (10 μM). d , Protein levels of the glutamine transporter SNAT1 were determined by immunoblot analysis where β-ACTIN was used as loading control. Representative image from five independent experiments. e , Densitometric quantification of SNAT1 ( n = 5). Results are presented as relative expression compared with untreated control T cells incubated in glucose-containing media. Data are shown as mean ± s.e.m ( b , c ). n values represent biologically independent samples ( c , e ). Two-tailed Student’s t -tests ( b ); Two-tailed paired Student’s t -tests ( e ); * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Incubation, Staining, Fluorescence, Software, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Two Tailed Test

    IRE1α-XBP1 activation in human OvCa-infiltrating T cells. a , XBP1 splicing assays for CD4 + or CD8 + T cells isolated from ascites or solid tumors of OvCa patients, or from blood of cancer-free female donors. XBP1s , spliced form; XBP1u , unspliced form. Data were generated from three independent experiments. b , Frequency of spliced XBP1/total XBP1 in T cells sorted from the indicated sources ( n = 8/group). c-e , Pairwise analyses for sorted tumor-associated CD4 + (circles) and CD8 + (squares) T cells ( n = 22 total). c, ER stress response gene expression. d , Proportion of CD45 + CD3 + OvCa-infiltrating T cells versus expression of the indicated genes in T cells from the same specimen. e , IFNG versus ER stress response genes in each sample. n -values correspond to biologically independent samples ( b-e ). One-way ANOVA with Tukey’s post-test, boxes represent median ± interquartile range and whiskers indicate minimum and maximum ( b ); Spearman’s rank correlation test, Spearman coefficient (r) with p-value (two-tailed), 95% confidence intervals (CI) for all correlation analyses ( c-e ) are described in Supplemental Table 2 .

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: IRE1α-XBP1 activation in human OvCa-infiltrating T cells. a , XBP1 splicing assays for CD4 + or CD8 + T cells isolated from ascites or solid tumors of OvCa patients, or from blood of cancer-free female donors. XBP1s , spliced form; XBP1u , unspliced form. Data were generated from three independent experiments. b , Frequency of spliced XBP1/total XBP1 in T cells sorted from the indicated sources ( n = 8/group). c-e , Pairwise analyses for sorted tumor-associated CD4 + (circles) and CD8 + (squares) T cells ( n = 22 total). c, ER stress response gene expression. d , Proportion of CD45 + CD3 + OvCa-infiltrating T cells versus expression of the indicated genes in T cells from the same specimen. e , IFNG versus ER stress response genes in each sample. n -values correspond to biologically independent samples ( b-e ). One-way ANOVA with Tukey’s post-test, boxes represent median ± interquartile range and whiskers indicate minimum and maximum ( b ); Spearman’s rank correlation test, Spearman coefficient (r) with p-value (two-tailed), 95% confidence intervals (CI) for all correlation analyses ( c-e ) are described in Supplemental Table 2 .

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Activation Assay, Isolation, Generated, Expressing, Two Tailed Test

    IRE1α-XBP1 signaling alters OvCa-associated T cell function and promotes malignant progression. a, Transcriptional profiling of splenic CD44 hi CD62L lo CD4 + T cells sorted from naïve WT versus XBP1-deficient mice. Expression of the differentially regulated genes identified in Fig. 4a is shown ( n = 3 per group). b - c and e - f , Analysis of WT versus XBP1-deficient CD4 + T cells isolated from mice bearing metastatic OvCa ( n = 4 per group). b , Expression of previously reported RIDD target genes. c , Relative gene expression of master transcription factors controlling helper T cell differentiation. d , Intracellular staining for FoxP3 (left) and proportion of FoxP3 + CD4 + T cells from WT ( n = 5) and XBP1-deficient ( n = 6) mice bearing metastatic OvCa for 21 days. e , Predicted upstream regulators associated with the transcriptional changes observed. f , Enriched cellular functions in XBP1-deficient CD4 + T cells at tumor sites. Z-scores greater than 2 indicate functions predicted to be significantly increased in XBP1-deficient CD4 + T cells. g , Intracellular staining for IFN-γ in CD45 + CD3 + CD8 + T cells from WT and conditional XBP1-deficient mice bearing metastatic OvCa for 20–23 days. Representative plots from three independent experiments. h - i , Intracellular staining for IFN-γ in CD45 + CD3 + CD4 + T cells ( h ) and for perforin in CD45 + CD3 + CD8 + T cells ( i ) from WT and conditional XBP1-deficient mice bearing metastatic OvCa for 29 days (late stage). Representative plots from two independent experiments. j , In vivo glucose uptake by CD44 hi CD4 + TILs in Xbp1 f/f ( n = 6) or Xbp1 f/f Cd4 cre ( n = 7) female mice bearing metastatic OvCa. k, Representative TMRE staining for OvCa-associated CD45 + TCRβ + CD44 + CD4 + T cells from tumor-bearing Xbp1 f/f ( n = 3) or Xbp1 f/f Cd4 cre ( n = 4) mice. l , Peritoneal wash samples were collected from mice at 24 days after tumor challenge and the proportion of OvCa-associated CD4 + T cells expressing PD-1, CTLA4, and TIM3 in WT ( n = 9) or XBP1-deficient ( n = 8) mice was determined. m , Female mice ( n = 4 per group) were implanted with ID8- Def29/Vegf-A OvCa cells in the flank and tumor growth was monitored over time (left). Tumors were resected at day 34 and final size was confirmed ex vivo (right). n , Ascites accumulation (left) in Ern1 f/f ( n = 5) or Ern1 f/f Cd4 cre ( n = 6) mice bearing ID8- Def29/Vegf-A OvCa and overall survival (right, n = 6 per group). Ern1 is the gene encoding IRE1α. Data are shown as mean ± s.e.m. ( c-d, j-l ). Boxes represent median ± interquartile range and whiskers indicate minimum and maximum ( m , n ). n values represent biologically independent mice ( a-d, j-n ). Two-tailed Student’s t -tests ( d , j - l, m-n ); Log-rank test for survival ( m - n ). * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: IRE1α-XBP1 signaling alters OvCa-associated T cell function and promotes malignant progression. a, Transcriptional profiling of splenic CD44 hi CD62L lo CD4 + T cells sorted from naïve WT versus XBP1-deficient mice. Expression of the differentially regulated genes identified in Fig. 4a is shown ( n = 3 per group). b - c and e - f , Analysis of WT versus XBP1-deficient CD4 + T cells isolated from mice bearing metastatic OvCa ( n = 4 per group). b , Expression of previously reported RIDD target genes. c , Relative gene expression of master transcription factors controlling helper T cell differentiation. d , Intracellular staining for FoxP3 (left) and proportion of FoxP3 + CD4 + T cells from WT ( n = 5) and XBP1-deficient ( n = 6) mice bearing metastatic OvCa for 21 days. e , Predicted upstream regulators associated with the transcriptional changes observed. f , Enriched cellular functions in XBP1-deficient CD4 + T cells at tumor sites. Z-scores greater than 2 indicate functions predicted to be significantly increased in XBP1-deficient CD4 + T cells. g , Intracellular staining for IFN-γ in CD45 + CD3 + CD8 + T cells from WT and conditional XBP1-deficient mice bearing metastatic OvCa for 20–23 days. Representative plots from three independent experiments. h - i , Intracellular staining for IFN-γ in CD45 + CD3 + CD4 + T cells ( h ) and for perforin in CD45 + CD3 + CD8 + T cells ( i ) from WT and conditional XBP1-deficient mice bearing metastatic OvCa for 29 days (late stage). Representative plots from two independent experiments. j , In vivo glucose uptake by CD44 hi CD4 + TILs in Xbp1 f/f ( n = 6) or Xbp1 f/f Cd4 cre ( n = 7) female mice bearing metastatic OvCa. k, Representative TMRE staining for OvCa-associated CD45 + TCRβ + CD44 + CD4 + T cells from tumor-bearing Xbp1 f/f ( n = 3) or Xbp1 f/f Cd4 cre ( n = 4) mice. l , Peritoneal wash samples were collected from mice at 24 days after tumor challenge and the proportion of OvCa-associated CD4 + T cells expressing PD-1, CTLA4, and TIM3 in WT ( n = 9) or XBP1-deficient ( n = 8) mice was determined. m , Female mice ( n = 4 per group) were implanted with ID8- Def29/Vegf-A OvCa cells in the flank and tumor growth was monitored over time (left). Tumors were resected at day 34 and final size was confirmed ex vivo (right). n , Ascites accumulation (left) in Ern1 f/f ( n = 5) or Ern1 f/f Cd4 cre ( n = 6) mice bearing ID8- Def29/Vegf-A OvCa and overall survival (right, n = 6 per group). Ern1 is the gene encoding IRE1α. Data are shown as mean ± s.e.m. ( c-d, j-l ). Boxes represent median ± interquartile range and whiskers indicate minimum and maximum ( m , n ). n values represent biologically independent mice ( a-d, j-n ). Two-tailed Student’s t -tests ( d , j - l, m-n ); Log-rank test for survival ( m - n ). * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Cell Function Assay, Mouse Assay, Expressing, Isolation, Cell Differentiation, Staining, In Vivo, Ex Vivo, Two Tailed Test

    T cell-intrinsic IRE1α-XBP1 signaling promotes OvCa progression. a , Transcriptional profiling of WT versus XBP1-deficient CD4 + T cells sorted from the peritoneal cavity of mice bearing metastatic ID8- Def29/VegfA OvCa for 20 days. Top upregulated genes in XBP1-deficient CD4 + T cells are shown ( n = 4). b , FACS analyses of OvCa-associated CD4 + T cells from the indicated mice bearing metastatic OvCa for 20–23 days. Representative intracellular staining for IFN-γ and IL-17 (left) and global IFN-γ analysis (right) in CD45 + CD3 + CD4 + T cells ( n = 9). c , IFN-γ secretion by CD4 + T cells isolated from the peritoneal cavity of the indicated OvCa-bearing mice upon ex vivo stimulation with OVA peptide ( n = 6 for all groups except for XBP1-deficient hosts challenged with ID8-ova). d , Growth of p53/K-ras-driven ovarian tumors in hosts reconstituted with bone marrow from the indicated genotypes ( n = 6). e , Ascites accumulation (left, n = 5–6) and overall survival (right, n = 8–13) for the indicated mice bearing ID8- Def29/Vegf-A OvCa. f , Imaging (left) and quantification (right) of peritoneal carcinomatosis in Ern1 f/f or Ern1 f/f Cd4 cre mice bearing luciferase-expressing ID8 OvCa for 20 days ( n = 5). g , Survival rates for mice depicted in panel f . n -values represent biologically independent mice ( a-g ). Data are shown as mean ± s.e.m. ( b, c, d, e ). Boxes represent median ± interquartile range and whiskers indicate minimum and maximum ( f ). Two-tailed Student’s t -test ( b ); One-way ANOVA with Tukey’s post-test ( c ); Two-tailed Mann-Whitney test ( d, e, f ); Log-rank test ( e , g ). * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: T cell-intrinsic IRE1α-XBP1 signaling promotes OvCa progression. a , Transcriptional profiling of WT versus XBP1-deficient CD4 + T cells sorted from the peritoneal cavity of mice bearing metastatic ID8- Def29/VegfA OvCa for 20 days. Top upregulated genes in XBP1-deficient CD4 + T cells are shown ( n = 4). b , FACS analyses of OvCa-associated CD4 + T cells from the indicated mice bearing metastatic OvCa for 20–23 days. Representative intracellular staining for IFN-γ and IL-17 (left) and global IFN-γ analysis (right) in CD45 + CD3 + CD4 + T cells ( n = 9). c , IFN-γ secretion by CD4 + T cells isolated from the peritoneal cavity of the indicated OvCa-bearing mice upon ex vivo stimulation with OVA peptide ( n = 6 for all groups except for XBP1-deficient hosts challenged with ID8-ova). d , Growth of p53/K-ras-driven ovarian tumors in hosts reconstituted with bone marrow from the indicated genotypes ( n = 6). e , Ascites accumulation (left, n = 5–6) and overall survival (right, n = 8–13) for the indicated mice bearing ID8- Def29/Vegf-A OvCa. f , Imaging (left) and quantification (right) of peritoneal carcinomatosis in Ern1 f/f or Ern1 f/f Cd4 cre mice bearing luciferase-expressing ID8 OvCa for 20 days ( n = 5). g , Survival rates for mice depicted in panel f . n -values represent biologically independent mice ( a-g ). Data are shown as mean ± s.e.m. ( b, c, d, e ). Boxes represent median ± interquartile range and whiskers indicate minimum and maximum ( f ). Two-tailed Student’s t -test ( b ); One-way ANOVA with Tukey’s post-test ( c ); Two-tailed Mann-Whitney test ( d, e, f ); Log-rank test ( e , g ). * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Mouse Assay, FACS, Staining, Isolation, Ex Vivo, Imaging, Luciferase, Expressing, Two Tailed Test, MANN-WHITNEY

    XBP1 inhibits glutamine influx in response to glucose deprivation. a - b , Naïve splenic CD4 + T cells isolated from WT mice were activated via CD3/CD28 stimulation for 48 h and then incubated for 6 h in the indicated media. a , Expression of ER stress-related gene markers ( n = 4 from two independent experiments). Data are shown as percent response change compared with control in the presence of glucose- and glutamine-containing media. b , Maximal OCR was measured in CD4 + T cells in the presence or absence of glucose, and treated with corresponding media (untreated, n = 5) or inhibitors blocking pyruvate (UK5099, n = 5), glutamine (BPTES, n = 4) or fatty acid (Etomoxir, n = 4) oxidation. Data are presented as percent response change compared with untreated control in the presence of glucose. c - i, Naïve splenic CD4 + T cells isolated from WT (solid bars) or XBP1-deficient (hatched bars) mice were activated via CD3/CD28 stimulation for 48 h, followed by culture in the presence or absence of glucose for 4.5 h, and then pulsed with [U- 13 C]-glutamine for an additional 1.5 h in the same culture condition. Relative abundance of 13 C-labeled metabolites and TCA intermediates including glutamine ( c ), glutamate ( d ), α-ketoglutarate ( e ), succinate ( f ), malate ( g ), citrate ( h ) and aspartate ( i ) was determined by LC-MS/MS. Data were normalized to cell number in all cases and are representative of two independent experiments with n = 2 biologically distinct samples per group. Data are shown as mean ± s.e.m. n values represent biologically independent samples ( a , b ). One-way ANOVA with Bonferroni’s multiple comparisons test ( a ); One-way ANOVA with Tukey’s multiple comparisons test ( b ); * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: XBP1 inhibits glutamine influx in response to glucose deprivation. a - b , Naïve splenic CD4 + T cells isolated from WT mice were activated via CD3/CD28 stimulation for 48 h and then incubated for 6 h in the indicated media. a , Expression of ER stress-related gene markers ( n = 4 from two independent experiments). Data are shown as percent response change compared with control in the presence of glucose- and glutamine-containing media. b , Maximal OCR was measured in CD4 + T cells in the presence or absence of glucose, and treated with corresponding media (untreated, n = 5) or inhibitors blocking pyruvate (UK5099, n = 5), glutamine (BPTES, n = 4) or fatty acid (Etomoxir, n = 4) oxidation. Data are presented as percent response change compared with untreated control in the presence of glucose. c - i, Naïve splenic CD4 + T cells isolated from WT (solid bars) or XBP1-deficient (hatched bars) mice were activated via CD3/CD28 stimulation for 48 h, followed by culture in the presence or absence of glucose for 4.5 h, and then pulsed with [U- 13 C]-glutamine for an additional 1.5 h in the same culture condition. Relative abundance of 13 C-labeled metabolites and TCA intermediates including glutamine ( c ), glutamate ( d ), α-ketoglutarate ( e ), succinate ( f ), malate ( g ), citrate ( h ) and aspartate ( i ) was determined by LC-MS/MS. Data were normalized to cell number in all cases and are representative of two independent experiments with n = 2 biologically distinct samples per group. Data are shown as mean ± s.e.m. n values represent biologically independent samples ( a , b ). One-way ANOVA with Bonferroni’s multiple comparisons test ( a ); One-way ANOVA with Tukey’s multiple comparisons test ( b ); * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Isolation, Mouse Assay, Incubation, Expressing, Blocking Assay, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Reduced glucose uptake and defective N -linked protein glycosylation promotes IRE1α-XBP1 activation in ascites-exposed human CD4 + T cells. a , Human CD4 + T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of OvCa ascites supernatants at the indicated concentrations. XBP1 expression was determined by qRT-PCR (10%, n = 58; 50%, n = 25; 100%, n = 30). Data were normalized to endogenous expression of ACTB in each sample. Results are presented as percent increase in expression compared with untreated controls. b , Histograms for FACS-based XBP1s staining in CD4 + T cells treated as indicated. Tm, tunicamycin; 4μ8C, inhibitor of the IRE1α RNase domain. Data were validated by three independent experiments. c-d, CD4 + T cells were treated with Vitamin E (VitE, 50μM) or vehicle (Ethanol 0.1%) for 1h and then stimulated with anti-CD3/CD28 beads for 16 h in the presence of ascites. Intracellular ROS staining by DCFDA ( c) and XBP1 expression ( d ) ( n = 10). Data are expressed as percent response change compared with untreated controls. e , Glucose concentration in regular culture media (cRPMI) and in seven independent OvCa ascites samples. Each dot represents the mean of two measurements. f , XBP1 expression in the samples described in panel a are displayed based on the final glucose concentration in the culture after addition of ascites. Three independent ascites samples were used: A10 (triangles), A15 (circles), A17 (squares) at three different concentrations (10, 50, and 100%). g , GLUT1 surface expression on the indicated cell types present in OvCa ascites from 6 independent patients was determined by GLUT1.RBD staining ( n = 6). Quantitative analysis (left) and representative histograms (right). h , Glucose uptake and XBP1s protein expression in activated CD4 + T cells exposed to three different ascites samples at 10 and 100% for 16 h ( n = 14). Results are presented as relative to untreated controls. i , CD4 + T cells were activated with anti-CD3/CD28 beads in the presence or absence of ascites for 16 h. Cells were lysed and the enriched glycoprotein fractions were analyzed for N -linked glycosylation events by LC-MS/MS. Total ion chromatograms for N -glycosylation at amino acid 315 in DPP2 are shown. Numbers in blue indicate abundance of each glycan via quantification of the corresponding peak area. Data are representative of two independent experiments with similar results. j , CD4 + T cells were treated with 10mM GlcNAc (N-Acetylglucosamine) for 1 h and stimulated via CD3/CD28 for 16 h in the presence of 10% ascites. Quantitative analyses for XBP1s protein by FACS (left, n = 6) and XBP1 gene expression by qRT-PCR (right, n = 15) are presented as percent response change compared with untreated controls. k . Relative basal and maximal OCR for CD4 + T cells exposed to 10% ascites analyzed in Fig. 2h ( n = 16 total from five independent experiments). Data are expressed as percent response change compared with untreated controls. l - m , CD4 + T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of Tm (tunicamycin) at the indicated concentrations ( n = 3). l, XBP1 expression was determined via qRT-PCR. m, OCR profile of Tm-treated CD4 + T cells are shown as relative to the untreated control. n - o , Relative quantification of basal (left) and maximal (right) OCR ( n ), and ECAR measurements ( o ) in all independent samples analyzed in Fig. 2j ( n = 9 total from three independent experiments). p - q , Relative quantification of basal (left) and maximal (right) OCR ( p ), and ECAR measurements ( q ) for the specimens described in panel Fig. 2k. ( n = 5 total from two independent experiments). Data are presented as relative expression compared with matching controls that were not exposed to ascites. Data are shown as mean ± s.e.m ( a , c-d , f-g , k-m ). n values represent biologically independent samples ( a , c - h , j - q ). One-way ANOVA with Bonferroni’s multiple comparisons test ( a, l ); Two-tailed Student’s t -test ( c , k ); One-way ANOVA with Tukey’s multiple comparisons test ( d, g ); Two-tailed paired Student’s t -test ( j, n-q ); Nonparametric Spearman’s rank correlation test Spearman coefficient (r) with p-value (two-tailed); 95% Confidence Interval (CI) −0.8811 to −0.1626 ( h ). * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: Reduced glucose uptake and defective N -linked protein glycosylation promotes IRE1α-XBP1 activation in ascites-exposed human CD4 + T cells. a , Human CD4 + T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of OvCa ascites supernatants at the indicated concentrations. XBP1 expression was determined by qRT-PCR (10%, n = 58; 50%, n = 25; 100%, n = 30). Data were normalized to endogenous expression of ACTB in each sample. Results are presented as percent increase in expression compared with untreated controls. b , Histograms for FACS-based XBP1s staining in CD4 + T cells treated as indicated. Tm, tunicamycin; 4μ8C, inhibitor of the IRE1α RNase domain. Data were validated by three independent experiments. c-d, CD4 + T cells were treated with Vitamin E (VitE, 50μM) or vehicle (Ethanol 0.1%) for 1h and then stimulated with anti-CD3/CD28 beads for 16 h in the presence of ascites. Intracellular ROS staining by DCFDA ( c) and XBP1 expression ( d ) ( n = 10). Data are expressed as percent response change compared with untreated controls. e , Glucose concentration in regular culture media (cRPMI) and in seven independent OvCa ascites samples. Each dot represents the mean of two measurements. f , XBP1 expression in the samples described in panel a are displayed based on the final glucose concentration in the culture after addition of ascites. Three independent ascites samples were used: A10 (triangles), A15 (circles), A17 (squares) at three different concentrations (10, 50, and 100%). g , GLUT1 surface expression on the indicated cell types present in OvCa ascites from 6 independent patients was determined by GLUT1.RBD staining ( n = 6). Quantitative analysis (left) and representative histograms (right). h , Glucose uptake and XBP1s protein expression in activated CD4 + T cells exposed to three different ascites samples at 10 and 100% for 16 h ( n = 14). Results are presented as relative to untreated controls. i , CD4 + T cells were activated with anti-CD3/CD28 beads in the presence or absence of ascites for 16 h. Cells were lysed and the enriched glycoprotein fractions were analyzed for N -linked glycosylation events by LC-MS/MS. Total ion chromatograms for N -glycosylation at amino acid 315 in DPP2 are shown. Numbers in blue indicate abundance of each glycan via quantification of the corresponding peak area. Data are representative of two independent experiments with similar results. j , CD4 + T cells were treated with 10mM GlcNAc (N-Acetylglucosamine) for 1 h and stimulated via CD3/CD28 for 16 h in the presence of 10% ascites. Quantitative analyses for XBP1s protein by FACS (left, n = 6) and XBP1 gene expression by qRT-PCR (right, n = 15) are presented as percent response change compared with untreated controls. k . Relative basal and maximal OCR for CD4 + T cells exposed to 10% ascites analyzed in Fig. 2h ( n = 16 total from five independent experiments). Data are expressed as percent response change compared with untreated controls. l - m , CD4 + T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of Tm (tunicamycin) at the indicated concentrations ( n = 3). l, XBP1 expression was determined via qRT-PCR. m, OCR profile of Tm-treated CD4 + T cells are shown as relative to the untreated control. n - o , Relative quantification of basal (left) and maximal (right) OCR ( n ), and ECAR measurements ( o ) in all independent samples analyzed in Fig. 2j ( n = 9 total from three independent experiments). p - q , Relative quantification of basal (left) and maximal (right) OCR ( p ), and ECAR measurements ( q ) for the specimens described in panel Fig. 2k. ( n = 5 total from two independent experiments). Data are presented as relative expression compared with matching controls that were not exposed to ascites. Data are shown as mean ± s.e.m ( a , c-d , f-g , k-m ). n values represent biologically independent samples ( a , c - h , j - q ). One-way ANOVA with Bonferroni’s multiple comparisons test ( a, l ); Two-tailed Student’s t -test ( c , k ); One-way ANOVA with Tukey’s multiple comparisons test ( d, g ); Two-tailed paired Student’s t -test ( j, n-q ); Nonparametric Spearman’s rank correlation test Spearman coefficient (r) with p-value (two-tailed); 95% Confidence Interval (CI) −0.8811 to −0.1626 ( h ). * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, FACS, Staining, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Two Tailed Test

    XBP1 limits glutamine influx in glucose-deprived CD4 + T cells. a, c-d, Naïve splenic CD4 + T cells isolated from WT (solid bars) or XBP1-deficient (hatched bars) mice were activated via CD3/CD28 stimulation for 48 h and then incubated for 6 h in the indicated media. a , Maximal OCR of T cells in the presence or absence of glucose ( n = 5). b , Glutamine tracing was performed as described in the methods and relative abundance of total 13 C-labeled metabolites was determined ( n = 4). Immunoblot ( c ) and quantification ( d ) of glutamine transporters in the indicated T cells ( n = 5 total from five independent experiments). OCR profile ( e ) and maximal OCR ( f ) in DMKG-treated WT T cells ( n = 14). Data are presented as relative expression compared with WT T cells incubated in the presence of glucose ( a, d, f ). OCR profile ( g ) and maximal OCR ( h ) for SNAT1-overexpressing human CD4 + T cells exposed to OvCa ascites ( n = 4). Data are shown as relative expression compared with control virus-transduced T cells that were not exposed to ascites. n -values represent biologically independent samples ( a , b , d , f , h ). Data are shown as mean ± s.e.m. One-way ANOVA with Tukey’s post-test ( a, b, d ); Two-tailed paired Student’s t -test ( f, h) ; * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: XBP1 limits glutamine influx in glucose-deprived CD4 + T cells. a, c-d, Naïve splenic CD4 + T cells isolated from WT (solid bars) or XBP1-deficient (hatched bars) mice were activated via CD3/CD28 stimulation for 48 h and then incubated for 6 h in the indicated media. a , Maximal OCR of T cells in the presence or absence of glucose ( n = 5). b , Glutamine tracing was performed as described in the methods and relative abundance of total 13 C-labeled metabolites was determined ( n = 4). Immunoblot ( c ) and quantification ( d ) of glutamine transporters in the indicated T cells ( n = 5 total from five independent experiments). OCR profile ( e ) and maximal OCR ( f ) in DMKG-treated WT T cells ( n = 14). Data are presented as relative expression compared with WT T cells incubated in the presence of glucose ( a, d, f ). OCR profile ( g ) and maximal OCR ( h ) for SNAT1-overexpressing human CD4 + T cells exposed to OvCa ascites ( n = 4). Data are shown as relative expression compared with control virus-transduced T cells that were not exposed to ascites. n -values represent biologically independent samples ( a , b , d , f , h ). Data are shown as mean ± s.e.m. One-way ANOVA with Tukey’s post-test ( a, b, d ); Two-tailed paired Student’s t -test ( f, h) ; * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Isolation, Mouse Assay, Incubation, Labeling, Expressing, Two Tailed Test

    OvCa ascites limits glucose uptake and causes IRE1α/XBP-mediated mitochondrial dysfunction in human CD4 + T cells. a - f , T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of OvCa ascites supernatants at the indicated concentrations. Histograms ( a ) and quantification ( b ) of XBP1s staining ( n = 16); Iso, isotype control. c , SLC2A1 expression was determined via qRT-PCR ( n = 48). Immunoblot and quantification ( d ) of GLUT1 in ascites-exposed CD4 + T cells. Density of GLUT1 was normalized to β-ACTIN, and data are shown as the relative expression compared with the untreated control ( n = 4 for 10% and 50% ascites; n = 2 for 100% ascites, all from two independent experiments). e , Glucose uptake was assessed using 2-NBDG and XBP1 was determined in the same sample. Symbols depict ascites from 3 independent patients tested at increasing concentrations on CD4 + T cells from multiple donors ( n = 37). Baseline ECAR ( f ) and OCR profile ( g ) of CD4 + T cells exposed to ascites ( n = 16). CD4 + T cells were treated with 4μ8C ( h, i ) or GlcNAc ( j ) for 1 h and then stimulated via CD3/CD28 for 16 h in the presence of 10% ascites. h , XBP1s determined by FACS ( n = 7). i , OCR profile in 4μ8C-treated T cells exposed to ascites ( n = 9). j , OCR for GlcNAc-treated T cells exposed to ascites ( n = 5). Data are shown as mean ± s.e.m ( b, c, d, f, g, i, j ). n -values represent biologically independent samples ( b-k ). One-way ANOVA with Tukey’s post-test ( b ); Two-tailed Student’s t -test ( c , f ); One-way ANOVA with Bonferroni’s post-test ( d ). Spearman’s rank correlation test, 95% CI −0.9076 to −0.6760 ( e ); Two-tailed paired Student’s t -test ( h ); * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: OvCa ascites limits glucose uptake and causes IRE1α/XBP-mediated mitochondrial dysfunction in human CD4 + T cells. a - f , T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of OvCa ascites supernatants at the indicated concentrations. Histograms ( a ) and quantification ( b ) of XBP1s staining ( n = 16); Iso, isotype control. c , SLC2A1 expression was determined via qRT-PCR ( n = 48). Immunoblot and quantification ( d ) of GLUT1 in ascites-exposed CD4 + T cells. Density of GLUT1 was normalized to β-ACTIN, and data are shown as the relative expression compared with the untreated control ( n = 4 for 10% and 50% ascites; n = 2 for 100% ascites, all from two independent experiments). e , Glucose uptake was assessed using 2-NBDG and XBP1 was determined in the same sample. Symbols depict ascites from 3 independent patients tested at increasing concentrations on CD4 + T cells from multiple donors ( n = 37). Baseline ECAR ( f ) and OCR profile ( g ) of CD4 + T cells exposed to ascites ( n = 16). CD4 + T cells were treated with 4μ8C ( h, i ) or GlcNAc ( j ) for 1 h and then stimulated via CD3/CD28 for 16 h in the presence of 10% ascites. h , XBP1s determined by FACS ( n = 7). i , OCR profile in 4μ8C-treated T cells exposed to ascites ( n = 9). j , OCR for GlcNAc-treated T cells exposed to ascites ( n = 5). Data are shown as mean ± s.e.m ( b, c, d, f, g, i, j ). n -values represent biologically independent samples ( b-k ). One-way ANOVA with Tukey’s post-test ( b ); Two-tailed Student’s t -test ( c , f ); One-way ANOVA with Bonferroni’s post-test ( d ). Spearman’s rank correlation test, 95% CI −0.9076 to −0.6760 ( e ); Two-tailed paired Student’s t -test ( h ); * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Staining, Expressing, Quantitative RT-PCR, FACS, Two Tailed Test

    Restoring glutamine influx enhances mitochondrial function in ascites-exposed CD4 + T cells. a - b , Immunoblot ( a ) and densitometric quantification ( b ) for ASCT2 and SNAT1 protein levels in human CD4 + T cells exposed to OvCa ascites at the indicated concentrations for 16 h. β-ACTIN was used as loading control. Data are shown as the relative expression compared with untreated (0%) controls. n = 4 for 10% ascites; n = 4 for 50% ascites; n = 2 for 100% ascites. Data were generated from two independent experiments. c-d , Human CD4 + T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of 25% OvCa ascites supernatants, and DMKG (5 mM) was added to the cell culture during the last 4 h of incubation. OCR profile ( c ) and quantification of maximal OCR ( d ). Data are presented as relative expression compared with untreated controls incubated in the absence of ascites ( n = 17 total from two independent experiments). e - f , Human CD4 + T cells activated via CD3/CD28 stimulation and IL-2 (50 U/ml) for 36 h were transduced with GFP-expressing retroviruses harboring no insert (control) or the gene encoding human SNAT1. GFP + cells were sorted 3 days post-transduction and expanded for an additional 48 h in the presence of CD3/CD28 stimulation and IL-2 (50 U/ml). After 20 h of resting, cells were restimulated with CD3/CD28 antibodies in the absence or presence of OvCa ascites supernatants for 16 h. e , Sorting plots showing GFP expression by CD4 + T cells that were left untreated or transduced with either control or SNAT1-expressing viruses. Representative plots from two experiments. f , Relative SLC38A1 expression levels in sorted cells after transduction ( n = 6 total from two independent experiments). n values represent biologically independent samples ( b, d, f ). Data are shown as mean ± s.e.m ( b , c , f ). One-way ANOVA with Bonferroni’s multiple comparisons test ( b ); Two-tailed paired Student’s t -test ( d ); Two-tailed Student’s t -test ( f ). * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: Restoring glutamine influx enhances mitochondrial function in ascites-exposed CD4 + T cells. a - b , Immunoblot ( a ) and densitometric quantification ( b ) for ASCT2 and SNAT1 protein levels in human CD4 + T cells exposed to OvCa ascites at the indicated concentrations for 16 h. β-ACTIN was used as loading control. Data are shown as the relative expression compared with untreated (0%) controls. n = 4 for 10% ascites; n = 4 for 50% ascites; n = 2 for 100% ascites. Data were generated from two independent experiments. c-d , Human CD4 + T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of 25% OvCa ascites supernatants, and DMKG (5 mM) was added to the cell culture during the last 4 h of incubation. OCR profile ( c ) and quantification of maximal OCR ( d ). Data are presented as relative expression compared with untreated controls incubated in the absence of ascites ( n = 17 total from two independent experiments). e - f , Human CD4 + T cells activated via CD3/CD28 stimulation and IL-2 (50 U/ml) for 36 h were transduced with GFP-expressing retroviruses harboring no insert (control) or the gene encoding human SNAT1. GFP + cells were sorted 3 days post-transduction and expanded for an additional 48 h in the presence of CD3/CD28 stimulation and IL-2 (50 U/ml). After 20 h of resting, cells were restimulated with CD3/CD28 antibodies in the absence or presence of OvCa ascites supernatants for 16 h. e , Sorting plots showing GFP expression by CD4 + T cells that were left untreated or transduced with either control or SNAT1-expressing viruses. Representative plots from two experiments. f , Relative SLC38A1 expression levels in sorted cells after transduction ( n = 6 total from two independent experiments). n values represent biologically independent samples ( b, d, f ). Data are shown as mean ± s.e.m ( b , c , f ). One-way ANOVA with Bonferroni’s multiple comparisons test ( b ); Two-tailed paired Student’s t -test ( d ); Two-tailed Student’s t -test ( f ). * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Expressing, Generated, Cell Culture, Incubation, Transduction, Two Tailed Test

    IRE1α-XBP1 regulates IFN-γ production in ascites-exposed human CD4 + T cells. a, IFNG expression in CD4 + T cells receiving CD3/CD28 activation for 16 h under increasing concentrations of OvCa ascites supernatants. 10% ( n = 12); 50% ( n = 3); 100% ( n = 3). b , CD4 + T cells were activated for 12 h, incubated for additional 36 h in the presence of 25% ascites, and IFN-γ in culture supernatants was determined by ELISA. Data were normalized to final viable cell counts in each case. n = 11 independent responder CD4 + T cells in all cases with the exception of A14 ( n = 9), A15 ( n = 10) and A17 ( n = 3). c-h , Pre-activated CD4 + T cells were treated with 4μ8C for 2 h, and 25% ascites was subsequently added to the culture for additional 12 h. c , IFN-γ in culture supernatants was quantified by ELISA ( n = 14). Data are presented as relative expression compared with matching controls that were not exposed to ascites. d , FACS histogram (left) and quantitative analysis (right) for intracellular IFN-γ in CD4 + T cells ( n = 16). e , Maximal OCR presented as percent response change compared with untreated controls ( n = 7). f , The frequency of viable cells among total cells was determined by staining with dead cell exclusion dye ( n = 12). The frequency of T-bet + ( g ) and RORγt + ( h ) populations among CD4 + T cells was determined by intracellular staining and presented as relative expression compared with ascites-untreated controls ( n = 12). i , IFNG expression by SNAT1-overexpressing human CD4 + T cells exposed to 10% OvCa ascites ( n = 6 from two independent experiments) or incubated in glucose-free media ( n = 3 from two independent experiments). Data were normalized to endogenous expression of GAPDH in each sample. Data are presented as relative expression compared with control virus-transduced T cells that were not exposed to ascites or glucose deprived. Data are shown as mean ± s.e.m. ( a-c, f-i ). n values represent biologically independent samples ( a - i ). One-way ANOVA with Bonferroni’s multiple comparisons test ( a , b ); One-way ANOVA with Tukey’s multiple comparisons test ( c , f , g , h ); Two-tailed paired Student’s t -tests ( d , e ); Two-tailed Student’s t -tests ( i ); * P

    Journal: Nature

    Article Title: IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

    doi: 10.1038/s41586-018-0597-x

    Figure Lengend Snippet: IRE1α-XBP1 regulates IFN-γ production in ascites-exposed human CD4 + T cells. a, IFNG expression in CD4 + T cells receiving CD3/CD28 activation for 16 h under increasing concentrations of OvCa ascites supernatants. 10% ( n = 12); 50% ( n = 3); 100% ( n = 3). b , CD4 + T cells were activated for 12 h, incubated for additional 36 h in the presence of 25% ascites, and IFN-γ in culture supernatants was determined by ELISA. Data were normalized to final viable cell counts in each case. n = 11 independent responder CD4 + T cells in all cases with the exception of A14 ( n = 9), A15 ( n = 10) and A17 ( n = 3). c-h , Pre-activated CD4 + T cells were treated with 4μ8C for 2 h, and 25% ascites was subsequently added to the culture for additional 12 h. c , IFN-γ in culture supernatants was quantified by ELISA ( n = 14). Data are presented as relative expression compared with matching controls that were not exposed to ascites. d , FACS histogram (left) and quantitative analysis (right) for intracellular IFN-γ in CD4 + T cells ( n = 16). e , Maximal OCR presented as percent response change compared with untreated controls ( n = 7). f , The frequency of viable cells among total cells was determined by staining with dead cell exclusion dye ( n = 12). The frequency of T-bet + ( g ) and RORγt + ( h ) populations among CD4 + T cells was determined by intracellular staining and presented as relative expression compared with ascites-untreated controls ( n = 12). i , IFNG expression by SNAT1-overexpressing human CD4 + T cells exposed to 10% OvCa ascites ( n = 6 from two independent experiments) or incubated in glucose-free media ( n = 3 from two independent experiments). Data were normalized to endogenous expression of GAPDH in each sample. Data are presented as relative expression compared with control virus-transduced T cells that were not exposed to ascites or glucose deprived. Data are shown as mean ± s.e.m. ( a-c, f-i ). n values represent biologically independent samples ( a - i ). One-way ANOVA with Bonferroni’s multiple comparisons test ( a , b ); One-way ANOVA with Tukey’s multiple comparisons test ( c , f , g , h ); Two-tailed paired Student’s t -tests ( d , e ); Two-tailed Student’s t -tests ( i ); * P

    Article Snippet: To stain human cells we utilized: anti-CD45 (HI30), anti-CD3 (HIT3), anti-CD4 (A161A1), anti-CD8 (HIT8a), anti-CD20 (2H7), anti-CD14 (63D3), anti-IFN-γ (4S.B3), anti-T-bet (4B10), TruStain FcX solution (422302); anti-XBP1s (Q3–695; BD Pharmingen); anti-RORγt (AFKJS-9, eBioscience).

    Techniques: Expressing, Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, FACS, Staining, Two Tailed Test

    Serum C-peptide levels of the mice at 48 h after anti-CD3 or isotype IgG treatment and their levels after IPGTT. Serum samples were collected from the mice in Figure 3 before IPGTT and collected again after IPGTT. ELISA was used to measure C-peptide levels of all mice in both groups. Compared to controls, C-peptide levels of anti-CD3 treated mice were significantly lower before and after IPGTT (* P

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Serum C-peptide levels of the mice at 48 h after anti-CD3 or isotype IgG treatment and their levels after IPGTT. Serum samples were collected from the mice in Figure 3 before IPGTT and collected again after IPGTT. ELISA was used to measure C-peptide levels of all mice in both groups. Compared to controls, C-peptide levels of anti-CD3 treated mice were significantly lower before and after IPGTT (* P

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Serum cytokines in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice were treated with anti-CD3 (4 mice) or isotype IgG (6 mice). Six hours later, serum samples were collected for cytokine measurement. The cytokines, IFN- γ , TNF- α , IL-1, and IL-6, were examined by Luminex and the statistic analysis data were shown in the figure.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Serum cytokines in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice were treated with anti-CD3 (4 mice) or isotype IgG (6 mice). Six hours later, serum samples were collected for cytokine measurement. The cytokines, IFN- γ , TNF- α , IL-1, and IL-6, were examined by Luminex and the statistic analysis data were shown in the figure.

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Mouse Assay, Luminex

    Effect of anti-CD3 treatment on blood glucose of normal strain of mice. C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) or isotype IgG (50 μ g/mouse). Four mice were included in each group. Blood glucose levels were measured at different time points as depicted in the figure. The blood glucose changes over 48 h observation were shown.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Effect of anti-CD3 treatment on blood glucose of normal strain of mice. C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) or isotype IgG (50 μ g/mouse). Four mice were included in each group. Blood glucose levels were measured at different time points as depicted in the figure. The blood glucose changes over 48 h observation were shown.

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Mouse Assay, Injection

    Anti-CD3 treatment enhances glucose intracellular transportation by the activated lymphocytes. (a) Freshly prepared spleen cells (1 × 10 7 /well) were cultured in a 24-well plate in 1 mL medium containing 10% fetal bovine serum with glucose concentration of 360 mg/dL. Anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) was added to the cultures. The culture wells were quadruplicated and incubated for 24 h. Glucose concentrations were measured before and 24 h after incubation. The results were reproduced in additional two independent experiments. (b) Freshly prepared spleen cells were stimulated with anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) for 3 hours; thereafter, different concentrations of 2-NBDG as indicated were added to the cultures for additional 2 hours. The fluorescent intensity of the cells was examined by BioTec plate reader using a laser filter with 480 nm excitation and 528 nm emission. The values of between anti-CD3 and isotype IgG were compared for each concentration of 2-NBDG (* P

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Anti-CD3 treatment enhances glucose intracellular transportation by the activated lymphocytes. (a) Freshly prepared spleen cells (1 × 10 7 /well) were cultured in a 24-well plate in 1 mL medium containing 10% fetal bovine serum with glucose concentration of 360 mg/dL. Anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) was added to the cultures. The culture wells were quadruplicated and incubated for 24 h. Glucose concentrations were measured before and 24 h after incubation. The results were reproduced in additional two independent experiments. (b) Freshly prepared spleen cells were stimulated with anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) for 3 hours; thereafter, different concentrations of 2-NBDG as indicated were added to the cultures for additional 2 hours. The fluorescent intensity of the cells was examined by BioTec plate reader using a laser filter with 480 nm excitation and 528 nm emission. The values of between anti-CD3 and isotype IgG were compared for each concentration of 2-NBDG (* P

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Cell Culture, Concentration Assay, Incubation

    Effect of anti-CD3 treatment on blood glucose in NOD-Rag −/− mice. T cell deficient NOD-Rag −/− mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 3 mice) and isotype IgG (50 μ g/mouse for 2 mice). Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Effect of anti-CD3 treatment on blood glucose in NOD-Rag −/− mice. T cell deficient NOD-Rag −/− mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 3 mice) and isotype IgG (50 μ g/mouse for 2 mice). Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Mouse Assay, Injection

    Effect of anti-CD3 antibody treatment on T and B cell activation in vitro and in vivo . (a) In vitro experiment . Spleen cells 1 × 10 6 /well in 24-well plate were stimulated with anti-CD3 antibodies (3 μ g/mL) or Isotype IgG (3 μ g/mL) for 24 hours. Thereafter, The cells were harvested and stained with CD4, CD8, B220, and CD69 and analyzed by flow cytometry. (b) In vivo experiment . C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibodies (50 μ g/mouse) or Isotype IgG (50 μ g/mouse). Three mice were included in each group. Twenty-four hours later, all mice were killed, and spleen cells of each individual mouse were prepared and stained for CD4, CD8, B220, and CD69. The expression of these markers was examined by flow cytometry.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Effect of anti-CD3 antibody treatment on T and B cell activation in vitro and in vivo . (a) In vitro experiment . Spleen cells 1 × 10 6 /well in 24-well plate were stimulated with anti-CD3 antibodies (3 μ g/mL) or Isotype IgG (3 μ g/mL) for 24 hours. Thereafter, The cells were harvested and stained with CD4, CD8, B220, and CD69 and analyzed by flow cytometry. (b) In vivo experiment . C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibodies (50 μ g/mouse) or Isotype IgG (50 μ g/mouse). Three mice were included in each group. Twenty-four hours later, all mice were killed, and spleen cells of each individual mouse were prepared and stained for CD4, CD8, B220, and CD69. The expression of these markers was examined by flow cytometry.

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Activation Assay, In Vitro, In Vivo, Staining, Flow Cytometry, Cytometry, Mouse Assay, Injection, Expressing

    Intraperitoneal glucose tolerance test (IPGTT) in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice under the treatment of anti-CD3 antibody or isotype IgG as described elsewhere for 48 h received intraperitoneal injection of glucose (1 g/kg body weight). Thereafter, the blood glucose was monitored at 10, 30, 60, and 120 min. The glucose level before glucose injection served as the baseline level for each mouse. The results of all animals at different time points were depicted individually.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Intraperitoneal glucose tolerance test (IPGTT) in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice under the treatment of anti-CD3 antibody or isotype IgG as described elsewhere for 48 h received intraperitoneal injection of glucose (1 g/kg body weight). Thereafter, the blood glucose was monitored at 10, 30, 60, and 120 min. The glucose level before glucose injection served as the baseline level for each mouse. The results of all animals at different time points were depicted individually.

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Mouse Assay, Injection

    Effect of anti-CD3 treatment on blood glucose of NOD mice with new onset disease. NOD mice with blood glucose over 200 mg/dL for two consecutive days were treated with intraperitoneal injection of anti-CD3 (50 μ g/mouse). Thereafter, blood glucose was monitored daily for 7 days. Eight diabetic NOD mice were included in this experiment.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Effect of anti-CD3 treatment on blood glucose of NOD mice with new onset disease. NOD mice with blood glucose over 200 mg/dL for two consecutive days were treated with intraperitoneal injection of anti-CD3 (50 μ g/mouse). Thereafter, blood glucose was monitored daily for 7 days. Eight diabetic NOD mice were included in this experiment.

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Mouse Assay, Injection

    The influence of cytokines on blood glucose. (a) C57BL/6 mice were treated with intraperitoneal injection of a high dose of IFN- γ (200 ng/mouse) or PBS. Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (b) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IFN- γ antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (c) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IL-6 antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (d) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 8 mice) or isotype IgG (50 μ g/mouse for 4 mice). Anti-CD3 group was further divided into two subgroups with one group (4 mice) receiving anti-TNF- α (50 μ g/mouse) and the other group (4 mice) receiving isotype IgG (50 μ g/mouse) at 0 and 24 h time points. Additionally, two control mice without any treatment were included in this experiment. Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: The influence of cytokines on blood glucose. (a) C57BL/6 mice were treated with intraperitoneal injection of a high dose of IFN- γ (200 ng/mouse) or PBS. Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (b) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IFN- γ antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (c) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IL-6 antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (d) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 8 mice) or isotype IgG (50 μ g/mouse for 4 mice). Anti-CD3 group was further divided into two subgroups with one group (4 mice) receiving anti-TNF- α (50 μ g/mouse) and the other group (4 mice) receiving isotype IgG (50 μ g/mouse) at 0 and 24 h time points. Additionally, two control mice without any treatment were included in this experiment. Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Article Snippet: Administration of Anti-CD3 Antibodies and Dynamic Observation of Blood Glucose Anti-CD3 antibodies (clone: 145-2C11, purchased from BD Bioscience) were diluted in PBS (1 μ g/μ L).

    Techniques: Mouse Assay, Injection

    17-DMAG induces Hsp70 expression. Analysis of Hsp70 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Hsp70 protein expression was analyzed in cell lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG induces Hsp70 expression. Analysis of Hsp70 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Hsp70 protein expression was analyzed in cell lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Expressing, Protein Concentration

    17-DMAG reduces frequencies of proinflammatory Th1 and Th17 cell subpopulations. PBMCs were stimulated with 1 μg/ml of plate-bound anti-CD3 antibody in absence or presence of 17-DMAG (0.1 μM) for 72 hours. GolgiStop™, PMA, and ionomycin were added in the last 5 hours of culture before permeabilization. Cells were washed, stained with anti-CD4, anti-IFN-γ, and anti-IL-17 antibodies, and analyzed by flow cytometry. The numbers in upper right quadrants of the representative dot blots show the percentage of positive cells of each CD4 + T cell subpopulation. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG reduces frequencies of proinflammatory Th1 and Th17 cell subpopulations. PBMCs were stimulated with 1 μg/ml of plate-bound anti-CD3 antibody in absence or presence of 17-DMAG (0.1 μM) for 72 hours. GolgiStop™, PMA, and ionomycin were added in the last 5 hours of culture before permeabilization. Cells were washed, stained with anti-CD4, anti-IFN-γ, and anti-IL-17 antibodies, and analyzed by flow cytometry. The numbers in upper right quadrants of the representative dot blots show the percentage of positive cells of each CD4 + T cell subpopulation. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Staining, Flow Cytometry, Cytometry

    17-DMAG arrests secretion of proinflammatory cytokines. ELISA-based evaluation of the cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, TNF-α, G-CSF, and TGF-β1 secreted into the culture medium by PBMCs which were stimulated with 1 μg/ml plate-bound anti-CD3 antibody and treated without or with 17-DMAG (0.1 μM) for 72 hours. The results are presented as mean values ± SEM of n = 3 healthy blood donors. The dotted line represents the mean detection limit of the assay. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG arrests secretion of proinflammatory cytokines. ELISA-based evaluation of the cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, TNF-α, G-CSF, and TGF-β1 secreted into the culture medium by PBMCs which were stimulated with 1 μg/ml plate-bound anti-CD3 antibody and treated without or with 17-DMAG (0.1 μM) for 72 hours. The results are presented as mean values ± SEM of n = 3 healthy blood donors. The dotted line represents the mean detection limit of the assay. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Enzyme-linked Immunosorbent Assay

    17-DMAG blunts NF κ B p65 activity. Analysis of NFκB p65 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. NFκB p65 activity and protein expression was analyzed in lysates of these cell cultures by ELISA and immunoblotting, respectively. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG blunts NF κ B p65 activity. Analysis of NFκB p65 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. NFκB p65 activity and protein expression was analyzed in lysates of these cell cultures by ELISA and immunoblotting, respectively. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Protein Concentration

    17-DMAG disrupts Lck activation. Analysis of Lck in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Lck phosphorylation at Tyr 394 position was analyzed in lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG disrupts Lck activation. Analysis of Lck in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Lck phosphorylation at Tyr 394 position was analyzed in lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Activation Assay, Protein Concentration

    17-DMAG inhibits proliferation of T cells. A Proliferation response of human PBMCs stimulated by plate-bound anti-CD3 antibody (1 μg/ml) in absence and presence of 17-DMAG. Cell cultures were exposed to different concentrations of 17-DMAG and proliferation was assayed by ELISA after BrdU incorporation on the 6th day of treatment followed by 24 hours incubation. B LDH-based cytotoxicity ELISA measurement in culture medium after 24-hour incubation of PBMCs with different concentrations of 17-DMAG. LDH release from cells lysed with 1% Triton X-100 was regarded as 100%. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG inhibits proliferation of T cells. A Proliferation response of human PBMCs stimulated by plate-bound anti-CD3 antibody (1 μg/ml) in absence and presence of 17-DMAG. Cell cultures were exposed to different concentrations of 17-DMAG and proliferation was assayed by ELISA after BrdU incorporation on the 6th day of treatment followed by 24 hours incubation. B LDH-based cytotoxicity ELISA measurement in culture medium after 24-hour incubation of PBMCs with different concentrations of 17-DMAG. LDH release from cells lysed with 1% Triton X-100 was regarded as 100%. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay, Incubation

    Effect of shikonin on CD25, CD69, and CD71 expression on human T lymphocytes. Human T lymphocytes (10 6 ) were pretreated with shikonin for 2 h and then stimulated by PMA (20 ng/mL)/ionomycin (1 μ M) (P/I) for 48 h or 72 h, respectively. The cells were double stained with PE-CD3 and FITC-CD69 (a), PE-CD3 and FITC-CD25 (b), PE-CD3 or FITC-CD71 (c) antibodies and then analyzed by flow cytometry. The unstimulated cells were served as negative control. Values represent percentages of the double stained cells.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Shikonin Suppresses Human T Lymphocyte Activation through Inhibition of IKKβ Activity and JNK Phosphorylation

    doi: 10.1155/2013/379536

    Figure Lengend Snippet: Effect of shikonin on CD25, CD69, and CD71 expression on human T lymphocytes. Human T lymphocytes (10 6 ) were pretreated with shikonin for 2 h and then stimulated by PMA (20 ng/mL)/ionomycin (1 μ M) (P/I) for 48 h or 72 h, respectively. The cells were double stained with PE-CD3 and FITC-CD69 (a), PE-CD3 and FITC-CD25 (b), PE-CD3 or FITC-CD71 (c) antibodies and then analyzed by flow cytometry. The unstimulated cells were served as negative control. Values represent percentages of the double stained cells.

    Article Snippet: Cells were then incubated with specific antibodies in the combination of anti-CD69-FITC and anti-CD3-PE, anti-CD25-FITC and anti-CD3-PE, or anti-CD71-FITC and anti-CD3-PE (BD Pharmingen, San Diego, CA, USA), stained for 30 min at room temperature in the dark, and then fixed with 4% PFA paraformaldehyde.

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Negative Control

    RASGRP2 Is Expressed in Cortical Neurons and Shows Reactivity for Memory T Cells in MS Patients (A) First column: HE and RASGRP2 immunohistochemical staining in the occipital lobe of a 53-year-old male patient. Scale bar: 1 mm. Second and third column: RASGRP2 reactivity is present in a punctate pattern (yellow asterisk) in the neuropil of the cortex (Cx) and diffusely throughout the cytoplasm of scattered neuronal cells (black arrow), whereas the white matter (WM) remains negative. Lx, leptomeninges. Scale bars: 100 µm and 10 µm. (B and C) Lymphocyte composition (B) and AP (C) before and after CD45RA depletion of PBMCs (mean ± SEM; Mann-Whitney U test). Proliferation was assessed by thymidine incorporation. (D) Proliferative responses of CD45RA-depleted PBMCs (NAT; n = 8) and TCC14 to overlapping 15-mer RASGRP2 peptide pools, covering the sequence of RASGRP2 from N (pool 1) to C terminus (pool 9), to the recombinant RASGRP2 protein, vehicle control (DMSO), and α-CD2/CD3/CD28 stimulation after 7 days. Dots represent replicate wells (NAT: mean ± SEM; TCC14: mean ± SEM of 6 replicate wells). Stimulatory index (SI) = ratio of the response of a given well to the mean response of the vehicle control. A SI > 2 is considered as positive response. See also Figure S7 and Table S7 .

    Journal: Cell

    Article Title: Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis

    doi: 10.1016/j.cell.2018.08.011

    Figure Lengend Snippet: RASGRP2 Is Expressed in Cortical Neurons and Shows Reactivity for Memory T Cells in MS Patients (A) First column: HE and RASGRP2 immunohistochemical staining in the occipital lobe of a 53-year-old male patient. Scale bar: 1 mm. Second and third column: RASGRP2 reactivity is present in a punctate pattern (yellow asterisk) in the neuropil of the cortex (Cx) and diffusely throughout the cytoplasm of scattered neuronal cells (black arrow), whereas the white matter (WM) remains negative. Lx, leptomeninges. Scale bars: 100 µm and 10 µm. (B and C) Lymphocyte composition (B) and AP (C) before and after CD45RA depletion of PBMCs (mean ± SEM; Mann-Whitney U test). Proliferation was assessed by thymidine incorporation. (D) Proliferative responses of CD45RA-depleted PBMCs (NAT; n = 8) and TCC14 to overlapping 15-mer RASGRP2 peptide pools, covering the sequence of RASGRP2 from N (pool 1) to C terminus (pool 9), to the recombinant RASGRP2 protein, vehicle control (DMSO), and α-CD2/CD3/CD28 stimulation after 7 days. Dots represent replicate wells (NAT: mean ± SEM; TCC14: mean ± SEM of 6 replicate wells). Stimulatory index (SI) = ratio of the response of a given well to the mean response of the vehicle control. A SI > 2 is considered as positive response. See also Figure S7 and Table S7 .

    Article Snippet: RNA sequencing For RNA sequencing analysis of non-proliferating (CFSEhi ) and AP (CFSEdim ) CD3+ CD4+ and CD19+ cells, we labeled 5x107 CFSE-labeled PBMCs of RRMS (REM) patients at day 7 of unstimulated in vitro culture with Live/Dead® Aqua and antibodies against CD3, CD4, CD8 and CD19 ( ) and subsequently sorted live CFSEhi and CFSEdim cells of CD3+ CD4+ and CD19+ subsets using a FACSAria III (BD Biosciences).

    Techniques: Mass Spectrometry, Immunohistochemistry, Staining, MANN-WHITNEY, Sequencing, Recombinant

    Th1-Th17 Reactivity to RASGRP2 Is Influenced by AP and Increases Stepwise from HD to REM to NAT, Related to Figures 1 , 2 , and 7 and Table S7 (A) Fluorospot images in PBMCs with cytokine reactivity (IFN-γ and/or IL-17) to the RASGRP2 peptide pool 1, as compared to the vehicle control. The positive control with anti-CD3 stimulation is included for comparison. (B) Fluorospot-based cytokine reactivity in PBMCs from HD (n = 11), REM (n = 9) or RRMS patients treated with NAT (n = 20). Number of IFN-γ + , IL17 + and IFN-γ + IL17 + spots is shown for the negative control (vehicle) and upon stimulation with RASGRP2 peptide pools, covering the sequence of RASGRP2 from N- (pool 1) to C terminus (pool 9). Each condition was performed for each donor in duplicate wells. The dots represent the response in a given well. Mean ± SEM is shown with red bars while the red dotted line depicts the mean + 3x standard deviation of the vehicle control. The latter is considered as threshold for positive responses. (C) The results from fluorospot were compared to the degree of AP measured by the CFSE assay in the same individuals (incl. HD and REM; n = 16). Number of IFN-γ + , IL17 + and IFN-γ + IL17 + spots is shown for the negative control (vehicle) and upon stimulation with RASGRP2 peptide pools. Orange dots correspond to samples with low AP (

    Journal: Cell

    Article Title: Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis

    doi: 10.1016/j.cell.2018.08.011

    Figure Lengend Snippet: Th1-Th17 Reactivity to RASGRP2 Is Influenced by AP and Increases Stepwise from HD to REM to NAT, Related to Figures 1 , 2 , and 7 and Table S7 (A) Fluorospot images in PBMCs with cytokine reactivity (IFN-γ and/or IL-17) to the RASGRP2 peptide pool 1, as compared to the vehicle control. The positive control with anti-CD3 stimulation is included for comparison. (B) Fluorospot-based cytokine reactivity in PBMCs from HD (n = 11), REM (n = 9) or RRMS patients treated with NAT (n = 20). Number of IFN-γ + , IL17 + and IFN-γ + IL17 + spots is shown for the negative control (vehicle) and upon stimulation with RASGRP2 peptide pools, covering the sequence of RASGRP2 from N- (pool 1) to C terminus (pool 9). Each condition was performed for each donor in duplicate wells. The dots represent the response in a given well. Mean ± SEM is shown with red bars while the red dotted line depicts the mean + 3x standard deviation of the vehicle control. The latter is considered as threshold for positive responses. (C) The results from fluorospot were compared to the degree of AP measured by the CFSE assay in the same individuals (incl. HD and REM; n = 16). Number of IFN-γ + , IL17 + and IFN-γ + IL17 + spots is shown for the negative control (vehicle) and upon stimulation with RASGRP2 peptide pools. Orange dots correspond to samples with low AP (

    Article Snippet: RNA sequencing For RNA sequencing analysis of non-proliferating (CFSEhi ) and AP (CFSEdim ) CD3+ CD4+ and CD19+ cells, we labeled 5x107 CFSE-labeled PBMCs of RRMS (REM) patients at day 7 of unstimulated in vitro culture with Live/Dead® Aqua and antibodies against CD3, CD4, CD8 and CD19 ( ) and subsequently sorted live CFSEhi and CFSEdim cells of CD3+ CD4+ and CD19+ subsets using a FACSAria III (BD Biosciences).

    Techniques: Positive Control, Negative Control, Sequencing, Standard Deviation, CFSE Assay

    Effector Memory T Cells Enrich in the Autoproliferating Compartment of RRMS Patients in Remission Independent of the Rounds of Cell Divisions, Related to Figure 1 and Table S1 (A) Cell proliferation measured by CFSE-labeling (CFSE dim ) was compared to thymidine incorporation assay using the same donors and equal amounts of PBMCs and incubating for 7 days in serum-free medium and absence of exogenous stimulus (Spearman’s rank correlation test). (B and C) Gating strategy of CFSE-labeled cells after 7 days of AP, analyzed by flow cytometry. (B) Gating on singlets, live- and then on CFSE dim cells including all cell divisions (div.), CFSE mid including 1-2 divisions, CFSE low including more than 2 divisions, or non-proliferating resting cells (CFSE hi ). (C) Exemplary cell subset phenotyping of the CFSE dim , CFSE mid , CFSE low and CFSE hi cell compartment showing increasing HLA-DR expression with increasing proliferation. (D) Frequency of AP T cells (CFSE dim CD3 + ) for each HD (n = 32), untreated RRMS in relapse (REL; n = 18) and RRMS in remission (REM; n = 32) (mean ± SEM; Kruskal-Wallis test). (E) CD4/CD8 ratio in the CFSE dim CD3 + compartment of HD (n = 32) and REM (n = 32) (whiskers: min - max). Dotted line at ratio of 1 indicates equal distribution of CD4 + and CD8 + T cells. (F and G) Ratio of naive/memory cell subset frequency in CFSE dim versus CFSE hi compartment of (F) CD4 + and (G) CD8 + T cells respectively is shown for HD (n = 15) and REM (n = 24) (whiskers: min - max; Mann-Whitney U test). A ratio of 1 (dotted line) indicates an equal proportion of the appropriate T cell subset in the CFSE dim and CFSE hi compartment. T cell subsets: T naive CD45RA + CCR7 + ; T CM CD45RA - CCR7 + ; T EM CD45RA - CCR7 - ; T TEMRA CD45RA + CCR7 - .

    Journal: Cell

    Article Title: Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis

    doi: 10.1016/j.cell.2018.08.011

    Figure Lengend Snippet: Effector Memory T Cells Enrich in the Autoproliferating Compartment of RRMS Patients in Remission Independent of the Rounds of Cell Divisions, Related to Figure 1 and Table S1 (A) Cell proliferation measured by CFSE-labeling (CFSE dim ) was compared to thymidine incorporation assay using the same donors and equal amounts of PBMCs and incubating for 7 days in serum-free medium and absence of exogenous stimulus (Spearman’s rank correlation test). (B and C) Gating strategy of CFSE-labeled cells after 7 days of AP, analyzed by flow cytometry. (B) Gating on singlets, live- and then on CFSE dim cells including all cell divisions (div.), CFSE mid including 1-2 divisions, CFSE low including more than 2 divisions, or non-proliferating resting cells (CFSE hi ). (C) Exemplary cell subset phenotyping of the CFSE dim , CFSE mid , CFSE low and CFSE hi cell compartment showing increasing HLA-DR expression with increasing proliferation. (D) Frequency of AP T cells (CFSE dim CD3 + ) for each HD (n = 32), untreated RRMS in relapse (REL; n = 18) and RRMS in remission (REM; n = 32) (mean ± SEM; Kruskal-Wallis test). (E) CD4/CD8 ratio in the CFSE dim CD3 + compartment of HD (n = 32) and REM (n = 32) (whiskers: min - max). Dotted line at ratio of 1 indicates equal distribution of CD4 + and CD8 + T cells. (F and G) Ratio of naive/memory cell subset frequency in CFSE dim versus CFSE hi compartment of (F) CD4 + and (G) CD8 + T cells respectively is shown for HD (n = 15) and REM (n = 24) (whiskers: min - max; Mann-Whitney U test). A ratio of 1 (dotted line) indicates an equal proportion of the appropriate T cell subset in the CFSE dim and CFSE hi compartment. T cell subsets: T naive CD45RA + CCR7 + ; T CM CD45RA - CCR7 + ; T EM CD45RA - CCR7 - ; T TEMRA CD45RA + CCR7 - .

    Article Snippet: RNA sequencing For RNA sequencing analysis of non-proliferating (CFSEhi ) and AP (CFSEdim ) CD3+ CD4+ and CD19+ cells, we labeled 5x107 CFSE-labeled PBMCs of RRMS (REM) patients at day 7 of unstimulated in vitro culture with Live/Dead® Aqua and antibodies against CD3, CD4, CD8 and CD19 ( ) and subsequently sorted live CFSEhi and CFSEdim cells of CD3+ CD4+ and CD19+ subsets using a FACSAria III (BD Biosciences).

    Techniques: Labeling, Thymidine Incorporation Assay, Flow Cytometry, Cytometry, Hi-C, Expressing, MANN-WHITNEY

    B Cells Participate in AP and Maintain and Drive AP T Helper Cells in MS, Related to Figure 3 and Table S1 (A) Proportion of CD3 + and CD19 + cells (pie charts) in the CFSE dim compartment of HD (n = 14; for PHA n = 10) and REM (n = 14; for PHA n = 12) after AP (unstimulated) and reactivities to conventional B- (α-IgM) or T cell stimulation with polyclonal/broad (MLR, PHA) or antigen-specific (TTx) activation using CFSE-labeling and subsequent co-culture for 7 days in serum-free medium. (B–D) Sorted and subsequently expanded (PHA, IL-2) AP cells (CFSE dim ) from DR15 + HD and REM were tested for their reactivity on autologous EBV-transformed B cells. For the co-culture experiments, we incubated 2x10 5 CFSE-labeled expanded bulk T cells either alone or with 5x10 5 irradiated (300 Gy) autologous EBV-transformed B cells in the absence of any stimulus or the presence of blocking HLA-DR (L243) antibody or PHA, respectively. After 72 hours, 4 replicate wells of each condition were pooled and analyzed for survival (live-dead marker) and proliferation (CFSE) by flow cytometry. Cells were gated first on CD4 + T cells to exclude B cells, prior to analysis of live cells and then on proliferating cells. (B) Exemplary dot plots of the different conditions shown for one HD and one REM. (C) Survival and (D) proliferation results of DR15 + HD (n = 5) and REM (n = 5) (mean ± SEM).

    Journal: Cell

    Article Title: Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis

    doi: 10.1016/j.cell.2018.08.011

    Figure Lengend Snippet: B Cells Participate in AP and Maintain and Drive AP T Helper Cells in MS, Related to Figure 3 and Table S1 (A) Proportion of CD3 + and CD19 + cells (pie charts) in the CFSE dim compartment of HD (n = 14; for PHA n = 10) and REM (n = 14; for PHA n = 12) after AP (unstimulated) and reactivities to conventional B- (α-IgM) or T cell stimulation with polyclonal/broad (MLR, PHA) or antigen-specific (TTx) activation using CFSE-labeling and subsequent co-culture for 7 days in serum-free medium. (B–D) Sorted and subsequently expanded (PHA, IL-2) AP cells (CFSE dim ) from DR15 + HD and REM were tested for their reactivity on autologous EBV-transformed B cells. For the co-culture experiments, we incubated 2x10 5 CFSE-labeled expanded bulk T cells either alone or with 5x10 5 irradiated (300 Gy) autologous EBV-transformed B cells in the absence of any stimulus or the presence of blocking HLA-DR (L243) antibody or PHA, respectively. After 72 hours, 4 replicate wells of each condition were pooled and analyzed for survival (live-dead marker) and proliferation (CFSE) by flow cytometry. Cells were gated first on CD4 + T cells to exclude B cells, prior to analysis of live cells and then on proliferating cells. (B) Exemplary dot plots of the different conditions shown for one HD and one REM. (C) Survival and (D) proliferation results of DR15 + HD (n = 5) and REM (n = 5) (mean ± SEM).

    Article Snippet: RNA sequencing For RNA sequencing analysis of non-proliferating (CFSEhi ) and AP (CFSEdim ) CD3+ CD4+ and CD19+ cells, we labeled 5x107 CFSE-labeled PBMCs of RRMS (REM) patients at day 7 of unstimulated in vitro culture with Live/Dead® Aqua and antibodies against CD3, CD4, CD8 and CD19 ( ) and subsequently sorted live CFSEhi and CFSEdim cells of CD3+ CD4+ and CD19+ subsets using a FACSAria III (BD Biosciences).

    Techniques: Mass Spectrometry, Cell Stimulation, Activation Assay, Labeling, Co-Culture Assay, Transformation Assay, Incubation, Irradiation, Blocking Assay, Marker, Flow Cytometry, Cytometry

    Variable ThPOK expression in γδ T cell subsets. A , ThPOK-GFP reporter expression in spleen γδ T cells and CD4 T cells WT and ThPOK GFP/WT . Numbers in quadrants indicate the percentage of γδ T cells that express GFP. B , GFP (ThPOK) and PLZF expression in spleen cells from ThPOK GFP/+ and ThPOK GFP/GFP mice. C , Expression of CD4 (APC-Cy7) and CD8 (Pacific Blue) in Vγ1.1 + Vδ6.3 + splenocytes from ThPOK GFP/WT and ThPOK GFP/KO reporter mice. D , INF-γ and IL-4 expression in spleen Vγ1.1 + Vδ6.3 + T cells from WT or ThPOK-deficient (ThPOK ko/ko ) mice following activation with PMA/ionomycin. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 3 × 10 6 events were acquired for these experiments. Doublet exclusion was done as indicated in the Materials and Methods. B–D , Numbers indicate the percentage of cells in each quadrant. Data are representative of at least two experiments. All flow cytometry plots are quantified in log10 fluorescence.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate ?? T Cells Is Controlled by Strength of TCR Signaling and Id3

    doi: 10.4049/jimmunol.0903218

    Figure Lengend Snippet: Variable ThPOK expression in γδ T cell subsets. A , ThPOK-GFP reporter expression in spleen γδ T cells and CD4 T cells WT and ThPOK GFP/WT . Numbers in quadrants indicate the percentage of γδ T cells that express GFP. B , GFP (ThPOK) and PLZF expression in spleen cells from ThPOK GFP/+ and ThPOK GFP/GFP mice. C , Expression of CD4 (APC-Cy7) and CD8 (Pacific Blue) in Vγ1.1 + Vδ6.3 + splenocytes from ThPOK GFP/WT and ThPOK GFP/KO reporter mice. D , INF-γ and IL-4 expression in spleen Vγ1.1 + Vδ6.3 + T cells from WT or ThPOK-deficient (ThPOK ko/ko ) mice following activation with PMA/ionomycin. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 3 × 10 6 events were acquired for these experiments. Doublet exclusion was done as indicated in the Materials and Methods. B–D , Numbers indicate the percentage of cells in each quadrant. Data are representative of at least two experiments. All flow cytometry plots are quantified in log10 fluorescence.

    Article Snippet: For cytokine-secretion assays, sorted γδ T cells were activated with plate-bound anti-CD3 (10 μg/ml) in round-bottom 96-well plates (complete media) for 72 h. Secreted cytokines were detected with cytokine bead arrays (BD Biosciences).

    Techniques: Expressing, Mouse Assay, Activation Assay, Flow Cytometry, Cytometry, Fluorescence

    Phenotype and function of PLZF-positive and -negative Vγ1.1 + Vδ6.3 + T cells. A , Expression of CD44 (APC), CD69 (PerCP-Cy5.5), NK1.1 (PerCP-Cy5.5), and CD62L (APC-Alexa Fluor 750) by WT PLZF-positive (red), PLZF-negative (blue or black), and PLZF-deficient (gray) γδ thymocytes. B , CD4 (APC-Cy7) and CD8 (Pacific Blue) expression on WT Vγ1.1 + Vδ6.3 + T cells compared with intracellular staining for PLZF. C , Intracellular staining for IFN-γ (PE-Cy7), IL-4 (APC), and PLZF (Alexa Fluor 488) in Vδ6.3 T cells following activation. Numbers indicate the percentage of cells in each quadrant. D , Cytokine analysis of supernatants of 5 × 10 4 FACS-sorted Vδ6.3 + and Vδ6.3 − T cells from WT and PLZF-deficient splenocytes with plate-bound anti-CD3 for 3 d. Error bars represent the SD. *** p = 0.05. Data are representative of six ( A and B ) or five ( C and D ) independent experiments. γδ T cell subsets were identified with anti-CD3 PerCP-Cy5.5, anti-γδ TCR biotin, and anti-Vδ6.3 TCR PE. Doublet exclusion was done as indicated in the Materials and Methods . All flow cytometry plots are quantified in log10 fluorescence.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate ?? T Cells Is Controlled by Strength of TCR Signaling and Id3

    doi: 10.4049/jimmunol.0903218

    Figure Lengend Snippet: Phenotype and function of PLZF-positive and -negative Vγ1.1 + Vδ6.3 + T cells. A , Expression of CD44 (APC), CD69 (PerCP-Cy5.5), NK1.1 (PerCP-Cy5.5), and CD62L (APC-Alexa Fluor 750) by WT PLZF-positive (red), PLZF-negative (blue or black), and PLZF-deficient (gray) γδ thymocytes. B , CD4 (APC-Cy7) and CD8 (Pacific Blue) expression on WT Vγ1.1 + Vδ6.3 + T cells compared with intracellular staining for PLZF. C , Intracellular staining for IFN-γ (PE-Cy7), IL-4 (APC), and PLZF (Alexa Fluor 488) in Vδ6.3 T cells following activation. Numbers indicate the percentage of cells in each quadrant. D , Cytokine analysis of supernatants of 5 × 10 4 FACS-sorted Vδ6.3 + and Vδ6.3 − T cells from WT and PLZF-deficient splenocytes with plate-bound anti-CD3 for 3 d. Error bars represent the SD. *** p = 0.05. Data are representative of six ( A and B ) or five ( C and D ) independent experiments. γδ T cell subsets were identified with anti-CD3 PerCP-Cy5.5, anti-γδ TCR biotin, and anti-Vδ6.3 TCR PE. Doublet exclusion was done as indicated in the Materials and Methods . All flow cytometry plots are quantified in log10 fluorescence.

    Article Snippet: For cytokine-secretion assays, sorted γδ T cells were activated with plate-bound anti-CD3 (10 μg/ml) in round-bottom 96-well plates (complete media) for 72 h. Secreted cytokines were detected with cytokine bead arrays (BD Biosciences).

    Techniques: Expressing, Staining, Activation Assay, FACS, Flow Cytometry, Cytometry, Fluorescence

    Reduced strength of TCR signal enhances the development of Vγ1.1 + Vδ6.3 + T cells. A , Frequency of γδ T cells in the thymuses from WT, SLP-76 Y112:128F (Y112:128F), and SLP-76 Y145F mutant mice (Y145). B , The absolute numbers of indicated γδ subsets in the thymus, spleen, and lymph nodes of the indicated mouse strains. C , The frequency of γδ subsets and PLZF expression analysis in Vγ1.1 + Vδ6.3 + T cells from thymocytes and splenocytes of WT, Y112:128F, and Y145F mice. D , Intracellular staining for IFN-γ (Pacific Blue) and IL-4 (Alexa Fluor 488; left panel ) or TNF-α (Alexa Fluor 488) and PLZF (Pacific Blue; right panel ) in indicated γδ subsets from pooled splenocytes and lymphocytes in WT ( top ), Y112:128F ( middle ), and Y145F ( bottom ) mice. E , The frequency of Vγ1.1 + Vδ6.3 + T cells that are SPs for IFN-γ ( left ) and IL-4 ( middle ) or DPs ( right ) for IFN-γ and IL-4 from WT, Y112:128F, and Y145F mice. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 6 × 10 6 events per file were collected for these experiments. Multiple files were concatenated in FloJo software. Doublet exclusion was done as indicated in the Materials and Methods . Each symbol represents an individual mouse. * p = 0.05. Error bars represent the SD. All flow cytometry plots are quantified in log10 fluorescence.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate ?? T Cells Is Controlled by Strength of TCR Signaling and Id3

    doi: 10.4049/jimmunol.0903218

    Figure Lengend Snippet: Reduced strength of TCR signal enhances the development of Vγ1.1 + Vδ6.3 + T cells. A , Frequency of γδ T cells in the thymuses from WT, SLP-76 Y112:128F (Y112:128F), and SLP-76 Y145F mutant mice (Y145). B , The absolute numbers of indicated γδ subsets in the thymus, spleen, and lymph nodes of the indicated mouse strains. C , The frequency of γδ subsets and PLZF expression analysis in Vγ1.1 + Vδ6.3 + T cells from thymocytes and splenocytes of WT, Y112:128F, and Y145F mice. D , Intracellular staining for IFN-γ (Pacific Blue) and IL-4 (Alexa Fluor 488; left panel ) or TNF-α (Alexa Fluor 488) and PLZF (Pacific Blue; right panel ) in indicated γδ subsets from pooled splenocytes and lymphocytes in WT ( top ), Y112:128F ( middle ), and Y145F ( bottom ) mice. E , The frequency of Vγ1.1 + Vδ6.3 + T cells that are SPs for IFN-γ ( left ) and IL-4 ( middle ) or DPs ( right ) for IFN-γ and IL-4 from WT, Y112:128F, and Y145F mice. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 6 × 10 6 events per file were collected for these experiments. Multiple files were concatenated in FloJo software. Doublet exclusion was done as indicated in the Materials and Methods . Each symbol represents an individual mouse. * p = 0.05. Error bars represent the SD. All flow cytometry plots are quantified in log10 fluorescence.

    Article Snippet: For cytokine-secretion assays, sorted γδ T cells were activated with plate-bound anti-CD3 (10 μg/ml) in round-bottom 96-well plates (complete media) for 72 h. Secreted cytokines were detected with cytokine bead arrays (BD Biosciences).

    Techniques: Mutagenesis, Mouse Assay, Expressing, Staining, Software, Flow Cytometry, Cytometry, Fluorescence

    Vγ1.1 + Vδ6.3 + T cells in Fyn- and SAP-deficient mice and SAP-deficient PLZF-transgenic mice. A , FACS analysis of indicated γδ subsets thymocytes ( top ) and splenocytes ( bottom ) in indicated mouse strains. B , Percent frequency of Vγ1.1 + Vδ6.3 + T cells among thymocytes ( left ) and splenocytes ( right ) from WT ( n = 5), Fyn-deficient ( n = 4), SAP-deficient ( n = 4), and SAP-deficient PLZF-transgenic ( n = 4) mice. Each symbol represents an individual mouse. * p = 0.05. C , PLZF expression in Vγ1.1 + Vδ6.3 + thymocytes ( left ) and splenocytes ( right ) in indicated mouse strains. D , Mean fluorescence intensity (W/m 2 ) of PLZF levels in Vγ1.1 + Vδ6.3 + thymocytes: WT = 2559 ±1049; Fyn KO = 3464 ± 1407; SAP KO = 517 ± 127; SAP KO-PLZF Tg = 856 ± 171; Vγ1.1 + Vδ6.3 + spleen cells: WT = 527 ±187; Fyn KO = 715 ± 186; SAP KO = 316 ± 69; SAP KO-PLZF Tg = 494 ± 72. Error bars represent the SD. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 3 × 10 6 events were acquired for these experiments. Doublet exclusion was done as indicated in the Materials and Methods . Data are representative of at least three experiments. All flow cytometry plots are quantified in log10 fluorescence.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate ?? T Cells Is Controlled by Strength of TCR Signaling and Id3

    doi: 10.4049/jimmunol.0903218

    Figure Lengend Snippet: Vγ1.1 + Vδ6.3 + T cells in Fyn- and SAP-deficient mice and SAP-deficient PLZF-transgenic mice. A , FACS analysis of indicated γδ subsets thymocytes ( top ) and splenocytes ( bottom ) in indicated mouse strains. B , Percent frequency of Vγ1.1 + Vδ6.3 + T cells among thymocytes ( left ) and splenocytes ( right ) from WT ( n = 5), Fyn-deficient ( n = 4), SAP-deficient ( n = 4), and SAP-deficient PLZF-transgenic ( n = 4) mice. Each symbol represents an individual mouse. * p = 0.05. C , PLZF expression in Vγ1.1 + Vδ6.3 + thymocytes ( left ) and splenocytes ( right ) in indicated mouse strains. D , Mean fluorescence intensity (W/m 2 ) of PLZF levels in Vγ1.1 + Vδ6.3 + thymocytes: WT = 2559 ±1049; Fyn KO = 3464 ± 1407; SAP KO = 517 ± 127; SAP KO-PLZF Tg = 856 ± 171; Vγ1.1 + Vδ6.3 + spleen cells: WT = 527 ±187; Fyn KO = 715 ± 186; SAP KO = 316 ± 69; SAP KO-PLZF Tg = 494 ± 72. Error bars represent the SD. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 3 × 10 6 events were acquired for these experiments. Doublet exclusion was done as indicated in the Materials and Methods . Data are representative of at least three experiments. All flow cytometry plots are quantified in log10 fluorescence.

    Article Snippet: For cytokine-secretion assays, sorted γδ T cells were activated with plate-bound anti-CD3 (10 μg/ml) in round-bottom 96-well plates (complete media) for 72 h. Secreted cytokines were detected with cytokine bead arrays (BD Biosciences).

    Techniques: Mouse Assay, Transgenic Assay, FACS, Expressing, Fluorescence, Flow Cytometry, Cytometry

    Id3 controls the development of PLZF-expressing Vγ1.1 + Vδ6.3 + T cells. A , The frequency of γδ T cells in the thymuses from WT and Id3-deficient (Id3 KO) mice. B , The absolute numbers of Vγ1.1 + Vδ6.3 + T cells in the thymus, spleen, and lymph nodes of WT and Id3 KO mice. * p = 0.0005. C , The frequency of γδ T cell subsets in WT and Id3 KO mice ( left ) and PLZF expression analysis of Vγ1.1 + Vδ6.3 + thymocytes and splenocytes of WT and Id3 KO mice (right). D , Intracellular staining for IFN-γ (Pacific Blue) and IL-4 (Alexa Fluor 488; left panel ) or TNF-α (Alexa Fluor 488) and PLZF (Pacific Blue; right panel ) in indicated γδ subsets from pooled splenocytes and lymphocytes in WT ( top ) and Id3 KO ( bottom ) mice. E , The frequency of Vγ1.1 + Vδ6.3 + T cells that are SPs for IFN-γ ( left ) and IL-4 ( middle ) or DPs for IFN-γ and IL-4 ( right ) in WT and Id3 KO mice. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 6 × 10 6 events per file were collected for these experiments. Multiple files were concatenated in FloJo software. Doublet exclusion was done as indicated in the Materials and Methods . Data are representative of at least four experiments. Error bars represent the SD. All flow cytometry plots are quantified in log10 fluorescence.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate ?? T Cells Is Controlled by Strength of TCR Signaling and Id3

    doi: 10.4049/jimmunol.0903218

    Figure Lengend Snippet: Id3 controls the development of PLZF-expressing Vγ1.1 + Vδ6.3 + T cells. A , The frequency of γδ T cells in the thymuses from WT and Id3-deficient (Id3 KO) mice. B , The absolute numbers of Vγ1.1 + Vδ6.3 + T cells in the thymus, spleen, and lymph nodes of WT and Id3 KO mice. * p = 0.0005. C , The frequency of γδ T cell subsets in WT and Id3 KO mice ( left ) and PLZF expression analysis of Vγ1.1 + Vδ6.3 + thymocytes and splenocytes of WT and Id3 KO mice (right). D , Intracellular staining for IFN-γ (Pacific Blue) and IL-4 (Alexa Fluor 488; left panel ) or TNF-α (Alexa Fluor 488) and PLZF (Pacific Blue; right panel ) in indicated γδ subsets from pooled splenocytes and lymphocytes in WT ( top ) and Id3 KO ( bottom ) mice. E , The frequency of Vγ1.1 + Vδ6.3 + T cells that are SPs for IFN-γ ( left ) and IL-4 ( middle ) or DPs for IFN-γ and IL-4 ( right ) in WT and Id3 KO mice. γδ T cell subsets were identified with anti-CD3 (PerCP-Cy5.5), anti-γδ TCR (APC), anti-Vδ6.3 TCR (PE), and anti-Vγ1.1 TCR (biotin/streptavidin PE-Cy7). Approximately 6 × 10 6 events per file were collected for these experiments. Multiple files were concatenated in FloJo software. Doublet exclusion was done as indicated in the Materials and Methods . Data are representative of at least four experiments. Error bars represent the SD. All flow cytometry plots are quantified in log10 fluorescence.

    Article Snippet: For cytokine-secretion assays, sorted γδ T cells were activated with plate-bound anti-CD3 (10 μg/ml) in round-bottom 96-well plates (complete media) for 72 h. Secreted cytokines were detected with cytokine bead arrays (BD Biosciences).

    Techniques: Expressing, Mouse Assay, Staining, Software, Flow Cytometry, Cytometry, Fluorescence

    The majority of PLZF is expressed by Vγ1.1 + Vδ6.3 + T cells. Intracellular staining for PLZF in γδ T cells in primary tissues, secondary tissues, and iIELs ( A ), in Vγ4 (FITC), Vδ4 (FITC), Vγ1.1 (Biotin), and Vδ6.3 (PE) γδ thymocytes ( B ), and in γδ T cell subsets bearing indicated γδ TCR heterodimers in thymus ( C ) and splenocytes ( D ) in WT (open curves) or PLZF-deficient mice (shaded curves). Numbers adjacent to outlined areas indicate the percentage of γδ T cells; bracketed lines next to graphs specify the percent total of PLZF-positive cells. γδ T cell subsets were identified with anti-CD3 PerCP-Cy5.5, anti-γδ TCR APC, anti-Vδ6.3 TCR PE, and anti-Vγ1.1 TCR biotin/streptavidin PE-Cy7. E , Absolute numbers of total γδ T cells and γδ subsets in the thymus and spleen of WT littermates ( n = 6) and PLZF-deficient mice ( n = 6; error bars represent the SD). Approximately 3 × 10 6 events were acquired for these experiments. Doublet exclusion was done as indicated in the Materials and Methods . Data are representative of more than six independent experiments. All flow cytometry plots are quantified in log10 fluorescence. Each symbol represents an individual mouse. * p = 0.05; ** p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate ?? T Cells Is Controlled by Strength of TCR Signaling and Id3

    doi: 10.4049/jimmunol.0903218

    Figure Lengend Snippet: The majority of PLZF is expressed by Vγ1.1 + Vδ6.3 + T cells. Intracellular staining for PLZF in γδ T cells in primary tissues, secondary tissues, and iIELs ( A ), in Vγ4 (FITC), Vδ4 (FITC), Vγ1.1 (Biotin), and Vδ6.3 (PE) γδ thymocytes ( B ), and in γδ T cell subsets bearing indicated γδ TCR heterodimers in thymus ( C ) and splenocytes ( D ) in WT (open curves) or PLZF-deficient mice (shaded curves). Numbers adjacent to outlined areas indicate the percentage of γδ T cells; bracketed lines next to graphs specify the percent total of PLZF-positive cells. γδ T cell subsets were identified with anti-CD3 PerCP-Cy5.5, anti-γδ TCR APC, anti-Vδ6.3 TCR PE, and anti-Vγ1.1 TCR biotin/streptavidin PE-Cy7. E , Absolute numbers of total γδ T cells and γδ subsets in the thymus and spleen of WT littermates ( n = 6) and PLZF-deficient mice ( n = 6; error bars represent the SD). Approximately 3 × 10 6 events were acquired for these experiments. Doublet exclusion was done as indicated in the Materials and Methods . Data are representative of more than six independent experiments. All flow cytometry plots are quantified in log10 fluorescence. Each symbol represents an individual mouse. * p = 0.05; ** p

    Article Snippet: For cytokine-secretion assays, sorted γδ T cells were activated with plate-bound anti-CD3 (10 μg/ml) in round-bottom 96-well plates (complete media) for 72 h. Secreted cytokines were detected with cytokine bead arrays (BD Biosciences).

    Techniques: Staining, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence

    Proliferation and apoptosis in lymphocytes from patients with PHTS. A, Gut tissue sections from patients with PHTS or control subjects were stained for Ki-67. Cell proliferation was analyzed within the T-cell areas. E , Epithelium. B, CD3, CD20, and CD10 costaining with terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) for apoptosis detection in control appendix sections. The graph demonstrates the percentage of TUNEL + cells that are CD3 + , CD20 + , or CD10 + . Differences were analyzed by using the Mann-Whitney test. Scale bars = 50 μm. Symbols represent individual patients. C and D, In vitro apoptosis assays. Apoptosis was induced in PHA-activated T-cell blasts by means of CD95L (FasL) stimulation (Fig E4, C ) or IL-2 deprivation (Fig E4, D ). Percentages of surviving cells are shown for 1 patient with PHTS (p.R130Q) and 2 healthy control subjects. Similar results have been obtained by using analysis of 6 further patients with PHTS with different mutations in an independent experiment (data not shown). Dapi , 4′, 6′-Diamidino-2-phenylindole.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: Proliferation and apoptosis in lymphocytes from patients with PHTS. A, Gut tissue sections from patients with PHTS or control subjects were stained for Ki-67. Cell proliferation was analyzed within the T-cell areas. E , Epithelium. B, CD3, CD20, and CD10 costaining with terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) for apoptosis detection in control appendix sections. The graph demonstrates the percentage of TUNEL + cells that are CD3 + , CD20 + , or CD10 + . Differences were analyzed by using the Mann-Whitney test. Scale bars = 50 μm. Symbols represent individual patients. C and D, In vitro apoptosis assays. Apoptosis was induced in PHA-activated T-cell blasts by means of CD95L (FasL) stimulation (Fig E4, C ) or IL-2 deprivation (Fig E4, D ). Percentages of surviving cells are shown for 1 patient with PHTS (p.R130Q) and 2 healthy control subjects. Similar results have been obtained by using analysis of 6 further patients with PHTS with different mutations in an independent experiment (data not shown). Dapi , 4′, 6′-Diamidino-2-phenylindole.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: Staining, TUNEL Assay, MANN-WHITNEY, In Vitro

    Phenotype of Treg cells in patients with PHTS. A, CD3 + CD4 + FOXP3 + CD25 + CD127 − cells (Treg cells) were compared with FOXP3 − CD25 − CD127 + (naive and central Tmem cells) and FOXP3 − CD25 − CD127 − cells (effector T cells). B, Expression levels of Helios are shown as a histogram and quantified in Treg cells. C, Expression levels of CTLA-4 are shown as a histogram and quantified in Treg cells. D, CD3 + CD4 + FOXP3 + CD127 − (Treg) cells were compared with FOXP3 − CD127 + (naive and central Tmem) and FOXP3 − CD127 − (effector T cells) cells. CD25 expression in CD4 + FOXP3 + Treg cells is shown as a histogram and quantified in Treg cells. No statistical differences were detected by using the Mann-Whitney U test.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: Phenotype of Treg cells in patients with PHTS. A, CD3 + CD4 + FOXP3 + CD25 + CD127 − cells (Treg cells) were compared with FOXP3 − CD25 − CD127 + (naive and central Tmem cells) and FOXP3 − CD25 − CD127 − cells (effector T cells). B, Expression levels of Helios are shown as a histogram and quantified in Treg cells. C, Expression levels of CTLA-4 are shown as a histogram and quantified in Treg cells. D, CD3 + CD4 + FOXP3 + CD127 − (Treg) cells were compared with FOXP3 − CD127 + (naive and central Tmem) and FOXP3 − CD127 − (effector T cells) cells. CD25 expression in CD4 + FOXP3 + Treg cells is shown as a histogram and quantified in Treg cells. No statistical differences were detected by using the Mann-Whitney U test.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: Expressing, MANN-WHITNEY

    Treg cells in patients with PHTS. A, Absolute counts and frequencies of Treg cells in blood from patients with PHTS or control subjects. B, Percentages of FOXP3 + cells among CD3 + cells were analyzed in MALT from control subjects and patients. Representative staining of tissue sections from patients with PHTS is shown. C, FOXP3 and Ki-67 expression in MALT. Percentages of Ki-67 + proliferating FOXP3 + Treg cells were analyzed in control and patient samples. Arrows mark Ki-67 + FOXP3 + Treg cells. Scale bars in all images represent 50 μm. Dapi , 4′, 6′-Diamidino-2-phenylindole; GC , location of a germinal center.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: Treg cells in patients with PHTS. A, Absolute counts and frequencies of Treg cells in blood from patients with PHTS or control subjects. B, Percentages of FOXP3 + cells among CD3 + cells were analyzed in MALT from control subjects and patients. Representative staining of tissue sections from patients with PHTS is shown. C, FOXP3 and Ki-67 expression in MALT. Percentages of Ki-67 + proliferating FOXP3 + Treg cells were analyzed in control and patient samples. Arrows mark Ki-67 + FOXP3 + Treg cells. Scale bars in all images represent 50 μm. Dapi , 4′, 6′-Diamidino-2-phenylindole; GC , location of a germinal center.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: Staining, Expressing

    Lymphocyte subsets of patients with PHTS (proportions). A, Frequencies of CD19 + , CD5 + , CD10 + immature, and IgM high CD38 high transitional B cells. B, Frequencies of CD3 + T cells and percentages of CD4 + and CD8 + T cells among CD3 + T cells. C, Frequencies of HLA-DR + activated T cells and CD45RO − naive T cells, CD45RO + CCR7 + central memory CD4 + T cells, and CD45RO + CCR7 − effector memory CD4 + T cells. D, Frequencies of CD45RO + CCR7 + central memory CD8 + T cells and CD45RO + CCR7 - effector memory CD8 + T cells. E, Frequencies of CD16 + CD56 + NK cells and CD3 + CD56 + NKT cells. Each dot represents 1 patient. The mean is presented by the red line . Statistical differences were analyzed by using the Mann-Whitney test.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: Lymphocyte subsets of patients with PHTS (proportions). A, Frequencies of CD19 + , CD5 + , CD10 + immature, and IgM high CD38 high transitional B cells. B, Frequencies of CD3 + T cells and percentages of CD4 + and CD8 + T cells among CD3 + T cells. C, Frequencies of HLA-DR + activated T cells and CD45RO − naive T cells, CD45RO + CCR7 + central memory CD4 + T cells, and CD45RO + CCR7 − effector memory CD4 + T cells. D, Frequencies of CD45RO + CCR7 + central memory CD8 + T cells and CD45RO + CCR7 - effector memory CD8 + T cells. E, Frequencies of CD16 + CD56 + NK cells and CD3 + CD56 + NKT cells. Each dot represents 1 patient. The mean is presented by the red line . Statistical differences were analyzed by using the Mann-Whitney test.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: MANN-WHITNEY

    FACS analysis of T-cell subsets, NK cells, and NKT cells in peripheral blood of patients with PHTS (absolute numbers). A, Levels of HLA-DR + activated T cells and CD45RO − naive T cells, CD45RO + CCR7 + central memory CD4 + T cells, and CD45RO + CCR7 − effector memory CD4 + T cells. B, Levels of CD45RO + CCR7 + central memory CD8 + T cells and CD45RO + CCR7 − effector memory CD8 + T cells. C, Levels of CD16 + CD56 + NK cells and CD3 + CD56 + NKT cells. Each dot represents 1 patient. The mean is presented by the red line . Statistical differences were analyzed by using the Mann-Whitney test.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: FACS analysis of T-cell subsets, NK cells, and NKT cells in peripheral blood of patients with PHTS (absolute numbers). A, Levels of HLA-DR + activated T cells and CD45RO − naive T cells, CD45RO + CCR7 + central memory CD4 + T cells, and CD45RO + CCR7 − effector memory CD4 + T cells. B, Levels of CD45RO + CCR7 + central memory CD8 + T cells and CD45RO + CCR7 − effector memory CD8 + T cells. C, Levels of CD16 + CD56 + NK cells and CD3 + CD56 + NKT cells. Each dot represents 1 patient. The mean is presented by the red line . Statistical differences were analyzed by using the Mann-Whitney test.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: FACS, MANN-WHITNEY

    iTreg assay and blockade of iTreg generation by phosphatase-specific inhibitors. A, In vitro differentiation of naive T cells into FOXP3 iTreg cells. B, Effects of TCR signal strength on FOXP3 induction. TCR stimulation was provided by T-cell activator beads or plate-bound anti-CD3 (10 μg/mL)/soluble anti-CD28 (1 μg/mL). C, In vitro T-cell proliferation suppression assay. Carboxyfluorescein succinimidyl ester (CFSE) –labeled naive (responder) T cells were cocultured with anti-CD3/anti-CD28 beads in the presence and absence of iTreg cells. Responder T-cell proliferation was measured by means of CFSE dilution. Data are representative of 2 independent experiments. D, Small-molecule inhibitors were used to block various phosphatases (PTEN, PHLPP1/2, SHIP1, SHIP2, and PP2A) that provide negative regulation at different points of the PI3K/AKT/mTOR signaling pathway.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: iTreg assay and blockade of iTreg generation by phosphatase-specific inhibitors. A, In vitro differentiation of naive T cells into FOXP3 iTreg cells. B, Effects of TCR signal strength on FOXP3 induction. TCR stimulation was provided by T-cell activator beads or plate-bound anti-CD3 (10 μg/mL)/soluble anti-CD28 (1 μg/mL). C, In vitro T-cell proliferation suppression assay. Carboxyfluorescein succinimidyl ester (CFSE) –labeled naive (responder) T cells were cocultured with anti-CD3/anti-CD28 beads in the presence and absence of iTreg cells. Responder T-cell proliferation was measured by means of CFSE dilution. Data are representative of 2 independent experiments. D, Small-molecule inhibitors were used to block various phosphatases (PTEN, PHLPP1/2, SHIP1, SHIP2, and PP2A) that provide negative regulation at different points of the PI3K/AKT/mTOR signaling pathway.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: In Vitro, Suppression Assay, Labeling, Blocking Assay

    PTEN activity and PI3K signaling in natural Treg cells. A, FACS-sorted Treg cells (CD4 + CD25 high CD127 low ) and Tmem cells (CD4 + CD45RO + CD127 high CD25 low ) were left unstimulated or subjected to anti-CD3/CD28 and IL-2 stimulation for 10 minutes. Cellular levels of pAKT, pS6, and phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in each cell type with or without stimulation were analyzed by using Phosflow. B and C, PBMCs from healthy donors were rested or stimulated for 24 hours with anti-CD3 and anti-CD28 T-cell activator beads in the presence of IL-2. PTEN expression in FOXP3 − nonregulatory and FOXP3 + Treg cell populations among total PBMCs was measured by using FACS to detect changes in PTEN protein levels after TCR and IL-2 receptor stimulation. D, MALT sections were stained for FOXP3 + cells, as well as phosphorylation of the S6 ribosomal protein (p[Ser235/236]S6). Scale bar = 50 μm. E, Percentage of pS6 + expressing cells among FOXP3 + cells in patients with PHTS versus control subjects determined by using immunohistochemical staining. Symbols represent individual patients, and lines represent means. Differences were analyzed by using the Mann-Whitney U test. F, Multiplex ligation-dependent probe amplification copy number analysis revealing a microdeletion spanning from exon 2 to exon 9 and a 3′ untranslated region (UTR) of PTEN. Protein representation of a wild-type (wt) subject and a patient with PHTS with the microdeletion (del E2-9) are shown for comparison. G, PTEN expression in CD3 + T cells from a control donor or a patient with PHTS (del E2-9, PTEN microdeletion) was determined by using FACS with an mAb that recognizes the C-terminal region of PTEN, allowing selective detection of the nonmutated copy of PTEN protein. PTEN levels were measured in cells with or without anti-CD3/CD28 and IL-2 stimulation for 24 hours.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: PTEN activity and PI3K signaling in natural Treg cells. A, FACS-sorted Treg cells (CD4 + CD25 high CD127 low ) and Tmem cells (CD4 + CD45RO + CD127 high CD25 low ) were left unstimulated or subjected to anti-CD3/CD28 and IL-2 stimulation for 10 minutes. Cellular levels of pAKT, pS6, and phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in each cell type with or without stimulation were analyzed by using Phosflow. B and C, PBMCs from healthy donors were rested or stimulated for 24 hours with anti-CD3 and anti-CD28 T-cell activator beads in the presence of IL-2. PTEN expression in FOXP3 − nonregulatory and FOXP3 + Treg cell populations among total PBMCs was measured by using FACS to detect changes in PTEN protein levels after TCR and IL-2 receptor stimulation. D, MALT sections were stained for FOXP3 + cells, as well as phosphorylation of the S6 ribosomal protein (p[Ser235/236]S6). Scale bar = 50 μm. E, Percentage of pS6 + expressing cells among FOXP3 + cells in patients with PHTS versus control subjects determined by using immunohistochemical staining. Symbols represent individual patients, and lines represent means. Differences were analyzed by using the Mann-Whitney U test. F, Multiplex ligation-dependent probe amplification copy number analysis revealing a microdeletion spanning from exon 2 to exon 9 and a 3′ untranslated region (UTR) of PTEN. Protein representation of a wild-type (wt) subject and a patient with PHTS with the microdeletion (del E2-9) are shown for comparison. G, PTEN expression in CD3 + T cells from a control donor or a patient with PHTS (del E2-9, PTEN microdeletion) was determined by using FACS with an mAb that recognizes the C-terminal region of PTEN, allowing selective detection of the nonmutated copy of PTEN protein. PTEN levels were measured in cells with or without anti-CD3/CD28 and IL-2 stimulation for 24 hours.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: Activity Assay, FACS, Expressing, Staining, Immunohistochemistry, MANN-WHITNEY, Multiplex Assay, Ligation, Amplification

    PHTS patient cohort and clinical phenotype. A, Representation of different pathogenic germline PTEN mutations in 79 patients with PHTS investigated. Symbols represent the mutation site of individual patients. Colored symbols represent patients who present with autoimmunity, lymphoid hyperplasia, or both. B, Immunologic conditions in the PHTS patient cohort. C, Peripheral blood leukocyte counts of adult patients with PHTS (n = 32) and blood donor control subjects (n = 216). Each dot represents 1 patient. Gray boxes mark the normal range. Statistical differences were analyzed by using the Mann-Whitney test. D, Numbers of CD19 + , CD5 + , CD10 + immature, and IgM high CD38 high transitional B cells. E, Numbers of CD3 + T cells, percentages of CD4 + and CD8 + T cells among CD3 + T cells, and CD4 + /CD8 + T-cell ratio.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: PHTS patient cohort and clinical phenotype. A, Representation of different pathogenic germline PTEN mutations in 79 patients with PHTS investigated. Symbols represent the mutation site of individual patients. Colored symbols represent patients who present with autoimmunity, lymphoid hyperplasia, or both. B, Immunologic conditions in the PHTS patient cohort. C, Peripheral blood leukocyte counts of adult patients with PHTS (n = 32) and blood donor control subjects (n = 216). Each dot represents 1 patient. Gray boxes mark the normal range. Statistical differences were analyzed by using the Mann-Whitney test. D, Numbers of CD19 + , CD5 + , CD10 + immature, and IgM high CD38 high transitional B cells. E, Numbers of CD3 + T cells, percentages of CD4 + and CD8 + T cells among CD3 + T cells, and CD4 + /CD8 + T-cell ratio.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: Mutagenesis, MANN-WHITNEY

    Phosphatase and NHERF1 assembly at the immunologic synapse. A-C, Laser-scanning microscopy of CD4 + CD25 + T cells stimulated on CD3- and ICAM-1–coated glass slides. Time-dependent accumulation of PHLLP, PTEN, and NHERF1 after anti-CD3–mediated TCR activation was assessed by measuring protein accumulation in the planar layer adjacent to the glass slide. Exposure to ICAM-1 without TCR engagement served as a negative control. Dots represent individual cells analyzed. D, PTEN, NHERF1, and PHLLP subcellular localization in relation to the central supramolecular activation complex (indicated by CD3 staining) and peripheral supramolecular activation complex (indicated by ICAM-1 staining) was analyzed by using TIRF microscopy. Differences were analyzed by using the Mann-Whitney U test.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

    doi: 10.1016/j.jaci.2016.03.059

    Figure Lengend Snippet: Phosphatase and NHERF1 assembly at the immunologic synapse. A-C, Laser-scanning microscopy of CD4 + CD25 + T cells stimulated on CD3- and ICAM-1–coated glass slides. Time-dependent accumulation of PHLLP, PTEN, and NHERF1 after anti-CD3–mediated TCR activation was assessed by measuring protein accumulation in the planar layer adjacent to the glass slide. Exposure to ICAM-1 without TCR engagement served as a negative control. Dots represent individual cells analyzed. D, PTEN, NHERF1, and PHLLP subcellular localization in relation to the central supramolecular activation complex (indicated by CD3 staining) and peripheral supramolecular activation complex (indicated by ICAM-1 staining) was analyzed by using TIRF microscopy. Differences were analyzed by using the Mann-Whitney U test.

    Article Snippet: Immunohistochemistry and fluorescence microscopy The following antibodies were used: anti-CD3 (polyclonal; Dako, Glostrup, Denmark), anti-CD10 (clone 56C6; Leica, Wetzlar, Germany), anti-CD20 (clone L26; Dako), anti–Ki-67 (clone Mib-1 or anti–Ki-67–FITC clone Mib-1 FITC labeled; Dako), anti-FOXP3 (clone 236A/E7; Abcam), anti–p(Ser235/236)-S6 (clone D57.2.2E; Cell Signaling), anti-NHERF1 (NB300-536; Novus Biologicals, Littleton, Colo), and anti-PHLPP (IHC-00382; Bethyl Laboratories, Montgomery, Tex).

    Techniques: Laser-Scanning Microscopy, Activation Assay, Negative Control, Staining, Microscopy, MANN-WHITNEY

    The ability to polarize Tfh cells is intrinsic for cDC2 but is tissue dependent for macrophages. (A–C) Purified human tonsil DC subsets and macrophages were cultured for 3 h with or without R848. After washing, preactivated DCs or macrophages were co-cultured with allogeneic naive CD4 + T cells for 6 d. T cells polarized with cDC1s, cDC2s, pDCs, or CD14 + macrophages are termed T cDC1 , T cDC2 , T pDC , and T MACRO , respectively. T Ø corresponds to T cells cultured without APCs. (A) T cell proliferation was assessed by dilution of a proliferation dye (CTV). Histogram representative of three independent experiments. Graphs show mean ± SEM ( n = 3). (B and C) Cytokine secretion was analyzed by cytometric bead array (CBA) (B) or ELISA (C) after restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor ( n = 9). (D–G) Purified cDC1 and cDC2 from blood (D) or skin-draining lymph node (E), or DCs and macrophages from peritoneal tumor ascites (F) or synovial fluid of rheumatoid arthritis patients (G) were co-cultured with allogeneic naive CD4 + T cells for 6 d. IFN-γ and CXCL13 secretion was measured in the supernatant after restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor ( n = 6 for blood, 8 for lymph node, 5–7 for ascites, and 2 for synovial fluid). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Human lymphoid organ cDC2 and macrophages play complementary roles in T follicular helper responses

    doi: 10.1084/jem.20181994

    Figure Lengend Snippet: The ability to polarize Tfh cells is intrinsic for cDC2 but is tissue dependent for macrophages. (A–C) Purified human tonsil DC subsets and macrophages were cultured for 3 h with or without R848. After washing, preactivated DCs or macrophages were co-cultured with allogeneic naive CD4 + T cells for 6 d. T cells polarized with cDC1s, cDC2s, pDCs, or CD14 + macrophages are termed T cDC1 , T cDC2 , T pDC , and T MACRO , respectively. T Ø corresponds to T cells cultured without APCs. (A) T cell proliferation was assessed by dilution of a proliferation dye (CTV). Histogram representative of three independent experiments. Graphs show mean ± SEM ( n = 3). (B and C) Cytokine secretion was analyzed by cytometric bead array (CBA) (B) or ELISA (C) after restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor ( n = 9). (D–G) Purified cDC1 and cDC2 from blood (D) or skin-draining lymph node (E), or DCs and macrophages from peritoneal tumor ascites (F) or synovial fluid of rheumatoid arthritis patients (G) were co-cultured with allogeneic naive CD4 + T cells for 6 d. IFN-γ and CXCL13 secretion was measured in the supernatant after restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor ( n = 6 for blood, 8 for lymph node, 5–7 for ascites, and 2 for synovial fluid). *, P

    Article Snippet: To analyze T cell polarization, total cells from each culture well were washed and incubated with anti-CD3/CD28 beads (Thermo Fisher Scientific) for 24 h in X-VIVO 15 serum free medium (Lonza).

    Techniques: Purification, Cell Culture, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay

    Tonsil cDC2 and macrophages are the best inducers of Tfh polarization. Purified human tonsil DC subsets and macrophages were co-cultured with allogeneic naive CD4 + T cells. T cells polarized with cDC1s, cDC2s, pDCs, or CD14 + macrophages are termed T cDC1 , T cDC2 , T pDC , and T MACRO , respectively. (A) T cell proliferation was assessed by dilution of a proliferation dye (CTV). Histogram representative of three independent experiments. Graphs show mean ± SEM ( n = 3). (B and C) Cytokine production was analyzed at day 5 (cDC1s and cDC2s) or day 6 (pDCs and macrophages), by intracellular staining after restimulation with PMA and ionomycin in presence of brefeldin A. (B) Representative staining, gated on live CD4 + T cells. T Ø corresponds to T cells cultured without APCs. (C) Percentage of divided cells (CTV − ) producing IFN-γ ( n = 22), IL-21 ( n = 22), and CXCL13 ( n = 13). Each symbol represents an individual donor. (D) Cytokine secretion was analyzed by ELISA after 6 d of culture and restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor ( n = 19). (E and F) Expression of PD-1 and CXCR5 was analyzed by flow cytometry at day 4 or day 5. Each symbol represents an individual donor ( n = 7). (E) Representative staining, gated on total live CD4 + T cells. (F) Percentage of PD-1 + CXCR5 + cells. (G) Mean fluorescence intensity (MFI) of Bcl6 staining in PD-1 + CXCR5 + cells at day 4 ( n = 7). Representative staining is shown for T cells cultured with cDC1 or cDC2. Dashed line represents isotype control staining. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Human lymphoid organ cDC2 and macrophages play complementary roles in T follicular helper responses

    doi: 10.1084/jem.20181994

    Figure Lengend Snippet: Tonsil cDC2 and macrophages are the best inducers of Tfh polarization. Purified human tonsil DC subsets and macrophages were co-cultured with allogeneic naive CD4 + T cells. T cells polarized with cDC1s, cDC2s, pDCs, or CD14 + macrophages are termed T cDC1 , T cDC2 , T pDC , and T MACRO , respectively. (A) T cell proliferation was assessed by dilution of a proliferation dye (CTV). Histogram representative of three independent experiments. Graphs show mean ± SEM ( n = 3). (B and C) Cytokine production was analyzed at day 5 (cDC1s and cDC2s) or day 6 (pDCs and macrophages), by intracellular staining after restimulation with PMA and ionomycin in presence of brefeldin A. (B) Representative staining, gated on live CD4 + T cells. T Ø corresponds to T cells cultured without APCs. (C) Percentage of divided cells (CTV − ) producing IFN-γ ( n = 22), IL-21 ( n = 22), and CXCL13 ( n = 13). Each symbol represents an individual donor. (D) Cytokine secretion was analyzed by ELISA after 6 d of culture and restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor ( n = 19). (E and F) Expression of PD-1 and CXCR5 was analyzed by flow cytometry at day 4 or day 5. Each symbol represents an individual donor ( n = 7). (E) Representative staining, gated on total live CD4 + T cells. (F) Percentage of PD-1 + CXCR5 + cells. (G) Mean fluorescence intensity (MFI) of Bcl6 staining in PD-1 + CXCR5 + cells at day 4 ( n = 7). Representative staining is shown for T cells cultured with cDC1 or cDC2. Dashed line represents isotype control staining. *, P

    Article Snippet: To analyze T cell polarization, total cells from each culture well were washed and incubated with anti-CD3/CD28 beads (Thermo Fisher Scientific) for 24 h in X-VIVO 15 serum free medium (Lonza).

    Techniques: Purification, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence

    IL-12 or activin A and TGFβ are involved in inducing IL-21 or CXCL13 production, respectively. Purified cDC2s and macrophages were co-cultured with naive CD4 + T cells in the presence of blocking antibodies, in combination or individually, against IL-12 (two different clones termed Ab1 and Ab2), activin A, and TGFβ1.2.3, or isotype control at the same total concentration. T cells polarized with cDC2 or CD14 + macrophages are termed T cDC2 and T MACRO , respectively. (A, B, D, and E) IFN-γ and IL-21 production was analyzed by intracellular staining after restimulation with PMA and ionomycin in presence of brefeldin A. (C, F, and G) CXCL13 secretion was analyzed by ELISA after restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor. (A–C) n = 11 for cDC2s and n = 10 for macrophages. (D and E) For cDC2: n = 10 for anti–IL-12 Ab1, n = 7 for anti–IL-12 Ab2, n = 9 for anti–activin A, and n = 8 for anti-TGFβ. For macrophages: n = 6 for anti–IL-12 Ab1, n = 7 for anti–IL-12 Ab2, n = 8 for anti–activin A, and n = 8 for anti-TGFβ. (F) For cDC2: n = 12 for anti–IL-12, n = 10 for anti–activin A, and n = 9 for anti-TGFβ. For macrophages: n = 7 for anti–IL-12, n = 10 for anti–activin A, and n = 8 for anti-TGFβ. (G) n = 7 for cDC2, n = 8 for macrophages. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Human lymphoid organ cDC2 and macrophages play complementary roles in T follicular helper responses

    doi: 10.1084/jem.20181994

    Figure Lengend Snippet: IL-12 or activin A and TGFβ are involved in inducing IL-21 or CXCL13 production, respectively. Purified cDC2s and macrophages were co-cultured with naive CD4 + T cells in the presence of blocking antibodies, in combination or individually, against IL-12 (two different clones termed Ab1 and Ab2), activin A, and TGFβ1.2.3, or isotype control at the same total concentration. T cells polarized with cDC2 or CD14 + macrophages are termed T cDC2 and T MACRO , respectively. (A, B, D, and E) IFN-γ and IL-21 production was analyzed by intracellular staining after restimulation with PMA and ionomycin in presence of brefeldin A. (C, F, and G) CXCL13 secretion was analyzed by ELISA after restimulation with anti-CD3/CD28 beads. Each symbol represents an individual donor. (A–C) n = 11 for cDC2s and n = 10 for macrophages. (D and E) For cDC2: n = 10 for anti–IL-12 Ab1, n = 7 for anti–IL-12 Ab2, n = 9 for anti–activin A, and n = 8 for anti-TGFβ. For macrophages: n = 6 for anti–IL-12 Ab1, n = 7 for anti–IL-12 Ab2, n = 8 for anti–activin A, and n = 8 for anti-TGFβ. (F) For cDC2: n = 12 for anti–IL-12, n = 10 for anti–activin A, and n = 9 for anti-TGFβ. For macrophages: n = 7 for anti–IL-12, n = 10 for anti–activin A, and n = 8 for anti-TGFβ. (G) n = 7 for cDC2, n = 8 for macrophages. *, P

    Article Snippet: To analyze T cell polarization, total cells from each culture well were washed and incubated with anti-CD3/CD28 beads (Thermo Fisher Scientific) for 24 h in X-VIVO 15 serum free medium (Lonza).

    Techniques: Purification, Cell Culture, Blocking Assay, Clone Assay, Concentration Assay, Staining, Enzyme-linked Immunosorbent Assay

    Structural and functional integrity of recombinant soluble NKT TCRs. (A) Purified bacterial NKT15 TCR was analyzed by SDS-PAGE under reducing (+DTT, dithiothreitol) and nonreducing (−DTT) conditions demonstrating αβ heterodimers. (B) Native gel electrophoresis of folded αβ heterodimeric NKT15 and control LC13 TCRs. (C) Recombinant soluble NKT12, NKT15, NKT18, and control LC13 TCRs were tested for their ability to block binding of mouse CD1d/α-GalCer tetramers to murine thymocytes. Phycoerythrin-conjugated mCD1d/α-GalCer tetramers were preincubated with the indicated soluble TCRs over a range of TCR concentrations before staining mouse thymocytes. Cells were analyzed by two-color flow cytometry showing mCD1d/α-GalCer tetramer staining on the vertical axis and FITC-CD3 (mAb 145-2C11) staining on the horizontal axis. Cells staining positively with mCD1d/α-GalCer tetramer and FITC-CD3 are indicated with a circle.

    Journal: The Journal of Experimental Medicine

    Article Title: A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition

    doi: 10.1084/jem.20051777

    Figure Lengend Snippet: Structural and functional integrity of recombinant soluble NKT TCRs. (A) Purified bacterial NKT15 TCR was analyzed by SDS-PAGE under reducing (+DTT, dithiothreitol) and nonreducing (−DTT) conditions demonstrating αβ heterodimers. (B) Native gel electrophoresis of folded αβ heterodimeric NKT15 and control LC13 TCRs. (C) Recombinant soluble NKT12, NKT15, NKT18, and control LC13 TCRs were tested for their ability to block binding of mouse CD1d/α-GalCer tetramers to murine thymocytes. Phycoerythrin-conjugated mCD1d/α-GalCer tetramers were preincubated with the indicated soluble TCRs over a range of TCR concentrations before staining mouse thymocytes. Cells were analyzed by two-color flow cytometry showing mCD1d/α-GalCer tetramer staining on the vertical axis and FITC-CD3 (mAb 145-2C11) staining on the horizontal axis. Cells staining positively with mCD1d/α-GalCer tetramer and FITC-CD3 are indicated with a circle.

    Article Snippet: Anti-CD3–FITC (clone 145-2C11) was purchased from BD Biosciences.

    Techniques: Functional Assay, Recombinant, Purification, SDS Page, Nucleic Acid Electrophoresis, Blocking Assay, Binding Assay, Staining, Flow Cytometry, Cytometry

    Prominent expansion of erythroid cells in the spleen and of myeloid cells in the liver of irradiated mice with a liver shield. Two-colour staining for TER119 and CD3 and that for Gr-1 and Mac-1 were conducted in control mice and irradiated mice with or without a liver shield (on day 14). Representative results of three experiments are depicted. Erythroid (TER119 + ) cells expanded prominently in the spleen of mice with a liver shield, whereas myeloid cells expanded in the liver of the same mice without a liver shield.

    Journal: Clinical and Experimental Immunology

    Article Title: Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

    doi: 10.1046/j.1365-2249.1998.00726.x

    Figure Lengend Snippet: Prominent expansion of erythroid cells in the spleen and of myeloid cells in the liver of irradiated mice with a liver shield. Two-colour staining for TER119 and CD3 and that for Gr-1 and Mac-1 were conducted in control mice and irradiated mice with or without a liver shield (on day 14). Representative results of three experiments are depicted. Erythroid (TER119 + ) cells expanded prominently in the spleen of mice with a liver shield, whereas myeloid cells expanded in the liver of the same mice without a liver shield.

    Article Snippet: The MoAbs used included FITC- or biotin-conjugated reagents of anti-CD3 (145-2C11), NK1.1 (PK136), TER119 (erythroid marker), anti-Gr-1 (RA3-8C5), anti-Mac-1 (M1/70), anti-H-2Kb , and anti-H-2Kk MoAbs, obtained from PharMingen (San Diego, CA).

    Techniques: Irradiation, Mouse Assay, Staining

    Expansion of granulocytes in irradiated mice with a liver shield, treatment with anti-NK1.1 MoAb, and subsequent allogeneic bone marrow transplantation (BMT). Two-colour staining for Gr-1 and Mac-1 was conducted. Representative results of three experiments are shown. Even after the elimination of NK and NK T cells, NK1.1 − CD3 int cells rejected allogeneic BM cells. In this case, granulocytes always expanded, especially in the BM.

    Journal: Clinical and Experimental Immunology

    Article Title: Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

    doi: 10.1046/j.1365-2249.1998.00726.x

    Figure Lengend Snippet: Expansion of granulocytes in irradiated mice with a liver shield, treatment with anti-NK1.1 MoAb, and subsequent allogeneic bone marrow transplantation (BMT). Two-colour staining for Gr-1 and Mac-1 was conducted. Representative results of three experiments are shown. Even after the elimination of NK and NK T cells, NK1.1 − CD3 int cells rejected allogeneic BM cells. In this case, granulocytes always expanded, especially in the BM.

    Article Snippet: The MoAbs used included FITC- or biotin-conjugated reagents of anti-CD3 (145-2C11), NK1.1 (PK136), TER119 (erythroid marker), anti-Gr-1 (RA3-8C5), anti-Mac-1 (M1/70), anti-H-2Kb , and anti-H-2Kk MoAbs, obtained from PharMingen (San Diego, CA).

    Techniques: Irradiation, Mouse Assay, Transplantation Assay, Staining

    Induction of potent cytotoxicity against syngeneic thymocytes, allogeneic thymocytes, and other targets in mice with a liver shield and which underwent allogeneic bone marrow transplantation (BMT). Effector cells were isolated from the liver of control C3H/He mice (•) or irradiated C3H/He mice (○) with a liver shield and subjected to allogeneic (B6) BMT. The mean and 1 s.d. from four experiments are depicted. Possibly due to the increased proportions of NK cells and CD3 int cells in the liver of those mice and the interaction of B6 cells, potent cytotoxicity against allogeneic thymocytes (B6), YAC-1 cells (NK-sensitive target), syngeneic thymocytes (C3H/He), and EL-4 (NK-resistant target) was induced.

    Journal: Clinical and Experimental Immunology

    Article Title: Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

    doi: 10.1046/j.1365-2249.1998.00726.x

    Figure Lengend Snippet: Induction of potent cytotoxicity against syngeneic thymocytes, allogeneic thymocytes, and other targets in mice with a liver shield and which underwent allogeneic bone marrow transplantation (BMT). Effector cells were isolated from the liver of control C3H/He mice (•) or irradiated C3H/He mice (○) with a liver shield and subjected to allogeneic (B6) BMT. The mean and 1 s.d. from four experiments are depicted. Possibly due to the increased proportions of NK cells and CD3 int cells in the liver of those mice and the interaction of B6 cells, potent cytotoxicity against allogeneic thymocytes (B6), YAC-1 cells (NK-sensitive target), syngeneic thymocytes (C3H/He), and EL-4 (NK-resistant target) was induced.

    Article Snippet: The MoAbs used included FITC- or biotin-conjugated reagents of anti-CD3 (145-2C11), NK1.1 (PK136), TER119 (erythroid marker), anti-Gr-1 (RA3-8C5), anti-Mac-1 (M1/70), anti-H-2Kb , and anti-H-2Kk MoAbs, obtained from PharMingen (San Diego, CA).

    Techniques: Mouse Assay, Transplantation Assay, Isolation, Irradiation

    (See also pp 444 and 445.) Injection of anti-NK1.1 MoAb or anti-IL-2Rβ MoAb in vivo . (a) Two-colour staining for CD3 and NK1.1. (b) Two-colour staining for H-2K b and H-2K k . (c) Two-colour staining for CD3 and IL-2Rβ. To eliminate NK, NK T cells and TCR int cells, in vivo injection of anti-NK1.1 MoAb (200 μg/mouse) or anti-IL-2Rβ MoAb (500 μg/mouse) was performed, respectively. A single in vivo injection of anti-NK1.1 MoAb was first performed. Every 3 days after in vivo injection of anti-IL-2Rβ MoAb was then continued at least 21 days from the beginning. Representative data of three experiments are depicted. The elimination of NK and NK T cells (or CD3 int cells) was complete.

    Journal: Clinical and Experimental Immunology

    Article Title: Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

    doi: 10.1046/j.1365-2249.1998.00726.x

    Figure Lengend Snippet: (See also pp 444 and 445.) Injection of anti-NK1.1 MoAb or anti-IL-2Rβ MoAb in vivo . (a) Two-colour staining for CD3 and NK1.1. (b) Two-colour staining for H-2K b and H-2K k . (c) Two-colour staining for CD3 and IL-2Rβ. To eliminate NK, NK T cells and TCR int cells, in vivo injection of anti-NK1.1 MoAb (200 μg/mouse) or anti-IL-2Rβ MoAb (500 μg/mouse) was performed, respectively. A single in vivo injection of anti-NK1.1 MoAb was first performed. Every 3 days after in vivo injection of anti-IL-2Rβ MoAb was then continued at least 21 days from the beginning. Representative data of three experiments are depicted. The elimination of NK and NK T cells (or CD3 int cells) was complete.

    Article Snippet: The MoAbs used included FITC- or biotin-conjugated reagents of anti-CD3 (145-2C11), NK1.1 (PK136), TER119 (erythroid marker), anti-Gr-1 (RA3-8C5), anti-Mac-1 (M1/70), anti-H-2Kb , and anti-H-2Kk MoAbs, obtained from PharMingen (San Diego, CA).

    Techniques: Injection, In Vivo, Staining

    (see also pp 438 and 439.) Phenotypic characterization of lymphocytes in various organs of irradiated mice with or without a liver shield, which were subsequently subjected to allogeneic bone marrow transplantation (BMT). (a) Two-colour staining for H-2K b and H-2K k . (b) Two-colour staining for CD3 and IL-2Rβ. (c) Two-colour staining for CD4 and CD8. In this experiment, irradiated mice were C3H/He and mice subjected to BMT were B6 mice. Representative results of three experiments are depicted. In mice with a liver shield, lymphocytes (mainly CD3 int CD8 + ) of recipient origin (H-2K k ) expanded. On the other hand, CD3 int CD8 + lymphocytes of donor origin (H-2K b ) expanded in mice without a liver shield.

    Journal: Clinical and Experimental Immunology

    Article Title: Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

    doi: 10.1046/j.1365-2249.1998.00726.x

    Figure Lengend Snippet: (see also pp 438 and 439.) Phenotypic characterization of lymphocytes in various organs of irradiated mice with or without a liver shield, which were subsequently subjected to allogeneic bone marrow transplantation (BMT). (a) Two-colour staining for H-2K b and H-2K k . (b) Two-colour staining for CD3 and IL-2Rβ. (c) Two-colour staining for CD4 and CD8. In this experiment, irradiated mice were C3H/He and mice subjected to BMT were B6 mice. Representative results of three experiments are depicted. In mice with a liver shield, lymphocytes (mainly CD3 int CD8 + ) of recipient origin (H-2K k ) expanded. On the other hand, CD3 int CD8 + lymphocytes of donor origin (H-2K b ) expanded in mice without a liver shield.

    Article Snippet: The MoAbs used included FITC- or biotin-conjugated reagents of anti-CD3 (145-2C11), NK1.1 (PK136), TER119 (erythroid marker), anti-Gr-1 (RA3-8C5), anti-Mac-1 (M1/70), anti-H-2Kb , and anti-H-2Kk MoAbs, obtained from PharMingen (San Diego, CA).

    Techniques: Irradiation, Mouse Assay, Transplantation Assay, Staining

    Migration of lymphocyte subsets in the liver to the spleen and bone marrow (BM) after irradiation in mice with a liver shield. B6 and C3H/He mice with a liver shield were irradiated (9.5 Gy) and phenotypic characterization was performed in various organs on days 7 and 21. Control indicates untreated B6 mice. Two-colour staining for CD3 and IL-2Rβ was conducted to identify NK cells (CD3 − IL-2Rβ + ), extrathymic T cells (CD3 int IL-2Rβ + ), and conventional T cells (CD3 high IL-2Rβ − ). Numbers indicate the proportion of fluorescence-positive cells in corresponding areas. Representative results of three experiments are depicted.

    Journal: Clinical and Experimental Immunology

    Article Title: Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

    doi: 10.1046/j.1365-2249.1998.00726.x

    Figure Lengend Snippet: Migration of lymphocyte subsets in the liver to the spleen and bone marrow (BM) after irradiation in mice with a liver shield. B6 and C3H/He mice with a liver shield were irradiated (9.5 Gy) and phenotypic characterization was performed in various organs on days 7 and 21. Control indicates untreated B6 mice. Two-colour staining for CD3 and IL-2Rβ was conducted to identify NK cells (CD3 − IL-2Rβ + ), extrathymic T cells (CD3 int IL-2Rβ + ), and conventional T cells (CD3 high IL-2Rβ − ). Numbers indicate the proportion of fluorescence-positive cells in corresponding areas. Representative results of three experiments are depicted.

    Article Snippet: The MoAbs used included FITC- or biotin-conjugated reagents of anti-CD3 (145-2C11), NK1.1 (PK136), TER119 (erythroid marker), anti-Gr-1 (RA3-8C5), anti-Mac-1 (M1/70), anti-H-2Kb , and anti-H-2Kk MoAbs, obtained from PharMingen (San Diego, CA).

    Techniques: Migration, Irradiation, Mouse Assay, Staining, Fluorescence

    Raltegravir intracellular accumulation analysis of CD3+CD4+P-gp high and P-gp low populations. (a) After Rho123 incubation (1 μM, 20 min, 37°C), PBMCs were put in culture (complete medium) during 2 h to let the dye be effluxed and then stained with anti-CD3-APC and anti-CD4-PerCP antibodies. CD3+CD4+ T cells were sorted based on their Rho123 staining: P-gp high (Rho123 very low) and P-gp low (Rho123 high). Cells were directly used in subsequent [ 3 H]raltegravir accumulation assays. (b) CD3+CD4+P-gp high and CD3+CD4+P-gp low cells were incubated in transport medium with 1 μM (1 μCi/mL) [ 3 H]raltegravir in the absence (mock) or presence of the P-gp-specific inhibitor XR9051 (1 μM). Unsorted (total) PBMCs were used as control of viability and transport, considering that they contain CD8+ T cells, NK and monocytes. The results represent the mean ± SEM of five independent experiments (each with a different blood donor) performed in duplicate. Statistical significance was assessed by paired t -test (* P

    Journal: Journal of Antimicrobial Chemotherapy

    Article Title: P-glycoprotein (ABCB1) activity decreases raltegravir disposition in primary CD4+P-gphigh cells and correlates with HIV-1 viral load

    doi: 10.1093/jac/dkw215

    Figure Lengend Snippet: Raltegravir intracellular accumulation analysis of CD3+CD4+P-gp high and P-gp low populations. (a) After Rho123 incubation (1 μM, 20 min, 37°C), PBMCs were put in culture (complete medium) during 2 h to let the dye be effluxed and then stained with anti-CD3-APC and anti-CD4-PerCP antibodies. CD3+CD4+ T cells were sorted based on their Rho123 staining: P-gp high (Rho123 very low) and P-gp low (Rho123 high). Cells were directly used in subsequent [ 3 H]raltegravir accumulation assays. (b) CD3+CD4+P-gp high and CD3+CD4+P-gp low cells were incubated in transport medium with 1 μM (1 μCi/mL) [ 3 H]raltegravir in the absence (mock) or presence of the P-gp-specific inhibitor XR9051 (1 μM). Unsorted (total) PBMCs were used as control of viability and transport, considering that they contain CD8+ T cells, NK and monocytes. The results represent the mean ± SEM of five independent experiments (each with a different blood donor) performed in duplicate. Statistical significance was assessed by paired t -test (* P

    Article Snippet: Cells were stained with anti-CD3-APC and anti-CD4-PerCP antibodies (BD Biosciences) for 20 min at 4°C in PBS with 1% FBS.

    Techniques: Incubation, Staining

    P-gp activity correlation with plasma viral load in different CD4+ subsets in HIV-1-infected subjects. HIV-1-infected blood samples were processed by Ficoll-Paque to obtain the PBMC fraction and loaded with Rho123. After 2 h of efflux time, cells were stained with two panels of antibodies (plus CD3-APC and CD4-PerCP, present in both panels): (i) HIV-1 co-receptors, CXCR4 and CCR5; and (ii) naive/memory populations (CD45RA, CD27, CCR7). CD3+CD4+, CD4+CCR5+ and double positive (CD4+CCR5+CXCR4+) populations and their correlations with plasma viral load (HIV-1 RNA log copies/mL) are shown in (a), (b) and (c), respectively. (d), (e) and (f) show CD4+CD45RA− (memory T cells), CD45RA−CD27+CCR7+ (T central memory, T CM ) and CD45RA−CD27+CCR7− (T transitional memory, T TM ) correlations. Statistical analyses were performed by Spearman correlations ( r ) of percentage of P-gp active cells versus viral load. Statistically significant P values

    Journal: Journal of Antimicrobial Chemotherapy

    Article Title: P-glycoprotein (ABCB1) activity decreases raltegravir disposition in primary CD4+P-gphigh cells and correlates with HIV-1 viral load

    doi: 10.1093/jac/dkw215

    Figure Lengend Snippet: P-gp activity correlation with plasma viral load in different CD4+ subsets in HIV-1-infected subjects. HIV-1-infected blood samples were processed by Ficoll-Paque to obtain the PBMC fraction and loaded with Rho123. After 2 h of efflux time, cells were stained with two panels of antibodies (plus CD3-APC and CD4-PerCP, present in both panels): (i) HIV-1 co-receptors, CXCR4 and CCR5; and (ii) naive/memory populations (CD45RA, CD27, CCR7). CD3+CD4+, CD4+CCR5+ and double positive (CD4+CCR5+CXCR4+) populations and their correlations with plasma viral load (HIV-1 RNA log copies/mL) are shown in (a), (b) and (c), respectively. (d), (e) and (f) show CD4+CD45RA− (memory T cells), CD45RA−CD27+CCR7+ (T central memory, T CM ) and CD45RA−CD27+CCR7− (T transitional memory, T TM ) correlations. Statistical analyses were performed by Spearman correlations ( r ) of percentage of P-gp active cells versus viral load. Statistically significant P values

    Article Snippet: Cells were stained with anti-CD3-APC and anti-CD4-PerCP antibodies (BD Biosciences) for 20 min at 4°C in PBS with 1% FBS.

    Techniques: Activity Assay, Infection, Staining

    Dex treatment does not affect ERK and JNK activation. Cell extracts were prepared from Dex-treated and control naive CD4+ T cells stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28. The phosphorylation of ( a ) ERK and ( b ) JNK was assessed by Western blotting using specific Ab’s. Results from a representative experiment are shown.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Dex treatment does not affect ERK and JNK activation. Cell extracts were prepared from Dex-treated and control naive CD4+ T cells stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28. The phosphorylation of ( a ) ERK and ( b ) JNK was assessed by Western blotting using specific Ab’s. Results from a representative experiment are shown.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Activation Assay, Western Blot

    Costimulation restores the Dex-mediated inhibition of c-Fos induction. Cell extracts were prepared from Dex-treated and control human naive CD4+ T cells stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28. The induction of ( a ) c-Jun and ( b ) c-Fos expression was assessed by Western blotting using specific Ab’s. Results from a representative experiment are shown.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Costimulation restores the Dex-mediated inhibition of c-Fos induction. Cell extracts were prepared from Dex-treated and control human naive CD4+ T cells stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28. The induction of ( a ) c-Jun and ( b ) c-Fos expression was assessed by Western blotting using specific Ab’s. Results from a representative experiment are shown.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Inhibition, Expressing, Western Blot

    Inhibition of MEK/ERK activity abrogates T cell resistance to Dex. Dex-treated and control murine naive CD4+ T cells were stimulated for 3 days with ( a ) anti-CD3 plus IL-2 or ( b ) anti-CD3 plus anti-CD28 in the presence of 10 μM (max) or 5 μM (half) of U0126. ( c ) Purified human naive CD4+ T cells were also stimulated with anti-CD3 plus anti-CD28 in the presence and absence of U0126. T cell proliferation was assessed by measuring 3H incorporation. Results are expressed as mean counts per minute (± SD) of triplicate cultures.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Inhibition of MEK/ERK activity abrogates T cell resistance to Dex. Dex-treated and control murine naive CD4+ T cells were stimulated for 3 days with ( a ) anti-CD3 plus IL-2 or ( b ) anti-CD3 plus anti-CD28 in the presence of 10 μM (max) or 5 μM (half) of U0126. ( c ) Purified human naive CD4+ T cells were also stimulated with anti-CD3 plus anti-CD28 in the presence and absence of U0126. T cell proliferation was assessed by measuring 3H incorporation. Results are expressed as mean counts per minute (± SD) of triplicate cultures.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Inhibition, Activity Assay, Purification

    Dex alters IL-2–induced activation of STAT5. ( a ) Dex-treated and control naive CD4+ T cells were stimulated with IL-2 for 20 minutes, and cell extracts were prepared and assessed for STAT5 DNA binding using EMSA. ( b ) Dex-treated and control naive CD4+ T cells stimulated with anti-CD3 or anti-CD3 plus IL-2 for 4 or 24 hours were harvested, washed, rested, and stimulated with IL-2 for 20 minutes. STAT5 DNA binding was assessed in the cell extracts by EMSA.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Dex alters IL-2–induced activation of STAT5. ( a ) Dex-treated and control naive CD4+ T cells were stimulated with IL-2 for 20 minutes, and cell extracts were prepared and assessed for STAT5 DNA binding using EMSA. ( b ) Dex-treated and control naive CD4+ T cells stimulated with anti-CD3 or anti-CD3 plus IL-2 for 4 or 24 hours were harvested, washed, rested, and stimulated with IL-2 for 20 minutes. STAT5 DNA binding was assessed in the cell extracts by EMSA.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Activation Assay, Binding Assay

    Costimulation promotes T cell resistance to Dex. Dex-treated and control T cells were stained with CFSE and stimulated with anti-CD3 or anti-CD3 plus IL-2 for 3 days. Cells were harvested before stimulation and on days 1, 2, and 3 and analyzed by flow cytometry. Results from a representative experiment are shown. Filled purple: before stimulation; dashed black line: after 24 hours; pink line: after 48 hours; blue line: after 72 hours.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Costimulation promotes T cell resistance to Dex. Dex-treated and control T cells were stained with CFSE and stimulated with anti-CD3 or anti-CD3 plus IL-2 for 3 days. Cells were harvested before stimulation and on days 1, 2, and 3 and analyzed by flow cytometry. Results from a representative experiment are shown. Filled purple: before stimulation; dashed black line: after 24 hours; pink line: after 48 hours; blue line: after 72 hours.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Staining, Flow Cytometry, Cytometry

    Stimulation of Dex-treated T cells is not associated with enhanced apoptosis. Dex-treated and control naive CD4+ T cells were stimulated with anti-CD3 or anti-CD3 plus IL-2 and stained with ( a ) annexin V and ( b ) propidium iodide 24, 48, and 72 hours later. The number of positively stained cells was assessed by flow cytometry. Results are shown as mean ± SD of duplicate samples. Control, medium alone.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Stimulation of Dex-treated T cells is not associated with enhanced apoptosis. Dex-treated and control naive CD4+ T cells were stimulated with anti-CD3 or anti-CD3 plus IL-2 and stained with ( a ) annexin V and ( b ) propidium iodide 24, 48, and 72 hours later. The number of positively stained cells was assessed by flow cytometry. Results are shown as mean ± SD of duplicate samples. Control, medium alone.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Staining, Flow Cytometry, Cytometry

    Costimulation restores the inhibition of AP-1 activation by Dex. Nuclear extracts were prepared from Dex-treated and control naive CD4+ T cells stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28 and analyzed by electrophoretic mobility shift assay (EMSA) using 32P-labeled oligonucleotide probes corresponding to ( a ) AP-1, ( b ) NF-AT, or ( c ) NF-κB DNA binding sites. As a control, the competition (Comp) for DNA binding with excess oligonucleotide was assessed. Results from a representative experiment are shown.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Costimulation restores the inhibition of AP-1 activation by Dex. Nuclear extracts were prepared from Dex-treated and control naive CD4+ T cells stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28 and analyzed by electrophoretic mobility shift assay (EMSA) using 32P-labeled oligonucleotide probes corresponding to ( a ) AP-1, ( b ) NF-AT, or ( c ) NF-κB DNA binding sites. As a control, the competition (Comp) for DNA binding with excess oligonucleotide was assessed. Results from a representative experiment are shown.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Inhibition, Activation Assay, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay

    Inhibition of JNK activation does not affect T cell resistance to Dex. Dex-treated and control murine naive CD4+ T cells were stimulated for 3 days with ( a ) anti-CD3 plus IL-2 or ( b ) anti-CD3 plus anti-CD28 in the presence of 10 μM (max) or 5 μM (half) of SP600125. T cell proliferation was assessed by measuring 3H incorporation. Results are expressed as mean counts per minute (± SD) of triplicate cultures.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Inhibition of JNK activation does not affect T cell resistance to Dex. Dex-treated and control murine naive CD4+ T cells were stimulated for 3 days with ( a ) anti-CD3 plus IL-2 or ( b ) anti-CD3 plus anti-CD28 in the presence of 10 μM (max) or 5 μM (half) of SP600125. T cell proliferation was assessed by measuring 3H incorporation. Results are expressed as mean counts per minute (± SD) of triplicate cultures.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques: Inhibition, Activation Assay

    Costimulation abrogates the inhibitory effect of Dex on TCR-induced proliferation. ( a ) Murine or ( b ) human Dex-treated and control naive CD4+ T cells were stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28 for 3 days, and T cell proliferation was assessed by measuring 3H incorporation. Results are expressed as mean counts per minute (± SD) of triplicate cultures. *Reduction in proliferation is statistically significant.

    Journal: Journal of Clinical Investigation

    Article Title: Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells

    doi: 10.1172/JCI200418975

    Figure Lengend Snippet: Costimulation abrogates the inhibitory effect of Dex on TCR-induced proliferation. ( a ) Murine or ( b ) human Dex-treated and control naive CD4+ T cells were stimulated with anti-CD3, anti-CD3 plus IL-2, or anti-CD3 plus anti-CD28 for 3 days, and T cell proliferation was assessed by measuring 3H incorporation. Results are expressed as mean counts per minute (± SD) of triplicate cultures. *Reduction in proliferation is statistically significant.

    Article Snippet: The anti-mouse and anti-human CD3 and CD28 mAb’s used for T cell stimulation were purchased from BD Pharmingen (San Diego, California, USA).

    Techniques:

    IL-23 induces IL-17A production in CD4 + and particularly in CD4 − CD8 − T cells. Cells were extracted from the lymph nodes of 5-mo-old B6, B6/ lpr , 4-mo-old MRL/MPJ, and MRL/ lpr mice. The cells were incubated in DMEM with plate-bound anti-CD3

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The Role of IL-23/IL-17 Axis in Lupus Nephritis 1

    doi: 10.4049/jimmunol.0900385

    Figure Lengend Snippet: IL-23 induces IL-17A production in CD4 + and particularly in CD4 − CD8 − T cells. Cells were extracted from the lymph nodes of 5-mo-old B6, B6/ lpr , 4-mo-old MRL/MPJ, and MRL/ lpr mice. The cells were incubated in DMEM with plate-bound anti-CD3

    Article Snippet: Cell surface marker and intracellular staining for murine lymphocytes isolated from the spleen and the lymph nodes were done with anti-mouse CD3, CD28, CD4, CD8, and IL-17 Abs (BD Pharmingen).

    Techniques: Mouse Assay, Incubation

    IL-17A + CD3 + cells are found in kidneys of Rag-1 −/− mice that were injected with IL-23-treated B6/ lpr -derived lymphocytes. Rag-1 −/− mice that were injected with lymphocytes derived from control B6 and B6/ lpr mice were sacrificed

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The Role of IL-23/IL-17 Axis in Lupus Nephritis 1

    doi: 10.4049/jimmunol.0900385

    Figure Lengend Snippet: IL-17A + CD3 + cells are found in kidneys of Rag-1 −/− mice that were injected with IL-23-treated B6/ lpr -derived lymphocytes. Rag-1 −/− mice that were injected with lymphocytes derived from control B6 and B6/ lpr mice were sacrificed

    Article Snippet: Cell surface marker and intracellular staining for murine lymphocytes isolated from the spleen and the lymph nodes were done with anti-mouse CD3, CD28, CD4, CD8, and IL-17 Abs (BD Pharmingen).

    Techniques: Mouse Assay, Injection, Derivative Assay

    IL-17A- expressing T cells infiltrate the kidneys of lupus-prone mice. Frozen kidney sections from both 5-mo-old MRL/MPJ and MRL/ lpr mice were stained with anti-CD3-FITC and anti-IL-17 Abs, followed by goat anti-rat IgG Texas Red fluorescent secondary

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The Role of IL-23/IL-17 Axis in Lupus Nephritis 1

    doi: 10.4049/jimmunol.0900385

    Figure Lengend Snippet: IL-17A- expressing T cells infiltrate the kidneys of lupus-prone mice. Frozen kidney sections from both 5-mo-old MRL/MPJ and MRL/ lpr mice were stained with anti-CD3-FITC and anti-IL-17 Abs, followed by goat anti-rat IgG Texas Red fluorescent secondary

    Article Snippet: Cell surface marker and intracellular staining for murine lymphocytes isolated from the spleen and the lymph nodes were done with anti-mouse CD3, CD28, CD4, CD8, and IL-17 Abs (BD Pharmingen).

    Techniques: Expressing, Mouse Assay, Staining

    IL-23 treatment leads to an increase in the number of IL- 17A + lymphocytes and does not affect the percentage of B cells. Lymphocytes from 5- mo-old control B6 and lupus-prone B6/lpr mice were extracted (three mice in each group) and stimulated with anti-CD3

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The Role of IL-23/IL-17 Axis in Lupus Nephritis 1

    doi: 10.4049/jimmunol.0900385

    Figure Lengend Snippet: IL-23 treatment leads to an increase in the number of IL- 17A + lymphocytes and does not affect the percentage of B cells. Lymphocytes from 5- mo-old control B6 and lupus-prone B6/lpr mice were extracted (three mice in each group) and stimulated with anti-CD3

    Article Snippet: Cell surface marker and intracellular staining for murine lymphocytes isolated from the spleen and the lymph nodes were done with anti-mouse CD3, CD28, CD4, CD8, and IL-17 Abs (BD Pharmingen).

    Techniques: Mouse Assay

    CD3 + CD4 − CD8 − T cells from lupus-prone MRL/ lpr mice express IL-17A at a higher level than CD3 + CD4 + T cells from both lupus-prone and control mice. Lymph nodes (LN) and spleens were harvested from lupus-prone MRL/ lpr and control MRL/MPJ

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The Role of IL-23/IL-17 Axis in Lupus Nephritis 1

    doi: 10.4049/jimmunol.0900385

    Figure Lengend Snippet: CD3 + CD4 − CD8 − T cells from lupus-prone MRL/ lpr mice express IL-17A at a higher level than CD3 + CD4 + T cells from both lupus-prone and control mice. Lymph nodes (LN) and spleens were harvested from lupus-prone MRL/ lpr and control MRL/MPJ

    Article Snippet: Cell surface marker and intracellular staining for murine lymphocytes isolated from the spleen and the lymph nodes were done with anti-mouse CD3, CD28, CD4, CD8, and IL-17 Abs (BD Pharmingen).

    Techniques: Mouse Assay

    IL-17A + CD3 + cells are found in the spleen of Rag-1 −/− mice that were injected with IL-23-treated B6/ lpr -derived lymphocytes. Rag-1 −/− mice that were injected with lymphocytes derived from control B6 and B6/ lpr mice were

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The Role of IL-23/IL-17 Axis in Lupus Nephritis 1

    doi: 10.4049/jimmunol.0900385

    Figure Lengend Snippet: IL-17A + CD3 + cells are found in the spleen of Rag-1 −/− mice that were injected with IL-23-treated B6/ lpr -derived lymphocytes. Rag-1 −/− mice that were injected with lymphocytes derived from control B6 and B6/ lpr mice were

    Article Snippet: Cell surface marker and intracellular staining for murine lymphocytes isolated from the spleen and the lymph nodes were done with anti-mouse CD3, CD28, CD4, CD8, and IL-17 Abs (BD Pharmingen).

    Techniques: Mouse Assay, Injection, Derivative Assay

    Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by anti-CD3/CD28 beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

    doi: 10.3389/fcell.2020.00803

    Figure Lengend Snippet: Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by anti-CD3/CD28 beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.

    Article Snippet: Detection of T Cell Proliferation and Activation T cells were labeled with CFSE (Sigma, Cat. No. 21888) and activated by anti-CD3/CD28 Dynabeads for 24 h. Then the activated T cells were mixed with PD-L1-overexpressing H322 cells, which had been fixed using paraformaldehyde (Sigma Aldrich), at the ratio of 1:1 and transferred into 0.4 μm transwell chambers.

    Techniques: Staining, FACS, Western Blot, Labeling, Flow Cytometry, Derivative Assay

    Effects of secreted α-PD-1 scFv on T cell activation. CD3+ T cells from healthy donors were stained with CFSE and then activated by anti-CD3/CD28 beads for 2 days. (A) PD-1 expressions were determined in resting and activated T cells by FACS assay. (B) 293T cells transfected with mock or α-PD-1 scFv-expressing vectors were plated at the bottom. 48 h later, activated T cells were added into the upper transwell chambers with fixed PD-L1-overexpressing A549 cells at the ratio of 1:1 for 24 h. (C) Then the dilutions of CFSE in CD3+ T cells were determined using FACS assay. (D–F) The expressions of Ki67 (D) , CD107a (E) and IFN-γ (F) were further detected in CD3+ T cells after incubation. T cells from 5 donors were tested and the representative results were depicted. * indicates P

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

    doi: 10.3389/fcell.2020.00803

    Figure Lengend Snippet: Effects of secreted α-PD-1 scFv on T cell activation. CD3+ T cells from healthy donors were stained with CFSE and then activated by anti-CD3/CD28 beads for 2 days. (A) PD-1 expressions were determined in resting and activated T cells by FACS assay. (B) 293T cells transfected with mock or α-PD-1 scFv-expressing vectors were plated at the bottom. 48 h later, activated T cells were added into the upper transwell chambers with fixed PD-L1-overexpressing A549 cells at the ratio of 1:1 for 24 h. (C) Then the dilutions of CFSE in CD3+ T cells were determined using FACS assay. (D–F) The expressions of Ki67 (D) , CD107a (E) and IFN-γ (F) were further detected in CD3+ T cells after incubation. T cells from 5 donors were tested and the representative results were depicted. * indicates P

    Article Snippet: Detection of T Cell Proliferation and Activation T cells were labeled with CFSE (Sigma, Cat. No. 21888) and activated by anti-CD3/CD28 Dynabeads for 24 h. Then the activated T cells were mixed with PD-L1-overexpressing H322 cells, which had been fixed using paraformaldehyde (Sigma Aldrich), at the ratio of 1:1 and transferred into 0.4 μm transwell chambers.

    Techniques: Activation Assay, Staining, FACS, Transfection, Expressing, Incubation

    Blockade of PD-1 suppression by self-delivered α-PD-1 scFv. (A,B) Activated CAR-T cells were stained with anti-PD-1 antibody and checked on image flow cytometer (A) and the expressing levels of PD-1 were determined (B) . (C–E) To check the effects of scFv on the cytotoxic activities, CAR-T cells were directly incubated with H322 cells without pre-activation by anti-CD3/CD28 Dynabeads. CAR-T cells were co-cultured with luciferase-expressing H322 cells at indicated E:T ratios for 18 h. Then the relative viabilities of tumor cells were determined and compared between treatments with CAR-meso or CAR-meso-α-PD-1 cells (C) . Additionally, CAR-T cells were co-incubated with H322 cells at the ratio of 1:1 for 24 h. Then the supernatants were checked for the secretions of IFN-γ (D) and IL2 (E) . The cells were collected and Ki67 expressions were determined in CD3+ T cells using FACS test (F) . CAR-T cells derived from 5 different donors were tested and the representative data were demonstrated. * indicates P

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

    doi: 10.3389/fcell.2020.00803

    Figure Lengend Snippet: Blockade of PD-1 suppression by self-delivered α-PD-1 scFv. (A,B) Activated CAR-T cells were stained with anti-PD-1 antibody and checked on image flow cytometer (A) and the expressing levels of PD-1 were determined (B) . (C–E) To check the effects of scFv on the cytotoxic activities, CAR-T cells were directly incubated with H322 cells without pre-activation by anti-CD3/CD28 Dynabeads. CAR-T cells were co-cultured with luciferase-expressing H322 cells at indicated E:T ratios for 18 h. Then the relative viabilities of tumor cells were determined and compared between treatments with CAR-meso or CAR-meso-α-PD-1 cells (C) . Additionally, CAR-T cells were co-incubated with H322 cells at the ratio of 1:1 for 24 h. Then the supernatants were checked for the secretions of IFN-γ (D) and IL2 (E) . The cells were collected and Ki67 expressions were determined in CD3+ T cells using FACS test (F) . CAR-T cells derived from 5 different donors were tested and the representative data were demonstrated. * indicates P

    Article Snippet: Detection of T Cell Proliferation and Activation T cells were labeled with CFSE (Sigma, Cat. No. 21888) and activated by anti-CD3/CD28 Dynabeads for 24 h. Then the activated T cells were mixed with PD-L1-overexpressing H322 cells, which had been fixed using paraformaldehyde (Sigma Aldrich), at the ratio of 1:1 and transferred into 0.4 μm transwell chambers.

    Techniques: Staining, Flow Cytometry, Expressing, Incubation, Activation Assay, Cell Culture, Luciferase, FACS, Derivative Assay

    Gating strategy for menstrual and peripheral blood cells. A. PBMC and B. MBC. Initial gating is performed from the SSC-A vs. FSC-A to determine the total lymphocyte gate (small gate) and the larger gate is used to further gate for monocytes (CD14). From the lymphocytes, live cells are gated, followed by gating B cells (CD19), T cells (CD3), CD4+CD3+ and CD8+CD3+ cells. From the CD3- gate, NK cell phenotypes are selected. CD16-CD56+(immunoregulatory cells) and CD16+CD56+(Cytotoxic cells). Percentages of positive cells for a given marker are indicated above/near gate.

    Journal: PLoS ONE

    Article Title: Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

    doi: 10.1371/journal.pone.0028894

    Figure Lengend Snippet: Gating strategy for menstrual and peripheral blood cells. A. PBMC and B. MBC. Initial gating is performed from the SSC-A vs. FSC-A to determine the total lymphocyte gate (small gate) and the larger gate is used to further gate for monocytes (CD14). From the lymphocytes, live cells are gated, followed by gating B cells (CD19), T cells (CD3), CD4+CD3+ and CD8+CD3+ cells. From the CD3- gate, NK cell phenotypes are selected. CD16-CD56+(immunoregulatory cells) and CD16+CD56+(Cytotoxic cells). Percentages of positive cells for a given marker are indicated above/near gate.

    Article Snippet: The surface phenotype of samples was determined by staining with anti-CD3-Pacific Blue, anti-CD4-PercpCy5.5, and anti-CD8-PE antibodies (BD Biosciences, San Jose, CA, USA) for 20 min. at room temperature.

    Techniques: Marker

    A representative example of surface antigen expression. Cell surface expression of CD62L, HLA-DR and αEβ7 on A. CD3+CD4+ and B. CD3+CD8+ T cells from PBMC, MBC and endometrial tissue (ENDO) is shown. Percent of positive cells for a given marker are indicated above the gate.

    Journal: PLoS ONE

    Article Title: Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

    doi: 10.1371/journal.pone.0028894

    Figure Lengend Snippet: A representative example of surface antigen expression. Cell surface expression of CD62L, HLA-DR and αEβ7 on A. CD3+CD4+ and B. CD3+CD8+ T cells from PBMC, MBC and endometrial tissue (ENDO) is shown. Percent of positive cells for a given marker are indicated above the gate.

    Article Snippet: The surface phenotype of samples was determined by staining with anti-CD3-Pacific Blue, anti-CD4-PercpCy5.5, and anti-CD8-PE antibodies (BD Biosciences, San Jose, CA, USA) for 20 min. at room temperature.

    Techniques: Expressing, Marker

    Memory marker expression. CCR7 and CD45RA surface staining was performed on both CD3+CD4+ and CD3+CD8+ T cells from PBMC and MBC. A–D represent samples gated on different populations of cells CCR7 and CD45RA expression. The median and interquartile range data is shown for 12 healthy women. Tcm = central memory, Tem = effector memory, and Temra = effector memory RA+. Comparisons were made using Wilcoxon Signed Rank test.

    Journal: PLoS ONE

    Article Title: Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

    doi: 10.1371/journal.pone.0028894

    Figure Lengend Snippet: Memory marker expression. CCR7 and CD45RA surface staining was performed on both CD3+CD4+ and CD3+CD8+ T cells from PBMC and MBC. A–D represent samples gated on different populations of cells CCR7 and CD45RA expression. The median and interquartile range data is shown for 12 healthy women. Tcm = central memory, Tem = effector memory, and Temra = effector memory RA+. Comparisons were made using Wilcoxon Signed Rank test.

    Article Snippet: The surface phenotype of samples was determined by staining with anti-CD3-Pacific Blue, anti-CD4-PercpCy5.5, and anti-CD8-PE antibodies (BD Biosciences, San Jose, CA, USA) for 20 min. at room temperature.

    Techniques: Marker, Expressing, Staining, Transmission Electron Microscopy

    Cell composition of menstrual blood cells (MBCs). The percentage of various cell types present in menstrual blood versus PBMC were resolved by staining with cell type-specific surface markers. A. The total lymphocytes, were determined by the percentage of cells within a lymphocyte gate (small cells from FSC vs SSC plots) and monocytes (N = 8), are CD14+ cell from a leuokocyte gate (all cells within the larger FSC vs SSC plot), refer to Figure 1 . B. Lymphocyte subsets present in menstrual versus peripheral blood. From the live cell gate, B cells were gated on CD19, T cells were gated on CD3 and T cell subsets were further divided into CD4+CD3+ and CD8+CD3+ T cells. C. Natural killer cell subsets (N = 8) gated from CD3- cells. CD16+CD56+ (cytotoxic NK cells) and CD16-CD56+ (immunoregulatory NK cells). The median is shown for data from 12 healthy women, except for monocytes and NK cells, where 8 samples were analyzed. Statistically significant differences (p

    Journal: PLoS ONE

    Article Title: Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

    doi: 10.1371/journal.pone.0028894

    Figure Lengend Snippet: Cell composition of menstrual blood cells (MBCs). The percentage of various cell types present in menstrual blood versus PBMC were resolved by staining with cell type-specific surface markers. A. The total lymphocytes, were determined by the percentage of cells within a lymphocyte gate (small cells from FSC vs SSC plots) and monocytes (N = 8), are CD14+ cell from a leuokocyte gate (all cells within the larger FSC vs SSC plot), refer to Figure 1 . B. Lymphocyte subsets present in menstrual versus peripheral blood. From the live cell gate, B cells were gated on CD19, T cells were gated on CD3 and T cell subsets were further divided into CD4+CD3+ and CD8+CD3+ T cells. C. Natural killer cell subsets (N = 8) gated from CD3- cells. CD16+CD56+ (cytotoxic NK cells) and CD16-CD56+ (immunoregulatory NK cells). The median is shown for data from 12 healthy women, except for monocytes and NK cells, where 8 samples were analyzed. Statistically significant differences (p

    Article Snippet: The surface phenotype of samples was determined by staining with anti-CD3-Pacific Blue, anti-CD4-PercpCy5.5, and anti-CD8-PE antibodies (BD Biosciences, San Jose, CA, USA) for 20 min. at room temperature.

    Techniques: Staining

    Surface marker expression on PBMC, MBC, and cells from endometrial tissue. Surface markers on CD3+CD4+ cells are shown for panels A–C and on CD3+CD8+ on panels D–F . The median data from peripheral and menstrual blood is shown for 12 healthy women. Endometrial tissue from 7 women was available for several analyses; however, for some samples, data from only four samples was obtained. Comparisons were made using Wilcoxon Signed Rank test and Mann Whitney U test for paired and unpaired samples, respectively.

    Journal: PLoS ONE

    Article Title: Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

    doi: 10.1371/journal.pone.0028894

    Figure Lengend Snippet: Surface marker expression on PBMC, MBC, and cells from endometrial tissue. Surface markers on CD3+CD4+ cells are shown for panels A–C and on CD3+CD8+ on panels D–F . The median data from peripheral and menstrual blood is shown for 12 healthy women. Endometrial tissue from 7 women was available for several analyses; however, for some samples, data from only four samples was obtained. Comparisons were made using Wilcoxon Signed Rank test and Mann Whitney U test for paired and unpaired samples, respectively.

    Article Snippet: The surface phenotype of samples was determined by staining with anti-CD3-Pacific Blue, anti-CD4-PercpCy5.5, and anti-CD8-PE antibodies (BD Biosciences, San Jose, CA, USA) for 20 min. at room temperature.

    Techniques: Marker, Expressing, MANN-WHITNEY

    Effects of CSE on Treg differentiation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Cells were co-stained for CD25 and FOXP3 expression and measured by flow cytometry; representative pseudocolour dot plots gated on CD4 + T cells are shown. (B) Summary data of CD25 + FOXP3 + Tregs and CD25 + T cells in each condition, from (A) Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Journal: International Journal of Molecular Medicine

    Article Title: Cytokine-induced alterations of BAMBI mediate the reciprocal regulation of human Th17/Treg cells in response to cigarette smoke extract

    doi: 10.3892/ijmm.2018.3919

    Figure Lengend Snippet: Effects of CSE on Treg differentiation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Cells were co-stained for CD25 and FOXP3 expression and measured by flow cytometry; representative pseudocolour dot plots gated on CD4 + T cells are shown. (B) Summary data of CD25 + FOXP3 + Tregs and CD25 + T cells in each condition, from (A) Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Article Snippet: Cell viability assay Purified naive CD4+ T cells (1×105 cells/well) were cultured and incubated at 37°C in 200 μ l of complete medium containing 10% heat‑inactivated (56°C for 30 min) foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 50 U/ml IL-2 (PeproTech, Inc., Rocky Hill, NJ, USA) in 96-well plates and stimulated with plate-bound anti-CD3 (monoclonal, OKT3; 5 μ g/ml) and anti‑CD28 (5 μ g/ml) antibodies (both purchased from eBioscience; Thermo Fisher Scientific, Inc.).

    Techniques: Isolation, Cell Culture, Staining, Expressing, Flow Cytometry, Cytometry

    BAMBI expression increases during activation and CSE exposure. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Expression levels of BAMBI were determined by flow cytometry, and representative histograms gated on CD4 + T cells are provided. (B) Summary data of BAMBI expression in each condition from (A). Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Journal: International Journal of Molecular Medicine

    Article Title: Cytokine-induced alterations of BAMBI mediate the reciprocal regulation of human Th17/Treg cells in response to cigarette smoke extract

    doi: 10.3892/ijmm.2018.3919

    Figure Lengend Snippet: BAMBI expression increases during activation and CSE exposure. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Expression levels of BAMBI were determined by flow cytometry, and representative histograms gated on CD4 + T cells are provided. (B) Summary data of BAMBI expression in each condition from (A). Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Article Snippet: Cell viability assay Purified naive CD4+ T cells (1×105 cells/well) were cultured and incubated at 37°C in 200 μ l of complete medium containing 10% heat‑inactivated (56°C for 30 min) foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 50 U/ml IL-2 (PeproTech, Inc., Rocky Hill, NJ, USA) in 96-well plates and stimulated with plate-bound anti-CD3 (monoclonal, OKT3; 5 μ g/ml) and anti‑CD28 (5 μ g/ml) antibodies (both purchased from eBioscience; Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Activation Assay, Isolation, Cell Culture, Flow Cytometry, Cytometry

    Effects of CSE on Smad2/Smad3 phosphorylation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Phosphorylation levels of Smad2/Smad3 were determined by flow cytometry, and representative histograms gated on CD4 + T cells are shown. (B) Summary data of Smad2/Smad3 phosphorylation in each condition from (A). Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Journal: International Journal of Molecular Medicine

    Article Title: Cytokine-induced alterations of BAMBI mediate the reciprocal regulation of human Th17/Treg cells in response to cigarette smoke extract

    doi: 10.3892/ijmm.2018.3919

    Figure Lengend Snippet: Effects of CSE on Smad2/Smad3 phosphorylation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Phosphorylation levels of Smad2/Smad3 were determined by flow cytometry, and representative histograms gated on CD4 + T cells are shown. (B) Summary data of Smad2/Smad3 phosphorylation in each condition from (A). Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Article Snippet: Cell viability assay Purified naive CD4+ T cells (1×105 cells/well) were cultured and incubated at 37°C in 200 μ l of complete medium containing 10% heat‑inactivated (56°C for 30 min) foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 50 U/ml IL-2 (PeproTech, Inc., Rocky Hill, NJ, USA) in 96-well plates and stimulated with plate-bound anti-CD3 (monoclonal, OKT3; 5 μ g/ml) and anti‑CD28 (5 μ g/ml) antibodies (both purchased from eBioscience; Thermo Fisher Scientific, Inc.).

    Techniques: Isolation, Cell Culture, Flow Cytometry, Cytometry

    Effects of CSE on cell viability. Naive CD4 + T cells isolated from peripheral blood were stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies in the presence of CSE at 0, 0.002, 0.02 and 0.2% for 5 days. Cell viability was examined by Cell Counting Kit-8 at an absorbance of 450 nm. The data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; * P

    Journal: International Journal of Molecular Medicine

    Article Title: Cytokine-induced alterations of BAMBI mediate the reciprocal regulation of human Th17/Treg cells in response to cigarette smoke extract

    doi: 10.3892/ijmm.2018.3919

    Figure Lengend Snippet: Effects of CSE on cell viability. Naive CD4 + T cells isolated from peripheral blood were stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies in the presence of CSE at 0, 0.002, 0.02 and 0.2% for 5 days. Cell viability was examined by Cell Counting Kit-8 at an absorbance of 450 nm. The data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; * P

    Article Snippet: Cell viability assay Purified naive CD4+ T cells (1×105 cells/well) were cultured and incubated at 37°C in 200 μ l of complete medium containing 10% heat‑inactivated (56°C for 30 min) foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 50 U/ml IL-2 (PeproTech, Inc., Rocky Hill, NJ, USA) in 96-well plates and stimulated with plate-bound anti-CD3 (monoclonal, OKT3; 5 μ g/ml) and anti‑CD28 (5 μ g/ml) antibodies (both purchased from eBioscience; Thermo Fisher Scientific, Inc.).

    Techniques: Isolation, Cell Counting

    Effects of cigarette smoke extract (CSE) on Th17 cell differentiation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Th17 cell counts were determined by flow cytometry, and representative histograms gated on lymphocytes are presented. (B) Summary data of Th17 cells in each condition from (A). Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Journal: International Journal of Molecular Medicine

    Article Title: Cytokine-induced alterations of BAMBI mediate the reciprocal regulation of human Th17/Treg cells in response to cigarette smoke extract

    doi: 10.3892/ijmm.2018.3919

    Figure Lengend Snippet: Effects of cigarette smoke extract (CSE) on Th17 cell differentiation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Th17 cell counts were determined by flow cytometry, and representative histograms gated on lymphocytes are presented. (B) Summary data of Th17 cells in each condition from (A). Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Article Snippet: Cell viability assay Purified naive CD4+ T cells (1×105 cells/well) were cultured and incubated at 37°C in 200 μ l of complete medium containing 10% heat‑inactivated (56°C for 30 min) foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 50 U/ml IL-2 (PeproTech, Inc., Rocky Hill, NJ, USA) in 96-well plates and stimulated with plate-bound anti-CD3 (monoclonal, OKT3; 5 μ g/ml) and anti‑CD28 (5 μ g/ml) antibodies (both purchased from eBioscience; Thermo Fisher Scientific, Inc.).

    Techniques: Cell Differentiation, Isolation, Cell Culture, Flow Cytometry, Cytometry

    Effects of CSE on Th17 cell differentiation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Th17 cell counts were determined by flow cytometry, and representative histograms gated on lymphocytes are presented. (B) Summary data of Th17 cells in each condition from (A) Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Journal: International Journal of Molecular Medicine

    Article Title: Cytokine-induced alterations of BAMBI mediate the reciprocal regulation of human Th17/Treg cells in response to cigarette smoke extract

    doi: 10.3892/ijmm.2018.3919

    Figure Lengend Snippet: Effects of CSE on Th17 cell differentiation. (A and B) Naive CD4 + T cells isolated from peripheral blood were cultured in complete medium and stimulated with plate-bound α-CD3 and α-CD28 monoclonal antibodies under the indicated conditions for 5 days. (A) Th17 cell counts were determined by flow cytometry, and representative histograms gated on lymphocytes are presented. (B) Summary data of Th17 cells in each condition from (A) Data are presented as the mean ± standard error of the mean (n=4), and are representative of three independent experiments; # P

    Article Snippet: Cell viability assay Purified naive CD4+ T cells (1×105 cells/well) were cultured and incubated at 37°C in 200 μ l of complete medium containing 10% heat‑inactivated (56°C for 30 min) foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 50 U/ml IL-2 (PeproTech, Inc., Rocky Hill, NJ, USA) in 96-well plates and stimulated with plate-bound anti-CD3 (monoclonal, OKT3; 5 μ g/ml) and anti‑CD28 (5 μ g/ml) antibodies (both purchased from eBioscience; Thermo Fisher Scientific, Inc.).

    Techniques: Cell Differentiation, Isolation, Cell Culture, Flow Cytometry, Cytometry

    The Effect of Protocol A and Protocol B on the Expansion and Function of Treg Lines (A) Tregs were expanded by stimulation with anti-CD3/CD28 beads and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. There was no difference in the fold expansion of Tregs between the different isolation strategies (protocol A and protocol B) or culture conditions. (B) The suppressive function of freshly isolated Tregs and expanded Treg lines. Suppressive ability of Tregs was measured as a decrease of proliferation of effector T cells in the presence of different concentrations of Tregs (Treg:Teff 1:1 and 1:10). Data represent the percentage Treg suppression at a Treg:Teff ratio of 1:1. Data represent mean ± SD of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA and revealed no significant difference.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

    doi: 10.1016/j.omtm.2018.01.006

    Figure Lengend Snippet: The Effect of Protocol A and Protocol B on the Expansion and Function of Treg Lines (A) Tregs were expanded by stimulation with anti-CD3/CD28 beads and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. There was no difference in the fold expansion of Tregs between the different isolation strategies (protocol A and protocol B) or culture conditions. (B) The suppressive function of freshly isolated Tregs and expanded Treg lines. Suppressive ability of Tregs was measured as a decrease of proliferation of effector T cells in the presence of different concentrations of Tregs (Treg:Teff 1:1 and 1:10). Data represent the percentage Treg suppression at a Treg:Teff ratio of 1:1. Data represent mean ± SD of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA and revealed no significant difference.

    Article Snippet: 1 × 105 /well of responder T cells were co-cultured with Tregs at different ratios (Treg:Teff 1:1, 1:5, and 1:10) in X-vivo 15 medium supplemented with 5% HS and activated by anti-CD3/CD28-coated beads (Invitrogen, Paisley, UK) in U-bottom 96-well plates.

    Techniques: Isolation

    Purity, Expansion, and Phenotypic Characterization of Tregs on Isolation and Expansion Tregs isolated in accordance with protocol B, stimulated with anti-CD3/CD28 beads, and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. (A) CD4 + CD25 + FOXP3 + expression of freshly isolated cells and expanded Treg lines. (B) Treg fold expansion by day 36 of culture. Rapa-treated and untreated cultures exhibited similar expansion profiles. (C) Total Treg numbers post expansion. (D) Expression of regulatory markers and homing receptor expression on CD4 + CD25 + FOXP3 + cells throughout culture. S- stimulation: S1, day 0; S2, day 12; S3, day 24; and final harvest, day 36. Data represent the mean ± SD of 25 independent experiments. Statistical analysis was performed using 1-way ANOVA, and where there was a significant difference, this is indicated. *p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

    doi: 10.1016/j.omtm.2018.01.006

    Figure Lengend Snippet: Purity, Expansion, and Phenotypic Characterization of Tregs on Isolation and Expansion Tregs isolated in accordance with protocol B, stimulated with anti-CD3/CD28 beads, and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. (A) CD4 + CD25 + FOXP3 + expression of freshly isolated cells and expanded Treg lines. (B) Treg fold expansion by day 36 of culture. Rapa-treated and untreated cultures exhibited similar expansion profiles. (C) Total Treg numbers post expansion. (D) Expression of regulatory markers and homing receptor expression on CD4 + CD25 + FOXP3 + cells throughout culture. S- stimulation: S1, day 0; S2, day 12; S3, day 24; and final harvest, day 36. Data represent the mean ± SD of 25 independent experiments. Statistical analysis was performed using 1-way ANOVA, and where there was a significant difference, this is indicated. *p

    Article Snippet: 1 × 105 /well of responder T cells were co-cultured with Tregs at different ratios (Treg:Teff 1:1, 1:5, and 1:10) in X-vivo 15 medium supplemented with 5% HS and activated by anti-CD3/CD28-coated beads (Invitrogen, Paisley, UK) in U-bottom 96-well plates.

    Techniques: Isolation, Expressing

    Schematic Representation of the GMP-Compliant Protocol for the Isolation and Expansion of Tregs for Clinical Application Blood is volume reduced using the Sepax 2 device (Biosafe) prior to Treg isolation. CD4 + CD25 + T cells are isolated using a combination of CD8 + depletion (CD8 reagent, Miltenyi Biotec) and enrichment step for CD25 + cells (CD25 reagent, Miltenyi Biotec) using the automated CliniMACS Plus System (Miltenyi Biotec). All processing steps are performed in closed systems. Tregs are stimulated with anti-CD3/CD28 beads in a 4:1 ratio (ExpAct Treg kit, Miltenyi Biotec) and cultured in TexMACS GMP media (Miltenyi Biotec) supplemented with 5% human serum (Lonza/Seralab). Rapamycin (Pfizer) (100 nM) is added at the beginning of the culture, whereas IL-2 (Novartis) (500 IU/mL) is added after 4 days. Both rapamycin and IL-2 are replenished every 2 to 3 days, and cells are rested for 4 days prior to restimulation. Cells are restimulated every 12 days by adding new activation beads, rapamycin, and IL-2. Phenotypic and functional characterization of the Tregs is carried out following final harvest at day 36 to ensure all final products meet the specified release criteria prior to cryopreservation and subsequent clinical application.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

    doi: 10.1016/j.omtm.2018.01.006

    Figure Lengend Snippet: Schematic Representation of the GMP-Compliant Protocol for the Isolation and Expansion of Tregs for Clinical Application Blood is volume reduced using the Sepax 2 device (Biosafe) prior to Treg isolation. CD4 + CD25 + T cells are isolated using a combination of CD8 + depletion (CD8 reagent, Miltenyi Biotec) and enrichment step for CD25 + cells (CD25 reagent, Miltenyi Biotec) using the automated CliniMACS Plus System (Miltenyi Biotec). All processing steps are performed in closed systems. Tregs are stimulated with anti-CD3/CD28 beads in a 4:1 ratio (ExpAct Treg kit, Miltenyi Biotec) and cultured in TexMACS GMP media (Miltenyi Biotec) supplemented with 5% human serum (Lonza/Seralab). Rapamycin (Pfizer) (100 nM) is added at the beginning of the culture, whereas IL-2 (Novartis) (500 IU/mL) is added after 4 days. Both rapamycin and IL-2 are replenished every 2 to 3 days, and cells are rested for 4 days prior to restimulation. Cells are restimulated every 12 days by adding new activation beads, rapamycin, and IL-2. Phenotypic and functional characterization of the Tregs is carried out following final harvest at day 36 to ensure all final products meet the specified release criteria prior to cryopreservation and subsequent clinical application.

    Article Snippet: 1 × 105 /well of responder T cells were co-cultured with Tregs at different ratios (Treg:Teff 1:1, 1:5, and 1:10) in X-vivo 15 medium supplemented with 5% HS and activated by anti-CD3/CD28-coated beads (Invitrogen, Paisley, UK) in U-bottom 96-well plates.

    Techniques: Isolation, Cell Culture, Activation Assay, Functional Assay