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  • 99
    Thermo Fisher anti cd4
    Ubc13-deficient T reg cells are sensitive to lymphopenic and inflammatory conditions for acquiring effector functions ( a, b ) Flow cytometry analysis of IL-17- and IFN-γ-producing MLN T reg cells (gated on CD45.2 + Foxp3 + <t>CD4</t> + cells) derived from Rag1 KO mice (6 weeks old) adoptively transferred, for 10 or 21 days, with WT CD45.1 + CD4 + CD25 − CD45RB hi T cells plus CD4 + YFP + T reg cells purified from WT-R26 YFP (WT) or Ube2n Treg-KO R26 YFP (KO) mice (6 weeks old, CD45.2 + ). Data are representative ( a ) or summary ( b , day 21 data only) of two independent experiments (n=3). *p=0.05. ( c ) Flow cytometry analysis of IL-17- and IFN-γ-producing MLN T reg cells (gated on CD45.2 + Foxp3 + cells) from Rag1 KO mice (5 weeks old) adoptively transferred, for 5 weeks, with CD45.1 + Foxp3 + WT and/or CD45.2 + Foxp3 + Ube2n Treg-KO (KO) T reg cells. ( d ) Flow cytometric analysis of Foxp3 expression in CD45.1 + Foxp3 + and CD45.2 + Foxp3 + T reg cells from the Rag1 KO recipients of WT plus KO T reg cells described in d . ( e ) Flow cytometry measuring the frequency of IL-17- and IFN-γ-expressing T reg cells in the spleen of 10 weeks old mice, gating on Foxp3 + or YFP + cells. ( f ) ELISA quantifying the secreted IFN-γ and IL-17 by YFP + CD4 + T reg cells (isolated from 10 weeks old mice), stimulated with PMA and ionomycin for 24 h. Data are presented as means±SD of two independent experiments. ( g, h ) Flow cytometric analysis of the frequency of Foxp3 + IL-17 + cells in sorted YFP + CD4 + T reg cells, derived from the indicated mice (6 weeks old) and activated in the absence or presence of TGF-β and IL-6. Data are representative ( e ) or summary ( f ) of four independent experiments (n=3). **p=0.01.
    Anti Cd4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson cd4
    Needle-free SL/B immunization generates vaccine-specific <t>CD4</t> and CD8 T cells in the blood. PMBCs were stimulated with HIV-1 consensus B Gag and Env peptides and analyzed by flow cytometry for cytokine production. a Representative flow plots for IFN-γ and TNF-α cytokine expression on Live CD3 + CD4 + cells in non-stimulated (NS), Gag, or Env stimulated PBMCs is shown. Kinetics of the total (Gag + Env) b IFN-γ, c TNF-α, and d IL-2 response in CD4 + T cells (mean ± S.D.), with the peak response (wk 16) highlighted for each animal (line denotes mean). e Kinetics of the total IFN-γ response in CD8 + cells (mean ± S.D.), with the peak response (wk 16) highlighted for each animal. White circle, topical SL/B ( n = 4); blue square, needle-free SL/B ( n = 5); gray triangle, ID/SC ( n = 6)
    Cd4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 27445 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson antibodies cd4
    Activation induces extensive metabolic reprogramming in T cells. Purified T cells were untreated (UT) or activated with anti-CD3/CD28 beads (CD3/28). (a) Geometric mean fluorescence intensity (gMFI) was measured for activation and metabolic proteins in <t>CD4</t> + T cells. Each dot represents one donor, data representative of n=8 donors, from 3 independent experiments. (b) FitSNE projection and corresponding expression of metabolic protein and activation markers in T cells, data acquired from n=5 samples, with 10,000 cells per donor. (c) Chord visualization of correlation between immune and metabolic proteins in activated CD4 + T cells, representative of n=8 donors. (d) Spearman correlation of GLUT1 and CD25 expression in untreated and (e) activated CD4 + T cells. (f) Heatmap of FitSNE projection of GLUT1 and CD25 expression in untreated and activated CD4 + T cells.
    Antibodies Cd4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher cd4
    Incubation of vehicle (veh), epinephrine (epine), and salbutamol (sal) of immature Ovalbumin loaded BMDC in a T cell proliferation assay. Cells were incubated during LPS maturation o/n. The cells were washed and freshly isolated naïve <t>CD4</t> OT-II cells were stained with CFSE. Cells were co-cultured for 3 days and CFSE dilution was determined by flow cytometry. Overlays shown are representative of 3 independent experiments.
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    94
    Becton Dickinson cd4 t cells
    DC-T cell interactions. ( A ) Productive and latent infection was quantified in eFluor670-labelled resting memory CD4 +  T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or anti-CD18 (dark grey) prior to infection, n = 5. ( B ) Latent infection was determined in eFluor670-labelled resting CD4 +  T cells that were cultured alone, with mDC or alternatively with soluble ICAM-1-fc and anti-IgG-fc, n = 2. ( C ) Latent infection was determined in sorted eFluor hi EGFP −  CD4 +  T cells following stimulation with anti-CD3/CD28, that were cultured alone or with mDC that were added prior to infection or post-infection, n = 5. Columns represent the median of 5 experiments and error bars the interquartile range. *P
    Cd4 T Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 9579 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec cd4 t cell isolation kit
    IL-17 responses are significantly elevated in the MLN and PPs of  H. pylori -infected IL-21 −/−  mice. Real-time RT-PCR was used to measure  Il-21  (A) and  Il-17a  (B) expression levels in  H. pylori -infected mice. (C) Intracellular cytokine staining was used to measure IFN-γ and IL-17A production by CD4 +  T cells and γδ-positive T cells from the Peyer’s patches of  H. pylori -infected mice (with PMA restimulation [stim] and without PMA restimulation [null]). Four to 7 mice were used for each experiment, and the data presented are representative of those from 3 experiments. (A, B) An unpaired  t  test was performed to test for statistical significance. (C) ANOVA followed by Dunnett’s correction for multiple comparisons was used. ns, not significant; *,  P
    Cd4 T Cell Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 7764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson cd4 fitc
    Effects of EA on percentage of CD3+, <t>CD4+,</t> and <t>CD8+</t> T lymphocytes in intestinal mucosa. Data were presented as means ± SD ( n = 5) and # p
    Cd4 Fitc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 3147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher cd4 monoclonal antibody
    Severe colitis in DSS treated CD69 −/− mice is associated with increased pro-inflammatory response and early influx of <t>CD4</t> T cells devoid of Foxp3 Treg cell into the colon. A. IL-17 concentration was determined by ELISA in the sera of control B6 mice and DSS treated B6 and CD69 −/− mice 7 days after the DSS administration. Mean (± SEM) of at least five mice per group is presented. B. RNA was isolated from frozen colon tissue samples of B6 and CD69 −/− mice treated with DSS, seven days after the DSS administration, or the control B6 mice and reverse transcribed to cDNA. Relative expression of IFN-γ gene was measured by qRT-PCR and presented as mean ± SEM of at least five mice per group. C. Mean (± SEM) total number of CD4 + T cells in blood, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) of 3 days DSS treated B6 and CD69 −/− mice are shown. At least five mice per each strain were analysed. D. The expression of Foxp3 by CD4 T cells from blood, MLN and cLP of B6 and CD69 −/− mice treated with DSS for 3 days was analysed by flow cytometry. Data from an individual, representative mouse (out of at least 5 mice per group analysed) are shown. Numbers indicate the percentage of CD4 T cells that express the transcriptional factor Foxp3. E. Mean (± SEM) total number of CD4 + Foxp3 + T cells in blood, MLN and cLP of 3 days DSS treated B6 and CD69 −/− mice are shown. At least five mice per each strain were analyzed. N.S. – not statistically significant; *p≤0.05; **p≤0.005.
    Cd4 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2094 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson cd4 percp
    CMV-specific <t>CD4</t> + T cells display a Th1 cytokine secretion pattern (arbitrary units for all axes). Dot plots of lymphocytes from patient 4, gated on positive <t>CD4-PerCP</t> and CD69-APC fluorescence. ( a ) Anti–IFNγ-PE fluorescence (X-axis) versus anti TNFα-FITC fluorescence (Y-axis). ( b ) Anti–IL4-PE fluorescence (X-axis) versus anti–IL2-FITC fluorescence (Y-axis).
    Cd4 Percp, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 2264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec cd4 microbeads
    Modulation of production of IFN-λ of pDC A) Cross-linking of <t>CD4</t> or BDCA-2 on pDC inhibits IFN-λ and IFN-α production of PDC: CD4 or BDCA-2 on pDC within PBMC was cross-linked with anti-CD4 or anti-BDCA-2 <t>microbeads,</t> and then PBMC were simulated for 7 hr with HSV. The expression of IFN-α (i) or IFN-λ1 (ii) by pDC was measured by flow cytometry as described above. Data represent mean ± 1 SD for 6 separate experiments. * P
    Cd4 Microbeads, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 2492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson cd4 apc
    Examples of flow cytometric gating strategies. (A) Gating strategy for regulatory T-cells. Gated for lymphocytes (FSC-A, SSC-A), singlets cells (FSC-A, FSC-H), live cells (FSC-A, <t>APC-Cy7A:</t> NIR), CD3 and <t>CD4</t> positive cells (Horizon V500-A: CD3, APC-A:
    Cd4 Apc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Becton Dickinson anti cd4 allophycocyanin
    SinΔC expression inhibits production of mature T cells. (A) Splenocytes (10 6 ) from 6- to-8-week-old wild-type (WT) and transgenic (Tg) animals were triply stained with <t>CD4-allophycocyanin/CD8-peridinin</t> chlorophyll-a protein and CD3-fluorescein isothiocyanate and analyzed by flow cytometry. In the dot plots, the numbers indicate the percentage of cells in each region. Histograms represent CD3 expression within the CD4 and CD8 SP populations in the dot plots. Two different transgenic founder lines, CR1 and MA2, with their respective wild-type controls, are shown. (B) Splenocytes were counted and stained, and the percentages of total CD3 T cells and CD4 + and CD8 + subpopulations were determined by fluorescence-activated cell sorting analysis and used to calculate the actual cell numbers for each population. Results from at least five wild-type and MA2 transgenic mice are represented as the mean ± the standard deviation (SD).
    Anti Cd4 Allophycocyanin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson anti cd4 percp
    <t>CD4</t> + T lymphocytes expressing effector/memory phenotype. Mononuclear cells were stained with mAb anti-CD44 FITC, anti-CD62L PE, anti-CD4 <t>PerCp,</t> anti-CD8 PerCp, and anti-CD3 APC and analyzed by flow cytometry. A high percentage of kidney and liver CD4 + T cells expresses effector/memory phenotype (CD44 high CD62L neg ) when compared with lung, spleen, and blood lymphocytes. Percentages are expressed from the gates: lymphocytes and CD3 + and CD4 + T cell areas. Dot plots are representative examples of three independent experiments ( n =8).
    Anti Cd4 Percp, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec cd4 cd25
    iTregs expanded in vitro by mtDCs retained Foxp3 expression and efficiently inhibited <t>CD4</t> + T cell proliferation. (a) Schematic overview of the induction/expansion strategy for iTregs. The two expansion cycles were established as described in the Materials and Methods. The differentiation of iTregs was first induced with an anti-CD3/CD28 mAb, TGF- β , and IL-2 for 5 days in vitro. Then, iTreg mtDC or iTreg mDC were generated by expanding iTregs for 4 days using mtDCs or mDCs, respectively. The expression of the transcription factor Foxp3 and <t>CD25</t> in cells was determined in each cycle using flow cytometry. Data are representative of three independent experiments. (b) Expansion of different types of iTregs was determined by counting the cells, and Foxp3 expression levels were determined by flow cytometry. All data are presented as means ± SEM ( n = 5), and ∗ P
    Cd4 Cd25, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 6443 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd4 fitc
    Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation. Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and <t>anti-CD4-FITC</t> mAb. Lymphocytes were isolated and stained ex-vivo for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8 + T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8 + T-cells both within the tissue and in the vasculature. C. Contour plots represent proliferation of CD4 + T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8 + T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4 + T-cells with and without DTx treatment is shown. **p
    Anti Cd4 Fitc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen cd4
    CCR9 low dendritic cells (DC) are potent stimulators of naïve T cells. (a) DC purified by magnetic antibody cell sorting (MACS) were sorted by flow cytometry into CCR9 low and CCR9 high fractions. Allogeneic C57BL/6J-purified spleen T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and added to 96-well flat-bottomed plates at a fixed number (2 × 10 5 cells/well). Sorted DC fractions were added to T cells at 1 × 10 5 cells /well or at 5 × 10 4 cells/well. Cultures were harvested 4 days later and labeled with <t>CD4</t> peridinin chlorophyll protein (PerCP) (FL3). Dead cells were gated out and > 25 000 events were collected on a flow cytometer. Histograms show CD4-stained T cells that were labeled for CFSE at a DC : T-cell ratio of 1:2, and are representative of three separate experiments. (b) Unfractionated DC and CCR9 low DC were used to stimulate T cells, as described above, at a DC : T-cell ratio of 1:2. CCR9 low DC were almost as potent stimulators of T cells as unfractionated DC.
    Cd4, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 1540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies cd4
    Immunohistochemical staining of liver specimens with liver injury resulting from osimertinib immediately after nivolumab therapy. In Case 4, CD3 and CD8 lymphocytes were predominantly expressed in the liver tissues compared to <t>CD4</t> and CD20 lymphocytes.
    Cd4, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 1547 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen anti cd4
    Differences in Th17 cell frequencies in the LP of IL-15 Tg or KO mice, but not in frequencies of Th1 or Treg cells. Cells from spleen and small intestine LP were stimulated for four hours with PMA and ionomycin and stained with surface markers CD3 and <t>CD4,</t> followed by intracellular staining of IL-17, IFNγ or Foxp3. A. Higher frequencies of Th17 cells were found in the LP of IL-15 KO mice than matched co-caged WT mice (p
    Anti Cd4, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 1160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson cd4 percp cy5 5
    Removal of <t>CD4+</t> T cells abrogates the effects of CTLA-4 blockade on HIV-specific CD8+ T Cells. PBMC (or CD4 negative PBMC) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, IL-10 APC, anti-CD3 Am Cyan, anti-CD4 <t>PerCP</t> <t>CY5.5,</t> anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. Representative plots of (A) Gag and (B) Nef-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. Data plots shown are representative of three volunteers examined in three independent experiments yielding similar results.
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    Image Search Results


    Ubc13-deficient T reg cells are sensitive to lymphopenic and inflammatory conditions for acquiring effector functions ( a, b ) Flow cytometry analysis of IL-17- and IFN-γ-producing MLN T reg cells (gated on CD45.2 + Foxp3 + CD4 + cells) derived from Rag1 KO mice (6 weeks old) adoptively transferred, for 10 or 21 days, with WT CD45.1 + CD4 + CD25 − CD45RB hi T cells plus CD4 + YFP + T reg cells purified from WT-R26 YFP (WT) or Ube2n Treg-KO R26 YFP (KO) mice (6 weeks old, CD45.2 + ). Data are representative ( a ) or summary ( b , day 21 data only) of two independent experiments (n=3). *p=0.05. ( c ) Flow cytometry analysis of IL-17- and IFN-γ-producing MLN T reg cells (gated on CD45.2 + Foxp3 + cells) from Rag1 KO mice (5 weeks old) adoptively transferred, for 5 weeks, with CD45.1 + Foxp3 + WT and/or CD45.2 + Foxp3 + Ube2n Treg-KO (KO) T reg cells. ( d ) Flow cytometric analysis of Foxp3 expression in CD45.1 + Foxp3 + and CD45.2 + Foxp3 + T reg cells from the Rag1 KO recipients of WT plus KO T reg cells described in d . ( e ) Flow cytometry measuring the frequency of IL-17- and IFN-γ-expressing T reg cells in the spleen of 10 weeks old mice, gating on Foxp3 + or YFP + cells. ( f ) ELISA quantifying the secreted IFN-γ and IL-17 by YFP + CD4 + T reg cells (isolated from 10 weeks old mice), stimulated with PMA and ionomycin for 24 h. Data are presented as means±SD of two independent experiments. ( g, h ) Flow cytometric analysis of the frequency of Foxp3 + IL-17 + cells in sorted YFP + CD4 + T reg cells, derived from the indicated mice (6 weeks old) and activated in the absence or presence of TGF-β and IL-6. Data are representative ( e ) or summary ( f ) of four independent experiments (n=3). **p=0.01.

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: Ubc13-deficient T reg cells are sensitive to lymphopenic and inflammatory conditions for acquiring effector functions ( a, b ) Flow cytometry analysis of IL-17- and IFN-γ-producing MLN T reg cells (gated on CD45.2 + Foxp3 + CD4 + cells) derived from Rag1 KO mice (6 weeks old) adoptively transferred, for 10 or 21 days, with WT CD45.1 + CD4 + CD25 − CD45RB hi T cells plus CD4 + YFP + T reg cells purified from WT-R26 YFP (WT) or Ube2n Treg-KO R26 YFP (KO) mice (6 weeks old, CD45.2 + ). Data are representative ( a ) or summary ( b , day 21 data only) of two independent experiments (n=3). *p=0.05. ( c ) Flow cytometry analysis of IL-17- and IFN-γ-producing MLN T reg cells (gated on CD45.2 + Foxp3 + cells) from Rag1 KO mice (5 weeks old) adoptively transferred, for 5 weeks, with CD45.1 + Foxp3 + WT and/or CD45.2 + Foxp3 + Ube2n Treg-KO (KO) T reg cells. ( d ) Flow cytometric analysis of Foxp3 expression in CD45.1 + Foxp3 + and CD45.2 + Foxp3 + T reg cells from the Rag1 KO recipients of WT plus KO T reg cells described in d . ( e ) Flow cytometry measuring the frequency of IL-17- and IFN-γ-expressing T reg cells in the spleen of 10 weeks old mice, gating on Foxp3 + or YFP + cells. ( f ) ELISA quantifying the secreted IFN-γ and IL-17 by YFP + CD4 + T reg cells (isolated from 10 weeks old mice), stimulated with PMA and ionomycin for 24 h. Data are presented as means±SD of two independent experiments. ( g, h ) Flow cytometric analysis of the frequency of Foxp3 + IL-17 + cells in sorted YFP + CD4 + T reg cells, derived from the indicated mice (6 weeks old) and activated in the absence or presence of TGF-β and IL-6. Data are representative ( e ) or summary ( f ) of four independent experiments (n=3). **p=0.01.

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

    Techniques: Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Purification, Expressing, Enzyme-linked Immunosorbent Assay, Isolation

    Ubc13 is dispensable for expression of T reg signature genes but regulates expression SOCS1 and IL-10 ( a ) Flow cytometric analysis of splenocytes derived from WT-R26 YFP and Ube2n Treg-KO R26 YFP mice (5 weeks old), measuring the expression of surface markers on T reg cells (gated on Foxp3 + CD4 + ). Data are representative of two independent experiments. ( b ) Real-time RT-PCR analysis of the relative mRNA expression level of the indicated genes in YFP + CD4 + T reg cells, sorted (based on YFP) from 5-week old WT-R26 YFP and Ube2n Treg-KO R26 YFP mice. Data were normalized to a reference gene, β-actin. ( c ) Flow cytometry measuring the frequency of IL-10-producing cells among the YFP + CD4 + T reg cells from MLN of WT-R26 YFP and Ube2n Treg-KO R26 YFP mice (5 weeks old). Data are representative (left) and summary (right) of three independent experiments (n=5/genotype). *p=0.05 (two-tailed unpaired t-test). ( d ) ELISA determining IL-10 production by purified YFP + CD4 + T reg cells stimulated with PMA and ionomycin for 24 h. Data are mean±SD of three independent experiments. ( e ) Flow cytometric analysis of SOCS1-expressing cells in Foxp3 + CD4 + T reg cells from MLN of WT-R26 YFP and Ube2n Treg-KO R26 YFP mice (6 weeks old). Data are representative (left) and summary (right) of three independent experiments (n=4–7/genotype in each experiment). **p=0.01 (two-tailed unpaired t-test). ( f ) Flow cytometric analysis of SOCS1-expressing T reg cells in the indicated littermates (6 weeks old), presented as a summary graph. **p=0.01 (two-tailed unpaired t-test).

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: Ubc13 is dispensable for expression of T reg signature genes but regulates expression SOCS1 and IL-10 ( a ) Flow cytometric analysis of splenocytes derived from WT-R26 YFP and Ube2n Treg-KO R26 YFP mice (5 weeks old), measuring the expression of surface markers on T reg cells (gated on Foxp3 + CD4 + ). Data are representative of two independent experiments. ( b ) Real-time RT-PCR analysis of the relative mRNA expression level of the indicated genes in YFP + CD4 + T reg cells, sorted (based on YFP) from 5-week old WT-R26 YFP and Ube2n Treg-KO R26 YFP mice. Data were normalized to a reference gene, β-actin. ( c ) Flow cytometry measuring the frequency of IL-10-producing cells among the YFP + CD4 + T reg cells from MLN of WT-R26 YFP and Ube2n Treg-KO R26 YFP mice (5 weeks old). Data are representative (left) and summary (right) of three independent experiments (n=5/genotype). *p=0.05 (two-tailed unpaired t-test). ( d ) ELISA determining IL-10 production by purified YFP + CD4 + T reg cells stimulated with PMA and ionomycin for 24 h. Data are mean±SD of three independent experiments. ( e ) Flow cytometric analysis of SOCS1-expressing cells in Foxp3 + CD4 + T reg cells from MLN of WT-R26 YFP and Ube2n Treg-KO R26 YFP mice (6 weeks old). Data are representative (left) and summary (right) of three independent experiments (n=4–7/genotype in each experiment). **p=0.01 (two-tailed unpaired t-test). ( f ) Flow cytometric analysis of SOCS1-expressing T reg cells in the indicated littermates (6 weeks old), presented as a summary graph. **p=0.01 (two-tailed unpaired t-test).

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

    Techniques: Expressing, Flow Cytometry, Derivative Assay, Mouse Assay, Quantitative RT-PCR, Cytometry, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Purification

    A SOCS1 mimetic peptide partially rescues the functional defect of Ubc13-deficient T reg cells both in vitro and in vivo ( a ) Flow cytometry measuring the frequency of IL-17-producting cells in sorted YFP + CD4 + T reg cells derived from WT-R26 YFP and Ube2n Treg-KO R26 YFP mice, activated under T H 17 polarizing conditions in the presence of DMSO, the SOCS1 mimetic peptide SOCS1-KIR, or the negative control peptide SOCS1-KIR2A. *p=0.05, **p=0.01 (two-tailed unpaired t-test). Data are representative of two independent experiments. ( b,c ) Disease phenotypes of Rag1 KO mice (5 weeks old) adoptively transferred with WT CD45.1 + CD4 + CD25 − CD45RB hi naïve T cells together with CD4 + YFP + T reg cells purified from WT-R26 YFP or Ube2n Treg-KO R26 YFP mice (6 weeks old). The recipient mice were either not treated or injected (i.p.) with SOCS1-KIR or SOCS1-KIR2A every other day. Bodyweight of recipient mice was measured at different times and presented as percentage of initial weight ( b ), and the frequency of IL-17 + or IFN-γ + cells among the transferred CD4 + YFP + T reg cells was determined by flow cytometry (gated on CD45.2 + cells) after 7 weeks of transfer ( c ).

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: A SOCS1 mimetic peptide partially rescues the functional defect of Ubc13-deficient T reg cells both in vitro and in vivo ( a ) Flow cytometry measuring the frequency of IL-17-producting cells in sorted YFP + CD4 + T reg cells derived from WT-R26 YFP and Ube2n Treg-KO R26 YFP mice, activated under T H 17 polarizing conditions in the presence of DMSO, the SOCS1 mimetic peptide SOCS1-KIR, or the negative control peptide SOCS1-KIR2A. *p=0.05, **p=0.01 (two-tailed unpaired t-test). Data are representative of two independent experiments. ( b,c ) Disease phenotypes of Rag1 KO mice (5 weeks old) adoptively transferred with WT CD45.1 + CD4 + CD25 − CD45RB hi naïve T cells together with CD4 + YFP + T reg cells purified from WT-R26 YFP or Ube2n Treg-KO R26 YFP mice (6 weeks old). The recipient mice were either not treated or injected (i.p.) with SOCS1-KIR or SOCS1-KIR2A every other day. Bodyweight of recipient mice was measured at different times and presented as percentage of initial weight ( b ), and the frequency of IL-17 + or IFN-γ + cells among the transferred CD4 + YFP + T reg cells was determined by flow cytometry (gated on CD45.2 + cells) after 7 weeks of transfer ( c ).

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

    Techniques: Functional Assay, In Vitro, In Vivo, Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Negative Control, Two Tailed Test, Purification, Injection

    T reg -specific ablation of Ubc13 impairs T-cell homeostasis ( a ) Flow cytometric analysis of T cells derived from the indicated lymphoid organs of WT and Ube2n Treg-KO mice (8 weeks old), showing the percentage of naïve (CD44 lo CD62L hi ) and memory-like (CD44 hi CD62L lo ) CD4 + T cells. Data are representative of five experiments with three mice per group. ( b, c ) Summary (mean ± SD value) of the frequency ( b ) and absolute numbers ( c ) of memory-like CD4 + T cells in the indicated lymphoid organs of WT and Ube2n Treg-KO mice (8 weeks old), determined by flow cytometry. *p=0.05 and **p=0.01 (two-tailed unpaired t-test). ( d ) ICS measuring the frequency of IL-17-, IFN-γ, and IL-4-producing CD4 + T cells (gated on CD3 + CD4 + cells) within the spleen of WT and Ube2n Treg-KO mice (8–10 weeks old). *p=0.05 and **p=0.01 (two-tailed unpaired t-test). Data are representative of three independent experiments (each circle represents one mouse). ( e, f ) Flow cytometric analyses of CD4 + or CD8 + ( e ) and IL-17- and IFN-γ-producing CD4 + T cells ( f ) from small intestine lamina propria (SI-LP) and large intestine lamina propria (LI-LP) of WT and Ube2n Treg-KO mice (8 weeks old). Numbers in quadrants indicate percentage of cells. Data are representative of two experiments with three mice per group.

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: T reg -specific ablation of Ubc13 impairs T-cell homeostasis ( a ) Flow cytometric analysis of T cells derived from the indicated lymphoid organs of WT and Ube2n Treg-KO mice (8 weeks old), showing the percentage of naïve (CD44 lo CD62L hi ) and memory-like (CD44 hi CD62L lo ) CD4 + T cells. Data are representative of five experiments with three mice per group. ( b, c ) Summary (mean ± SD value) of the frequency ( b ) and absolute numbers ( c ) of memory-like CD4 + T cells in the indicated lymphoid organs of WT and Ube2n Treg-KO mice (8 weeks old), determined by flow cytometry. *p=0.05 and **p=0.01 (two-tailed unpaired t-test). ( d ) ICS measuring the frequency of IL-17-, IFN-γ, and IL-4-producing CD4 + T cells (gated on CD3 + CD4 + cells) within the spleen of WT and Ube2n Treg-KO mice (8–10 weeks old). *p=0.05 and **p=0.01 (two-tailed unpaired t-test). Data are representative of three independent experiments (each circle represents one mouse). ( e, f ) Flow cytometric analyses of CD4 + or CD8 + ( e ) and IL-17- and IFN-γ-producing CD4 + T cells ( f ) from small intestine lamina propria (SI-LP) and large intestine lamina propria (LI-LP) of WT and Ube2n Treg-KO mice (8 weeks old). Numbers in quadrants indicate percentage of cells. Data are representative of two experiments with three mice per group.

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

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

    Ubc13 is dispensable for T reg homeostasis and in vitro suppressive activity ( a ) Flow cytometric analysis of the frequency of YFP + T reg cells (among CD3 + CD4 + cells) in the indicated lymphoid organs of 6 weeks old mice. Data are representative of five experiments with three mice per group. ( b–d ) Frequency ( b ) and absolute number ( c ) of YFP + cells (among CD3 + CD4 + cells) and absolute number of total CD4 + T cells (d) in the spleen of the indicated ages of mice, measured by flow cytometry and shown as the mean±SD value. *p=0.05 (n=5) (two-tailed unpaired t-test). ( e ) In vitro suppressive activity of T reg cells, measured based on the proliferation (CFSE dilution) of naïve CD4 + T cells activated by anti-CD3 plus antigen-presenting cells (irradiated CD3-depleted splenocytes from WT mice) in the presence of the indicated ratios of sorted T reg cells derived from WT or Ube2n Treg-KO mice (6 weeks old). The percentage of undivided cells is indicated.

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: Ubc13 is dispensable for T reg homeostasis and in vitro suppressive activity ( a ) Flow cytometric analysis of the frequency of YFP + T reg cells (among CD3 + CD4 + cells) in the indicated lymphoid organs of 6 weeks old mice. Data are representative of five experiments with three mice per group. ( b–d ) Frequency ( b ) and absolute number ( c ) of YFP + cells (among CD3 + CD4 + cells) and absolute number of total CD4 + T cells (d) in the spleen of the indicated ages of mice, measured by flow cytometry and shown as the mean±SD value. *p=0.05 (n=5) (two-tailed unpaired t-test). ( e ) In vitro suppressive activity of T reg cells, measured based on the proliferation (CFSE dilution) of naïve CD4 + T cells activated by anti-CD3 plus antigen-presenting cells (irradiated CD3-depleted splenocytes from WT mice) in the presence of the indicated ratios of sorted T reg cells derived from WT or Ube2n Treg-KO mice (6 weeks old). The percentage of undivided cells is indicated.

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

    Techniques: In Vitro, Activity Assay, Flow Cytometry, Mouse Assay, Cytometry, Two Tailed Test, Irradiation, Derivative Assay

    T reg -specific Ubc13 ablation impairs the in vivo immunosuppressive function of T reg cells ( a–d ) Disease phenotypes of Rag1 KO mice (6 weeks old) adoptively transferred with WT CD45.1 + congenic CD45RB hi naïve CD4 + T cells together with either PBS or sorted T reg cells derived from WT or Ube2n Treg-KO mice (6 weeks old). Bodyweight was measured at the indicated times and presented as percentage of initial weight (mean±SD) ( a ). A representative lymphoid organ picture ( b ), frequency of the indicated cytokine-producing effector CD4 + T cells in the MLN (measured by flow cytometry and gated on CD45.1 + cells) ( c ), and H E staining of colon ( d ) were obtained from recipient mice at 12 weeks after adoptive transfer. Data are from a total of three independent experiments (3 recipient mice per group in each experiment). ( e–h ) Disease phenotypes of Rag1 KO mice (5 weeks old) adoptively transferred with CD45.1 + Foxp3 + WT T reg (from B6.SJL congenic mice) and/or CD45.2 + Foxp3 + Ube2n Treg-KO (KO) T reg cells. Bodyweight was measured weekly after transfer ( e ). A representative picture of spleen ( f ), absolute number of total splenocytes ( g , presented as mean±SD value), and absolute number of CD45.2 + Foxp3 + cells from the spleen ( h , presented as the mean±SD value) were determined 5 weeks after transfer. **p=0.01 (two-tailed unpaired t-test).

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: T reg -specific Ubc13 ablation impairs the in vivo immunosuppressive function of T reg cells ( a–d ) Disease phenotypes of Rag1 KO mice (6 weeks old) adoptively transferred with WT CD45.1 + congenic CD45RB hi naïve CD4 + T cells together with either PBS or sorted T reg cells derived from WT or Ube2n Treg-KO mice (6 weeks old). Bodyweight was measured at the indicated times and presented as percentage of initial weight (mean±SD) ( a ). A representative lymphoid organ picture ( b ), frequency of the indicated cytokine-producing effector CD4 + T cells in the MLN (measured by flow cytometry and gated on CD45.1 + cells) ( c ), and H E staining of colon ( d ) were obtained from recipient mice at 12 weeks after adoptive transfer. Data are from a total of three independent experiments (3 recipient mice per group in each experiment). ( e–h ) Disease phenotypes of Rag1 KO mice (5 weeks old) adoptively transferred with CD45.1 + Foxp3 + WT T reg (from B6.SJL congenic mice) and/or CD45.2 + Foxp3 + Ube2n Treg-KO (KO) T reg cells. Bodyweight was measured weekly after transfer ( e ). A representative picture of spleen ( f ), absolute number of total splenocytes ( g , presented as mean±SD value), and absolute number of CD45.2 + Foxp3 + cells from the spleen ( h , presented as the mean±SD value) were determined 5 weeks after transfer. **p=0.01 (two-tailed unpaired t-test).

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

    Techniques: In Vivo, Mouse Assay, Derivative Assay, Flow Cytometry, Cytometry, Staining, Adoptive Transfer Assay, Two Tailed Test

    The T reg -specific function of Ubc13 involves its downstream target IKK ( a ) Flow cytometric analysis of RelA phosphorylation in sorted T reg cells from WT or Ube2n Treg-KO mice, stimulated with anti-CD3 and anti-CD28 for 15 min using a crosslinking method 45 . ( b ) Flow cytometric analysis of CD44 hi CD62L lo memory-like T cells (gated on CD3 + CD4 + cells) within different lymphoid organs of the indicated mice (6–8 weeks old). Data are presented as mean ± SD value and representative of three independent experiments. *p=0.05 and **p=0.01 (two-tailed unpaired t-test). ( c ) Flow cytometric analysis of CD4 + T cells derived from the indicated lymphoid organs of WT or IKK2 Treg-KO mice (7 weeks old), measuring the percentage (numbers in quadrangles) of naïve (CD44 lo CD62L hi ) and memory-like (CD44 hi CD62L lo ) T cells. Data are representative of three experiments. ( d, e ) Flow cytometric analysis of the frequency ( d ) and absolute number ( e ) of CD44 hi CD62 lo memory-like CD4 + T cells in the indicated lymphoid organs of WT and IKK2 Treg-KO littermates (7 weeks old). Data are representative of two independent experiments.

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: The T reg -specific function of Ubc13 involves its downstream target IKK ( a ) Flow cytometric analysis of RelA phosphorylation in sorted T reg cells from WT or Ube2n Treg-KO mice, stimulated with anti-CD3 and anti-CD28 for 15 min using a crosslinking method 45 . ( b ) Flow cytometric analysis of CD44 hi CD62L lo memory-like T cells (gated on CD3 + CD4 + cells) within different lymphoid organs of the indicated mice (6–8 weeks old). Data are presented as mean ± SD value and representative of three independent experiments. *p=0.05 and **p=0.01 (two-tailed unpaired t-test). ( c ) Flow cytometric analysis of CD4 + T cells derived from the indicated lymphoid organs of WT or IKK2 Treg-KO mice (7 weeks old), measuring the percentage (numbers in quadrangles) of naïve (CD44 lo CD62L hi ) and memory-like (CD44 hi CD62L lo ) T cells. Data are representative of three experiments. ( d, e ) Flow cytometric analysis of the frequency ( d ) and absolute number ( e ) of CD44 hi CD62 lo memory-like CD4 + T cells in the indicated lymphoid organs of WT and IKK2 Treg-KO littermates (7 weeks old). Data are representative of two independent experiments.

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

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

    T reg -specific ablation of Ubc13 causes systemic autoimmunity ( a ) immunoblot of Ubc13 expression in CD4 + T cells and T reg cells sorted from Ube2n fl/fl Foxp3 GFP-Cre (KO) and Ube2n +/+ Foxp3 GFP-Cre (WT) mice. Loading control: protein Hsp60. Data are representative of two independent experiments. ( b ) Bodyweight of Ube2n Treg-KO and Ube2n +/+ Foxp3 GFP-Cre (WT) measured at the indicated times and presented as mean±SD. Results were from a total of three independent experiments (5–7 mice/genotype in each experiment). *p=0.05; **p=0.01 (two-tailed unpaired t-test). ( c ) Number of total cells from spleen, mesenteric lymph node (MLN) and peripheral lymph node (PLN) of age- and sex-matched Ube2n Treg-KO and Ube2n +/+ Foxp3 GFP-Cre (WT) 10 week old mice presented as the mean±SD value. **p=0.01 and ***p=0.001 (two-tailed unpaired t-test). Data are representative of two independent experiments. ( d ) H E staining of various non-lymphoid tissue sections from 20 weeks old Ube2n Treg-KO and Ube2n +/+ Foxp3 GFP-Cre (WT) mice. Data are representative of 3–4 mice/genotype.

    Journal: Nature Immunology

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    doi: 10.1038/ni.2267

    Figure Lengend Snippet: T reg -specific ablation of Ubc13 causes systemic autoimmunity ( a ) immunoblot of Ubc13 expression in CD4 + T cells and T reg cells sorted from Ube2n fl/fl Foxp3 GFP-Cre (KO) and Ube2n +/+ Foxp3 GFP-Cre (WT) mice. Loading control: protein Hsp60. Data are representative of two independent experiments. ( b ) Bodyweight of Ube2n Treg-KO and Ube2n +/+ Foxp3 GFP-Cre (WT) measured at the indicated times and presented as mean±SD. Results were from a total of three independent experiments (5–7 mice/genotype in each experiment). *p=0.05; **p=0.01 (two-tailed unpaired t-test). ( c ) Number of total cells from spleen, mesenteric lymph node (MLN) and peripheral lymph node (PLN) of age- and sex-matched Ube2n Treg-KO and Ube2n +/+ Foxp3 GFP-Cre (WT) 10 week old mice presented as the mean±SD value. **p=0.01 and ***p=0.001 (two-tailed unpaired t-test). Data are representative of two independent experiments. ( d ) H E staining of various non-lymphoid tissue sections from 20 weeks old Ube2n Treg-KO and Ube2n +/+ Foxp3 GFP-Cre (WT) mice. Data are representative of 3–4 mice/genotype.

    Article Snippet: Fluorescence-labeled antibodies used include phycoerythrin (PE)-conjugated anti-IL-17, anti-IL-10, anti-CD8, anti-CD25, anti-CD127, anti-CD137, anti-ICOS (eBioscience); FITC-conjugated anti-Foxp3 (eBioscience) and anti-CD62L (BD Bioscience); APC-conjugated anti-Foxp3, anti-CD44, anti-IFN-γ and anti-CD4 (eBioscience); PE-conjugated anti-CD103, anti-CD122, anti-CD126, anti-OX40, anti-GITR (BD Bioscience); Biotin-conjugated anti-SOCS1 (MBL); anti-phospho-NF-κB p65(Ser536) and APC-conjugated anti-Rabbit IgG (Cell Signaling).

    Techniques: Expressing, Mouse Assay, Two Tailed Test, Staining

    Needle-free SL/B immunization generates vaccine-specific CD4 and CD8 T cells in the blood. PMBCs were stimulated with HIV-1 consensus B Gag and Env peptides and analyzed by flow cytometry for cytokine production. a Representative flow plots for IFN-γ and TNF-α cytokine expression on Live CD3 + CD4 + cells in non-stimulated (NS), Gag, or Env stimulated PBMCs is shown. Kinetics of the total (Gag + Env) b IFN-γ, c TNF-α, and d IL-2 response in CD4 + T cells (mean ± S.D.), with the peak response (wk 16) highlighted for each animal (line denotes mean). e Kinetics of the total IFN-γ response in CD8 + cells (mean ± S.D.), with the peak response (wk 16) highlighted for each animal. White circle, topical SL/B ( n = 4); blue square, needle-free SL/B ( n = 5); gray triangle, ID/SC ( n = 6)

    Journal: Nature Communications

    Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge

    doi: 10.1038/s41467-019-08739-4

    Figure Lengend Snippet: Needle-free SL/B immunization generates vaccine-specific CD4 and CD8 T cells in the blood. PMBCs were stimulated with HIV-1 consensus B Gag and Env peptides and analyzed by flow cytometry for cytokine production. a Representative flow plots for IFN-γ and TNF-α cytokine expression on Live CD3 + CD4 + cells in non-stimulated (NS), Gag, or Env stimulated PBMCs is shown. Kinetics of the total (Gag + Env) b IFN-γ, c TNF-α, and d IL-2 response in CD4 + T cells (mean ± S.D.), with the peak response (wk 16) highlighted for each animal (line denotes mean). e Kinetics of the total IFN-γ response in CD8 + cells (mean ± S.D.), with the peak response (wk 16) highlighted for each animal. White circle, topical SL/B ( n = 4); blue square, needle-free SL/B ( n = 5); gray triangle, ID/SC ( n = 6)

    Article Snippet: Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis.

    Techniques: Flow Cytometry, Cytometry, Expressing

    Probability of achieving CD4 count normalization (i.e. CD4 > 400 cells/µL) and number at risk by gender. The full line represents women. The long dash line represents men.

    Journal: PLoS ONE

    Article Title: Among Patients with Sustained Viral Suppression in a Resource-Limited Setting, CD4 Gains Are Continuous Although Gender-Based Differences Occur

    doi: 10.1371/journal.pone.0073190

    Figure Lengend Snippet: Probability of achieving CD4 count normalization (i.e. CD4 > 400 cells/µL) and number at risk by gender. The full line represents women. The long dash line represents men.

    Article Snippet: A multivariable time series regression model was used to determine factors associated with CD4 count increases in these patients.

    Techniques:

    Median CD4 count over time stratified by baseline CD4 count. The full line with diamonds represents people with baseline CD4 count ≤100 cells/µL. The long dash line with ‘x’ s represents people with baseline CD4 count 101-200 cells/µL. The long dash-dotted line with triangles represents people with baseline CD4 count > 200 cells/µL.

    Journal: PLoS ONE

    Article Title: Among Patients with Sustained Viral Suppression in a Resource-Limited Setting, CD4 Gains Are Continuous Although Gender-Based Differences Occur

    doi: 10.1371/journal.pone.0073190

    Figure Lengend Snippet: Median CD4 count over time stratified by baseline CD4 count. The full line with diamonds represents people with baseline CD4 count ≤100 cells/µL. The long dash line with ‘x’ s represents people with baseline CD4 count 101-200 cells/µL. The long dash-dotted line with triangles represents people with baseline CD4 count > 200 cells/µL.

    Article Snippet: A multivariable time series regression model was used to determine factors associated with CD4 count increases in these patients.

    Techniques:

    Probability of achieving CD4 count > 400 cells/µL and number at risk by baseline CD4 count. The full line represents people with baseline CD4 count ≤100 cells/µL. The long dash line represents people with baseline CD4 count 101-200 cells/µL. The long dash-dotted line represents people with baseline CD4 count > 200 cells/µL.

    Journal: PLoS ONE

    Article Title: Among Patients with Sustained Viral Suppression in a Resource-Limited Setting, CD4 Gains Are Continuous Although Gender-Based Differences Occur

    doi: 10.1371/journal.pone.0073190

    Figure Lengend Snippet: Probability of achieving CD4 count > 400 cells/µL and number at risk by baseline CD4 count. The full line represents people with baseline CD4 count ≤100 cells/µL. The long dash line represents people with baseline CD4 count 101-200 cells/µL. The long dash-dotted line represents people with baseline CD4 count > 200 cells/µL.

    Article Snippet: A multivariable time series regression model was used to determine factors associated with CD4 count increases in these patients.

    Techniques:

    Median CD4 count trajectory stratified by gender within baseline CD4 count groups. The full line represents women. The long dash line represents men.

    Journal: PLoS ONE

    Article Title: Among Patients with Sustained Viral Suppression in a Resource-Limited Setting, CD4 Gains Are Continuous Although Gender-Based Differences Occur

    doi: 10.1371/journal.pone.0073190

    Figure Lengend Snippet: Median CD4 count trajectory stratified by gender within baseline CD4 count groups. The full line represents women. The long dash line represents men.

    Article Snippet: A multivariable time series regression model was used to determine factors associated with CD4 count increases in these patients.

    Techniques:

    CD1b-restricted T cell populations express the αβ TCR and CD4. PBMCs from four subjects infected with Mycobacterium tuberculosis were subjected to multicolor FACS analysis. Cells were stained with CD3, violet viability dye, CD14 and CD19-PercP-Cy5.5, loaded CD1b tetramers, and anti–TCR-αβ (a) and CD4 (b).

    Journal: The Journal of Experimental Medicine

    Article Title: CD1b tetramers bind ?? T cell receptors to identify a mycobacterial glycolipid-reactive T cell repertoire in humans

    doi: 10.1084/jem.20110665

    Figure Lengend Snippet: CD1b-restricted T cell populations express the αβ TCR and CD4. PBMCs from four subjects infected with Mycobacterium tuberculosis were subjected to multicolor FACS analysis. Cells were stained with CD3, violet viability dye, CD14 and CD19-PercP-Cy5.5, loaded CD1b tetramers, and anti–TCR-αβ (a) and CD4 (b).

    Article Snippet: Tetramer-positive cells were stained with TCR-αβ FITC (BD) or CD4-PE (BD).

    Techniques: Infection, FACS, Staining

    Correlation of CD4+T cells and viral load as indicators of HIV progression. Box plots representing (A) the distribution of CD4+T cell/mm 3 of blood in Healthy, HIV and HIV-TB. (B) The distribution of viral RNA copies measured in terms of IU/ml of sample for the HIV and HIV-TB patients. (C) Analyses of the CD4+T cell count of HIV and HIV-TB samples with low viral load (VL≤10 5 IU/ml) and high viral load (VL > 10 5 IU/ml). The threshold for significance was set at p≤0.05. Bars above the plots represent the statistical significance (p value) between the groups.

    Journal: PLoS ONE

    Article Title: Discordance in CD4+T-Cell Levels and Viral Loads with Co-Occurrence of Elevated Peripheral TNF-? and IL-4 in Newly Diagnosed HIV-TB Co-Infected Cases

    doi: 10.1371/journal.pone.0070250

    Figure Lengend Snippet: Correlation of CD4+T cells and viral load as indicators of HIV progression. Box plots representing (A) the distribution of CD4+T cell/mm 3 of blood in Healthy, HIV and HIV-TB. (B) The distribution of viral RNA copies measured in terms of IU/ml of sample for the HIV and HIV-TB patients. (C) Analyses of the CD4+T cell count of HIV and HIV-TB samples with low viral load (VL≤10 5 IU/ml) and high viral load (VL > 10 5 IU/ml). The threshold for significance was set at p≤0.05. Bars above the plots represent the statistical significance (p value) between the groups.

    Article Snippet: CD4+T Cell Count by FACS Analysis CD4+T cells were counted by FACS using a monoclonal antibody cocktail of CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC (MultiTEST, BD Biosciences, San Jose, CA).

    Techniques: Cell Counting, Significance Assay

    Plasma levels of TNF-α in relation to CD4+T cells and viral load. Box plots representing (A) comparison of plasma TNF-α levels between Healthy, TB, HIV and HIV-TB categories. (B) Plasma levels of TNF-α in HIV patients categorized on the basis of low CD4+T cells (≤200/mm 3 ) and high CD4+T cells ( > 200/mm 3 ). (C) Plasma levels of TNF-α in HIV-TB patients categorized on the basis of low CD4+T cells (≤200/mm 3 ) and high CD4+T cells ( > 200/mm 3 ). (D) Plasma levels of TNF-α in HIV and HIV-TB patients whose CD4+T cells are below ≤200/mm 3 of blood. (E) Plasma levels of TNF-α in HIV-TB patients with low viral load (VL≤10 5 IU/ml) and high viral load (VL > 10 5 IU/ml). The threshold for significance was set at p≤0.05. Bars above and below the plots represent the statistical significance (p value) between the groups.

    Journal: PLoS ONE

    Article Title: Discordance in CD4+T-Cell Levels and Viral Loads with Co-Occurrence of Elevated Peripheral TNF-? and IL-4 in Newly Diagnosed HIV-TB Co-Infected Cases

    doi: 10.1371/journal.pone.0070250

    Figure Lengend Snippet: Plasma levels of TNF-α in relation to CD4+T cells and viral load. Box plots representing (A) comparison of plasma TNF-α levels between Healthy, TB, HIV and HIV-TB categories. (B) Plasma levels of TNF-α in HIV patients categorized on the basis of low CD4+T cells (≤200/mm 3 ) and high CD4+T cells ( > 200/mm 3 ). (C) Plasma levels of TNF-α in HIV-TB patients categorized on the basis of low CD4+T cells (≤200/mm 3 ) and high CD4+T cells ( > 200/mm 3 ). (D) Plasma levels of TNF-α in HIV and HIV-TB patients whose CD4+T cells are below ≤200/mm 3 of blood. (E) Plasma levels of TNF-α in HIV-TB patients with low viral load (VL≤10 5 IU/ml) and high viral load (VL > 10 5 IU/ml). The threshold for significance was set at p≤0.05. Bars above and below the plots represent the statistical significance (p value) between the groups.

    Article Snippet: CD4+T Cell Count by FACS Analysis CD4+T cells were counted by FACS using a monoclonal antibody cocktail of CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC (MultiTEST, BD Biosciences, San Jose, CA).

    Techniques: Significance Assay

    Plasma levels of IFN-γ in Healthy, TB, HIV and HIV-TB subjects and its relation to CD4+T cell and viral load under HIV and HIV-TB categories. Box plots representing (A) comparison of plasma IFN-γ levels among Healthy, TB, HIV and HIV-TB categories. (B) Plasma levels of IFN-γ in HIV and HIV-TB patients categorized on the basis of low CD4+T cells (≤200/mm 3 ) and high CD4+T cells ( > 200/mm 3 ). (C) Plasma levels of IFN-γ for HIV and HIV-TB patients with low viral load (VL≤10 5 IU/ml) and high viral load (VL > 10 5 IU/ml). The threshold for significance was set at p≤0.05. Bars above the plots represent the statistical significance (p value) between the groups.

    Journal: PLoS ONE

    Article Title: Discordance in CD4+T-Cell Levels and Viral Loads with Co-Occurrence of Elevated Peripheral TNF-? and IL-4 in Newly Diagnosed HIV-TB Co-Infected Cases

    doi: 10.1371/journal.pone.0070250

    Figure Lengend Snippet: Plasma levels of IFN-γ in Healthy, TB, HIV and HIV-TB subjects and its relation to CD4+T cell and viral load under HIV and HIV-TB categories. Box plots representing (A) comparison of plasma IFN-γ levels among Healthy, TB, HIV and HIV-TB categories. (B) Plasma levels of IFN-γ in HIV and HIV-TB patients categorized on the basis of low CD4+T cells (≤200/mm 3 ) and high CD4+T cells ( > 200/mm 3 ). (C) Plasma levels of IFN-γ for HIV and HIV-TB patients with low viral load (VL≤10 5 IU/ml) and high viral load (VL > 10 5 IU/ml). The threshold for significance was set at p≤0.05. Bars above the plots represent the statistical significance (p value) between the groups.

    Article Snippet: CD4+T Cell Count by FACS Analysis CD4+T cells were counted by FACS using a monoclonal antibody cocktail of CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC (MultiTEST, BD Biosciences, San Jose, CA).

    Techniques: Significance Assay

    Activation induces extensive metabolic reprogramming in T cells. Purified T cells were untreated (UT) or activated with anti-CD3/CD28 beads (CD3/28). (a) Geometric mean fluorescence intensity (gMFI) was measured for activation and metabolic proteins in CD4 + T cells. Each dot represents one donor, data representative of n=8 donors, from 3 independent experiments. (b) FitSNE projection and corresponding expression of metabolic protein and activation markers in T cells, data acquired from n=5 samples, with 10,000 cells per donor. (c) Chord visualization of correlation between immune and metabolic proteins in activated CD4 + T cells, representative of n=8 donors. (d) Spearman correlation of GLUT1 and CD25 expression in untreated and (e) activated CD4 + T cells. (f) Heatmap of FitSNE projection of GLUT1 and CD25 expression in untreated and activated CD4 + T cells.

    Journal: bioRxiv

    Article Title: A novel strategy for single-cell metabolic analysis highlights dynamic changes in immune subpopulations

    doi: 10.1101/2020.01.21.914663

    Figure Lengend Snippet: Activation induces extensive metabolic reprogramming in T cells. Purified T cells were untreated (UT) or activated with anti-CD3/CD28 beads (CD3/28). (a) Geometric mean fluorescence intensity (gMFI) was measured for activation and metabolic proteins in CD4 + T cells. Each dot represents one donor, data representative of n=8 donors, from 3 independent experiments. (b) FitSNE projection and corresponding expression of metabolic protein and activation markers in T cells, data acquired from n=5 samples, with 10,000 cells per donor. (c) Chord visualization of correlation between immune and metabolic proteins in activated CD4 + T cells, representative of n=8 donors. (d) Spearman correlation of GLUT1 and CD25 expression in untreated and (e) activated CD4 + T cells. (f) Heatmap of FitSNE projection of GLUT1 and CD25 expression in untreated and activated CD4 + T cells.

    Article Snippet: After permeablization, cells were stained with an antibody cocktail mix for 1 hour at room temperature in Perm/Wash Buffer I, including the antibodies CD4 (clone SK3, BD, BV480, 566104), CD8 (clone SK1, BD, BUV805, 564912), CD3 (clone UCTH1, BD, BB630, 624294), HLA-DR (clone G46-6, BD, BV786, 564041), CD16 (clone 3G8, BD, BV750, 624380), CD45 (clone 2D1, BD, BUV563, 624284), CD69 (clone FN50, BD, APC-H7, 560737), CCR7 (clone G043H7, Biolegend, BV650, 353134), CD45RA (cloneHI100, BD, PE, 561883), CD25 (clone 2A3, BD, PE-Cy7, 335789), CD14 (M5E2, BD, BV570, 624298), the phosphorylated ribosomal protein S6 (Ser240/244, clone D68F8, AF647, 5044), as well as the abovementioned 9 metabolic antibodies.

    Techniques: Activation Assay, Purification, Fluorescence, Expressing

    Respiration and downstream AKT signaling increase with T cell activation. (a) Glycolytic function across untreated (UT), 2-FDG treated, CD3/28 activated and combination treated (2-FDG+CD3/28) donor samples. Graph depicts one representative sample from a single donor. (b) Glycolytic parameters measured by extracellular acidification rate (ECAR) across treatments. (c) Mitochondrial respiration measured by oxygen consumption rate (OCR) in purified T cells across treatments and its associated (d) mitochondrial parameters. Data represents n=6 donor samples from 2 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. (e) CD4+ T cells phosphorylation status of phospho-S6 (pS6) and respective levels of metabolic and activation markers. Data shown represents n=6 by FitSNE analysis. (f) Phosphorylation status across different treatments. (g) Phosphorylation status across memory subsets with treatment. Statistical analysis was performed using multiple t-test and Wilcoxon Signed Rank test.

    Journal: bioRxiv

    Article Title: A novel strategy for single-cell metabolic analysis highlights dynamic changes in immune subpopulations

    doi: 10.1101/2020.01.21.914663

    Figure Lengend Snippet: Respiration and downstream AKT signaling increase with T cell activation. (a) Glycolytic function across untreated (UT), 2-FDG treated, CD3/28 activated and combination treated (2-FDG+CD3/28) donor samples. Graph depicts one representative sample from a single donor. (b) Glycolytic parameters measured by extracellular acidification rate (ECAR) across treatments. (c) Mitochondrial respiration measured by oxygen consumption rate (OCR) in purified T cells across treatments and its associated (d) mitochondrial parameters. Data represents n=6 donor samples from 2 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. (e) CD4+ T cells phosphorylation status of phospho-S6 (pS6) and respective levels of metabolic and activation markers. Data shown represents n=6 by FitSNE analysis. (f) Phosphorylation status across different treatments. (g) Phosphorylation status across memory subsets with treatment. Statistical analysis was performed using multiple t-test and Wilcoxon Signed Rank test.

    Article Snippet: After permeablization, cells were stained with an antibody cocktail mix for 1 hour at room temperature in Perm/Wash Buffer I, including the antibodies CD4 (clone SK3, BD, BV480, 566104), CD8 (clone SK1, BD, BUV805, 564912), CD3 (clone UCTH1, BD, BB630, 624294), HLA-DR (clone G46-6, BD, BV786, 564041), CD16 (clone 3G8, BD, BV750, 624380), CD45 (clone 2D1, BD, BUV563, 624284), CD69 (clone FN50, BD, APC-H7, 560737), CCR7 (clone G043H7, Biolegend, BV650, 353134), CD45RA (cloneHI100, BD, PE, 561883), CD25 (clone 2A3, BD, PE-Cy7, 335789), CD14 (M5E2, BD, BV570, 624298), the phosphorylated ribosomal protein S6 (Ser240/244, clone D68F8, AF647, 5044), as well as the abovementioned 9 metabolic antibodies.

    Techniques: Activation Assay, Purification

    The activation and metabolic states of CD4+ T cells are altered by glycolytic inhibition. Fold change of metabolic protein and activation markers (gMFI) was measured in CD4 + T cells with no treatment (UT), 2-FDG, CD3/28, and combination of 2-FDG with CD3/28 (Combi). Metabolic proteins are grouped by (a) anabolic pathways, including fatty acid synthesis and arginine metabolism, and (b) catabolic pathways, including glycolysis, oxidative PPP, TCA cycle and fatty acid oxidation. (c) Activation markers and (d) the ATP synthase protein critical for OXPHOS, glucose transporter and the antioxidant protein were measured. Each dot represents one donor sample, total n=8 donors from 3 independent experiments.

    Journal: bioRxiv

    Article Title: A novel strategy for single-cell metabolic analysis highlights dynamic changes in immune subpopulations

    doi: 10.1101/2020.01.21.914663

    Figure Lengend Snippet: The activation and metabolic states of CD4+ T cells are altered by glycolytic inhibition. Fold change of metabolic protein and activation markers (gMFI) was measured in CD4 + T cells with no treatment (UT), 2-FDG, CD3/28, and combination of 2-FDG with CD3/28 (Combi). Metabolic proteins are grouped by (a) anabolic pathways, including fatty acid synthesis and arginine metabolism, and (b) catabolic pathways, including glycolysis, oxidative PPP, TCA cycle and fatty acid oxidation. (c) Activation markers and (d) the ATP synthase protein critical for OXPHOS, glucose transporter and the antioxidant protein were measured. Each dot represents one donor sample, total n=8 donors from 3 independent experiments.

    Article Snippet: After permeablization, cells were stained with an antibody cocktail mix for 1 hour at room temperature in Perm/Wash Buffer I, including the antibodies CD4 (clone SK3, BD, BV480, 566104), CD8 (clone SK1, BD, BUV805, 564912), CD3 (clone UCTH1, BD, BB630, 624294), HLA-DR (clone G46-6, BD, BV786, 564041), CD16 (clone 3G8, BD, BV750, 624380), CD45 (clone 2D1, BD, BUV563, 624284), CD69 (clone FN50, BD, APC-H7, 560737), CCR7 (clone G043H7, Biolegend, BV650, 353134), CD45RA (cloneHI100, BD, PE, 561883), CD25 (clone 2A3, BD, PE-Cy7, 335789), CD14 (M5E2, BD, BV570, 624298), the phosphorylated ribosomal protein S6 (Ser240/244, clone D68F8, AF647, 5044), as well as the abovementioned 9 metabolic antibodies.

    Techniques: Activation Assay, Inhibition

    T cell memory subsets differentially respond to glycolytic inhibition (a) FitSNE projection of resting state CD4 + memory populations, data represents n=5 donor samples. (b) Metabolic protein expression of resting state CD4 + memory subsets by gMFI, data represents n=8 donor samples. (c) Gating strategy of CD4 + memory subsets by CCR7 and CD45RA. (d) Cell count of CD4 + T memory populations across treatments. (e) FitSNE of CD4 + CM populations across treatments, data represents 5 donor samples from 2 independent experiments, with 20,000 cells per samples.

    Journal: bioRxiv

    Article Title: A novel strategy for single-cell metabolic analysis highlights dynamic changes in immune subpopulations

    doi: 10.1101/2020.01.21.914663

    Figure Lengend Snippet: T cell memory subsets differentially respond to glycolytic inhibition (a) FitSNE projection of resting state CD4 + memory populations, data represents n=5 donor samples. (b) Metabolic protein expression of resting state CD4 + memory subsets by gMFI, data represents n=8 donor samples. (c) Gating strategy of CD4 + memory subsets by CCR7 and CD45RA. (d) Cell count of CD4 + T memory populations across treatments. (e) FitSNE of CD4 + CM populations across treatments, data represents 5 donor samples from 2 independent experiments, with 20,000 cells per samples.

    Article Snippet: After permeablization, cells were stained with an antibody cocktail mix for 1 hour at room temperature in Perm/Wash Buffer I, including the antibodies CD4 (clone SK3, BD, BV480, 566104), CD8 (clone SK1, BD, BUV805, 564912), CD3 (clone UCTH1, BD, BB630, 624294), HLA-DR (clone G46-6, BD, BV786, 564041), CD16 (clone 3G8, BD, BV750, 624380), CD45 (clone 2D1, BD, BUV563, 624284), CD69 (clone FN50, BD, APC-H7, 560737), CCR7 (clone G043H7, Biolegend, BV650, 353134), CD45RA (cloneHI100, BD, PE, 561883), CD25 (clone 2A3, BD, PE-Cy7, 335789), CD14 (M5E2, BD, BV570, 624298), the phosphorylated ribosomal protein S6 (Ser240/244, clone D68F8, AF647, 5044), as well as the abovementioned 9 metabolic antibodies.

    Techniques: Inhibition, Expressing, Cell Counting

    Glucose restriction and metabolic remodeling drive the expansion of inflammatory memory T subpopulation. (a) FitSNE projection of GM-CSF producing total CD4+ T cells. (b) GM-CSF producing frequency (%) of CD4+ T cells across all treatments and memory subsets. (c) Comparison of activation and combi (CD3/28+2-FDG) treated memory subset frequency. (d) Differential expression of metabolic proteins across T cell memory subsets with glycolytic inhibition during CD3/28 activation (combi). All data represents n=8 donors, and statistical analysis was performed using T test or Friedman’s test with Dunn’s multiple comparisons.

    Journal: bioRxiv

    Article Title: A novel strategy for single-cell metabolic analysis highlights dynamic changes in immune subpopulations

    doi: 10.1101/2020.01.21.914663

    Figure Lengend Snippet: Glucose restriction and metabolic remodeling drive the expansion of inflammatory memory T subpopulation. (a) FitSNE projection of GM-CSF producing total CD4+ T cells. (b) GM-CSF producing frequency (%) of CD4+ T cells across all treatments and memory subsets. (c) Comparison of activation and combi (CD3/28+2-FDG) treated memory subset frequency. (d) Differential expression of metabolic proteins across T cell memory subsets with glycolytic inhibition during CD3/28 activation (combi). All data represents n=8 donors, and statistical analysis was performed using T test or Friedman’s test with Dunn’s multiple comparisons.

    Article Snippet: After permeablization, cells were stained with an antibody cocktail mix for 1 hour at room temperature in Perm/Wash Buffer I, including the antibodies CD4 (clone SK3, BD, BV480, 566104), CD8 (clone SK1, BD, BUV805, 564912), CD3 (clone UCTH1, BD, BB630, 624294), HLA-DR (clone G46-6, BD, BV786, 564041), CD16 (clone 3G8, BD, BV750, 624380), CD45 (clone 2D1, BD, BUV563, 624284), CD69 (clone FN50, BD, APC-H7, 560737), CCR7 (clone G043H7, Biolegend, BV650, 353134), CD45RA (cloneHI100, BD, PE, 561883), CD25 (clone 2A3, BD, PE-Cy7, 335789), CD14 (M5E2, BD, BV570, 624298), the phosphorylated ribosomal protein S6 (Ser240/244, clone D68F8, AF647, 5044), as well as the abovementioned 9 metabolic antibodies.

    Techniques: Activation Assay, Expressing, Inhibition

    Representative gating strategy for the FACS analysis of lamina propria CD3+, CD4+, CD8+ and regulatory T-cells from pinch biopsies obtained from the appendiceal orifice region of one healthy subject.

    Journal: PLoS ONE

    Article Title: Distribution of CD4pos -, CD8pos - and Regulatory T Cells in the Upper and Lower Gastrointestinal Tract in Healthy Young Subjects

    doi: 10.1371/journal.pone.0080362

    Figure Lengend Snippet: Representative gating strategy for the FACS analysis of lamina propria CD3+, CD4+, CD8+ and regulatory T-cells from pinch biopsies obtained from the appendiceal orifice region of one healthy subject.

    Article Snippet: FACS staining The following fluorochrome-conjugated monoclonal antibodies were used for surface staining: anti-CD3 PerCp-Cy5.5, anti-CD4 PE, anti-CD8 V500, anti-CD25 PE-Cy7 and anti-CD127 FITC (BD Biosciences, USA).

    Techniques: FACS

    Relative abundance of CD3 pos cells, CD8 pos -, CD4 pos -T cells and Tregs in the intestinal mucosa. A) T cells are more or less evenly distributed in the gut B) CD8 pos T cells are enriched in the human gastric corpus and antrum. C) CD3 pos CD4 pos T cells are predominantly found in the lower intestinal tract. D) The relative abundance of CD4 pos CD25 high CD127 low/neg FOXP3 cells was highest in the appendiceal orifice region and the ascending colon. The figures represent cumulative flow cytometry data from all study participants (N=16) from all seven biopsy regions including the gastric corpus (GC), gastric antrum (GA), duodenum (DD), terminal ileum (TI), appendiceal orifice (AO), ascending colon (AC) and sigmoid colon (SC). Unless otherwise indicated, differences were not significant. *P

    Journal: PLoS ONE

    Article Title: Distribution of CD4pos -, CD8pos - and Regulatory T Cells in the Upper and Lower Gastrointestinal Tract in Healthy Young Subjects

    doi: 10.1371/journal.pone.0080362

    Figure Lengend Snippet: Relative abundance of CD3 pos cells, CD8 pos -, CD4 pos -T cells and Tregs in the intestinal mucosa. A) T cells are more or less evenly distributed in the gut B) CD8 pos T cells are enriched in the human gastric corpus and antrum. C) CD3 pos CD4 pos T cells are predominantly found in the lower intestinal tract. D) The relative abundance of CD4 pos CD25 high CD127 low/neg FOXP3 cells was highest in the appendiceal orifice region and the ascending colon. The figures represent cumulative flow cytometry data from all study participants (N=16) from all seven biopsy regions including the gastric corpus (GC), gastric antrum (GA), duodenum (DD), terminal ileum (TI), appendiceal orifice (AO), ascending colon (AC) and sigmoid colon (SC). Unless otherwise indicated, differences were not significant. *P

    Article Snippet: FACS staining The following fluorochrome-conjugated monoclonal antibodies were used for surface staining: anti-CD3 PerCp-Cy5.5, anti-CD4 PE, anti-CD8 V500, anti-CD25 PE-Cy7 and anti-CD127 FITC (BD Biosciences, USA).

    Techniques: Flow Cytometry, Cytometry

    Incubation of vehicle (veh), epinephrine (epine), and salbutamol (sal) of immature Ovalbumin loaded BMDC in a T cell proliferation assay. Cells were incubated during LPS maturation o/n. The cells were washed and freshly isolated naïve CD4 OT-II cells were stained with CFSE. Cells were co-cultured for 3 days and CFSE dilution was determined by flow cytometry. Overlays shown are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Adrenergic ?2 Receptor Activation Stimulates Anti-Inflammatory Properties of Dendritic Cells In Vitro

    doi: 10.1371/journal.pone.0085086

    Figure Lengend Snippet: Incubation of vehicle (veh), epinephrine (epine), and salbutamol (sal) of immature Ovalbumin loaded BMDC in a T cell proliferation assay. Cells were incubated during LPS maturation o/n. The cells were washed and freshly isolated naïve CD4 OT-II cells were stained with CFSE. Cells were co-cultured for 3 days and CFSE dilution was determined by flow cytometry. Overlays shown are representative of 3 independent experiments.

    Article Snippet: Cells were stained for CD4 (eBiosciences) and fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) permeabilized using 0.5% Saponin (Sigma-Aldrich) for intracellular staining for IL-4, IL-17A (eBioscience) and IFNγ (BD Biosciences) or fixed and permeabilized with Foxp3 staining kit (eBiosciences) for Foxp3 expression according to manufacturer's instructions and characterized by flow cytometry.

    Techniques: Incubation, Proliferation Assay, Isolation, Staining, Cell Culture, Flow Cytometry, Cytometry

    Adrenergic agonists exposure stimulates skewing of a Treg population. Panel A shows flow cytometry analyses of intracellular staining of Foxp3 positive T cells skewed by BMDC treated with indicated AR agonist. Panel B, histograms of Foxp3 positive T cells (left) and IL-10 (right) concentrations in supernatant representative of three independent experiments. Panel C, FACS analyses and, panel D histograms of T cells skewed as described in fig. 7A with added TGF-β, to stimulate Foxp3 differentiation. Panel E shows the concentrations of TGF-β analyses of intracellular staining of Foxp3 posVeh), epinephrine (Epine) and salbutamol (Sal). Data are expressed in % positive of CD4 gated T cells (left) or as pg/ml (right) and represent the mean ± SEM of three independent experiments representative of 4 experiments. *p

    Journal: PLoS ONE

    Article Title: Adrenergic ?2 Receptor Activation Stimulates Anti-Inflammatory Properties of Dendritic Cells In Vitro

    doi: 10.1371/journal.pone.0085086

    Figure Lengend Snippet: Adrenergic agonists exposure stimulates skewing of a Treg population. Panel A shows flow cytometry analyses of intracellular staining of Foxp3 positive T cells skewed by BMDC treated with indicated AR agonist. Panel B, histograms of Foxp3 positive T cells (left) and IL-10 (right) concentrations in supernatant representative of three independent experiments. Panel C, FACS analyses and, panel D histograms of T cells skewed as described in fig. 7A with added TGF-β, to stimulate Foxp3 differentiation. Panel E shows the concentrations of TGF-β analyses of intracellular staining of Foxp3 posVeh), epinephrine (Epine) and salbutamol (Sal). Data are expressed in % positive of CD4 gated T cells (left) or as pg/ml (right) and represent the mean ± SEM of three independent experiments representative of 4 experiments. *p

    Article Snippet: Cells were stained for CD4 (eBiosciences) and fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) permeabilized using 0.5% Saponin (Sigma-Aldrich) for intracellular staining for IL-4, IL-17A (eBioscience) and IFNγ (BD Biosciences) or fixed and permeabilized with Foxp3 staining kit (eBiosciences) for Foxp3 expression according to manufacturer's instructions and characterized by flow cytometry.

    Techniques: Flow Cytometry, Cytometry, Staining, FACS

    The effect of adrenergic agonists on BMDC potential to skew Tcells. Panel A. FACS plots of intracellular IFNγFand IL-4 in -OT-II T cells co-cultured with BMDC pre-treated with vehicle (Veh), epinephrine (Epine) or salbutamol (Sal) (all at 1 µM). Intracellular IFNγ FNr salarerer affected, while IL-4 production is increased after AR-βaffected, while IL-4 productB, histograms of % IFNγ positive T cells by FACS and by ELISA of day 4 of co-culture supernatant. Panel C, histograms of % IL-4 positive T cells by FACS and by ELISA. Data are expressed as % positive of CD4 gated T cells or as pg/ml and represent the mean ± SEM of three independent experiments representative of 4 experiments. ***p

    Journal: PLoS ONE

    Article Title: Adrenergic ?2 Receptor Activation Stimulates Anti-Inflammatory Properties of Dendritic Cells In Vitro

    doi: 10.1371/journal.pone.0085086

    Figure Lengend Snippet: The effect of adrenergic agonists on BMDC potential to skew Tcells. Panel A. FACS plots of intracellular IFNγFand IL-4 in -OT-II T cells co-cultured with BMDC pre-treated with vehicle (Veh), epinephrine (Epine) or salbutamol (Sal) (all at 1 µM). Intracellular IFNγ FNr salarerer affected, while IL-4 production is increased after AR-βaffected, while IL-4 productB, histograms of % IFNγ positive T cells by FACS and by ELISA of day 4 of co-culture supernatant. Panel C, histograms of % IL-4 positive T cells by FACS and by ELISA. Data are expressed as % positive of CD4 gated T cells or as pg/ml and represent the mean ± SEM of three independent experiments representative of 4 experiments. ***p

    Article Snippet: Cells were stained for CD4 (eBiosciences) and fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) permeabilized using 0.5% Saponin (Sigma-Aldrich) for intracellular staining for IL-4, IL-17A (eBioscience) and IFNγ (BD Biosciences) or fixed and permeabilized with Foxp3 staining kit (eBiosciences) for Foxp3 expression according to manufacturer's instructions and characterized by flow cytometry.

    Techniques: FACS, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

    DC-T cell interactions. ( A ) Productive and latent infection was quantified in eFluor670-labelled resting memory CD4 +  T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or anti-CD18 (dark grey) prior to infection, n = 5. ( B ) Latent infection was determined in eFluor670-labelled resting CD4 +  T cells that were cultured alone, with mDC or alternatively with soluble ICAM-1-fc and anti-IgG-fc, n = 2. ( C ) Latent infection was determined in sorted eFluor hi EGFP −  CD4 +  T cells following stimulation with anti-CD3/CD28, that were cultured alone or with mDC that were added prior to infection or post-infection, n = 5. Columns represent the median of 5 experiments and error bars the interquartile range. *P

    Journal: PLoS Pathogens

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    doi: 10.1371/journal.ppat.1003799

    Figure Lengend Snippet: DC-T cell interactions. ( A ) Productive and latent infection was quantified in eFluor670-labelled resting memory CD4 + T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or anti-CD18 (dark grey) prior to infection, n = 5. ( B ) Latent infection was determined in eFluor670-labelled resting CD4 + T cells that were cultured alone, with mDC or alternatively with soluble ICAM-1-fc and anti-IgG-fc, n = 2. ( C ) Latent infection was determined in sorted eFluor hi EGFP − CD4 + T cells following stimulation with anti-CD3/CD28, that were cultured alone or with mDC that were added prior to infection or post-infection, n = 5. Columns represent the median of 5 experiments and error bars the interquartile range. *P

    Article Snippet: Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber.

    Techniques: Infection, Cell Culture

    Myeloid DC induce post-integration latency in non-proliferating memory CD4 +  T cells. SNARF-labelled resting CD4 +  T cells were cultured alone (light grey) or with syngeneic plasmacytoid (pDC; grey) or myeloid DC (mDC; dark grey). ( A ) Productive infection (EGFP +  cells) was determined by flow cytometry on day 5 post-infection. ( B ) Latent infection was quantified in SNARF hi EGFP −  cells following either addition of PHA-activated PBMC, n = 5; or ( C ) direct activation with anti-CD3/CD28 in the presence or absence of the integrase inhibitor L8. ( D ) Integrated HIV DNA was quantified in the sorted SNARF hi EGFP −  T cells by Alu-LTR real-time PCR, n = 3. ( E ) Productive and latent infection was determined in SNARF hi EGFP −  CD4 +  T cells from mDC-T cell co-cultures following infection with nef-deficient (-nef) or nef-competent EGFP HIV. ( F ) Latent infection was determined in sorted SNARF hi EGFP -  CD4 +  T cells, cultured alone or with mDC, following activation with PHA-PBMC. ( G ) Productive and latent infection was determined in SNARF hi EGFP −  CD4 +  T cells from mDC-T cell co-cultures with and without Staphylococcus Enterotoxin B (SEB), n = 4. ( H ) SNARF-labelled resting CD4 +  T cells were cultured alone (light grey) or with syngeneic mDC (grey) at decreasing DC∶T cell ratios and latent infection quantified in sorted SNARF hi EGFP −  T cells following addition of PHA-activated PBMC, n = 5. ( I ) Resting CD4 +  T cells were cultured either alone or in the presence of mDC. At day 5 post-infection, SNARF hi EGFP −  cells were sorted into naïve (light grey) or memory (grey) CD4 +  T cells and latent infection quantified, n = 5. The lower limit of detection is represented by a dotted line. Columns represent the median of 3–7 donors and error bars indicate the interquartile range. * P

    Journal: PLoS Pathogens

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    doi: 10.1371/journal.ppat.1003799

    Figure Lengend Snippet: Myeloid DC induce post-integration latency in non-proliferating memory CD4 + T cells. SNARF-labelled resting CD4 + T cells were cultured alone (light grey) or with syngeneic plasmacytoid (pDC; grey) or myeloid DC (mDC; dark grey). ( A ) Productive infection (EGFP + cells) was determined by flow cytometry on day 5 post-infection. ( B ) Latent infection was quantified in SNARF hi EGFP − cells following either addition of PHA-activated PBMC, n = 5; or ( C ) direct activation with anti-CD3/CD28 in the presence or absence of the integrase inhibitor L8. ( D ) Integrated HIV DNA was quantified in the sorted SNARF hi EGFP − T cells by Alu-LTR real-time PCR, n = 3. ( E ) Productive and latent infection was determined in SNARF hi EGFP − CD4 + T cells from mDC-T cell co-cultures following infection with nef-deficient (-nef) or nef-competent EGFP HIV. ( F ) Latent infection was determined in sorted SNARF hi EGFP - CD4 + T cells, cultured alone or with mDC, following activation with PHA-PBMC. ( G ) Productive and latent infection was determined in SNARF hi EGFP − CD4 + T cells from mDC-T cell co-cultures with and without Staphylococcus Enterotoxin B (SEB), n = 4. ( H ) SNARF-labelled resting CD4 + T cells were cultured alone (light grey) or with syngeneic mDC (grey) at decreasing DC∶T cell ratios and latent infection quantified in sorted SNARF hi EGFP − T cells following addition of PHA-activated PBMC, n = 5. ( I ) Resting CD4 + T cells were cultured either alone or in the presence of mDC. At day 5 post-infection, SNARF hi EGFP − cells were sorted into naïve (light grey) or memory (grey) CD4 + T cells and latent infection quantified, n = 5. The lower limit of detection is represented by a dotted line. Columns represent the median of 3–7 donors and error bars indicate the interquartile range. * P

    Article Snippet: Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber.

    Techniques: Cell Culture, Infection, Flow Cytometry, Cytometry, Activation Assay, Real-time Polymerase Chain Reaction

    DC-induced latency in resting CD4 +  T cells. ( A ) Resting CD4 +  T cells were isolated from the blood of healthy donors and labelled with the proliferation dye SNARF, which decreases in intensity following each round of cell division allowing identification of non-proliferating cells. SNARF-labelled resting CD4 +  T cells were cultured either alone or with syngeneic blood DC. Following 24 hours of culture, cells were infected with NL(AD8)-nef/EGFP at an MOI of 0.5. All culture media was supplemented with IL-2 (2 U/mL). ( B ) At day 5 post-infection, the number of productively infected (EGFP + ) cells was determined and the non-proliferating (SNARF hi ) cells that were not productively infected (EGFP − ) were sorted. The sorted SNARF hi EGFP −  cells were stimulated with PHA/IL-2 in the presence of PBMC and cultured for 5 days to amplify any replication competent latent infection. ( C ) Productive infection and ( D ) latent infection following infection of T cells cultured alone (light grey) or in the presence of DC (grey) is shown. ( E ) Latent infection in the presence of DC cultured with (grey) or without (light grey) 0.1 µM Indinavir. ( F ) Expression of the early (CD69; black) and late (HLA-DR; grey) surface activation markers and the intracellular proliferation marker Ki67 (light grey) was quantified by flow cytometry on sorted SNARF hi EGFP +  CD4 +  T cells following HIV infection of T cells cultured alone or in the presence of DC. The lower limit of detection of each assay is represented by a dotted line. Columns represent the median of 5 independent experiments and error bars indicate the interquartile range. * P

    Journal: PLoS Pathogens

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    doi: 10.1371/journal.ppat.1003799

    Figure Lengend Snippet: DC-induced latency in resting CD4 + T cells. ( A ) Resting CD4 + T cells were isolated from the blood of healthy donors and labelled with the proliferation dye SNARF, which decreases in intensity following each round of cell division allowing identification of non-proliferating cells. SNARF-labelled resting CD4 + T cells were cultured either alone or with syngeneic blood DC. Following 24 hours of culture, cells were infected with NL(AD8)-nef/EGFP at an MOI of 0.5. All culture media was supplemented with IL-2 (2 U/mL). ( B ) At day 5 post-infection, the number of productively infected (EGFP + ) cells was determined and the non-proliferating (SNARF hi ) cells that were not productively infected (EGFP − ) were sorted. The sorted SNARF hi EGFP − cells were stimulated with PHA/IL-2 in the presence of PBMC and cultured for 5 days to amplify any replication competent latent infection. ( C ) Productive infection and ( D ) latent infection following infection of T cells cultured alone (light grey) or in the presence of DC (grey) is shown. ( E ) Latent infection in the presence of DC cultured with (grey) or without (light grey) 0.1 µM Indinavir. ( F ) Expression of the early (CD69; black) and late (HLA-DR; grey) surface activation markers and the intracellular proliferation marker Ki67 (light grey) was quantified by flow cytometry on sorted SNARF hi EGFP + CD4 + T cells following HIV infection of T cells cultured alone or in the presence of DC. The lower limit of detection of each assay is represented by a dotted line. Columns represent the median of 5 independent experiments and error bars indicate the interquartile range. * P

    Article Snippet: Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber.

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

    Acquisition of latently infected cells for microarray analysis. ( A ) SNARF-labelled resting CD4 +  T cells were cultured with or without syngeneic blood DC. Following 24 hours of culture, cells were mock-infected with media alone (control) or infected with NL(AD8)-nef/EGFP (MOI of 5). Cells were cultured with IL-7 (10 ng/mL) throughout the culture period. On day 5 post-infection, SNARF hi EGFP − CD4 +  T cells were sorted and lysed for either microarray or ( B ) qPCR, where total HIV DNA was quantified.

    Journal: PLoS Pathogens

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    doi: 10.1371/journal.ppat.1003799

    Figure Lengend Snippet: Acquisition of latently infected cells for microarray analysis. ( A ) SNARF-labelled resting CD4 + T cells were cultured with or without syngeneic blood DC. Following 24 hours of culture, cells were mock-infected with media alone (control) or infected with NL(AD8)-nef/EGFP (MOI of 5). Cells were cultured with IL-7 (10 ng/mL) throughout the culture period. On day 5 post-infection, SNARF hi EGFP − CD4 + T cells were sorted and lysed for either microarray or ( B ) qPCR, where total HIV DNA was quantified.

    Article Snippet: Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber.

    Techniques: Infection, Microarray, Cell Culture, Real-time Polymerase Chain Reaction

    Changes in gene expression in DC-induced latently infected CD4 +  T cells. ( A ) Fold change scatterplot comparing gene expression between HIV T (+DC) (CD4 +  T cells cultured with DC and HIV), and Mock T (+DC) (CD4 +  T cells cultured with only DC) relative to their controls, HIV T (CD4 +  T cells cultured with HIV) and Mock T (CD4 +  T cells cultured in media alone) respectively. The solid line indicates absolute 2-fold change. ( B  and  C ) Top two gene interaction networks as ranked by Ingenuity Pathway Analysis. The networks were built from the list of differentially expressed genes induced by HIV T (+DC), relative to Mock T (+DC) after subtracting HIV T and Mock T from each group respectively. Genes highlighted in red were up-regulated and those in blue were down-regulated. The different node shapes indicate genes in different functional categories according to the legend. The interactions between the different nodes are shown as solid (direct interaction) or dashed (indirect interaction) lines (edges). ( D ) Fold change in gene expression values for selected genes from the Illumina BeadArrays plotted against Real-Time PCR (qPCR) deltaCt values for each target gene. PCR targets were mapped to BeadArray probes by matching the official gene symbols.

    Journal: PLoS Pathogens

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    doi: 10.1371/journal.ppat.1003799

    Figure Lengend Snippet: Changes in gene expression in DC-induced latently infected CD4 + T cells. ( A ) Fold change scatterplot comparing gene expression between HIV T (+DC) (CD4 + T cells cultured with DC and HIV), and Mock T (+DC) (CD4 + T cells cultured with only DC) relative to their controls, HIV T (CD4 + T cells cultured with HIV) and Mock T (CD4 + T cells cultured in media alone) respectively. The solid line indicates absolute 2-fold change. ( B and C ) Top two gene interaction networks as ranked by Ingenuity Pathway Analysis. The networks were built from the list of differentially expressed genes induced by HIV T (+DC), relative to Mock T (+DC) after subtracting HIV T and Mock T from each group respectively. Genes highlighted in red were up-regulated and those in blue were down-regulated. The different node shapes indicate genes in different functional categories according to the legend. The interactions between the different nodes are shown as solid (direct interaction) or dashed (indirect interaction) lines (edges). ( D ) Fold change in gene expression values for selected genes from the Illumina BeadArrays plotted against Real-Time PCR (qPCR) deltaCt values for each target gene. PCR targets were mapped to BeadArray probes by matching the official gene symbols.

    Article Snippet: Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber.

    Techniques: Expressing, Infection, Cell Culture, Functional Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Depletion of CD69+ cells has no effect on mDC-induced latency. ( A ) Expression of CD69 was determined in sorted SNARF hi EGFP −  CD4 +  T cells, n = 6. ( B ) Latent infection was determined for SNARF hi EGFP −  CD4 +  T cells sorted from mDC-T cell co-cultures that either contained (+CD69, light grey) or were depleted of CD69-expressing cells (−CD69, black), n = 4. The lower limit of detection is represented by a dotted line. Columns represent the median of 4–6 donors and error bars indicate the interquartile range. * P

    Journal: PLoS Pathogens

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    doi: 10.1371/journal.ppat.1003799

    Figure Lengend Snippet: Depletion of CD69+ cells has no effect on mDC-induced latency. ( A ) Expression of CD69 was determined in sorted SNARF hi EGFP − CD4 + T cells, n = 6. ( B ) Latent infection was determined for SNARF hi EGFP − CD4 + T cells sorted from mDC-T cell co-cultures that either contained (+CD69, light grey) or were depleted of CD69-expressing cells (−CD69, black), n = 4. The lower limit of detection is represented by a dotted line. Columns represent the median of 4–6 donors and error bars indicate the interquartile range. * P

    Article Snippet: Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber.

    Techniques: Expressing, Infection

    Soluble factors in DC-induced HIV latency. ( A ) Resting CD4 +  T cells were cultured alone (light grey) or with sorted pDC (grey) or mDC (dark grey). At day 5 post-infection, cytokines and chemokines were quantified in culture supernatants using cytometric bead arrays, n = 3. * P

    Journal: PLoS Pathogens

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    doi: 10.1371/journal.ppat.1003799

    Figure Lengend Snippet: Soluble factors in DC-induced HIV latency. ( A ) Resting CD4 + T cells were cultured alone (light grey) or with sorted pDC (grey) or mDC (dark grey). At day 5 post-infection, cytokines and chemokines were quantified in culture supernatants using cytometric bead arrays, n = 3. * P

    Article Snippet: Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber.

    Techniques: Cell Culture, Infection

    IL-17 responses are significantly elevated in the MLN and PPs of  H. pylori -infected IL-21 −/−  mice. Real-time RT-PCR was used to measure  Il-21  (A) and  Il-17a  (B) expression levels in  H. pylori -infected mice. (C) Intracellular cytokine staining was used to measure IFN-γ and IL-17A production by CD4 +  T cells and γδ-positive T cells from the Peyer’s patches of  H. pylori -infected mice (with PMA restimulation [stim] and without PMA restimulation [null]). Four to 7 mice were used for each experiment, and the data presented are representative of those from 3 experiments. (A, B) An unpaired  t  test was performed to test for statistical significance. (C) ANOVA followed by Dunnett’s correction for multiple comparisons was used. ns, not significant; *,  P

    Journal: Infection and Immunity

    Article Title: Interleukin-21 (IL-21) Downregulates Dendritic Cell Cytokine Responses to Helicobacter pylori and Modulates T Lymphocyte IL-17A Expression in Peyer’s Patches during Infection

    doi: 10.1128/IAI.00237-19

    Figure Lengend Snippet: IL-17 responses are significantly elevated in the MLN and PPs of H. pylori -infected IL-21 −/− mice. Real-time RT-PCR was used to measure Il-21 (A) and Il-17a (B) expression levels in H. pylori -infected mice. (C) Intracellular cytokine staining was used to measure IFN-γ and IL-17A production by CD4 + T cells and γδ-positive T cells from the Peyer’s patches of H. pylori -infected mice (with PMA restimulation [stim] and without PMA restimulation [null]). Four to 7 mice were used for each experiment, and the data presented are representative of those from 3 experiments. (A, B) An unpaired t test was performed to test for statistical significance. (C) ANOVA followed by Dunnett’s correction for multiple comparisons was used. ns, not significant; *, P

    Article Snippet: T cells were then isolated using a CD4+ T cell isolation kit, mouse (catalog no. 130-104-454; Miltenyi Biotec, Bergisch Gladbach, Germany), according to the protocol provided by the manufacturer.

    Techniques: Infection, Mouse Assay, Quantitative RT-PCR, Expressing, Staining

    CD4 + T cell activation is enhanced by a co-culture with BMDCs stimulated with the combination of M-M and CpG 1826 in vitro. ( A ) The isolation of CD4 + T cells from the spleen samples from immunized mice and BMDCs from untreated mice. The CD4 + T cell and DC percentage was analyzed by flow cytometry. The purity of the CD4 + T cells was 96.9%. The purity of the DCs was 96.6%. ( B ) M-M and CpG 1826 synergistically increased the proliferation of co-cultured CD4 + T cells and DCs. ( C – E ) The production of IFN-γ, IL-12p70, and IL-4 in the CD4 + T cells cocultured with DCs, as detected by ELISA. The CD4 T cells were co-cultured with the DCs at a ratio of 50:1. All the experiments were repeated three times, and all the data are expressed as the mean ± SD ( n = 3). # represents production

    Journal: International Journal of Molecular Sciences

    Article Title: CpG ODN1826 as a Promising Mucin1-Maltose-Binding Protein Vaccine Adjuvant Induced DC Maturation and Enhanced Antitumor Immunity

    doi: 10.3390/ijms19030920

    Figure Lengend Snippet: CD4 + T cell activation is enhanced by a co-culture with BMDCs stimulated with the combination of M-M and CpG 1826 in vitro. ( A ) The isolation of CD4 + T cells from the spleen samples from immunized mice and BMDCs from untreated mice. The CD4 + T cell and DC percentage was analyzed by flow cytometry. The purity of the CD4 + T cells was 96.9%. The purity of the DCs was 96.6%. ( B ) M-M and CpG 1826 synergistically increased the proliferation of co-cultured CD4 + T cells and DCs. ( C – E ) The production of IFN-γ, IL-12p70, and IL-4 in the CD4 + T cells cocultured with DCs, as detected by ELISA. The CD4 T cells were co-cultured with the DCs at a ratio of 50:1. All the experiments were repeated three times, and all the data are expressed as the mean ± SD ( n = 3). # represents production

    Article Snippet: CD4+ T cells were isolated from this single-cell suspension using the CD4+ T Cell Isolation Kit II, an LS column, and a MidiMACS™ separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

    Techniques: Activation Assay, Co-Culture Assay, In Vitro, Isolation, Mouse Assay, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of EA on percentage of CD3+, CD4+, and CD8+ T lymphocytes in intestinal mucosa. Data were presented as means ± SD ( n = 5) and # p

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture at Bilateral Zusanli Points (ST36) Protects Intestinal Mucosal Immune Barrier in Sepsis

    doi: 10.1155/2015/639412

    Figure Lengend Snippet: Effects of EA on percentage of CD3+, CD4+, and CD8+ T lymphocytes in intestinal mucosa. Data were presented as means ± SD ( n = 5) and # p

    Article Snippet: For cytofluorometric analyses cells were preincubated in PBS and stained for 30 min at 4°C using saturating concentrations of the following antibodies: CD3-FITC+γ /δ T-PE and CD4-FITC+CD8-PE antibody (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

    Techniques:

    SBE treatment down-regulated the population of Treg cells. Tumors from control and SBE treated groups were digested with collagenase and hyaluronidase. a Tumor-infiltrating lymphocytes (TILs) were isolated, stained with anti-Foxp3-APC after surface staining with anti-CD25-PE and anti-CD4-FITC. b The amount of Treg cells in tumor microenvironment between the two groups is shown. Data are representative of three independent experiments. * P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Scutellaria barbata D. Don extract inhibits the tumor growth through down-regulating of Treg cells and manipulating Th1/Th17 immune response in hepatoma H22-bearing mice

    doi: 10.1186/s12906-016-1551-9

    Figure Lengend Snippet: SBE treatment down-regulated the population of Treg cells. Tumors from control and SBE treated groups were digested with collagenase and hyaluronidase. a Tumor-infiltrating lymphocytes (TILs) were isolated, stained with anti-Foxp3-APC after surface staining with anti-CD25-PE and anti-CD4-FITC. b The amount of Treg cells in tumor microenvironment between the two groups is shown. Data are representative of three independent experiments. * P

    Article Snippet: After incubation of TILs at 4 °C with anti-mouse CD16/CD32mAb (2.4G2) in a staining buffer (phosphate-buffered saline containing 2% FCS and 0.1% sodium azide) on ice for 15 min. To detect regulatory T cells, cells were stained with anti-Foxp3-APC after surface staining with anti-CD25-PE and anti-CD4-FITC according to the the manufacturer’s protocols (BD Bioscience, San Jose, CA, USA).

    Techniques: Isolation, Staining

    SBE treatment down-regulated the amount of CD4 + IL-17 + Th17 cells. a Tumor-infiltrating lymphocytes (TILs) were isolated and stimulated, and the cells were stained with anti-CD4-FITC followed by labeling with anti-IL-17-PE cytokine Abs. b The amount of Th17 cells in tumor microenvironment between the two groups is shown. Data are representative of three independent experiments. * P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Scutellaria barbata D. Don extract inhibits the tumor growth through down-regulating of Treg cells and manipulating Th1/Th17 immune response in hepatoma H22-bearing mice

    doi: 10.1186/s12906-016-1551-9

    Figure Lengend Snippet: SBE treatment down-regulated the amount of CD4 + IL-17 + Th17 cells. a Tumor-infiltrating lymphocytes (TILs) were isolated and stimulated, and the cells were stained with anti-CD4-FITC followed by labeling with anti-IL-17-PE cytokine Abs. b The amount of Th17 cells in tumor microenvironment between the two groups is shown. Data are representative of three independent experiments. * P

    Article Snippet: After incubation of TILs at 4 °C with anti-mouse CD16/CD32mAb (2.4G2) in a staining buffer (phosphate-buffered saline containing 2% FCS and 0.1% sodium azide) on ice for 15 min. To detect regulatory T cells, cells were stained with anti-Foxp3-APC after surface staining with anti-CD25-PE and anti-CD4-FITC according to the the manufacturer’s protocols (BD Bioscience, San Jose, CA, USA).

    Techniques: Isolation, Staining, Labeling

    Severe colitis in DSS treated CD69 −/− mice is associated with increased pro-inflammatory response and early influx of CD4 T cells devoid of Foxp3 Treg cell into the colon. A. IL-17 concentration was determined by ELISA in the sera of control B6 mice and DSS treated B6 and CD69 −/− mice 7 days after the DSS administration. Mean (± SEM) of at least five mice per group is presented. B. RNA was isolated from frozen colon tissue samples of B6 and CD69 −/− mice treated with DSS, seven days after the DSS administration, or the control B6 mice and reverse transcribed to cDNA. Relative expression of IFN-γ gene was measured by qRT-PCR and presented as mean ± SEM of at least five mice per group. C. Mean (± SEM) total number of CD4 + T cells in blood, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) of 3 days DSS treated B6 and CD69 −/− mice are shown. At least five mice per each strain were analysed. D. The expression of Foxp3 by CD4 T cells from blood, MLN and cLP of B6 and CD69 −/− mice treated with DSS for 3 days was analysed by flow cytometry. Data from an individual, representative mouse (out of at least 5 mice per group analysed) are shown. Numbers indicate the percentage of CD4 T cells that express the transcriptional factor Foxp3. E. Mean (± SEM) total number of CD4 + Foxp3 + T cells in blood, MLN and cLP of 3 days DSS treated B6 and CD69 −/− mice are shown. At least five mice per each strain were analyzed. N.S. – not statistically significant; *p≤0.05; **p≤0.005.

    Journal: PLoS ONE

    Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    doi: 10.1371/journal.pone.0065413

    Figure Lengend Snippet: Severe colitis in DSS treated CD69 −/− mice is associated with increased pro-inflammatory response and early influx of CD4 T cells devoid of Foxp3 Treg cell into the colon. A. IL-17 concentration was determined by ELISA in the sera of control B6 mice and DSS treated B6 and CD69 −/− mice 7 days after the DSS administration. Mean (± SEM) of at least five mice per group is presented. B. RNA was isolated from frozen colon tissue samples of B6 and CD69 −/− mice treated with DSS, seven days after the DSS administration, or the control B6 mice and reverse transcribed to cDNA. Relative expression of IFN-γ gene was measured by qRT-PCR and presented as mean ± SEM of at least five mice per group. C. Mean (± SEM) total number of CD4 + T cells in blood, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) of 3 days DSS treated B6 and CD69 −/− mice are shown. At least five mice per each strain were analysed. D. The expression of Foxp3 by CD4 T cells from blood, MLN and cLP of B6 and CD69 −/− mice treated with DSS for 3 days was analysed by flow cytometry. Data from an individual, representative mouse (out of at least 5 mice per group analysed) are shown. Numbers indicate the percentage of CD4 T cells that express the transcriptional factor Foxp3. E. Mean (± SEM) total number of CD4 + Foxp3 + T cells in blood, MLN and cLP of 3 days DSS treated B6 and CD69 −/− mice are shown. At least five mice per each strain were analyzed. N.S. – not statistically significant; *p≤0.05; **p≤0.005.

    Article Snippet: The following antibodies were used: APC-conjugated mAb binding CD4 GK1.5 (cat. no. 17-0041-83; eBioscience, Frankfurt, Germany), CXCR3 CXCR3-173 (cat. no. 17-1831-80; eBioscience) and CCR7 4B12 (cat. no. FAB3477A; R & D systems); FITC-conjugated mAb binding CD4 GK1.5 (cat. no. 11-0041-86; eBioscience) and CD44 IM7 (cat. no. 553133; BD Biosciences); PE-conjugated mAb binding CD103 M290 (cat. no. 557495; BD Bioscience) and α4β7 DATK32 (cat. no. 553811; BD Bioscience); biotin-conjugated mAb binding CD62L MEL-14 (cat.no.

    Techniques: Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry

    Increased susceptibility of CD69 −/− mice to DSS induced colitis is partially due to increased chemokine-dependent CD4 T cell migration to cLP. B6 or CD69 −/− mice are administrated 2% dextrane sodium sulphate (DSS) in the drinking water for 5 days and then provided with normal sterile water. One group of DSS-administrated CD69 −/− mice were treated with the mixture of CCL-1, CXCL-10 and CCL-19 blocking Abs by i.p. injection at days 0, 2, 4 and 6 (CD69 −/− +Ch-block group). The control group represents non-treated B6 animals. A. Mean (± SEM) of body weight loss (%) of at least 5 mice per group is shown. B. Colitis scores of the histological colon sections of DSS treated B6 and CD69 −/− mice, control and CD69 −/− +Ch-block groups. Mean (± SEM) for at least five mice per group are presented. C. Histopathological colon tissue samples taken from control B6 mice, DSS treated B6 or CD69 −/− animals and CD69 −/− +Ch-block group were embedded in paraffin, sectioned on a microtome, mounted on slides, and stained with H E. Representative images of one individual mouse per group (from at least five mice per group analyzed) are shown (original magnification×20). RNA was isolated from the frozen colon tissue samples of control B6 animals and DSS treated B6 or CD69 −/− mice and reverse transcribed to complementary DNA. Relative expression of CCL-1 ( D ), CXCL-10 ( E ) and CCL-19 ( F ) genes compared to β-actin gene is measured by qRT-PCR. Mean (± SEM) for at least five mice per group are presented. N.S. – not statistically significant; *p≤0.05; **p≤0.005.

    Journal: PLoS ONE

    Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    doi: 10.1371/journal.pone.0065413

    Figure Lengend Snippet: Increased susceptibility of CD69 −/− mice to DSS induced colitis is partially due to increased chemokine-dependent CD4 T cell migration to cLP. B6 or CD69 −/− mice are administrated 2% dextrane sodium sulphate (DSS) in the drinking water for 5 days and then provided with normal sterile water. One group of DSS-administrated CD69 −/− mice were treated with the mixture of CCL-1, CXCL-10 and CCL-19 blocking Abs by i.p. injection at days 0, 2, 4 and 6 (CD69 −/− +Ch-block group). The control group represents non-treated B6 animals. A. Mean (± SEM) of body weight loss (%) of at least 5 mice per group is shown. B. Colitis scores of the histological colon sections of DSS treated B6 and CD69 −/− mice, control and CD69 −/− +Ch-block groups. Mean (± SEM) for at least five mice per group are presented. C. Histopathological colon tissue samples taken from control B6 mice, DSS treated B6 or CD69 −/− animals and CD69 −/− +Ch-block group were embedded in paraffin, sectioned on a microtome, mounted on slides, and stained with H E. Representative images of one individual mouse per group (from at least five mice per group analyzed) are shown (original magnification×20). RNA was isolated from the frozen colon tissue samples of control B6 animals and DSS treated B6 or CD69 −/− mice and reverse transcribed to complementary DNA. Relative expression of CCL-1 ( D ), CXCL-10 ( E ) and CCL-19 ( F ) genes compared to β-actin gene is measured by qRT-PCR. Mean (± SEM) for at least five mice per group are presented. N.S. – not statistically significant; *p≤0.05; **p≤0.005.

    Article Snippet: The following antibodies were used: APC-conjugated mAb binding CD4 GK1.5 (cat. no. 17-0041-83; eBioscience, Frankfurt, Germany), CXCR3 CXCR3-173 (cat. no. 17-1831-80; eBioscience) and CCR7 4B12 (cat. no. FAB3477A; R & D systems); FITC-conjugated mAb binding CD4 GK1.5 (cat. no. 11-0041-86; eBioscience) and CD44 IM7 (cat. no. 553133; BD Biosciences); PE-conjugated mAb binding CD103 M290 (cat. no. 557495; BD Bioscience) and α4β7 DATK32 (cat. no. 553811; BD Bioscience); biotin-conjugated mAb binding CD62L MEL-14 (cat.no.

    Techniques: Mouse Assay, Migration, Blocking Assay, Injection, Staining, Isolation, Expressing, Quantitative RT-PCR

    Increased migratory potential of CD69-deficient CD4 T cells to the mucosal intestinal tissues in vivo . CD4 T cells were enriched from the spleen of red fluorescent DsRed or CD69 −/− animals, both on the CD45.2 + B6 background. CD69-deficient CD4 T cells were labelled with green fluorescent CFSE. These cells were mixed in the approximate ratio 1∶1 and injected i.v. into CD45.1 + B6 mice. 18 or 72 h later cells were isolated from blood, mesenteric lymph nodes (MLN), small intestinal lamina propria (siLP) and colonic lamina propria (cLP) of the hosts and analyzed for the surface expression of CD4, CD45.2, DsRed and CFSE by multicolour flow cytometry (FCM). Numbers of DsRed and CFSE expressing CD45.2 + CD4 + cells recovered 18 h ( A ) or 72 h ( D ) after the transfer from the host tissues were determined with FCM and presented as mean (± SEM) total number of recovered cells per tissue for four mice analyzed. N.S. – not statistically significant; *p≤0.05. Homing index (HI) for every tissue 18 h ( B ) or 72 h ( E ) post-injection is calculated as: HI = number of CD4 + CD45.2 + CFSE + cells/number of CD4 + CD45.2 + DsRed + cells : IR (where IR is input ratio calculated before the injection as: IR = number of CD4 + CD45.2 + CFSE + cells/number of CD4 + CD45.2 + DsRed + cells). This value was normalized to the HI in the blood, so the potential retention of the injected cells in some of the periphery organs is eliminated. Mean (± SEM) of blood-normalized HI per tissue for four mice is presented. The deviation from the theoretical mean (TM = 1) was assessed (*p≤0.05). C. 18 h after the cell transfer sections of small intestine and colon of the host were stained with Alexa Fluor 350-conjugated wheat germ agglutinin (blue) analyzed for the number of green (CFSE + CD69 −/− ) and red (DsRed + B6) CD4 T cells by confocal microscope (original magnification×40).

    Journal: PLoS ONE

    Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    doi: 10.1371/journal.pone.0065413

    Figure Lengend Snippet: Increased migratory potential of CD69-deficient CD4 T cells to the mucosal intestinal tissues in vivo . CD4 T cells were enriched from the spleen of red fluorescent DsRed or CD69 −/− animals, both on the CD45.2 + B6 background. CD69-deficient CD4 T cells were labelled with green fluorescent CFSE. These cells were mixed in the approximate ratio 1∶1 and injected i.v. into CD45.1 + B6 mice. 18 or 72 h later cells were isolated from blood, mesenteric lymph nodes (MLN), small intestinal lamina propria (siLP) and colonic lamina propria (cLP) of the hosts and analyzed for the surface expression of CD4, CD45.2, DsRed and CFSE by multicolour flow cytometry (FCM). Numbers of DsRed and CFSE expressing CD45.2 + CD4 + cells recovered 18 h ( A ) or 72 h ( D ) after the transfer from the host tissues were determined with FCM and presented as mean (± SEM) total number of recovered cells per tissue for four mice analyzed. N.S. – not statistically significant; *p≤0.05. Homing index (HI) for every tissue 18 h ( B ) or 72 h ( E ) post-injection is calculated as: HI = number of CD4 + CD45.2 + CFSE + cells/number of CD4 + CD45.2 + DsRed + cells : IR (where IR is input ratio calculated before the injection as: IR = number of CD4 + CD45.2 + CFSE + cells/number of CD4 + CD45.2 + DsRed + cells). This value was normalized to the HI in the blood, so the potential retention of the injected cells in some of the periphery organs is eliminated. Mean (± SEM) of blood-normalized HI per tissue for four mice is presented. The deviation from the theoretical mean (TM = 1) was assessed (*p≤0.05). C. 18 h after the cell transfer sections of small intestine and colon of the host were stained with Alexa Fluor 350-conjugated wheat germ agglutinin (blue) analyzed for the number of green (CFSE + CD69 −/− ) and red (DsRed + B6) CD4 T cells by confocal microscope (original magnification×40).

    Article Snippet: The following antibodies were used: APC-conjugated mAb binding CD4 GK1.5 (cat. no. 17-0041-83; eBioscience, Frankfurt, Germany), CXCR3 CXCR3-173 (cat. no. 17-1831-80; eBioscience) and CCR7 4B12 (cat. no. FAB3477A; R & D systems); FITC-conjugated mAb binding CD4 GK1.5 (cat. no. 11-0041-86; eBioscience) and CD44 IM7 (cat. no. 553133; BD Biosciences); PE-conjugated mAb binding CD103 M290 (cat. no. 557495; BD Bioscience) and α4β7 DATK32 (cat. no. 553811; BD Bioscience); biotin-conjugated mAb binding CD62L MEL-14 (cat.no.

    Techniques: In Vivo, Injection, Mouse Assay, Isolation, Expressing, Flow Cytometry, Cytometry, Staining, Microscopy

    Increased in vitro functionality of CD4 T cell chemokine receptors for CCL-1, CXCL-10 and CCL-5 in the absence of CD69. CD4 T cells were enriched from the spleen of B6 or CD69 −/− mice and migration of these cells was analysed in vitro in transwell system. Medium alone or containing indicated concentrations of CCL-1, CXCL-10, CCL-5 and CCL-4 were loaded in the lower chamber of the transwell system, while CD4 T cells in the medium were loaded in the upper chamber. Cell number migrating from the upper chamber to the chemokine containing chamber trough polycarbonate membrane after 1 h at 37°C, 5% CO 2 was counted by flow cytometry. All the reactions were done in at least four repeats and average cell number per chemokine concentration per cell type was calculated. Results are presented as the number of cells migrating to the wells containing medium alone ( A ) or indicated concentration of CCL-1 ( B ), CXCL-10 ( C ), CCL-5 ( D ) or CCL-4 ( E ). Mean (± SEM) for at least four repeats per each chemokine concentration and cell type is presented. The lower graph set represents chemotactic indexes of B6 and CD69 −/− CD4 T cells for CCL-1 ( F) , CXCL-10 ( G) , CCL-5 ( H ) and CCL-4 ( I ). The chemotactic index was calculated as: the number of cells migrating to the media with chemokine divided by the average number of cells migrating to the media alone. *p≤0.05.

    Journal: PLoS ONE

    Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    doi: 10.1371/journal.pone.0065413

    Figure Lengend Snippet: Increased in vitro functionality of CD4 T cell chemokine receptors for CCL-1, CXCL-10 and CCL-5 in the absence of CD69. CD4 T cells were enriched from the spleen of B6 or CD69 −/− mice and migration of these cells was analysed in vitro in transwell system. Medium alone or containing indicated concentrations of CCL-1, CXCL-10, CCL-5 and CCL-4 were loaded in the lower chamber of the transwell system, while CD4 T cells in the medium were loaded in the upper chamber. Cell number migrating from the upper chamber to the chemokine containing chamber trough polycarbonate membrane after 1 h at 37°C, 5% CO 2 was counted by flow cytometry. All the reactions were done in at least four repeats and average cell number per chemokine concentration per cell type was calculated. Results are presented as the number of cells migrating to the wells containing medium alone ( A ) or indicated concentration of CCL-1 ( B ), CXCL-10 ( C ), CCL-5 ( D ) or CCL-4 ( E ). Mean (± SEM) for at least four repeats per each chemokine concentration and cell type is presented. The lower graph set represents chemotactic indexes of B6 and CD69 −/− CD4 T cells for CCL-1 ( F) , CXCL-10 ( G) , CCL-5 ( H ) and CCL-4 ( I ). The chemotactic index was calculated as: the number of cells migrating to the media with chemokine divided by the average number of cells migrating to the media alone. *p≤0.05.

    Article Snippet: The following antibodies were used: APC-conjugated mAb binding CD4 GK1.5 (cat. no. 17-0041-83; eBioscience, Frankfurt, Germany), CXCR3 CXCR3-173 (cat. no. 17-1831-80; eBioscience) and CCR7 4B12 (cat. no. FAB3477A; R & D systems); FITC-conjugated mAb binding CD4 GK1.5 (cat. no. 11-0041-86; eBioscience) and CD44 IM7 (cat. no. 553133; BD Biosciences); PE-conjugated mAb binding CD103 M290 (cat. no. 557495; BD Bioscience) and α4β7 DATK32 (cat. no. 553811; BD Bioscience); biotin-conjugated mAb binding CD62L MEL-14 (cat.no.

    Techniques: In Vitro, Mouse Assay, Migration, Flow Cytometry, Cytometry, Concentration Assay

    B6 and CD69 −/− CD4 T cells differ in the expression of naïve and memory surface cell markers. Cells were isolated from the spleen (spl), mesenteric lymph nodes (MLN), small intestinal lamina propria (siLP), colonic lamina propria (cLP) and blood (bld) of non-treated B6 and CD69 −/− mice and analyzed by flow cytometry. The surface expression of CD44 ( A ), CD103 ( B ), CD62L ( C ), CCR-7 ( D ), CCR-9 ( E ) and α4β7 integrin ( F ) by CD4 T cells were analysed. Graphs represent mean (± SEM) of CD4 T cell fraction expressing the indicated molecule for four mice per each strain per tissue. Black bars represent data for B6 and white bars for CD69 −/− cells. *p≤0.05.

    Journal: PLoS ONE

    Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    doi: 10.1371/journal.pone.0065413

    Figure Lengend Snippet: B6 and CD69 −/− CD4 T cells differ in the expression of naïve and memory surface cell markers. Cells were isolated from the spleen (spl), mesenteric lymph nodes (MLN), small intestinal lamina propria (siLP), colonic lamina propria (cLP) and blood (bld) of non-treated B6 and CD69 −/− mice and analyzed by flow cytometry. The surface expression of CD44 ( A ), CD103 ( B ), CD62L ( C ), CCR-7 ( D ), CCR-9 ( E ) and α4β7 integrin ( F ) by CD4 T cells were analysed. Graphs represent mean (± SEM) of CD4 T cell fraction expressing the indicated molecule for four mice per each strain per tissue. Black bars represent data for B6 and white bars for CD69 −/− cells. *p≤0.05.

    Article Snippet: The following antibodies were used: APC-conjugated mAb binding CD4 GK1.5 (cat. no. 17-0041-83; eBioscience, Frankfurt, Germany), CXCR3 CXCR3-173 (cat. no. 17-1831-80; eBioscience) and CCR7 4B12 (cat. no. FAB3477A; R & D systems); FITC-conjugated mAb binding CD4 GK1.5 (cat. no. 11-0041-86; eBioscience) and CD44 IM7 (cat. no. 553133; BD Biosciences); PE-conjugated mAb binding CD103 M290 (cat. no. 557495; BD Bioscience) and α4β7 DATK32 (cat. no. 553811; BD Bioscience); biotin-conjugated mAb binding CD62L MEL-14 (cat.no.

    Techniques: Expressing, Isolation, Mouse Assay, Flow Cytometry, Cytometry

    Absence of CD69 leads to the increased expression of CCL-1 , CXCL-10 and CCL-19 . RNA was isolated from the frozen mesenteric lymph nodes (MLN), small intestinal (si) and colonic (co) tissue samples or from sorted CD4 + and CD4 − spleen cells of non-treated B6 or CD69 −/− animals and reverse transcribed to complementary DNA. Relative expression of CCL-1 ( A and D ), CXCL-10 ( B and E ) and CCL-19 ( C and F ) genes compared to β-actin gene is analyzed by qRT-PCR. Mean (± SEM) for at least six mice per each strain is presented. *p≤0.05; **p≤0.005; ***p≤0.0005; N.S. – not significant.

    Journal: PLoS ONE

    Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    doi: 10.1371/journal.pone.0065413

    Figure Lengend Snippet: Absence of CD69 leads to the increased expression of CCL-1 , CXCL-10 and CCL-19 . RNA was isolated from the frozen mesenteric lymph nodes (MLN), small intestinal (si) and colonic (co) tissue samples or from sorted CD4 + and CD4 − spleen cells of non-treated B6 or CD69 −/− animals and reverse transcribed to complementary DNA. Relative expression of CCL-1 ( A and D ), CXCL-10 ( B and E ) and CCL-19 ( C and F ) genes compared to β-actin gene is analyzed by qRT-PCR. Mean (± SEM) for at least six mice per each strain is presented. *p≤0.05; **p≤0.005; ***p≤0.0005; N.S. – not significant.

    Article Snippet: The following antibodies were used: APC-conjugated mAb binding CD4 GK1.5 (cat. no. 17-0041-83; eBioscience, Frankfurt, Germany), CXCR3 CXCR3-173 (cat. no. 17-1831-80; eBioscience) and CCR7 4B12 (cat. no. FAB3477A; R & D systems); FITC-conjugated mAb binding CD4 GK1.5 (cat. no. 11-0041-86; eBioscience) and CD44 IM7 (cat. no. 553133; BD Biosciences); PE-conjugated mAb binding CD103 M290 (cat. no. 557495; BD Bioscience) and α4β7 DATK32 (cat. no. 553811; BD Bioscience); biotin-conjugated mAb binding CD62L MEL-14 (cat.no.

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Mouse Assay

    CD69-defficient OT-II CD4 T cells accumulate in cLP of RAG −/− hosts during an antigen specific transfer colitis. Antigen specific colitis was induced by the transfer of CD45RB high CD4 T cells, isolated from the spleen of OT-II or OT-II×CD69 −/− mice, into the RAG −/− animals. Cell transfer was in some groups followed by the intragastrical feeding of the hosts with 1 mg OVA protein every second day. A. Mean (± SEM) of body weight loss (%) of at least five mice per group is shown for non-treated RAG −/− animals (control group), hosts transferred with OT-II or OT-II×CD69 −/− cells and not fed with OVA (OT-II and OT-II×CD69 −/− groups) and host transferred with OT-II or OT-II×CD69 −/− cells and fed with OVA (OT-II+OVA and OT-II×CD69 −/− +OVA groups). B. Histopathological colon tissue samples taken from the control, OT-II, OT-II×CD69 −/− , OT-II+OVA and OT-II×CD69 −/− +OVA groups were embedded in paraffin, sectioned on a microtome, mounted on slides, and stained with H E. Representative images of one individual mouse from at least five mice per group analysed are shown (original magnification×20). C. Colitis scores of the histological colon sections of control animals and cell transferred hosts. Mean (± SEM) for at least five mice per group are presented. D. Cells recovered from the colonic lamina propria of the control mice and cell transferred hosts were analysed for the number of CD4 + T cells by flow cytometry. Mean (± SEM) for at least five mice per group are presented. *p≤0.05; **p≤0.005; N.S. – not significant.

    Journal: PLoS ONE

    Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    doi: 10.1371/journal.pone.0065413

    Figure Lengend Snippet: CD69-defficient OT-II CD4 T cells accumulate in cLP of RAG −/− hosts during an antigen specific transfer colitis. Antigen specific colitis was induced by the transfer of CD45RB high CD4 T cells, isolated from the spleen of OT-II or OT-II×CD69 −/− mice, into the RAG −/− animals. Cell transfer was in some groups followed by the intragastrical feeding of the hosts with 1 mg OVA protein every second day. A. Mean (± SEM) of body weight loss (%) of at least five mice per group is shown for non-treated RAG −/− animals (control group), hosts transferred with OT-II or OT-II×CD69 −/− cells and not fed with OVA (OT-II and OT-II×CD69 −/− groups) and host transferred with OT-II or OT-II×CD69 −/− cells and fed with OVA (OT-II+OVA and OT-II×CD69 −/− +OVA groups). B. Histopathological colon tissue samples taken from the control, OT-II, OT-II×CD69 −/− , OT-II+OVA and OT-II×CD69 −/− +OVA groups were embedded in paraffin, sectioned on a microtome, mounted on slides, and stained with H E. Representative images of one individual mouse from at least five mice per group analysed are shown (original magnification×20). C. Colitis scores of the histological colon sections of control animals and cell transferred hosts. Mean (± SEM) for at least five mice per group are presented. D. Cells recovered from the colonic lamina propria of the control mice and cell transferred hosts were analysed for the number of CD4 + T cells by flow cytometry. Mean (± SEM) for at least five mice per group are presented. *p≤0.05; **p≤0.005; N.S. – not significant.

    Article Snippet: The following antibodies were used: APC-conjugated mAb binding CD4 GK1.5 (cat. no. 17-0041-83; eBioscience, Frankfurt, Germany), CXCR3 CXCR3-173 (cat. no. 17-1831-80; eBioscience) and CCR7 4B12 (cat. no. FAB3477A; R & D systems); FITC-conjugated mAb binding CD4 GK1.5 (cat. no. 11-0041-86; eBioscience) and CD44 IM7 (cat. no. 553133; BD Biosciences); PE-conjugated mAb binding CD103 M290 (cat. no. 557495; BD Bioscience) and α4β7 DATK32 (cat. no. 553811; BD Bioscience); biotin-conjugated mAb binding CD62L MEL-14 (cat.no.

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

    CMV-specific CD4 + T cells display a Th1 cytokine secretion pattern (arbitrary units for all axes). Dot plots of lymphocytes from patient 4, gated on positive CD4-PerCP and CD69-APC fluorescence. ( a ) Anti–IFNγ-PE fluorescence (X-axis) versus anti TNFα-FITC fluorescence (Y-axis). ( b ) Anti–IL4-PE fluorescence (X-axis) versus anti–IL2-FITC fluorescence (Y-axis).

    Journal: Journal of Clinical Investigation

    Article Title: Development of virus-specific CD4+ T cells during primary cytomegalovirus infection

    doi:

    Figure Lengend Snippet: CMV-specific CD4 + T cells display a Th1 cytokine secretion pattern (arbitrary units for all axes). Dot plots of lymphocytes from patient 4, gated on positive CD4-PerCP and CD69-APC fluorescence. ( a ) Anti–IFNγ-PE fluorescence (X-axis) versus anti TNFα-FITC fluorescence (Y-axis). ( b ) Anti–IL4-PE fluorescence (X-axis) versus anti–IL2-FITC fluorescence (Y-axis).

    Article Snippet: For surface staining, CD4-APC, CD4-PerCP, CD8-FITC, CD8-PE, CD27-PE, CD62L-PE, CD49d-PE (anti–VLA-4), CD38-PE, CD154(CD40L)-PE, CD45RA-FITC, CD45RO-PE (all from Becton Dickinson), and CD11a-FITC (DAKO Corp., Glostrup, Denmark) were used.

    Techniques: Fluorescence

    Modulation of production of IFN-λ of pDC A) Cross-linking of CD4 or BDCA-2 on pDC inhibits IFN-λ and IFN-α production of PDC: CD4 or BDCA-2 on pDC within PBMC was cross-linked with anti-CD4 or anti-BDCA-2 microbeads, and then PBMC were simulated for 7 hr with HSV. The expression of IFN-α (i) or IFN-λ1 (ii) by pDC was measured by flow cytometry as described above. Data represent mean ± 1 SD for 6 separate experiments. * P

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

    Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

    doi: 10.4049/jimmunol.1102038

    Figure Lengend Snippet: Modulation of production of IFN-λ of pDC A) Cross-linking of CD4 or BDCA-2 on pDC inhibits IFN-λ and IFN-α production of PDC: CD4 or BDCA-2 on pDC within PBMC was cross-linked with anti-CD4 or anti-BDCA-2 microbeads, and then PBMC were simulated for 7 hr with HSV. The expression of IFN-α (i) or IFN-λ1 (ii) by pDC was measured by flow cytometry as described above. Data represent mean ± 1 SD for 6 separate experiments. * P

    Article Snippet: Briefly, PBMC (2×106 cells/sample) were washed with MACS buffer (PBS with 0.5% BSA and 2 mM EDTA), incubated for 20 mins at 4°C with mouse anti-human BDCA-2 Ab (Miltenyi, clone AC114, 1µg/sample) or CD4 MicroBeads (Miltenyi, M-T466, 20 µl/sample) and washed again with MACS buffer.

    Techniques: Expressing, Flow Cytometry, Cytometry

    Examples of flow cytometric gating strategies. (A) Gating strategy for regulatory T-cells. Gated for lymphocytes (FSC-A, SSC-A), singlets cells (FSC-A, FSC-H), live cells (FSC-A, APC-Cy7A: NIR), CD3 and CD4 positive cells (Horizon V500-A: CD3, APC-A:

    Journal: Stem Cell Investigation

    Article Title: Indoleamine 2,3-dioxygenase and survivin peptide vaccine combined with temozolomide in metastatic melanoma

    doi: 10.21037/sci.2017.08.06

    Figure Lengend Snippet: Examples of flow cytometric gating strategies. (A) Gating strategy for regulatory T-cells. Gated for lymphocytes (FSC-A, SSC-A), singlets cells (FSC-A, FSC-H), live cells (FSC-A, APC-Cy7A: NIR), CD3 and CD4 positive cells (Horizon V500-A: CD3, APC-A:

    Article Snippet: For phenotyping of CD3+ T-cells, the following antibodies were used: CD45RA-FITC, CCR7-PE-Cy7, CD4-APC (purchased from BD biosciences), CD27-PerCP, CD8-BV421 (purchased from Biolegend) and CD3-HV500 (purchased from BD Horizon).

    Techniques: Flow Cytometry

    SinΔC expression inhibits production of mature T cells. (A) Splenocytes (10 6 ) from 6- to-8-week-old wild-type (WT) and transgenic (Tg) animals were triply stained with CD4-allophycocyanin/CD8-peridinin chlorophyll-a protein and CD3-fluorescein isothiocyanate and analyzed by flow cytometry. In the dot plots, the numbers indicate the percentage of cells in each region. Histograms represent CD3 expression within the CD4 and CD8 SP populations in the dot plots. Two different transgenic founder lines, CR1 and MA2, with their respective wild-type controls, are shown. (B) Splenocytes were counted and stained, and the percentages of total CD3 T cells and CD4 + and CD8 + subpopulations were determined by fluorescence-activated cell sorting analysis and used to calculate the actual cell numbers for each population. Results from at least five wild-type and MA2 transgenic mice are represented as the mean ± the standard deviation (SD).

    Journal: Molecular and Cellular Biology

    Article Title: The Adapter Molecule Sin Regulates T-Cell-Receptor-Mediated Signal Transduction by Modulating Signaling Substrate Availability

    doi: 10.1128/MCB.24.10.4581-4592.2004

    Figure Lengend Snippet: SinΔC expression inhibits production of mature T cells. (A) Splenocytes (10 6 ) from 6- to-8-week-old wild-type (WT) and transgenic (Tg) animals were triply stained with CD4-allophycocyanin/CD8-peridinin chlorophyll-a protein and CD3-fluorescein isothiocyanate and analyzed by flow cytometry. In the dot plots, the numbers indicate the percentage of cells in each region. Histograms represent CD3 expression within the CD4 and CD8 SP populations in the dot plots. Two different transgenic founder lines, CR1 and MA2, with their respective wild-type controls, are shown. (B) Splenocytes were counted and stained, and the percentages of total CD3 T cells and CD4 + and CD8 + subpopulations were determined by fluorescence-activated cell sorting analysis and used to calculate the actual cell numbers for each population. Results from at least five wild-type and MA2 transgenic mice are represented as the mean ± the standard deviation (SD).

    Article Snippet: Anti-CD4-allophycocyanin-, anti-CD8-peridinin chlorophyll-a protein-, and anti-CD3-fluorescein isothiocyanate-conjugated antibodies were purchased from BD Pharmingen.

    Techniques: Expressing, Transgenic Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, FACS, Mouse Assay, Standard Deviation

    CD4 + T lymphocytes expressing effector/memory phenotype. Mononuclear cells were stained with mAb anti-CD44 FITC, anti-CD62L PE, anti-CD4 PerCp, anti-CD8 PerCp, and anti-CD3 APC and analyzed by flow cytometry. A high percentage of kidney and liver CD4 + T cells expresses effector/memory phenotype (CD44 high CD62L neg ) when compared with lung, spleen, and blood lymphocytes. Percentages are expressed from the gates: lymphocytes and CD3 + and CD4 + T cell areas. Dot plots are representative examples of three independent experiments ( n =8).

    Journal: Journal of Leukocyte Biology

    Article Title: Normal mouse kidneys contain activated and CD3+CD4−CD8− double-negative T lymphocytes with a distinct TCR repertoire

    doi: 10.1189/jlb.0907651

    Figure Lengend Snippet: CD4 + T lymphocytes expressing effector/memory phenotype. Mononuclear cells were stained with mAb anti-CD44 FITC, anti-CD62L PE, anti-CD4 PerCp, anti-CD8 PerCp, and anti-CD3 APC and analyzed by flow cytometry. A high percentage of kidney and liver CD4 + T cells expresses effector/memory phenotype (CD44 high CD62L neg ) when compared with lung, spleen, and blood lymphocytes. Percentages are expressed from the gates: lymphocytes and CD3 + and CD4 + T cell areas. Dot plots are representative examples of three independent experiments ( n =8).

    Article Snippet: The fluorochrome-conjugated mAb to mouse antigens used for flow cytometry analysis were: anti-CD16/CD32 (2.4G2), anti-CD3 allophycocyanin (APC; 145-2C11), anti-TCRαβ (H57-597), anti-IL-2Rβ (TM-B1), anti-CD4 PerCP (RM4-5), anti-CD8b FITC (53-5.8), anti-CD19 PE (1D3), anti-B220 PE (RA3-6B2), anti-CD69 PE (H1.2F3), anti-CD25 PE (3C7), anti-NK1.1 PE (PK136), anti-CD44 FITC (IM7), and anti-CD62 ligand (anti-CD62L) PE (MEL-14; BD PharMingen, San Diego, CA, USA).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry

    TCR Vβ profile of kidneys and spleen, CD4 + , CD8 + , and DN T cell subsets. Mononuclear cells obtained from kidneys and spleens of three mice were stained with mAb anti-TCR Vβ FITC, anti-CD8 PE, anti-CD4 PerCp, and anti-CD3 APC for flow cytometry analysis. Bar grafts show the percentage of TCR Vβ-positive CD4 + T cells from the R1 gate, CD3 + CD4 − CD8 − DN T cells from the R2 gate, and CD8 + T cells from the R3 gate.

    Journal: Journal of Leukocyte Biology

    Article Title: Normal mouse kidneys contain activated and CD3+CD4−CD8− double-negative T lymphocytes with a distinct TCR repertoire

    doi: 10.1189/jlb.0907651

    Figure Lengend Snippet: TCR Vβ profile of kidneys and spleen, CD4 + , CD8 + , and DN T cell subsets. Mononuclear cells obtained from kidneys and spleens of three mice were stained with mAb anti-TCR Vβ FITC, anti-CD8 PE, anti-CD4 PerCp, and anti-CD3 APC for flow cytometry analysis. Bar grafts show the percentage of TCR Vβ-positive CD4 + T cells from the R1 gate, CD3 + CD4 − CD8 − DN T cells from the R2 gate, and CD8 + T cells from the R3 gate.

    Article Snippet: The fluorochrome-conjugated mAb to mouse antigens used for flow cytometry analysis were: anti-CD16/CD32 (2.4G2), anti-CD3 allophycocyanin (APC; 145-2C11), anti-TCRαβ (H57-597), anti-IL-2Rβ (TM-B1), anti-CD4 PerCP (RM4-5), anti-CD8b FITC (53-5.8), anti-CD19 PE (1D3), anti-B220 PE (RA3-6B2), anti-CD69 PE (H1.2F3), anti-CD25 PE (3C7), anti-NK1.1 PE (PK136), anti-CD44 FITC (IM7), and anti-CD62 ligand (anti-CD62L) PE (MEL-14; BD PharMingen, San Diego, CA, USA).

    Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry

    iTregs expanded in vitro by mtDCs retained Foxp3 expression and efficiently inhibited CD4 + T cell proliferation. (a) Schematic overview of the induction/expansion strategy for iTregs. The two expansion cycles were established as described in the Materials and Methods. The differentiation of iTregs was first induced with an anti-CD3/CD28 mAb, TGF- β , and IL-2 for 5 days in vitro. Then, iTreg mtDC or iTreg mDC were generated by expanding iTregs for 4 days using mtDCs or mDCs, respectively. The expression of the transcription factor Foxp3 and CD25 in cells was determined in each cycle using flow cytometry. Data are representative of three independent experiments. (b) Expansion of different types of iTregs was determined by counting the cells, and Foxp3 expression levels were determined by flow cytometry. All data are presented as means ± SEM ( n = 5), and ∗ P

    Journal: Journal of Immunology Research

    Article Title: Adoptive Cell Therapy of Induced Regulatory T Cells Expanded by Tolerogenic Dendritic Cells on Murine Autoimmune Arthritis

    doi: 10.1155/2017/7573154

    Figure Lengend Snippet: iTregs expanded in vitro by mtDCs retained Foxp3 expression and efficiently inhibited CD4 + T cell proliferation. (a) Schematic overview of the induction/expansion strategy for iTregs. The two expansion cycles were established as described in the Materials and Methods. The differentiation of iTregs was first induced with an anti-CD3/CD28 mAb, TGF- β , and IL-2 for 5 days in vitro. Then, iTreg mtDC or iTreg mDC were generated by expanding iTregs for 4 days using mtDCs or mDCs, respectively. The expression of the transcription factor Foxp3 and CD25 in cells was determined in each cycle using flow cytometry. Data are representative of three independent experiments. (b) Expansion of different types of iTregs was determined by counting the cells, and Foxp3 expression levels were determined by flow cytometry. All data are presented as means ± SEM ( n = 5), and ∗ P

    Article Snippet: Ex Vivo Generation of iTregmtDC iTregs were derived from CD4+ CD25− T cells that were purified from the splenocytes of D1 mice using a CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Germany) and stimulated with an anti-CD3/CD28 mAb (50 ng/ml, PeproTech) in the presence of TGF-β 1 (5 ng/ml, PeproTech) and IL-2 (100 ng/ml, PeproTech) for 5 days [ ].

    Techniques: In Vitro, Expressing, Generated, Flow Cytometry, Cytometry

    Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation. Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and anti-CD4-FITC mAb. Lymphocytes were isolated and stained ex-vivo for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8 + T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8 + T-cells both within the tissue and in the vasculature. C. Contour plots represent proliferation of CD4 + T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8 + T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4 + T-cells with and without DTx treatment is shown. **p

    Journal: PLoS ONE

    Article Title: Tregs Modulate Lymphocyte Proliferation, Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection

    doi: 10.1371/journal.pone.0145457

    Figure Lengend Snippet: Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation. Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and anti-CD4-FITC mAb. Lymphocytes were isolated and stained ex-vivo for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8 + T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8 + T-cells both within the tissue and in the vasculature. C. Contour plots represent proliferation of CD4 + T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8 + T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4 + T-cells with and without DTx treatment is shown. **p

    Article Snippet: For the procedure, 3 μg of anti–CD8 α -PE (clone 53–6.7 from eBioscience) and anti–CD4-FITC (clone RM4-4 from eBioscience) in 300 μl of DPBS per mouse were used for intravenous injection.

    Techniques: Infection, Mouse Assay, Injection, Isolation, Staining, Ex Vivo, Clone Assay

    Increased proliferation of CD4 + T-lymphocytes in the infected brains following Treg depletion. (A) Brain tissue from MCMV-infected, Foxp3-DTR transgenic mice was collected at 7, 14, and 30 dpi. Brain-infiltrating leukocytes were isolated and stained for flow cytometry with PE-Cy5-conjugated Abs specific for CD45, PE-Cy7-labeled for CD4-e-F 450, and Ki67 FITC–conjugated Abs. Isotype Abs for Ki67 FITC were used as gating control. Contour plots showing the proliferation frequency of CD4 + T-cells from infected, untreated (-DTx), and DTx-treated (+DTx) animals at the indicated time points are representative of 2 independent experiments. (B) Pooled data show percentage proliferation of CD4 + T-cells (mean ±SD) in infected brains with and without treatment at the indicated time points. ***p

    Journal: PLoS ONE

    Article Title: Tregs Modulate Lymphocyte Proliferation, Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection

    doi: 10.1371/journal.pone.0145457

    Figure Lengend Snippet: Increased proliferation of CD4 + T-lymphocytes in the infected brains following Treg depletion. (A) Brain tissue from MCMV-infected, Foxp3-DTR transgenic mice was collected at 7, 14, and 30 dpi. Brain-infiltrating leukocytes were isolated and stained for flow cytometry with PE-Cy5-conjugated Abs specific for CD45, PE-Cy7-labeled for CD4-e-F 450, and Ki67 FITC–conjugated Abs. Isotype Abs for Ki67 FITC were used as gating control. Contour plots showing the proliferation frequency of CD4 + T-cells from infected, untreated (-DTx), and DTx-treated (+DTx) animals at the indicated time points are representative of 2 independent experiments. (B) Pooled data show percentage proliferation of CD4 + T-cells (mean ±SD) in infected brains with and without treatment at the indicated time points. ***p

    Article Snippet: For the procedure, 3 μg of anti–CD8 α -PE (clone 53–6.7 from eBioscience) and anti–CD4-FITC (clone RM4-4 from eBioscience) in 300 μl of DPBS per mouse were used for intravenous injection.

    Techniques: Infection, Transgenic Assay, Mouse Assay, Isolation, Staining, Flow Cytometry, Cytometry, Labeling

    CCR9 low dendritic cells (DC) are potent stimulators of naïve T cells. (a) DC purified by magnetic antibody cell sorting (MACS) were sorted by flow cytometry into CCR9 low and CCR9 high fractions. Allogeneic C57BL/6J-purified spleen T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and added to 96-well flat-bottomed plates at a fixed number (2 × 10 5 cells/well). Sorted DC fractions were added to T cells at 1 × 10 5 cells /well or at 5 × 10 4 cells/well. Cultures were harvested 4 days later and labeled with CD4 peridinin chlorophyll protein (PerCP) (FL3). Dead cells were gated out and > 25 000 events were collected on a flow cytometer. Histograms show CD4-stained T cells that were labeled for CFSE at a DC : T-cell ratio of 1:2, and are representative of three separate experiments. (b) Unfractionated DC and CCR9 low DC were used to stimulate T cells, as described above, at a DC : T-cell ratio of 1:2. CCR9 low DC were almost as potent stimulators of T cells as unfractionated DC.

    Journal: Immunology

    Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

    doi: 10.1111/j.1365-2567.2009.03043.x

    Figure Lengend Snippet: CCR9 low dendritic cells (DC) are potent stimulators of naïve T cells. (a) DC purified by magnetic antibody cell sorting (MACS) were sorted by flow cytometry into CCR9 low and CCR9 high fractions. Allogeneic C57BL/6J-purified spleen T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and added to 96-well flat-bottomed plates at a fixed number (2 × 10 5 cells/well). Sorted DC fractions were added to T cells at 1 × 10 5 cells /well or at 5 × 10 4 cells/well. Cultures were harvested 4 days later and labeled with CD4 peridinin chlorophyll protein (PerCP) (FL3). Dead cells were gated out and > 25 000 events were collected on a flow cytometer. Histograms show CD4-stained T cells that were labeled for CFSE at a DC : T-cell ratio of 1:2, and are representative of three separate experiments. (b) Unfractionated DC and CCR9 low DC were used to stimulate T cells, as described above, at a DC : T-cell ratio of 1:2. CCR9 low DC were almost as potent stimulators of T cells as unfractionated DC.

    Article Snippet: Briefly, BALB/c spleen T cells were dual labeled with CD4 (PE; L3T4 GK.1.5; PharMingen) and CD45RB (FITC; C363.16A; PharMingen) mAbs, then sorted on a BD FACS Aria (Becton Dickinson, San Jose, CA), collecting the 35% most double-positive CD4− CD45RBhigh T cells.

    Techniques: Purification, FACS, Magnetic Cell Separation, Flow Cytometry, Cytometry, Labeling, Staining

    Immunohistochemical staining of liver specimens with liver injury resulting from osimertinib immediately after nivolumab therapy. In Case 4, CD3 and CD8 lymphocytes were predominantly expressed in the liver tissues compared to CD4 and CD20 lymphocytes.

    Journal: Thoracic Cancer

    Article Title: Severe hepatotoxicity due to osimertinib after nivolumab therapy in patients with non‐small cell lung cancer harboring EGFR mutation

    doi: 10.1111/1759-7714.13363

    Figure Lengend Snippet: Immunohistochemical staining of liver specimens with liver injury resulting from osimertinib immediately after nivolumab therapy. In Case 4, CD3 and CD8 lymphocytes were predominantly expressed in the liver tissues compared to CD4 and CD20 lymphocytes.

    Article Snippet: Immunohistochemical staining Immunohistochemical staining was performed to detect CD4‐ (1:200 dilution; Dako, Tokyo, Japan), CD8‐ (1:1000 dilution; Abcam, Tokyo, Japan), CD3‐ (1:200 dilution; Abcam), and CD20‐positive (1:200 dilution; Abcam) cells in the liver specimens.

    Techniques: Immunohistochemistry, Staining

    Differences in Th17 cell frequencies in the LP of IL-15 Tg or KO mice, but not in frequencies of Th1 or Treg cells. Cells from spleen and small intestine LP were stimulated for four hours with PMA and ionomycin and stained with surface markers CD3 and CD4, followed by intracellular staining of IL-17, IFNγ or Foxp3. A. Higher frequencies of Th17 cells were found in the LP of IL-15 KO mice than matched co-caged WT mice (p

    Journal: PLoS ONE

    Article Title: Interleukin-15 Constrains Mucosal T Helper 17 Cell Generation: Influence of Mononuclear Phagocytes

    doi: 10.1371/journal.pone.0143001

    Figure Lengend Snippet: Differences in Th17 cell frequencies in the LP of IL-15 Tg or KO mice, but not in frequencies of Th1 or Treg cells. Cells from spleen and small intestine LP were stimulated for four hours with PMA and ionomycin and stained with surface markers CD3 and CD4, followed by intracellular staining of IL-17, IFNγ or Foxp3. A. Higher frequencies of Th17 cells were found in the LP of IL-15 KO mice than matched co-caged WT mice (p

    Article Snippet: Intracellular staining and cell surface staining After cell surface staining with anti-CD4 and anti-CD3, cells were permeabilized with Cytofix/Cytoperm (PharMingen, San Diego, CA) in accordance with the manufacturer’s recommendations.

    Techniques: Mouse Assay, Staining

    Removal of CD4+ T cells abrogates the effects of CTLA-4 blockade on HIV-specific CD8+ T Cells. PBMC (or CD4 negative PBMC) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, IL-10 APC, anti-CD3 Am Cyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. Representative plots of (A) Gag and (B) Nef-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. Data plots shown are representative of three volunteers examined in three independent experiments yielding similar results.

    Journal: PLoS ONE

    Article Title: TGF-? and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

    doi: 10.1371/journal.pone.0008194

    Figure Lengend Snippet: Removal of CD4+ T cells abrogates the effects of CTLA-4 blockade on HIV-specific CD8+ T Cells. PBMC (or CD4 negative PBMC) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, IL-10 APC, anti-CD3 Am Cyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. Representative plots of (A) Gag and (B) Nef-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. Data plots shown are representative of three volunteers examined in three independent experiments yielding similar results.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD27 FITC, CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, CD3 AmCyan (BD Pharmigen).

    Techniques: Staining, Flow Cytometry, Expressing

    CTLA-4 blockade decreases TGF-β and IL-10 expression by HIV-specific CD8+ T Cells. PBMC (n = 6) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. (A) Representative plots of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. (B-D) Dashed line represents the cutoff for significant TGF-β (B), IL-10 (C), and IFN-γ (D) expression. Percentages in between brackets are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t- test.

    Journal: PLoS ONE

    Article Title: TGF-? and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

    doi: 10.1371/journal.pone.0008194

    Figure Lengend Snippet: CTLA-4 blockade decreases TGF-β and IL-10 expression by HIV-specific CD8+ T Cells. PBMC (n = 6) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. (A) Representative plots of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. (B-D) Dashed line represents the cutoff for significant TGF-β (B), IL-10 (C), and IFN-γ (D) expression. Percentages in between brackets are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t- test.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD27 FITC, CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, CD3 AmCyan (BD Pharmigen).

    Techniques: Expressing, Staining, Flow Cytometry

    HIV-specific TGF-β and IL-10 positive CD8+ T cells are CTLA-4 negative. PBMC were stimulated with HIV peptides then stained with anti-TGF-β PE (or IL-10 PE), anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 PE Cy7, anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4 positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. (A) Representative plots of samples that were first gated on the CD3+/CD4+ and CD3+/CD8+ lymphocyte population and then the percentages of CTLA-4 positive cells were determined. (B) Representative plots of samples that were first gated on the CD3+/CD8+ lymphocyte population and then the percent of TGF-β and IL-10 positive cells that express CTLA-4 was determined after subtraction of the back ground values. The values marked with an asterisk represent the fraction of TGF-β (or IL-10) positive cells that express CTLA-4 over the total number of TGF-β (or IL-10) positive cells (equivalent to 100%). Plots are from three independent experiments yielding similar results.

    Journal: PLoS ONE

    Article Title: TGF-? and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

    doi: 10.1371/journal.pone.0008194

    Figure Lengend Snippet: HIV-specific TGF-β and IL-10 positive CD8+ T cells are CTLA-4 negative. PBMC were stimulated with HIV peptides then stained with anti-TGF-β PE (or IL-10 PE), anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 PE Cy7, anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4 positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. (A) Representative plots of samples that were first gated on the CD3+/CD4+ and CD3+/CD8+ lymphocyte population and then the percentages of CTLA-4 positive cells were determined. (B) Representative plots of samples that were first gated on the CD3+/CD8+ lymphocyte population and then the percent of TGF-β and IL-10 positive cells that express CTLA-4 was determined after subtraction of the back ground values. The values marked with an asterisk represent the fraction of TGF-β (or IL-10) positive cells that express CTLA-4 over the total number of TGF-β (or IL-10) positive cells (equivalent to 100%). Plots are from three independent experiments yielding similar results.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD27 FITC, CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, CD3 AmCyan (BD Pharmigen).

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence

    TGF-β positive, IL-10 positive, and IFN-γ positive HIV-specific CD8+ T cell populations are distinct. PBMC were stimulated with HIV peptides then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, and anti-CD8 PE Cy7, anti-IL-10 APC, and analyzed by flow cytometry. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IFN-γ, and IL-10 positive CD8+ T cells were determined. (A) Data from individuals with significant cytokine expression and analysis were performed by Mann-Whitney U test. (B) Representative plots of the number of HIV-specific CD8+ T cells expressing TGF-β, IFN-γ, and IL-10 after subtraction of the back ground values.

    Journal: PLoS ONE

    Article Title: TGF-? and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

    doi: 10.1371/journal.pone.0008194

    Figure Lengend Snippet: TGF-β positive, IL-10 positive, and IFN-γ positive HIV-specific CD8+ T cell populations are distinct. PBMC were stimulated with HIV peptides then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, and anti-CD8 PE Cy7, anti-IL-10 APC, and analyzed by flow cytometry. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IFN-γ, and IL-10 positive CD8+ T cells were determined. (A) Data from individuals with significant cytokine expression and analysis were performed by Mann-Whitney U test. (B) Representative plots of the number of HIV-specific CD8+ T cells expressing TGF-β, IFN-γ, and IL-10 after subtraction of the back ground values.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD27 FITC, CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, CD3 AmCyan (BD Pharmigen).

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY