human recombinant il-2 Search Results


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  • 99
    Thermo Fisher recombinant human il 2
    Tregs can modulate <t>IL-2</t> mediated inter-clonal co-optation of weak T cell clones
    Recombinant Human Il 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore recombinant human il 2
    Impaired B cell differentiation in XLP is associated with reduced expression of ICOS by CD4 + T cells. PBMCs from a healthy donor and XLP no. 1 were unstimulated ( A ) or cultured with PHA and <t>IL-2</t> (stimulated; B ). After 24 hours, the cells were
    Recombinant Human Il 2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 578 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PeproTech recombinant human il 2
    CFSE can be used to obtain proliferative history and trace cells of interest in complex cultures by mass cytometry. (a) A strategy for adapting CFSE dye dilution assay to mass cytometry. Since both CFSE and FITC are derivatives of fluorescein, CFSE can be quantified by mass cytometry using intracellular staining with an anti-FITC antibody conjugated to a reporter metal isotope. With each division, daughter cells inherit ~50% of CFSE, providing a proxy for estimating the number of cell divisions (proliferative history). (b) Mass cytometry titration of a polyclonal anti-FITC- 172 Yb antibody on human CD8 + T cells, with the optimal concentration highlighted (red box). (c) Equivalent CFSE signal obtained from human CD8 + T cells analyzed in parallel by flow cytometry and mass cytometry, with the near-zero anti-FITC- 172 Yb antibody background highlighted (red box). (d) Experimental outline for tracing proliferative history of naïve CD8 + T cells in REP as a model system. CFSE-labeled naïve human T lymphocytes are induced to proliferate by CFSE-negative accessory cells, including monocytes (Mo), that present an anti-CD3ε antibody via Fcγ receptors (FcγRs) and express co-stimulatory molecules. <t>Interleukin-2</t> <t>(IL-2)</t> is added after 48 hours. (e) Proliferative history of CD8 + T cells was similar when measured directly by flow cytometry, or indirectly using a 172 Yb-labeled anti-FITC antibody by mass cytometry. A division ID (red arrows) was assigned to each cell falling into the ≥80% confidence region (blue), or division ID: −1 otherwise. Division IDs were added to the original file, enabling downstream analysis in software of choice, such as Cytobank . (f) Spearman correlation analysis comparing percentage of cells falling into each division state in samples analyzed in parallel by flow cytometry and mass cytometry (n = 26 time-series samples from 6 REP cultures). (g) CFSE signal reduction per division was calculated based on geometric means from cells in (f). Boxplots show quartiles with a band at median, whiskers indicating 1.5 interquartile range (IQR), and outliers shown separately. A red dashed line indicates the expected 50% reduction. The antibody was titrated once; results in (c,e-g) are from 1 experiment representative of 3 experiments. Results in (b-c) and (e-g) were obtained using antibody panels in Supplementary Data 1 and Supplementary Data 2 , respectively.
    Recombinant Human Il 2, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 2552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human interleukin 2
    ( A ) The cytotoxicity of T cells was evaluated by the LDH assay at different ( E ) T ratios. ( C ) Human Th1/Th2 Cytokine Kit determined the expression level of <t>IL-2,</t> IL-4, IL-6, TNF-α, and IFN-γ of DMSO or YN968D1-treated T cells. ( B, D ) The frequency of IFN-γ-producing T cells after co-incubation with gastric cancer cell MKN-45 for 18 hrs was evaluated by IFN-γ ELISPOT kit (Dakewei, China). T cells and MKN-45 were co-incubated at different E: T ratios (10:1, 5:1, 3:1). * means P value
    Recombinant Human Interleukin 2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novartis recombinant human il 2
    Experimental layout and expansion of PBMC in cultures supplemented with TG . Panel A : PBMC from consented healthy donors were initially exposed to IFN-γ (day 0), followed by different concentrations of either TG or αCD3 mAb (day +1) and <t>IL-2</t> every 3 days. Further details are provided in Materials and Methods. Panel B : The frequency of CD3 + CD8 + T cells, NK cells (CD3 - CD16 + CD56 + ) and CD3 + CD56 + T cells from a representative PBMC sample at baseline is shown. Quadrant markers were set according to the proper isotypic control (not shown). The percentage of cells staining positively for a given antigen is indicated. Panel C : Cells were harvested weekly and counted. The number of cells was significantly higher after challenging with TG either at 250 ( int TG; *p
    Recombinant Human Il 2, supplied by Novartis, used in various techniques. Bioz Stars score: 93/100, based on 771 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    R&D Systems human il 2
    Calcitriol response of TAGAP and IL2RA in CD4+ T cells is not influenced by MS or MS-risk genotype. CD4+ T cells from 8 RRMS patients (MS) and 10 healthy controls (HC) genotyped for MS-risk SNPs in TAGAP (rs1738074) and IL2RA (rs7090512) were cultivated for 7–8 days with αCD3/CD28 and <t>IL-2,</t> treated as described in Materials and Methods before harvesting after 6, 24 and 48 h, with 1,25(OH) 2 D 3 or vehicle control. The box plots with whiskers defining minimum and maximum show the log2 values of relative expression of indicted genes (relative to TBP ) in 1,25(OH) 2 D 3 -treated cells divided by relative expression in vehicle-treated cells, that is, log2 of fold induction by 1,25(OH) 2 D 3 at each time point for ( a ) MS patients compared with HCs and ( b ) the same samples sorted on IL2RA and TAGAP genotype, that is, samples carrying the minor allele ( IL2RA : N =10, MAF (G)=0.3480; TAGAP : N =11, MAF (G)=0.46563) compared with samples homozygous for major allele ( N =8 ( IL2RA ); N =7 ( TAGAP )). The horizontal lines within the boxes represent the median of the groups, and Mann–Whitney U -test was performed to compare the groups (MS vs HC, AA vs AG/GG). The horizontal line at 0 indicates no induction by 1,25(OH) 2 D 3 .
    Human Il 2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson recombinant human il 2
    Antigen-experienced “CD4lo” T cells give rise to “CD4hi” T cells. “CD4lo” T cells were enriched from the Con A blasts of NOD spleen cells as described in Figure 6 . The “CD4lo” T cells or Con A blasts were cultured overnight in <t>IL-2-containing</t> complete medium, then each was labeled with CFSE before incubation on anti-CD3-coated plates. Twenty-four and 48 hrs later the cells were labeled with anti-CD4-PE and analyzed by flow cytometry to reveal cell divisions. Proliferating “CD4hi” T cells arising from the blasts are indicated in the encircled population the numbers indicate the percentage in the total cell population. For each analysis, 100,000 events were collected, and the results are representative of four experiments.
    Recombinant Human Il 2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher human il 2
    IFNβ affects chromatin remodeling of the <t>IL-2</t> promoter through histone deacetylase activity and CREM
    Human Il 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Chiron Corporation recombinant human il 2
    Induction of high output NO synthesis in Meth A ascites tumor cells by <t>IL-2</t> therapy. Meth A ascites cells harvested from three IL-2 treated mice were cultured at 1.5×10 5 cells/well in microtiter plates in triplicate. After a 48h culture, nitrite was measured in 50 μ l culture supernatants by a colorimetric assay. Results are mean±SD. Meth A ascites cells harvested from three untreated mice served as controls.
    Recombinant Human Il 2, supplied by Chiron Corporation, used in various techniques. Bioz Stars score: 93/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Shionogi recombinant human il 2
    Macroscopic and microscopic appearance of a TARS-1 tumor. (a) Subcutaneous tumor at the injected site on day 14 following TARS-1 inoculation in an anti-CD80/CD86 MAb-treated rat (right) but not an untreated rat (left). (b) Regression of the tumor on day 35 in a rat treated with MAbs only during the initial 14 days (right). The TARS-1-inoculated MAb-untreated rat never developed a tumor (left). (c) Frozen section of subcutaneous tumor of a MAb-treated TARS-1-inoculated rat sacrificed on day 14, stained with MAb to <t>IL-2</t> receptor (brown) and hematoxylin (blue). (d) Hematoxylin-and-eosin-stained frozen section of the lung of the rat shown in panel c.
    Recombinant Human Il 2, supplied by Shionogi, used in various techniques. Bioz Stars score: 92/100, based on 168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher human recombinant il 2
    Interleukin-15 (IL)-15 + alendronate (ALN) or <t>IL-2</t> + ALN select for Jγ1.2 + chains, cytokine alone increases the proportion of Vγ2-Jγ1.2/2.3 + chains. (a) Vγ2 chain length distributions were determined by spectratyping for
    Human Recombinant Il 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioLegend recombinant human il 2
    PD-L1-dependent suppression of T cell proliferation and cytokine production by PD-L1 expressing B cells from HC and untreated RA patients. CD19 + B cells, CD8 + and CD4 + T cells were magnetic bead-purified from PBMC of HC and untreated RA patients. CD19 + B cells were incubated with <t>CpG+IL-2</t> for 72 h and then were washed and cultured 2:1 with autologous CFSE-labeled CD8 + or CD4 + T cells incubated in a plate-bound anti-CD3/anti-CD28 mAb in the presence of anti-PD-L1 mAb or control isotype. After 72 h of co-culture, T cell proliferation and intracellular cytokine production were analyzed by flow cytometry on CD3 + CD19 − lived cells. Percentage of proliferation, TNF and IFN-g production in (A) CD8 + T cells and (B) CD4 + T cells cultured alone or in the presence of CpG-activated CD19 + B cells with anti-PD-L1 mAb or control isotype. Mean ± SEM of five independent experiments (5 healthy controls and 5 untreated RA patients). * p
    Recombinant Human Il 2, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim recombinant human il 2
    B. bigemina RAP-1 CT-1-specific T cells respond to aa 418 to 480. Three CT-1-specific Th cell clones (A to C) and one RAP-1-specific Th cell clone specific for an epitope within aa 144 to 187 (D) were cultured in a 3-day proliferation assay with autologous APC, 2 U of recombinant human <t>IL-2</t> per ml, and 1, 5, or 25 μg of recombinant MBP fusion proteins per ml, consisting of the whole RAP-1 protein (closed circles), the N terminal fragment of CT-1 (CT-1N) consisting of aa 386 to 448 (closed triangles), the C-terminal fragment of CT-1 (CT-1C) consisting of aa 418 to 480 (open triangles), or control MBP protein (open circles). Cells were radiolabeled for 6 h with [ 3 H]thymidine, harvested, and counted. The results are presented as the mean ± range of variation around the mean of duplicate cultures. Background proliferative responses of cells in medium plus IL-2 for the clones were as follows: 2216.2B2, 15,305 ± 639 cpm; 2216.1G8, 12,652 ± 2,376 cpm; 2234.1E3, 12,635 ± 775 cpm; and 2216.1H4, 16,353 ± 3,758 cpm.
    Recombinant Human Il 2, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Tregs can modulate IL-2 mediated inter-clonal co-optation of weak T cell clones

    Journal: Cell reports

    Article Title: “T cells integrate Local and Global cues to discriminate between structurally similar antigens”

    doi: 10.1016/j.celrep.2015.04.051

    Figure Lengend Snippet: Tregs can modulate IL-2 mediated inter-clonal co-optation of weak T cell clones

    Article Snippet: Recombinant human IL-2 and mouse IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-21 and IFN-γ were obtained from eBioscience (San Diego, CA, USA).

    Techniques: Clone Assay

    The inter-clonal co-optation of weakly activated CD8+ T cells is dependent on IL-2

    Journal: Cell reports

    Article Title: “T cells integrate Local and Global cues to discriminate between structurally similar antigens”

    doi: 10.1016/j.celrep.2015.04.051

    Figure Lengend Snippet: The inter-clonal co-optation of weakly activated CD8+ T cells is dependent on IL-2

    Article Snippet: Recombinant human IL-2 and mouse IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-21 and IFN-γ were obtained from eBioscience (San Diego, CA, USA).

    Techniques:

    IL-2 modulation of cell cycle entry relies on PI3K activation and is independent of MAPK activity

    Journal: Cell reports

    Article Title: “T cells integrate Local and Global cues to discriminate between structurally similar antigens”

    doi: 10.1016/j.celrep.2015.04.051

    Figure Lengend Snippet: IL-2 modulation of cell cycle entry relies on PI3K activation and is independent of MAPK activity

    Article Snippet: Recombinant human IL-2 and mouse IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-21 and IFN-γ were obtained from eBioscience (San Diego, CA, USA).

    Techniques: Activation Assay, Activity Assay

    The model for CD8+ T cell activation recapitulates IL-2 modulation of cell cycle entry

    Journal: Cell reports

    Article Title: “T cells integrate Local and Global cues to discriminate between structurally similar antigens”

    doi: 10.1016/j.celrep.2015.04.051

    Figure Lengend Snippet: The model for CD8+ T cell activation recapitulates IL-2 modulation of cell cycle entry

    Article Snippet: Recombinant human IL-2 and mouse IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-21 and IFN-γ were obtained from eBioscience (San Diego, CA, USA).

    Techniques: Activation Assay

    General strategy for the design of the reporter cell line: [a] signal through most of the immunologically relevant receptors results in the activation of NF-κB 23 , 30 , 39 , 40 [b] the reporter construct encoding a secreted luciferase (NanoLuc) and mCherry separated by a T2A sequence downstream of an NF-κB response element (NF-κB-RE) was used for the viral transduction of cell lines of T and B cell origin (TCR: T cell receptor, TNFR: TNF receptor, IL4 R: IL-4 receptor, IL2 R: IL-2 receptor, BCR: B cell receptor, IL12 R: IL-12 receptor, TLR: Toll like receptor)

    Journal: bioRxiv

    Article Title: A universal reporter cell line for bioactivity evaluation of engineered cytokine products

    doi: 10.1101/739987

    Figure Lengend Snippet: General strategy for the design of the reporter cell line: [a] signal through most of the immunologically relevant receptors results in the activation of NF-κB 23 , 30 , 39 , 40 [b] the reporter construct encoding a secreted luciferase (NanoLuc) and mCherry separated by a T2A sequence downstream of an NF-κB response element (NF-κB-RE) was used for the viral transduction of cell lines of T and B cell origin (TCR: T cell receptor, TNFR: TNF receptor, IL4 R: IL-4 receptor, IL2 R: IL-2 receptor, BCR: B cell receptor, IL12 R: IL-12 receptor, TLR: Toll like receptor)

    Article Snippet: The NK-92 cells were obtained from DSMZ (ACC 488) and grown in in MEM Alpha medium (Gibco, #22571-020) supplemented with 5 ng/mL recombinant human interleukin 2 (Gibco, #PHC0027), 2 mM L-glutamine (Lonza, #17-605E), 12.5% Fetal Bovine Serum (Gibco, #10099-141) and 12.5% Horse serum (Sigma Aldrich, #H1270).

    Techniques: Activation Assay, Construct, Luciferase, Sequencing, Transduction

    Comparison of the expression of luciferase and mCherry by the newly generated reporter cell lines with the response obtained with the same proteins in the conventional assay. The immunocytokines that were used for this assay are schematically depicted (the antibody moiety is depicted as empty symbols and the cytokine moieties as hatched areas) [a] the proliferation of CTLL-2 and the response of the CTLL-2 reporter cell line triggered by L19-IL2 [b] the production of interferon-γ by NK-92 cells and the response of the A20 reporter cell line triggered by L19-IL12 [c] the cytotoxicity of F8-TNF for L-M fibroblasts compared to the response of the CTLL-2 reporter cell line to F8-TNF. In the cases where a strong hook effect was observed, only the sigmoidal part was used for curve fitting as indicated by the solid line.

    Journal: bioRxiv

    Article Title: A universal reporter cell line for bioactivity evaluation of engineered cytokine products

    doi: 10.1101/739987

    Figure Lengend Snippet: Comparison of the expression of luciferase and mCherry by the newly generated reporter cell lines with the response obtained with the same proteins in the conventional assay. The immunocytokines that were used for this assay are schematically depicted (the antibody moiety is depicted as empty symbols and the cytokine moieties as hatched areas) [a] the proliferation of CTLL-2 and the response of the CTLL-2 reporter cell line triggered by L19-IL2 [b] the production of interferon-γ by NK-92 cells and the response of the A20 reporter cell line triggered by L19-IL12 [c] the cytotoxicity of F8-TNF for L-M fibroblasts compared to the response of the CTLL-2 reporter cell line to F8-TNF. In the cases where a strong hook effect was observed, only the sigmoidal part was used for curve fitting as indicated by the solid line.

    Article Snippet: The NK-92 cells were obtained from DSMZ (ACC 488) and grown in in MEM Alpha medium (Gibco, #22571-020) supplemented with 5 ng/mL recombinant human interleukin 2 (Gibco, #PHC0027), 2 mM L-glutamine (Lonza, #17-605E), 12.5% Fetal Bovine Serum (Gibco, #10099-141) and 12.5% Horse serum (Sigma Aldrich, #H1270).

    Techniques: Expressing, Luciferase, Generated

    Impaired B cell differentiation in XLP is associated with reduced expression of ICOS by CD4 + T cells. PBMCs from a healthy donor and XLP no. 1 were unstimulated ( A ) or cultured with PHA and IL-2 (stimulated; B ). After 24 hours, the cells were

    Journal:

    Article Title: Impaired humoral immunity in X-linked lymphoproliferative disease is associated with defective IL-10 production by CD4+ T cells

    doi: 10.1172/JCI200523139

    Figure Lengend Snippet: Impaired B cell differentiation in XLP is associated with reduced expression of ICOS by CD4 + T cells. PBMCs from a healthy donor and XLP no. 1 were unstimulated ( A ) or cultured with PHA and IL-2 (stimulated; B ). After 24 hours, the cells were

    Article Snippet: Recombinant human IL-2 (rIL-2) was purchased from Chemicon; anti-CD3 mAb (Spv-T3b) was provided by Hergen Spits (Netherlands Cancer Institute, Amsterdam, The Netherlands) , human rIL-4, rIL-10, and neutralizing anti–IL-10 mAb were provided by Rene de Waal Malefyt (DNAX Research Institute, Palo Alto, California, USA).

    Techniques: Cell Differentiation, Expressing, Cell Culture

    Primary mink cells as well as the mink Mv.1.Lu cell line support all postentry steps in the HIV-1 replication cycle. (A) ConA-IL-2-activated mink splenocytes, mink fibroblasts, and the Mv.1.Lu cell line were inoculated with 15 ng of the p24 equivalent NL4-3 Luc E − R − reporter viruses pseudotyped with VSV-G envelope glycoprotein, and luciferase activity in the infected cells were measured at 3 days postinfection (in relative light units) as a marker of early HIV-1 gene expression. The data are representative of those from three independent experiments. (B) The cells were also infected with VSV-G-pseudotyped R7/3/162P3. The p24 antigen content in culture supernatants was determined at 3 days postinfection as a marker for expression and egress of a late, fully processed HIV-1 gene product. PHA-P-IL-2-activated human PBMC, HeLa cells, and HOS cells and ConA-IL-2-activated primary mouse splenocytes and NIH 3T3 cells served as control. Bars represent mean values from triplicate sample, with error bars representing standard deviations.

    Journal: Journal of Virology

    Article Title: Susceptibility of Mink (Mustera vision)-Derived Cells to Replication by Human Immunodeficiency Virus Type 1

    doi: 10.1128/JVI.77.9.5109-5117.2003

    Figure Lengend Snippet: Primary mink cells as well as the mink Mv.1.Lu cell line support all postentry steps in the HIV-1 replication cycle. (A) ConA-IL-2-activated mink splenocytes, mink fibroblasts, and the Mv.1.Lu cell line were inoculated with 15 ng of the p24 equivalent NL4-3 Luc E − R − reporter viruses pseudotyped with VSV-G envelope glycoprotein, and luciferase activity in the infected cells were measured at 3 days postinfection (in relative light units) as a marker of early HIV-1 gene expression. The data are representative of those from three independent experiments. (B) The cells were also infected with VSV-G-pseudotyped R7/3/162P3. The p24 antigen content in culture supernatants was determined at 3 days postinfection as a marker for expression and egress of a late, fully processed HIV-1 gene product. PHA-P-IL-2-activated human PBMC, HeLa cells, and HOS cells and ConA-IL-2-activated primary mouse splenocytes and NIH 3T3 cells served as control. Bars represent mean values from triplicate sample, with error bars representing standard deviations.

    Article Snippet: Human peripheral blood mononuclear cells (PBMC) from healthy donors were prepared by using Ficoll-Paque (Ficoll-Paque PLUS; Amersham Pharmacia Biotech AB, Uppsala, Sweden) density centrifugation and then cultured in RPMI1640 containing 10% FCS and 40 IU of human recombinant IL-2 per ml after activation with 3 μg of phytohemagglutinin (PHA) P (Sigma) per ml for 2 days.

    Techniques: Luciferase, Activity Assay, Infection, Marker, Expressing

    CFSE can be used to obtain proliferative history and trace cells of interest in complex cultures by mass cytometry. (a) A strategy for adapting CFSE dye dilution assay to mass cytometry. Since both CFSE and FITC are derivatives of fluorescein, CFSE can be quantified by mass cytometry using intracellular staining with an anti-FITC antibody conjugated to a reporter metal isotope. With each division, daughter cells inherit ~50% of CFSE, providing a proxy for estimating the number of cell divisions (proliferative history). (b) Mass cytometry titration of a polyclonal anti-FITC- 172 Yb antibody on human CD8 + T cells, with the optimal concentration highlighted (red box). (c) Equivalent CFSE signal obtained from human CD8 + T cells analyzed in parallel by flow cytometry and mass cytometry, with the near-zero anti-FITC- 172 Yb antibody background highlighted (red box). (d) Experimental outline for tracing proliferative history of naïve CD8 + T cells in REP as a model system. CFSE-labeled naïve human T lymphocytes are induced to proliferate by CFSE-negative accessory cells, including monocytes (Mo), that present an anti-CD3ε antibody via Fcγ receptors (FcγRs) and express co-stimulatory molecules. Interleukin-2 (IL-2) is added after 48 hours. (e) Proliferative history of CD8 + T cells was similar when measured directly by flow cytometry, or indirectly using a 172 Yb-labeled anti-FITC antibody by mass cytometry. A division ID (red arrows) was assigned to each cell falling into the ≥80% confidence region (blue), or division ID: −1 otherwise. Division IDs were added to the original file, enabling downstream analysis in software of choice, such as Cytobank . (f) Spearman correlation analysis comparing percentage of cells falling into each division state in samples analyzed in parallel by flow cytometry and mass cytometry (n = 26 time-series samples from 6 REP cultures). (g) CFSE signal reduction per division was calculated based on geometric means from cells in (f). Boxplots show quartiles with a band at median, whiskers indicating 1.5 interquartile range (IQR), and outliers shown separately. A red dashed line indicates the expected 50% reduction. The antibody was titrated once; results in (c,e-g) are from 1 experiment representative of 3 experiments. Results in (b-c) and (e-g) were obtained using antibody panels in Supplementary Data 1 and Supplementary Data 2 , respectively.

    Journal: Nature biotechnology

    Article Title: Proliferative tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells

    doi: 10.1038/s41587-019-0033-2

    Figure Lengend Snippet: CFSE can be used to obtain proliferative history and trace cells of interest in complex cultures by mass cytometry. (a) A strategy for adapting CFSE dye dilution assay to mass cytometry. Since both CFSE and FITC are derivatives of fluorescein, CFSE can be quantified by mass cytometry using intracellular staining with an anti-FITC antibody conjugated to a reporter metal isotope. With each division, daughter cells inherit ~50% of CFSE, providing a proxy for estimating the number of cell divisions (proliferative history). (b) Mass cytometry titration of a polyclonal anti-FITC- 172 Yb antibody on human CD8 + T cells, with the optimal concentration highlighted (red box). (c) Equivalent CFSE signal obtained from human CD8 + T cells analyzed in parallel by flow cytometry and mass cytometry, with the near-zero anti-FITC- 172 Yb antibody background highlighted (red box). (d) Experimental outline for tracing proliferative history of naïve CD8 + T cells in REP as a model system. CFSE-labeled naïve human T lymphocytes are induced to proliferate by CFSE-negative accessory cells, including monocytes (Mo), that present an anti-CD3ε antibody via Fcγ receptors (FcγRs) and express co-stimulatory molecules. Interleukin-2 (IL-2) is added after 48 hours. (e) Proliferative history of CD8 + T cells was similar when measured directly by flow cytometry, or indirectly using a 172 Yb-labeled anti-FITC antibody by mass cytometry. A division ID (red arrows) was assigned to each cell falling into the ≥80% confidence region (blue), or division ID: −1 otherwise. Division IDs were added to the original file, enabling downstream analysis in software of choice, such as Cytobank . (f) Spearman correlation analysis comparing percentage of cells falling into each division state in samples analyzed in parallel by flow cytometry and mass cytometry (n = 26 time-series samples from 6 REP cultures). (g) CFSE signal reduction per division was calculated based on geometric means from cells in (f). Boxplots show quartiles with a band at median, whiskers indicating 1.5 interquartile range (IQR), and outliers shown separately. A red dashed line indicates the expected 50% reduction. The antibody was titrated once; results in (c,e-g) are from 1 experiment representative of 3 experiments. Results in (b-c) and (e-g) were obtained using antibody panels in Supplementary Data 1 and Supplementary Data 2 , respectively.

    Article Snippet: Starting at 48 hours post-activation, cells were maintained at ~2×106 cells/mL in CCM containing 300 ng/mL anti-CD3ε antibody and 50 U/mL (5 ng/mL) recombinant human IL-2 (PeproTech #200-02, Rocky Hill, NJ, USA) and relevant concentrations of chemical inhibitors (ibrutinib: 700 ng/mL or 1.59 µM on days 0-2, and physiological concentrations , of 140 ng/mL or 318 nM on days 3-7; rapamycin: 50 ng/mL or 54.70 nM on days 0-2, and physiological concentrations , of 10 ng/mL or 10.94 nM on days 3-7).

    Techniques: Mass Cytometry, Dilution Assay, Staining, Titration, Concentration Assay, Flow Cytometry, Cytometry, Labeling, Software

    Serine leucocyte proteinase inhibitor (SLPI) inhibits human lymphocyte proliferation. Peripheral blood mononuclear cells (PBMC; 10 5 cells/well) were cultured for 5 days in the presence of well-coated OKT3 monoclonal antibody (mAb) (a, c) or 8 ng/ml interleukin-2

    Journal: Immunology

    Article Title: Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

    doi: 10.1111/j.1365-2567.2011.03451.x

    Figure Lengend Snippet: Serine leucocyte proteinase inhibitor (SLPI) inhibits human lymphocyte proliferation. Peripheral blood mononuclear cells (PBMC; 10 5 cells/well) were cultured for 5 days in the presence of well-coated OKT3 monoclonal antibody (mAb) (a, c) or 8 ng/ml interleukin-2

    Article Snippet: Human recombinant IL-2, IL-4, IL-6, IL-10 and interferon-γ (IFN-γ) were purchased from Peprotech (Rochy Hill, NJ).

    Techniques: Cell Culture

    Serine leucocyte proteinase inhibitor (SLPI) -treated monocyte culture supernatants (CS) inhibits human lymphocyte proliferation. (a) Peripheral blood mononuclear cells (PBMC; 10 5 cells/well) were cultured, for 5 days with or without 8 ng/ml of interleukin-2

    Journal: Immunology

    Article Title: Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

    doi: 10.1111/j.1365-2567.2011.03451.x

    Figure Lengend Snippet: Serine leucocyte proteinase inhibitor (SLPI) -treated monocyte culture supernatants (CS) inhibits human lymphocyte proliferation. (a) Peripheral blood mononuclear cells (PBMC; 10 5 cells/well) were cultured, for 5 days with or without 8 ng/ml of interleukin-2

    Article Snippet: Human recombinant IL-2, IL-4, IL-6, IL-10 and interferon-γ (IFN-γ) were purchased from Peprotech (Rochy Hill, NJ).

    Techniques: Cell Culture

    Co-culture of primary NK cells with K562 cells. A) Flow cytometry analysis of purity of isolated peripheral NK cells 24hrs after IL-2 stimulation. B) Flow cytometry analysis of activation status of CD3-CD56+ NK cells after 24hrs of IL-2 stimulation.

    Journal: bioRxiv

    Article Title: Systematic identification of cancer cell vulnerabilities to natural killer cell-mediated immune surveillance

    doi: 10.1101/597567

    Figure Lengend Snippet: Co-culture of primary NK cells with K562 cells. A) Flow cytometry analysis of purity of isolated peripheral NK cells 24hrs after IL-2 stimulation. B) Flow cytometry analysis of activation status of CD3-CD56+ NK cells after 24hrs of IL-2 stimulation.

    Article Snippet: NK-92 cells were grown in Myelocult H5100 (Stem cell Technologies) supplemented with 100U/ml human IL-2 (Peprotech cat#200-02).

    Techniques: Co-Culture Assay, Flow Cytometry, Isolation, Activation Assay

    High frequencies of DENV E-specific memory B cells in immune donors. ( A ) Frequencies of antigen-specific B cells were measured by ELISpot assay in triplicate in the PBMCs of DENV-immune donors after in vitro stimulation for 7 d with R848 and IL-2. The

    Journal: Viral Immunology

    Article Title: Analysis of Human Monoclonal Antibodies Generated by Dengue Virus-Specific Memory B Cells

    doi: 10.1089/vim.2012.0010

    Figure Lengend Snippet: High frequencies of DENV E-specific memory B cells in immune donors. ( A ) Frequencies of antigen-specific B cells were measured by ELISpot assay in triplicate in the PBMCs of DENV-immune donors after in vitro stimulation for 7 d with R848 and IL-2. The

    Article Snippet: PBMCs were stimulated with 2.5 μg/mL R848 (InvivoGen, San Diego, CA) and 1000 U/mL recombinant human (rh) IL-2 (Peprotech, Rocky Hill, NJ).

    Techniques: Enzyme-linked Immunospot, In Vitro

    PEG-IL2 is effective in expanding BiVax generated antigen-specific T cells. A, WT mice (3/group) were vaccinated with BiVax prime. Five days later the mice received boosters with BiVax, BiVax/IL2, BiVax/IL2Cx 122 or BiVax/PEG-IL2. Seven days later the numbers of Trp1 tetramer + T cells were evaluated in spleens. B, WT mice were inoculated s.c. with B16F10 cells and 7 days later, received a BiVax prime followed 5 days later by booster vaccines with BiVax, BiVax/IL2 or BiVax/PEG-IL2. Tumor size growth curves showing means with SD for each group. ( n = 10 mice/group). C , purified CD8 + T cells from WT mice receiving BiVax prime-boost (5 days apart) and treated or not with PEG-IL2 were incubated with either B16F10 (PD-L1 low) cells, IFNγ-treated (PD-L1 high) B16F10 cells (pulsed with 1 µg Trp1) with or without PD-L1 mAb (10 µg/ml) and the numbers of IFNγ-producing cells were evaluated by EliSpot. Representative results are shown from at least 3 independent experiments.

    Journal: Cancer immunology research

    Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

    doi: 10.1158/2326-6066.CIR-17-0549

    Figure Lengend Snippet: PEG-IL2 is effective in expanding BiVax generated antigen-specific T cells. A, WT mice (3/group) were vaccinated with BiVax prime. Five days later the mice received boosters with BiVax, BiVax/IL2, BiVax/IL2Cx 122 or BiVax/PEG-IL2. Seven days later the numbers of Trp1 tetramer + T cells were evaluated in spleens. B, WT mice were inoculated s.c. with B16F10 cells and 7 days later, received a BiVax prime followed 5 days later by booster vaccines with BiVax, BiVax/IL2 or BiVax/PEG-IL2. Tumor size growth curves showing means with SD for each group. ( n = 10 mice/group). C , purified CD8 + T cells from WT mice receiving BiVax prime-boost (5 days apart) and treated or not with PEG-IL2 were incubated with either B16F10 (PD-L1 low) cells, IFNγ-treated (PD-L1 high) B16F10 cells (pulsed with 1 µg Trp1) with or without PD-L1 mAb (10 µg/ml) and the numbers of IFNγ-producing cells were evaluated by EliSpot. Representative results are shown from at least 3 independent experiments.

    Article Snippet: The combination of recombinant human IL2 (hu-IL2) and anti-hu-IL2 (MAB602) (hu-IL2Cx) was as effective as the mouse IL2Cx122 (m-IL2Cx) in expanding antigen-specific cells ( ).

    Techniques: Generated, Mouse Assay, Purification, Incubation, Enzyme-linked Immunospot

    Sustained IL2 stimulation is responsible for the adjuvant effect of IL2Cx. A, TgTR1 cells were incubated with Trp1 peptide (1 µg/ml) and IL2Cx 122 or IL2Cx 25 (100 ng IL2/ml) in the presence of increasing concentrations of homologue IL2 mAb (JES6-5H4 or JES6-1A12, respectively). Seven days later T-cell expansion was evaluated. B, CD45.1 mice were adoptively transferred with 1 × 10 5 TgTR1 cells followed by BiVax prime. Five days later the mice were boosted with BiVax, BiVax/IL2Cx 25(10 µg) (2 µg IL2 + 10 µg IL2 mAb) or BiVax/IL2Cx 25(300 µg) (2 µg IL2 + 300 µg IL2 mAb). On day 12 the total numbers of TgTR1 cells were evaluated in spleens. C, Representative flow dot plots from the experiment in panel B , showing the percentage of TgTR1 cells (MHCII-CD45.2+) in spleen gating in total live cells. ( n = 3 mice/group). Representative results are shown from at least 3 independent experiments.

    Journal: Cancer immunology research

    Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

    doi: 10.1158/2326-6066.CIR-17-0549

    Figure Lengend Snippet: Sustained IL2 stimulation is responsible for the adjuvant effect of IL2Cx. A, TgTR1 cells were incubated with Trp1 peptide (1 µg/ml) and IL2Cx 122 or IL2Cx 25 (100 ng IL2/ml) in the presence of increasing concentrations of homologue IL2 mAb (JES6-5H4 or JES6-1A12, respectively). Seven days later T-cell expansion was evaluated. B, CD45.1 mice were adoptively transferred with 1 × 10 5 TgTR1 cells followed by BiVax prime. Five days later the mice were boosted with BiVax, BiVax/IL2Cx 25(10 µg) (2 µg IL2 + 10 µg IL2 mAb) or BiVax/IL2Cx 25(300 µg) (2 µg IL2 + 300 µg IL2 mAb). On day 12 the total numbers of TgTR1 cells were evaluated in spleens. C, Representative flow dot plots from the experiment in panel B , showing the percentage of TgTR1 cells (MHCII-CD45.2+) in spleen gating in total live cells. ( n = 3 mice/group). Representative results are shown from at least 3 independent experiments.

    Article Snippet: The combination of recombinant human IL2 (hu-IL2) and anti-hu-IL2 (MAB602) (hu-IL2Cx) was as effective as the mouse IL2Cx122 (m-IL2Cx) in expanding antigen-specific cells ( ).

    Techniques: Incubation, Mouse Assay, Flow Cytometry

    Selenite and MSA sensitizes tumor cells to NK cell mediated killing. Direct effects on immune cells (activation, shift in population) assessed after 24 h treatment with selenite and MSA. (A) HLA-DR expression on immune cell subsets following treatment with selenite and MSA (10 μM) ( n = 3). (B,C) Specific killing of tumor cells monitored after co incubation with non-activated or overnight IL-2 activated NK cells with A2780 cells pretreated with selenite or MSA (10 μM) ( n = 3). Columns represent mean specific killing (%); bar indicates SD . MSA and/or selenite treated samples were compared to the control ( * p ≤ 0.05).

    Journal: Frontiers in Oncology

    Article Title: Methylseleninic Acid Sensitizes Ovarian Cancer Cells to T-Cell Mediated Killing by Decreasing PDL1 and VEGF Levels

    doi: 10.3389/fonc.2018.00407

    Figure Lengend Snippet: Selenite and MSA sensitizes tumor cells to NK cell mediated killing. Direct effects on immune cells (activation, shift in population) assessed after 24 h treatment with selenite and MSA. (A) HLA-DR expression on immune cell subsets following treatment with selenite and MSA (10 μM) ( n = 3). (B,C) Specific killing of tumor cells monitored after co incubation with non-activated or overnight IL-2 activated NK cells with A2780 cells pretreated with selenite or MSA (10 μM) ( n = 3). Columns represent mean specific killing (%); bar indicates SD . MSA and/or selenite treated samples were compared to the control ( * p ≤ 0.05).

    Article Snippet: Cytolytic assays When indicated, recombinant human Interleukin-2 (IL-2) (PeproTech) was used for NK cell activation at a concentration of 1,000 IU/ml for 24 h prior to the lysis assay.

    Techniques: Activation Assay, Expressing, Incubation

    ( A ) The cytotoxicity of T cells was evaluated by the LDH assay at different ( E ) T ratios. ( C ) Human Th1/Th2 Cytokine Kit determined the expression level of IL-2, IL-4, IL-6, TNF-α, and IFN-γ of DMSO or YN968D1-treated T cells. ( B, D ) The frequency of IFN-γ-producing T cells after co-incubation with gastric cancer cell MKN-45 for 18 hrs was evaluated by IFN-γ ELISPOT kit (Dakewei, China). T cells and MKN-45 were co-incubated at different E: T ratios (10:1, 5:1, 3:1). * means P value

    Journal: OncoTargets and therapy

    Article Title: Immune-Mediated Antitumor Effect By VEGFR2 Selective Inhibitor For Gastric Cancer

    doi: 10.2147/OTT.S233496

    Figure Lengend Snippet: ( A ) The cytotoxicity of T cells was evaluated by the LDH assay at different ( E ) T ratios. ( C ) Human Th1/Th2 Cytokine Kit determined the expression level of IL-2, IL-4, IL-6, TNF-α, and IFN-γ of DMSO or YN968D1-treated T cells. ( B, D ) The frequency of IFN-γ-producing T cells after co-incubation with gastric cancer cell MKN-45 for 18 hrs was evaluated by IFN-γ ELISPOT kit (Dakewei, China). T cells and MKN-45 were co-incubated at different E: T ratios (10:1, 5:1, 3:1). * means P value

    Article Snippet: We used human anti-CD3 antibody (R & D systems, Minneapolis) at a final concentration of 25ng/mL and recombinant human interleukin-2 (rhIL-2, R & D systems) at a final concentration of 200IU/mL on day 1 for the stimulation and proliferation of T cells.

    Techniques: Lactate Dehydrogenase Assay, Expressing, Incubation, Enzyme-linked Immunospot

    Reduced proliferative response of CD4 + cells to hOKT3γ1(Ala-Ala) stimulation occurs only in the presence of CD8 + cells but is not due to lack of IL-2. CFSE-labeled cells were cultured in the presence of hOKT3γ1(Ala-Ala)

    Journal: Journal of Clinical Investigation

    Article Title: TCR stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs

    doi: 10.1172/JCI23961

    Figure Lengend Snippet: Reduced proliferative response of CD4 + cells to hOKT3γ1(Ala-Ala) stimulation occurs only in the presence of CD8 + cells but is not due to lack of IL-2. CFSE-labeled cells were cultured in the presence of hOKT3γ1(Ala-Ala)

    Article Snippet: Human recombinant IL-2, purified neutralizing human anti-FasL, anti–TNF–α, anti–TGF-β antibodies were obtained from R & D Systems; CTLA4-Ig, anti–IL-10 and Th1/Th2 multiplex microspheres (Luminex Corp.) from BioSource International.

    Techniques: Labeling, Cell Culture

    Highly efficient target gene KO in nonactivated human and mouse primary T cells with an optimized CRISPR/Cas9 RNP transfection approach. (A–D) KO efficiency by flow cytometry of CXCR4, CD127, and CCR7 in nonactivated human CD4 T cells 72 h after transfection (A); PD1, TIGIT, and CTLA4 in nonactivated human CD8 T cells 72 h after transfection followed by 72 h of stimulation with anti-CD3/anti-CD28 (B); CD90 and CTLA4 in IL-7–preconditioned nonactivated mouse CD4 T cells 5 d after transfection and incubation with IL-7 (C); and CD8α and CTLA4 in IL-7–preconditioned nonactivated mouse CD8 + T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (D; all compared with nontargeting control Cas9 RNP-transfected T cells). Data are presented as mean ± SD ( n = 2) and representative of three (A) or two (B–D) independent experiments. (E) IFN-γ expression by flow cytometry in crIFNγ or NTC-transfected nonactivated human CD8 + T cells cultured for 3 d and restimulated for 4 h with PMA/ionomycin. Data are presented as mean ± SD ( n = 2) and representative of two independent experiments. (F) FoxP3 and c-Maf expression by flow cytometry in crFoxp3 or NTC transfected IL-7–preconditioned nonactivated mouse CD4 + T cells 5 d after transfection with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2. Data are presented as mean ± SD ( n = 2) and representative of two independent experiments. (G–I) KO efficiency by flow cytometry of double KOs targeting CD127 and CCR7 in nonactivated human CD4 + T cells 72 h after transfection (G), CD90 and CTLA4 in nonactivated mouse CD8 T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (H), or CD90 and FoxP3 in nonactivated mouse CD4 + T cells after transfection and 5 d of incubation with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2 with anti-CD3/anti-CD28 (I). Data are presented as mean ± SD ( n = 2) and representative of two independent experiments. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells

    doi: 10.1084/jem.20171626

    Figure Lengend Snippet: Highly efficient target gene KO in nonactivated human and mouse primary T cells with an optimized CRISPR/Cas9 RNP transfection approach. (A–D) KO efficiency by flow cytometry of CXCR4, CD127, and CCR7 in nonactivated human CD4 T cells 72 h after transfection (A); PD1, TIGIT, and CTLA4 in nonactivated human CD8 T cells 72 h after transfection followed by 72 h of stimulation with anti-CD3/anti-CD28 (B); CD90 and CTLA4 in IL-7–preconditioned nonactivated mouse CD4 T cells 5 d after transfection and incubation with IL-7 (C); and CD8α and CTLA4 in IL-7–preconditioned nonactivated mouse CD8 + T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (D; all compared with nontargeting control Cas9 RNP-transfected T cells). Data are presented as mean ± SD ( n = 2) and representative of three (A) or two (B–D) independent experiments. (E) IFN-γ expression by flow cytometry in crIFNγ or NTC-transfected nonactivated human CD8 + T cells cultured for 3 d and restimulated for 4 h with PMA/ionomycin. Data are presented as mean ± SD ( n = 2) and representative of two independent experiments. (F) FoxP3 and c-Maf expression by flow cytometry in crFoxp3 or NTC transfected IL-7–preconditioned nonactivated mouse CD4 + T cells 5 d after transfection with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2. Data are presented as mean ± SD ( n = 2) and representative of two independent experiments. (G–I) KO efficiency by flow cytometry of double KOs targeting CD127 and CCR7 in nonactivated human CD4 + T cells 72 h after transfection (G), CD90 and CTLA4 in nonactivated mouse CD8 T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (H), or CD90 and FoxP3 in nonactivated mouse CD4 + T cells after transfection and 5 d of incubation with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2 with anti-CD3/anti-CD28 (I). Data are presented as mean ± SD ( n = 2) and representative of two independent experiments. ***, P

    Article Snippet: For CD8+ T cells, 10 ng/ml recombinant human IL-2 (catalog number 202-IL-010; R & D Systems) was added.

    Techniques: CRISPR, Transfection, Flow Cytometry, Cytometry, Incubation, Expressing, Cell Culture

    Experimental layout and expansion of PBMC in cultures supplemented with TG . Panel A : PBMC from consented healthy donors were initially exposed to IFN-γ (day 0), followed by different concentrations of either TG or αCD3 mAb (day +1) and IL-2 every 3 days. Further details are provided in Materials and Methods. Panel B : The frequency of CD3 + CD8 + T cells, NK cells (CD3 - CD16 + CD56 + ) and CD3 + CD56 + T cells from a representative PBMC sample at baseline is shown. Quadrant markers were set according to the proper isotypic control (not shown). The percentage of cells staining positively for a given antigen is indicated. Panel C : Cells were harvested weekly and counted. The number of cells was significantly higher after challenging with TG either at 250 ( int TG; *p

    Journal: Journal of Translational Medicine

    Article Title: Thymoglobulin, interferon-? and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures

    doi: 10.1186/1479-5876-8-129

    Figure Lengend Snippet: Experimental layout and expansion of PBMC in cultures supplemented with TG . Panel A : PBMC from consented healthy donors were initially exposed to IFN-γ (day 0), followed by different concentrations of either TG or αCD3 mAb (day +1) and IL-2 every 3 days. Further details are provided in Materials and Methods. Panel B : The frequency of CD3 + CD8 + T cells, NK cells (CD3 - CD16 + CD56 + ) and CD3 + CD56 + T cells from a representative PBMC sample at baseline is shown. Quadrant markers were set according to the proper isotypic control (not shown). The percentage of cells staining positively for a given antigen is indicated. Panel C : Cells were harvested weekly and counted. The number of cells was significantly higher after challenging with TG either at 250 ( int TG; *p

    Article Snippet: The following day, cells were stimulated with either αCD3 mAb (UCHT1 clone; 50-500 ng/ml, BD Biosciences, San Diego, CA) or Thymoglobulin® (50-500 ng/ml, Genzyme Corp., Cambridge, MA) and recombinant human IL-2 (rHuIL-2, 300 IU/ml; Proleukin® , Novartis Pharma, Milan, Italy).

    Techniques: Staining

    IL-2-associated changes in biomarker levels

    Journal:

    Article Title: IL-2 cycling causes transient increases in hsCRP and D-dimer that are not associated with plasma HIV-RNA levels

    doi: 10.1097/QAD.0b013e32832d72c6

    Figure Lengend Snippet: IL-2-associated changes in biomarker levels

    Article Snippet: Cycles of human recombinant IL-2 (Novartis, Emeryville, CA) were administered no more frequently than every 8 weeks for 5 consecutive days as continuous infusions of 6–18 million international units per day.

    Techniques: Biomarker Assay

    Calcitriol response of TAGAP and IL2RA in CD4+ T cells is not influenced by MS or MS-risk genotype. CD4+ T cells from 8 RRMS patients (MS) and 10 healthy controls (HC) genotyped for MS-risk SNPs in TAGAP (rs1738074) and IL2RA (rs7090512) were cultivated for 7–8 days with αCD3/CD28 and IL-2, treated as described in Materials and Methods before harvesting after 6, 24 and 48 h, with 1,25(OH) 2 D 3 or vehicle control. The box plots with whiskers defining minimum and maximum show the log2 values of relative expression of indicted genes (relative to TBP ) in 1,25(OH) 2 D 3 -treated cells divided by relative expression in vehicle-treated cells, that is, log2 of fold induction by 1,25(OH) 2 D 3 at each time point for ( a ) MS patients compared with HCs and ( b ) the same samples sorted on IL2RA and TAGAP genotype, that is, samples carrying the minor allele ( IL2RA : N =10, MAF (G)=0.3480; TAGAP : N =11, MAF (G)=0.46563) compared with samples homozygous for major allele ( N =8 ( IL2RA ); N =7 ( TAGAP )). The horizontal lines within the boxes represent the median of the groups, and Mann–Whitney U -test was performed to compare the groups (MS vs HC, AA vs AG/GG). The horizontal line at 0 indicates no induction by 1,25(OH) 2 D 3 .

    Journal: Genes and Immunity

    Article Title: The multiple sclerosis susceptibility genes TAGAP and IL2RA are regulated by vitamin D in CD4+ T cells

    doi: 10.1038/gene.2015.61

    Figure Lengend Snippet: Calcitriol response of TAGAP and IL2RA in CD4+ T cells is not influenced by MS or MS-risk genotype. CD4+ T cells from 8 RRMS patients (MS) and 10 healthy controls (HC) genotyped for MS-risk SNPs in TAGAP (rs1738074) and IL2RA (rs7090512) were cultivated for 7–8 days with αCD3/CD28 and IL-2, treated as described in Materials and Methods before harvesting after 6, 24 and 48 h, with 1,25(OH) 2 D 3 or vehicle control. The box plots with whiskers defining minimum and maximum show the log2 values of relative expression of indicted genes (relative to TBP ) in 1,25(OH) 2 D 3 -treated cells divided by relative expression in vehicle-treated cells, that is, log2 of fold induction by 1,25(OH) 2 D 3 at each time point for ( a ) MS patients compared with HCs and ( b ) the same samples sorted on IL2RA and TAGAP genotype, that is, samples carrying the minor allele ( IL2RA : N =10, MAF (G)=0.3480; TAGAP : N =11, MAF (G)=0.46563) compared with samples homozygous for major allele ( N =8 ( IL2RA ); N =7 ( TAGAP )). The horizontal lines within the boxes represent the median of the groups, and Mann–Whitney U -test was performed to compare the groups (MS vs HC, AA vs AG/GG). The horizontal line at 0 indicates no induction by 1,25(OH) 2 D 3 .

    Article Snippet: Freshly purified CD4+ T cells were either frozen on liquid nitrogen for later use or resuspended in X-VIVO medium (Lonza) with human IL-2 (R & D Systems, Minneapolis, MS, USA) and αCD3/CD28 beads (1:1) (Human T-activator CD3/CD28 Dynabeads for cell expansion and activation, Life Technologies) for activation and 1,25(OH)2 D3 treatment.

    Techniques: Mass Spectrometry, Expressing, MANN-WHITNEY

    Antigen-experienced “CD4lo” T cells give rise to “CD4hi” T cells. “CD4lo” T cells were enriched from the Con A blasts of NOD spleen cells as described in Figure 6 . The “CD4lo” T cells or Con A blasts were cultured overnight in IL-2-containing complete medium, then each was labeled with CFSE before incubation on anti-CD3-coated plates. Twenty-four and 48 hrs later the cells were labeled with anti-CD4-PE and analyzed by flow cytometry to reveal cell divisions. Proliferating “CD4hi” T cells arising from the blasts are indicated in the encircled population the numbers indicate the percentage in the total cell population. For each analysis, 100,000 events were collected, and the results are representative of four experiments.

    Journal: Autoimmune Diseases

    Article Title: Antigen-Experienced CD4lo T Cells Are Linked to Deficient Contraction of the Immune Response in Autoimmune Diabetes

    doi: 10.4061/2010/920148

    Figure Lengend Snippet: Antigen-experienced “CD4lo” T cells give rise to “CD4hi” T cells. “CD4lo” T cells were enriched from the Con A blasts of NOD spleen cells as described in Figure 6 . The “CD4lo” T cells or Con A blasts were cultured overnight in IL-2-containing complete medium, then each was labeled with CFSE before incubation on anti-CD3-coated plates. Twenty-four and 48 hrs later the cells were labeled with anti-CD4-PE and analyzed by flow cytometry to reveal cell divisions. Proliferating “CD4hi” T cells arising from the blasts are indicated in the encircled population the numbers indicate the percentage in the total cell population. For each analysis, 100,000 events were collected, and the results are representative of four experiments.

    Article Snippet: For secondary stimulation and the induction of AICD, Con A blasts were washed in complete medium supplemented with 10 ng/mL of recombinant human IL-2 (rIL-2), resuspended to 2.5 × 106 cells/mL in medium supplemented with rIL-2, rested at 37°C for < 3 hours, and then plated in triplicate on antimouse CD3ε -coated 96-well plates (50 μ l/well for 2 hrs at 37°C; clone 145-2C11; BD Biosciences, San Diego, CA).

    Techniques: Cell Culture, Labeling, Incubation, Flow Cytometry, Cytometry

    (a) Secondary NOD T cell blasts are resistant to AICD upon tertiary restimulation. NOD or BALB/c Con A blasts were restimulated on anti-CD3-coated plates, washed, and cultured in IL-2-containing medium for 3 days then stimulated again on anti-CD3-coated plates (0.003–3.0 μ g/mL; 50 μ l/well). Tritium-labeled thymidine was added for the last 16 hrs of a 72-hour culture. The data are expressed as percent change versus control wells [(mean cpm anti-CD3 wells/mean cpm IgG wells) × 100]. The standard deviation of triplicate wells was less than 10%, and the results are representative of four experiments. (b) Culture supernatants were collected at various times (24–96 hrs) following primary stimulation (Con A) of NOD or BALB/c spleen cells or secondary and tertiary stimulations of T cells blasts with plate-bound anti-CD3 (1.0 μ g/mL). IFN- γ levels were measured by ELISA, and the results are expressed as ng/mL. The results represent the mean and SD of triplicate wells and are representative of 3 independent experiments. Only P -values that are

    Journal: Autoimmune Diseases

    Article Title: Antigen-Experienced CD4lo T Cells Are Linked to Deficient Contraction of the Immune Response in Autoimmune Diabetes

    doi: 10.4061/2010/920148

    Figure Lengend Snippet: (a) Secondary NOD T cell blasts are resistant to AICD upon tertiary restimulation. NOD or BALB/c Con A blasts were restimulated on anti-CD3-coated plates, washed, and cultured in IL-2-containing medium for 3 days then stimulated again on anti-CD3-coated plates (0.003–3.0 μ g/mL; 50 μ l/well). Tritium-labeled thymidine was added for the last 16 hrs of a 72-hour culture. The data are expressed as percent change versus control wells [(mean cpm anti-CD3 wells/mean cpm IgG wells) × 100]. The standard deviation of triplicate wells was less than 10%, and the results are representative of four experiments. (b) Culture supernatants were collected at various times (24–96 hrs) following primary stimulation (Con A) of NOD or BALB/c spleen cells or secondary and tertiary stimulations of T cells blasts with plate-bound anti-CD3 (1.0 μ g/mL). IFN- γ levels were measured by ELISA, and the results are expressed as ng/mL. The results represent the mean and SD of triplicate wells and are representative of 3 independent experiments. Only P -values that are

    Article Snippet: For secondary stimulation and the induction of AICD, Con A blasts were washed in complete medium supplemented with 10 ng/mL of recombinant human IL-2 (rIL-2), resuspended to 2.5 × 106 cells/mL in medium supplemented with rIL-2, rested at 37°C for < 3 hours, and then plated in triplicate on antimouse CD3ε -coated 96-well plates (50 μ l/well for 2 hrs at 37°C; clone 145-2C11; BD Biosciences, San Diego, CA).

    Techniques: Cell Culture, Labeling, Standard Deviation, Enzyme-linked Immunosorbent Assay

    “CD4lo” T cells are present in Con A blasts produced from the spleen cells of NOD.BDC2.5 TCR transgenic mice. (a) Spleen cells from BDC2.5 TCR transgenic mice were stimulated with Con A for 72 hrs. The cells were then layered onto Ficol-Histopaque and centrifuged to recover an enriched population of the smaller “CD4lo” population. The Con A blasts and the enriched “CD4lo” population were cultured overnight in IL-2-supplemented medium and then stained with anti-CD4-PE and anti-CD69 FITC for FACS analysis. The numbers in the dot plots indicate the percentage of total cells that are within a region. A total of 20,000 events were collected for each plot. (b) 5 × 10 6 “CD4lo”-enriched or “CD4hi”-enriched T cells were each adoptively transferred into 6 NOD.scid mice. As a control, untreated BDC2.5 TCR Tg spleen cells were also transferred into a control group of NOD.scid mice. Ten days later the pancreas was recovered from at least two mice per group, fixed, embedded, and cut into 5 micron sections that were subsequently stained with hematoxylin and eosin. The sections were viewed and photographed by a blinded observer. At least 50 islets were counted for each group to evaluate the pattern of insulitis. The arrows point to areas of inflammation, peri-insulits, which was predominant in “CD4lo” T cell recipients, and invasive insulitis, which was representative of most islets in recipients of “CD4hi” T cell recipients. The remaining mice were monitored for the development of diabetes as described in the Methods. (c) Spleen cells were recovered from the recipients in (b), stained with anti-CD4-PE and anti-TCR V beta 4-FITC, and then 100,000 events were collected during FACS analysis. The data in the dot plot were gated on viable cells (FSC × SSC). The numbers indicate the percentage of cells in the region. The number in the parentheses for the “CD4lo” recipients indicates the percentage of “CD4lo” cells in the total CD4 + population.

    Journal: Autoimmune Diseases

    Article Title: Antigen-Experienced CD4lo T Cells Are Linked to Deficient Contraction of the Immune Response in Autoimmune Diabetes

    doi: 10.4061/2010/920148

    Figure Lengend Snippet: “CD4lo” T cells are present in Con A blasts produced from the spleen cells of NOD.BDC2.5 TCR transgenic mice. (a) Spleen cells from BDC2.5 TCR transgenic mice were stimulated with Con A for 72 hrs. The cells were then layered onto Ficol-Histopaque and centrifuged to recover an enriched population of the smaller “CD4lo” population. The Con A blasts and the enriched “CD4lo” population were cultured overnight in IL-2-supplemented medium and then stained with anti-CD4-PE and anti-CD69 FITC for FACS analysis. The numbers in the dot plots indicate the percentage of total cells that are within a region. A total of 20,000 events were collected for each plot. (b) 5 × 10 6 “CD4lo”-enriched or “CD4hi”-enriched T cells were each adoptively transferred into 6 NOD.scid mice. As a control, untreated BDC2.5 TCR Tg spleen cells were also transferred into a control group of NOD.scid mice. Ten days later the pancreas was recovered from at least two mice per group, fixed, embedded, and cut into 5 micron sections that were subsequently stained with hematoxylin and eosin. The sections were viewed and photographed by a blinded observer. At least 50 islets were counted for each group to evaluate the pattern of insulitis. The arrows point to areas of inflammation, peri-insulits, which was predominant in “CD4lo” T cell recipients, and invasive insulitis, which was representative of most islets in recipients of “CD4hi” T cell recipients. The remaining mice were monitored for the development of diabetes as described in the Methods. (c) Spleen cells were recovered from the recipients in (b), stained with anti-CD4-PE and anti-TCR V beta 4-FITC, and then 100,000 events were collected during FACS analysis. The data in the dot plot were gated on viable cells (FSC × SSC). The numbers indicate the percentage of cells in the region. The number in the parentheses for the “CD4lo” recipients indicates the percentage of “CD4lo” cells in the total CD4 + population.

    Article Snippet: For secondary stimulation and the induction of AICD, Con A blasts were washed in complete medium supplemented with 10 ng/mL of recombinant human IL-2 (rIL-2), resuspended to 2.5 × 106 cells/mL in medium supplemented with rIL-2, rested at 37°C for < 3 hours, and then plated in triplicate on antimouse CD3ε -coated 96-well plates (50 μ l/well for 2 hrs at 37°C; clone 145-2C11; BD Biosciences, San Diego, CA).

    Techniques: Produced, Transgenic Assay, Mouse Assay, Cell Culture, Staining, FACS

    Comparison of CTL Memory and Recall Responses in Lung and Spleen after SQ or IN Prime-boost Vaccination with OVA. C57BL/6 mice were immunized intranasally with 10 μg of OVA in 50 μl PBS with 5% ADJ (SQ) or 10% ADJ (IN) twice at 3 week intervals. Lung and spleen were collected 3 weeks after the second vaccine. ( A-F) Lung cells and splenocytes were stained with anti-CD8, anti-CD44, anti-CD62L, anti-CD103, anti-CD69, anti-CXCR3 and K b /SIINFEKL tetramers; cells were acquired on a flow cytometer. ( A ) Absolute numbers of CD8 T cells in the lung and spleen. ( B ) Absolute numbers of SIINFEKL-specific tetramer-binding CD8 T cells in the lung and spleen. ( C ) CD103 + SIINFEKL-specific CD8 T cells in lung and spleen. ( D ) Absolute numbers of SIINFEKL-specific tetramer-binding resident memory-like CD103 + CD69 + CXCR3 + CD8 T cells in the lung and spleen. ( E-H ) Lung cells and splenocytes were stimulated with the SIINFEKL peptide and the percentages of IFN-γ + IL-2 + TNF-α + CD8 T cells were quantified by flow cytometry. ( E ) Frequency of SIINFEKL-specific IFN-γ + CTLs among all CD8 + cells in lungs and spleen. ( F) Absolute numbers of SIINFEKL-specific IFN-γ + CTLs. ( G ) Frequency of SIINFEKL-specific IFN-γ + IL-2 + TNF-α + CTLs among all CD8 + cells. ( H) Absolute numbers SIINFEKL-specific IFN-γ + IL-2 + TNF-α + CTLs. ( I-J) C57BL/6 mice were immunized intranasally with 10 μg of OVA in 50 μl PBS with 5% ADJ (SQ) or 10% ADJ (IN) twice at 3 week intervals. 3 weeks after the second immunization, mice were challenged by intranasal administration of 500 PFU of recombinant influenza A/PR/8/34-OT-I H1N1 expressing the OVA SIINFEKL peptide. 6 days after challenge 5 mice/group were sacrificed and lungs were collected for quantification of CTLs and viral titration. ( I) Frequency of SIINFEKL-specific IFN-γ + CD8 + T cells among all CD8 + lung mononuclear cells. ( J) Lung viral titers expressed as plaque-forming-units (PFU) per gram of lung. * indicates p

    Journal: PLoS Pathogens

    Article Title: Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines

    doi: 10.1371/journal.ppat.1006064

    Figure Lengend Snippet: Comparison of CTL Memory and Recall Responses in Lung and Spleen after SQ or IN Prime-boost Vaccination with OVA. C57BL/6 mice were immunized intranasally with 10 μg of OVA in 50 μl PBS with 5% ADJ (SQ) or 10% ADJ (IN) twice at 3 week intervals. Lung and spleen were collected 3 weeks after the second vaccine. ( A-F) Lung cells and splenocytes were stained with anti-CD8, anti-CD44, anti-CD62L, anti-CD103, anti-CD69, anti-CXCR3 and K b /SIINFEKL tetramers; cells were acquired on a flow cytometer. ( A ) Absolute numbers of CD8 T cells in the lung and spleen. ( B ) Absolute numbers of SIINFEKL-specific tetramer-binding CD8 T cells in the lung and spleen. ( C ) CD103 + SIINFEKL-specific CD8 T cells in lung and spleen. ( D ) Absolute numbers of SIINFEKL-specific tetramer-binding resident memory-like CD103 + CD69 + CXCR3 + CD8 T cells in the lung and spleen. ( E-H ) Lung cells and splenocytes were stimulated with the SIINFEKL peptide and the percentages of IFN-γ + IL-2 + TNF-α + CD8 T cells were quantified by flow cytometry. ( E ) Frequency of SIINFEKL-specific IFN-γ + CTLs among all CD8 + cells in lungs and spleen. ( F) Absolute numbers of SIINFEKL-specific IFN-γ + CTLs. ( G ) Frequency of SIINFEKL-specific IFN-γ + IL-2 + TNF-α + CTLs among all CD8 + cells. ( H) Absolute numbers SIINFEKL-specific IFN-γ + IL-2 + TNF-α + CTLs. ( I-J) C57BL/6 mice were immunized intranasally with 10 μg of OVA in 50 μl PBS with 5% ADJ (SQ) or 10% ADJ (IN) twice at 3 week intervals. 3 weeks after the second immunization, mice were challenged by intranasal administration of 500 PFU of recombinant influenza A/PR/8/34-OT-I H1N1 expressing the OVA SIINFEKL peptide. 6 days after challenge 5 mice/group were sacrificed and lungs were collected for quantification of CTLs and viral titration. ( I) Frequency of SIINFEKL-specific IFN-γ + CD8 + T cells among all CD8 + lung mononuclear cells. ( J) Lung viral titers expressed as plaque-forming-units (PFU) per gram of lung. * indicates p

    Article Snippet: Cells were stimulated for 5 hours at 37C in the presence of human recombinant IL-2 (10 U/well), and brefeldin A (1 μl/ml, GolgiPlug, BD Biosciences), with one of the following peptides: SIINFEKL, NP366, PA224 (thinkpeptides® , ProImmune Ltd. Oxford, UK) at 0.1ug/ml, or without peptide.

    Techniques: CTL Assay, Mouse Assay, Staining, Flow Cytometry, Cytometry, Binding Assay, Recombinant, Expressing, Titration

    Kinetics of Primary CTL Responses and Viral Control after Subcutaneous Vaccination with OVA. ( A-D ) Prime-only Vaccination. C57BL/6 mice were vaccinated by subcutaneous injection of 10 μg ovalbumin in PBS alone or supplemented with 5% Adjuplex or 10 μg CpG. 3–5 mice/group were sacrificed at 8 and 90 days after vaccination. (A) The percentages of SIINFEKL-specific IFN-γ + TNF-α + and IL-2 + CD8 T cells in spleens were quantified by intracellular cytokine staining at day 8 and 90 after vaccination. ( B ) At day 7 after vaccination, splenocytes were stained with anti-CD8, K b /SIINFEKL tetramers, anti-CD127 and anti-KLRG-1. The percentages of short-lived effector cells (SLECs; CD127 LO /KLRG-1 HI ) and memory precursor effector cells (MPECs; CD127 HI /KLRG-1 LO ) among tetramer-binding SIINFEKL-specific CD8 T cells were quantified by flow cytometry. Data shows the total numbers of SLECs and MPECs in spleens of vaccinated mice. ( C-D ) Secondary CTL recall responses and lung viral titers after prime-only SQ vaccination and influenza challenge. C57BL/6 mice were vaccinated by SQ injection of 10 μg ovalbumin in PBS alone or supplemented with 5% Adjuplex or 10 μg CpG. 90 days after vaccination, mice were challenged by IN administration of 500 PFU of recombinant influenza A/PR/8/34-OT-I H1N1 expressing the OVA SIINFEKL peptide. 6 days after challenge, 3–5 mice/group were sacrificed and lungs were collected to quantify SIINFEKL-specific CTLs and viral titers. ( C ) Frequency of SIINFEKL-specific IFN-γ + CD8 + T cells among all CD8 + lung mononuclear cells at 6 days after challenge. ( D ) Lung viral titers expressed as plaque-forming-units (PFU) per gram of lung at 6 days after challenge. * indicates p

    Journal: PLoS Pathogens

    Article Title: Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines

    doi: 10.1371/journal.ppat.1006064

    Figure Lengend Snippet: Kinetics of Primary CTL Responses and Viral Control after Subcutaneous Vaccination with OVA. ( A-D ) Prime-only Vaccination. C57BL/6 mice were vaccinated by subcutaneous injection of 10 μg ovalbumin in PBS alone or supplemented with 5% Adjuplex or 10 μg CpG. 3–5 mice/group were sacrificed at 8 and 90 days after vaccination. (A) The percentages of SIINFEKL-specific IFN-γ + TNF-α + and IL-2 + CD8 T cells in spleens were quantified by intracellular cytokine staining at day 8 and 90 after vaccination. ( B ) At day 7 after vaccination, splenocytes were stained with anti-CD8, K b /SIINFEKL tetramers, anti-CD127 and anti-KLRG-1. The percentages of short-lived effector cells (SLECs; CD127 LO /KLRG-1 HI ) and memory precursor effector cells (MPECs; CD127 HI /KLRG-1 LO ) among tetramer-binding SIINFEKL-specific CD8 T cells were quantified by flow cytometry. Data shows the total numbers of SLECs and MPECs in spleens of vaccinated mice. ( C-D ) Secondary CTL recall responses and lung viral titers after prime-only SQ vaccination and influenza challenge. C57BL/6 mice were vaccinated by SQ injection of 10 μg ovalbumin in PBS alone or supplemented with 5% Adjuplex or 10 μg CpG. 90 days after vaccination, mice were challenged by IN administration of 500 PFU of recombinant influenza A/PR/8/34-OT-I H1N1 expressing the OVA SIINFEKL peptide. 6 days after challenge, 3–5 mice/group were sacrificed and lungs were collected to quantify SIINFEKL-specific CTLs and viral titers. ( C ) Frequency of SIINFEKL-specific IFN-γ + CD8 + T cells among all CD8 + lung mononuclear cells at 6 days after challenge. ( D ) Lung viral titers expressed as plaque-forming-units (PFU) per gram of lung at 6 days after challenge. * indicates p

    Article Snippet: Cells were stimulated for 5 hours at 37C in the presence of human recombinant IL-2 (10 U/well), and brefeldin A (1 μl/ml, GolgiPlug, BD Biosciences), with one of the following peptides: SIINFEKL, NP366, PA224 (thinkpeptides® , ProImmune Ltd. Oxford, UK) at 0.1ug/ml, or without peptide.

    Techniques: CTL Assay, Mouse Assay, Injection, Staining, Binding Assay, Flow Cytometry, Cytometry, Recombinant, Expressing

    Primary Antigen-Specific CTL Responses Correlate with Adjuplex Dose and Tissue Inflammation. C57BL/6 mice were vaccinated by intramuscular or subcutaneous injection of 100 μg ovalbumin in PBS alone or supplemented with 1, 5, 10 or 20% ADJ. 3–4 mice/group were sacrificed after 7 days. Injection sites were collected for routine histopathology, and spleens were collected for flow cytometry. Prior to FACS analysis, splenocytes were stimulated for 5 hours with SIINFEKL peptide in the presence of IL-2 and brefeldin-A. ( A) Frequency of SIINFEKL-specific IFN-γ + CD8 T cells among all splenocytes after IM vaccination . (B-F) Photomicrographs of histologic changes in the skeletal muscle at the injection site 7 days after IM injection at the indicated concentrations of Adjuplex. Images depict a progressive increase in the cellularity and extent of the inflammatory infiltrate at the injection site. ( G) Frequency of SIINFEKL-specific IFN-γ + CD8 T cells among all splenocytes after SQ vaccination . ( H-L) Photomicrographs of histologic changes in the subcutaneous adipose tissue and skeletal muscle at the injection site 7 days after SQ injection at the indicated concentrations of Adjuplex. Images depict a progressive increase in the cellularity and extent of the inflammatory infiltrate at the injection site. Data represents two independent experiments.

    Journal: PLoS Pathogens

    Article Title: Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines

    doi: 10.1371/journal.ppat.1006064

    Figure Lengend Snippet: Primary Antigen-Specific CTL Responses Correlate with Adjuplex Dose and Tissue Inflammation. C57BL/6 mice were vaccinated by intramuscular or subcutaneous injection of 100 μg ovalbumin in PBS alone or supplemented with 1, 5, 10 or 20% ADJ. 3–4 mice/group were sacrificed after 7 days. Injection sites were collected for routine histopathology, and spleens were collected for flow cytometry. Prior to FACS analysis, splenocytes were stimulated for 5 hours with SIINFEKL peptide in the presence of IL-2 and brefeldin-A. ( A) Frequency of SIINFEKL-specific IFN-γ + CD8 T cells among all splenocytes after IM vaccination . (B-F) Photomicrographs of histologic changes in the skeletal muscle at the injection site 7 days after IM injection at the indicated concentrations of Adjuplex. Images depict a progressive increase in the cellularity and extent of the inflammatory infiltrate at the injection site. ( G) Frequency of SIINFEKL-specific IFN-γ + CD8 T cells among all splenocytes after SQ vaccination . ( H-L) Photomicrographs of histologic changes in the subcutaneous adipose tissue and skeletal muscle at the injection site 7 days after SQ injection at the indicated concentrations of Adjuplex. Images depict a progressive increase in the cellularity and extent of the inflammatory infiltrate at the injection site. Data represents two independent experiments.

    Article Snippet: Cells were stimulated for 5 hours at 37C in the presence of human recombinant IL-2 (10 U/well), and brefeldin A (1 μl/ml, GolgiPlug, BD Biosciences), with one of the following peptides: SIINFEKL, NP366, PA224 (thinkpeptides® , ProImmune Ltd. Oxford, UK) at 0.1ug/ml, or without peptide.

    Techniques: CTL Assay, Mouse Assay, Injection, Histopathology, Flow Cytometry, Cytometry, FACS

    IFNβ affects chromatin remodeling of the IL-2 promoter through histone deacetylase activity and CREM

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

    Article Title: Interferon β selectively Inhibits Interleukin-2 (IL-2) production through cAMP responsive element modulator (CREM)-mediated chromatin remodeling

    doi: 10.4049/jimmunol.1403181

    Figure Lengend Snippet: IFNβ affects chromatin remodeling of the IL-2 promoter through histone deacetylase activity and CREM

    Article Snippet: DNase-treated RNA was isolated from activated T cells by the RNeasy method (Qiagen). cDNA was prepared with the high capacity cDNA reverse transcription kit (Applied Biosystems) and QT-PCR was performed using Taqman primers for mouse and human IL-2, mouse IL-4, and mouse IFN-γ (Applied Biosystems).

    Techniques: Histone Deacetylase Assay, Activity Assay

    Inhibition of IL-2 expression requires pre-treatment with IFNβ

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

    Article Title: Interferon β selectively Inhibits Interleukin-2 (IL-2) production through cAMP responsive element modulator (CREM)-mediated chromatin remodeling

    doi: 10.4049/jimmunol.1403181

    Figure Lengend Snippet: Inhibition of IL-2 expression requires pre-treatment with IFNβ

    Article Snippet: DNase-treated RNA was isolated from activated T cells by the RNeasy method (Qiagen). cDNA was prepared with the high capacity cDNA reverse transcription kit (Applied Biosystems) and QT-PCR was performed using Taqman primers for mouse and human IL-2, mouse IL-4, and mouse IFN-γ (Applied Biosystems).

    Techniques: Inhibition, Expressing

    IFNβ inhibition of IL-2 expression is independent of Treg cells and is reproduced in human peripheral blood leukocytes

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

    Article Title: Interferon β selectively Inhibits Interleukin-2 (IL-2) production through cAMP responsive element modulator (CREM)-mediated chromatin remodeling

    doi: 10.4049/jimmunol.1403181

    Figure Lengend Snippet: IFNβ inhibition of IL-2 expression is independent of Treg cells and is reproduced in human peripheral blood leukocytes

    Article Snippet: DNase-treated RNA was isolated from activated T cells by the RNeasy method (Qiagen). cDNA was prepared with the high capacity cDNA reverse transcription kit (Applied Biosystems) and QT-PCR was performed using Taqman primers for mouse and human IL-2, mouse IL-4, and mouse IFN-γ (Applied Biosystems).

    Techniques: Inhibition, Expressing

    Inhibition of IL-2 by IFNβ in naive T cells is dependent on STAT1 but does not affect proximal signaling through the T cell receptor

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

    Article Title: Interferon β selectively Inhibits Interleukin-2 (IL-2) production through cAMP responsive element modulator (CREM)-mediated chromatin remodeling

    doi: 10.4049/jimmunol.1403181

    Figure Lengend Snippet: Inhibition of IL-2 by IFNβ in naive T cells is dependent on STAT1 but does not affect proximal signaling through the T cell receptor

    Article Snippet: DNase-treated RNA was isolated from activated T cells by the RNeasy method (Qiagen). cDNA was prepared with the high capacity cDNA reverse transcription kit (Applied Biosystems) and QT-PCR was performed using Taqman primers for mouse and human IL-2, mouse IL-4, and mouse IFN-γ (Applied Biosystems).

    Techniques: Inhibition

    Inhibition of IL-2 production in T cells from mice injected with IFNβ or infected with LCMV Cl13

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

    Article Title: Interferon β selectively Inhibits Interleukin-2 (IL-2) production through cAMP responsive element modulator (CREM)-mediated chromatin remodeling

    doi: 10.4049/jimmunol.1403181

    Figure Lengend Snippet: Inhibition of IL-2 production in T cells from mice injected with IFNβ or infected with LCMV Cl13

    Article Snippet: DNase-treated RNA was isolated from activated T cells by the RNeasy method (Qiagen). cDNA was prepared with the high capacity cDNA reverse transcription kit (Applied Biosystems) and QT-PCR was performed using Taqman primers for mouse and human IL-2, mouse IL-4, and mouse IFN-γ (Applied Biosystems).

    Techniques: Inhibition, Mouse Assay, Injection, Infection

    Specific inhibition of IL-2 expression by IFNβ

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

    Article Title: Interferon β selectively Inhibits Interleukin-2 (IL-2) production through cAMP responsive element modulator (CREM)-mediated chromatin remodeling

    doi: 10.4049/jimmunol.1403181

    Figure Lengend Snippet: Specific inhibition of IL-2 expression by IFNβ

    Article Snippet: DNase-treated RNA was isolated from activated T cells by the RNeasy method (Qiagen). cDNA was prepared with the high capacity cDNA reverse transcription kit (Applied Biosystems) and QT-PCR was performed using Taqman primers for mouse and human IL-2, mouse IL-4, and mouse IFN-γ (Applied Biosystems).

    Techniques: Inhibition, Expressing

    Induction of high output NO synthesis in Meth A ascites tumor cells by IL-2 therapy. Meth A ascites cells harvested from three IL-2 treated mice were cultured at 1.5×10 5 cells/well in microtiter plates in triplicate. After a 48h culture, nitrite was measured in 50 μ l culture supernatants by a colorimetric assay. Results are mean±SD. Meth A ascites cells harvested from three untreated mice served as controls.

    Journal: The Korean Journal of Internal Medicine

    Article Title: Effects of Nitric Oxide (NO) Synthesis Inhibition on Antitumor Responses during Interleukin-2 (IL-2) Treatment of Mice

    doi: 10.3904/kjim.1996.11.2.93

    Figure Lengend Snippet: Induction of high output NO synthesis in Meth A ascites tumor cells by IL-2 therapy. Meth A ascites cells harvested from three IL-2 treated mice were cultured at 1.5×10 5 cells/well in microtiter plates in triplicate. After a 48h culture, nitrite was measured in 50 μ l culture supernatants by a colorimetric assay. Results are mean±SD. Meth A ascites cells harvested from three untreated mice served as controls.

    Article Snippet: The recombinant human IL-2 was a generous gift from Chiron Corporation, Emeryville, CA.

    Techniques: Mouse Assay, Cell Culture, Colorimetric Assay

    Evaluation of the role of IL-2 therapy-induced NO synthesis in the treatment of intraperitoneal Meth A tumor. Four groups of BALB/c mice bearing Meth A ascites tumor were treated (untreated control, MLA therapy only, IL-2 therapy only, and IL-2/MLA therapy). Survival (Panel A) and the rate of body weight increment (Panel B) (Bars express mean±SD, p

    Journal: The Korean Journal of Internal Medicine

    Article Title: Effects of Nitric Oxide (NO) Synthesis Inhibition on Antitumor Responses during Interleukin-2 (IL-2) Treatment of Mice

    doi: 10.3904/kjim.1996.11.2.93

    Figure Lengend Snippet: Evaluation of the role of IL-2 therapy-induced NO synthesis in the treatment of intraperitoneal Meth A tumor. Four groups of BALB/c mice bearing Meth A ascites tumor were treated (untreated control, MLA therapy only, IL-2 therapy only, and IL-2/MLA therapy). Survival (Panel A) and the rate of body weight increment (Panel B) (Bars express mean±SD, p

    Article Snippet: The recombinant human IL-2 was a generous gift from Chiron Corporation, Emeryville, CA.

    Techniques: Mouse Assay

    Effect of MLA on NO synthesis of Meth A ascites cells from IL-2 treated mice. Meth A ascites cells harvested from IL-2 treated mice were cultured at 1.5×10 5 cells/well in microtiter plates in triplicate with varying concentrations (0–500 μ M) of MLA. After a 48h culture, nitrite was measured in 50 μ l culture supernatants by a colorimetric assay. Results are mean±SD. Ascites tumor cells harvested from untreated mice served as controls.

    Journal: The Korean Journal of Internal Medicine

    Article Title: Effects of Nitric Oxide (NO) Synthesis Inhibition on Antitumor Responses during Interleukin-2 (IL-2) Treatment of Mice

    doi: 10.3904/kjim.1996.11.2.93

    Figure Lengend Snippet: Effect of MLA on NO synthesis of Meth A ascites cells from IL-2 treated mice. Meth A ascites cells harvested from IL-2 treated mice were cultured at 1.5×10 5 cells/well in microtiter plates in triplicate with varying concentrations (0–500 μ M) of MLA. After a 48h culture, nitrite was measured in 50 μ l culture supernatants by a colorimetric assay. Results are mean±SD. Ascites tumor cells harvested from untreated mice served as controls.

    Article Snippet: The recombinant human IL-2 was a generous gift from Chiron Corporation, Emeryville, CA.

    Techniques: Mouse Assay, Cell Culture, Colorimetric Assay

    Analysis of FoxP3, IFN-γ, IL-2 and Ki-67 expression in CD4 + cells from anti-CD3 activated human PBMC. Freshly isolated PBMC on day 0 (a–c) were incubated in the absence (d–f) or presence of soluble anti-CD3 at 100 (g–i),

    Journal: Immunology

    Article Title: Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses

    doi: 10.1111/j.1365-2567.2010.03280.x

    Figure Lengend Snippet: Analysis of FoxP3, IFN-γ, IL-2 and Ki-67 expression in CD4 + cells from anti-CD3 activated human PBMC. Freshly isolated PBMC on day 0 (a–c) were incubated in the absence (d–f) or presence of soluble anti-CD3 at 100 (g–i),

    Article Snippet: Where indicated, IFN-I [human leucocyte IFN (predominantly IFN-α) (R & D Systems, Minneapolis, MN) or human recombinant IFN-β 1b (PBL InterferonSource, Piscataway, NJ)] was added at 100–500 (IFN-β) or 100–1000 (IFN-α) units/ml, or 100 units/ml human recombinant IL-2 (Proleukin; Chiron Corp, Emeryville, CA) was added.

    Techniques: Expressing, Isolation, Incubation

    IFN-α suppresses Treg expansion by inhibiting IL-2 production. (a) PBMC from three separated donors were activated with anti-CD3 without (control) or with 1000 U/ml of IFN-α, and IL-2 was determined in the supernatants by ELISA at 24 and

    Journal: Immunology

    Article Title: Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses

    doi: 10.1111/j.1365-2567.2010.03280.x

    Figure Lengend Snippet: IFN-α suppresses Treg expansion by inhibiting IL-2 production. (a) PBMC from three separated donors were activated with anti-CD3 without (control) or with 1000 U/ml of IFN-α, and IL-2 was determined in the supernatants by ELISA at 24 and

    Article Snippet: Where indicated, IFN-I [human leucocyte IFN (predominantly IFN-α) (R & D Systems, Minneapolis, MN) or human recombinant IFN-β 1b (PBL InterferonSource, Piscataway, NJ)] was added at 100–500 (IFN-β) or 100–1000 (IFN-α) units/ml, or 100 units/ml human recombinant IL-2 (Proleukin; Chiron Corp, Emeryville, CA) was added.

    Techniques: Enzyme-linked Immunosorbent Assay

    Macroscopic and microscopic appearance of a TARS-1 tumor. (a) Subcutaneous tumor at the injected site on day 14 following TARS-1 inoculation in an anti-CD80/CD86 MAb-treated rat (right) but not an untreated rat (left). (b) Regression of the tumor on day 35 in a rat treated with MAbs only during the initial 14 days (right). The TARS-1-inoculated MAb-untreated rat never developed a tumor (left). (c) Frozen section of subcutaneous tumor of a MAb-treated TARS-1-inoculated rat sacrificed on day 14, stained with MAb to IL-2 receptor (brown) and hematoxylin (blue). (d) Hematoxylin-and-eosin-stained frozen section of the lung of the rat shown in panel c.

    Journal: Journal of Virology

    Article Title: Development of Human T-Cell Leukemia Virus Type 1-Transformed Tumors in Rats following Suppression of T-Cell Immunity by CD80 and CD86 Blockade

    doi:

    Figure Lengend Snippet: Macroscopic and microscopic appearance of a TARS-1 tumor. (a) Subcutaneous tumor at the injected site on day 14 following TARS-1 inoculation in an anti-CD80/CD86 MAb-treated rat (right) but not an untreated rat (left). (b) Regression of the tumor on day 35 in a rat treated with MAbs only during the initial 14 days (right). The TARS-1-inoculated MAb-untreated rat never developed a tumor (left). (c) Frozen section of subcutaneous tumor of a MAb-treated TARS-1-inoculated rat sacrificed on day 14, stained with MAb to IL-2 receptor (brown) and hematoxylin (blue). (d) Hematoxylin-and-eosin-stained frozen section of the lung of the rat shown in panel c.

    Article Snippet: For induction of HTLV-1-specific CTL in long-term cultivation, splenic T cells (2.5 × 106 cells/well) were cocultured with the same number of MMC-treated TARS-1 cells in 10% FCS–RPMI 1640 in the presence of 20 U of recombinant human IL-2 (rhIL-2; Shionogi Pharmaceutical Co., Osaka, Japan) per ml, with periodical stimulation using MMC-treated TARS-1 cells every 2 weeks.

    Techniques: Injection, Staining

    Minimal recovery of T-cell response to TARS-1 by exogenously added IL-2. Splenic T cells were isolated from age-matched naive rats (a) or TARS-1-inoculated rats treated without (b) or with (c) anti-CD80/CD86 MAbs on day 14 and were cocultured without (□) or with MMC-treated W7KSV (▧) or TARS-1 (■) cells in the absence or presence of 50 or 100 U of rhIL-2 per ml for 4 days. Gray bars represent positive control wells containing concanavalin A (Con A; 10 μg/ml). [ 3 H]TdR incorporation was measured during the last 18 h. Data represent the mean ± SD of triplicate wells. Similar results were obtained in two independent experiments.

    Journal: Journal of Virology

    Article Title: Development of Human T-Cell Leukemia Virus Type 1-Transformed Tumors in Rats following Suppression of T-Cell Immunity by CD80 and CD86 Blockade

    doi:

    Figure Lengend Snippet: Minimal recovery of T-cell response to TARS-1 by exogenously added IL-2. Splenic T cells were isolated from age-matched naive rats (a) or TARS-1-inoculated rats treated without (b) or with (c) anti-CD80/CD86 MAbs on day 14 and were cocultured without (□) or with MMC-treated W7KSV (▧) or TARS-1 (■) cells in the absence or presence of 50 or 100 U of rhIL-2 per ml for 4 days. Gray bars represent positive control wells containing concanavalin A (Con A; 10 μg/ml). [ 3 H]TdR incorporation was measured during the last 18 h. Data represent the mean ± SD of triplicate wells. Similar results were obtained in two independent experiments.

    Article Snippet: For induction of HTLV-1-specific CTL in long-term cultivation, splenic T cells (2.5 × 106 cells/well) were cocultured with the same number of MMC-treated TARS-1 cells in 10% FCS–RPMI 1640 in the presence of 20 U of recombinant human IL-2 (rhIL-2; Shionogi Pharmaceutical Co., Osaka, Japan) per ml, with periodical stimulation using MMC-treated TARS-1 cells every 2 weeks.

    Techniques: Isolation, Positive Control

    Interleukin-15 (IL)-15 + alendronate (ALN) or IL-2 + ALN select for Jγ1.2 + chains, cytokine alone increases the proportion of Vγ2-Jγ1.2/2.3 + chains. (a) Vγ2 chain length distributions were determined by spectratyping for

    Journal: Immunology

    Article Title: Human cord blood ?? T cells expressing public V?2 chains dominate the response to bisphosphonate plus interleukin-15

    doi: 10.1111/imm.12039

    Figure Lengend Snippet: Interleukin-15 (IL)-15 + alendronate (ALN) or IL-2 + ALN select for Jγ1.2 + chains, cytokine alone increases the proportion of Vγ2-Jγ1.2/2.3 + chains. (a) Vγ2 chain length distributions were determined by spectratyping for

    Article Snippet: To expand Vγ2Vδ2 T lymphocytes, cultures were treated with 4-amino-1-hydroxy-1-phosphonobutyl phosphonic acid [alendronate sodium trihydrate (ALN); Sigma, St Louis, MO] at 5 μ m , in the presence of 100 IU/ml human recombinant IL-2 (Tecin, NHI reagent program; NIH, Bethesda, MD) or 10 ng/ml human recombinant IL-15 (ThermoScientific, Rockford, IL).

    Techniques:

    Interleukin-15 (IL-15) + Alendronate (ALN) or IL-2 + ALN preferentially expand cells expressing public Jγ1.2 + clonotypes, and reduce the proportion of public Jγ1.3/2.3 + clonotypes. The proportions of Vγ2 chains coding public clonotypes

    Journal: Immunology

    Article Title: Human cord blood ?? T cells expressing public V?2 chains dominate the response to bisphosphonate plus interleukin-15

    doi: 10.1111/imm.12039

    Figure Lengend Snippet: Interleukin-15 (IL-15) + Alendronate (ALN) or IL-2 + ALN preferentially expand cells expressing public Jγ1.2 + clonotypes, and reduce the proportion of public Jγ1.3/2.3 + clonotypes. The proportions of Vγ2 chains coding public clonotypes

    Article Snippet: To expand Vγ2Vδ2 T lymphocytes, cultures were treated with 4-amino-1-hydroxy-1-phosphonobutyl phosphonic acid [alendronate sodium trihydrate (ALN); Sigma, St Louis, MO] at 5 μ m , in the presence of 100 IU/ml human recombinant IL-2 (Tecin, NHI reagent program; NIH, Bethesda, MD) or 10 ng/ml human recombinant IL-15 (ThermoScientific, Rockford, IL).

    Techniques: Expressing

    Interleukin-15 (IL-15) sustains the survival of CB Vδ2 cell lines expanded with bisphosphonate. (a) Cord blood mononuclear cells (CBMC) were stimulated with alendronate (5 μ m ) + IL-2 (100 U/ml), Alendronate + IL-15 (10 ng/ml), or IL-2

    Journal: Immunology

    Article Title: Human cord blood ?? T cells expressing public V?2 chains dominate the response to bisphosphonate plus interleukin-15

    doi: 10.1111/imm.12039

    Figure Lengend Snippet: Interleukin-15 (IL-15) sustains the survival of CB Vδ2 cell lines expanded with bisphosphonate. (a) Cord blood mononuclear cells (CBMC) were stimulated with alendronate (5 μ m ) + IL-2 (100 U/ml), Alendronate + IL-15 (10 ng/ml), or IL-2

    Article Snippet: To expand Vγ2Vδ2 T lymphocytes, cultures were treated with 4-amino-1-hydroxy-1-phosphonobutyl phosphonic acid [alendronate sodium trihydrate (ALN); Sigma, St Louis, MO] at 5 μ m , in the presence of 100 IU/ml human recombinant IL-2 (Tecin, NHI reagent program; NIH, Bethesda, MD) or 10 ng/ml human recombinant IL-15 (ThermoScientific, Rockford, IL).

    Techniques:

    Interleukin -15 (IL-15) + ALN treatment elicits Vγ2Vδ2 T cells to produce tumour necrosis factor-α (TNFα) and mobilize granules in response to T-cell receptor (TCR) re-stimulation. Sixteen days after IL-15 + ALN or IL-2

    Journal: Immunology

    Article Title: Human cord blood ?? T cells expressing public V?2 chains dominate the response to bisphosphonate plus interleukin-15

    doi: 10.1111/imm.12039

    Figure Lengend Snippet: Interleukin -15 (IL-15) + ALN treatment elicits Vγ2Vδ2 T cells to produce tumour necrosis factor-α (TNFα) and mobilize granules in response to T-cell receptor (TCR) re-stimulation. Sixteen days after IL-15 + ALN or IL-2

    Article Snippet: To expand Vγ2Vδ2 T lymphocytes, cultures were treated with 4-amino-1-hydroxy-1-phosphonobutyl phosphonic acid [alendronate sodium trihydrate (ALN); Sigma, St Louis, MO] at 5 μ m , in the presence of 100 IU/ml human recombinant IL-2 (Tecin, NHI reagent program; NIH, Bethesda, MD) or 10 ng/ml human recombinant IL-15 (ThermoScientific, Rockford, IL).

    Techniques:

    Interleukin-15 (IL-15) or IL-2 up-regulate natural killer (NK) receptors on Vγ2Vδ2 T cells. CD56 expression was assessed (a) 14 or (b) 28 days after stimulation. Expression of (c) NKG2D and (d) NKG2A was analysed 14 days after stimulation.

    Journal: Immunology

    Article Title: Human cord blood ?? T cells expressing public V?2 chains dominate the response to bisphosphonate plus interleukin-15

    doi: 10.1111/imm.12039

    Figure Lengend Snippet: Interleukin-15 (IL-15) or IL-2 up-regulate natural killer (NK) receptors on Vγ2Vδ2 T cells. CD56 expression was assessed (a) 14 or (b) 28 days after stimulation. Expression of (c) NKG2D and (d) NKG2A was analysed 14 days after stimulation.

    Article Snippet: To expand Vγ2Vδ2 T lymphocytes, cultures were treated with 4-amino-1-hydroxy-1-phosphonobutyl phosphonic acid [alendronate sodium trihydrate (ALN); Sigma, St Louis, MO] at 5 μ m , in the presence of 100 IU/ml human recombinant IL-2 (Tecin, NHI reagent program; NIH, Bethesda, MD) or 10 ng/ml human recombinant IL-15 (ThermoScientific, Rockford, IL).

    Techniques: Expressing

    PD-L1-dependent suppression of T cell proliferation and cytokine production by PD-L1 expressing B cells from HC and untreated RA patients. CD19 + B cells, CD8 + and CD4 + T cells were magnetic bead-purified from PBMC of HC and untreated RA patients. CD19 + B cells were incubated with CpG+IL-2 for 72 h and then were washed and cultured 2:1 with autologous CFSE-labeled CD8 + or CD4 + T cells incubated in a plate-bound anti-CD3/anti-CD28 mAb in the presence of anti-PD-L1 mAb or control isotype. After 72 h of co-culture, T cell proliferation and intracellular cytokine production were analyzed by flow cytometry on CD3 + CD19 − lived cells. Percentage of proliferation, TNF and IFN-g production in (A) CD8 + T cells and (B) CD4 + T cells cultured alone or in the presence of CpG-activated CD19 + B cells with anti-PD-L1 mAb or control isotype. Mean ± SEM of five independent experiments (5 healthy controls and 5 untreated RA patients). * p

    Journal: Frontiers in Immunology

    Article Title: PD-L1+ Regulatory B Cells Are Significantly Decreased in Rheumatoid Arthritis Patients and Increase After Successful Treatment

    doi: 10.3389/fimmu.2018.02241

    Figure Lengend Snippet: PD-L1-dependent suppression of T cell proliferation and cytokine production by PD-L1 expressing B cells from HC and untreated RA patients. CD19 + B cells, CD8 + and CD4 + T cells were magnetic bead-purified from PBMC of HC and untreated RA patients. CD19 + B cells were incubated with CpG+IL-2 for 72 h and then were washed and cultured 2:1 with autologous CFSE-labeled CD8 + or CD4 + T cells incubated in a plate-bound anti-CD3/anti-CD28 mAb in the presence of anti-PD-L1 mAb or control isotype. After 72 h of co-culture, T cell proliferation and intracellular cytokine production were analyzed by flow cytometry on CD3 + CD19 − lived cells. Percentage of proliferation, TNF and IFN-g production in (A) CD8 + T cells and (B) CD4 + T cells cultured alone or in the presence of CpG-activated CD19 + B cells with anti-PD-L1 mAb or control isotype. Mean ± SEM of five independent experiments (5 healthy controls and 5 untreated RA patients). * p

    Article Snippet: Freshly isolated B cells were cultured in complete medium [RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml of penicillin, 100 ug/ml of streptomycin, 1 mM L-glutamine, 10 mM HEPES (all from Gibco) and 2-mercaptoethanol (Sigma)] for 3 days in 96-well plates at 1 × 105 B cells/well in the presence of recombinant human IL-2 (40 ng/ml; Biolegend) and CpG-ODN 2006 (1 ug/ml; Invivogen), at 37°C in a fully humidified atmosphere containing 5% CO2 .

    Techniques: Expressing, Purification, Incubation, Cell Culture, Labeling, Co-Culture Assay, Flow Cytometry, Cytometry

    B. bigemina RAP-1 CT-1-specific T cells respond to aa 418 to 480. Three CT-1-specific Th cell clones (A to C) and one RAP-1-specific Th cell clone specific for an epitope within aa 144 to 187 (D) were cultured in a 3-day proliferation assay with autologous APC, 2 U of recombinant human IL-2 per ml, and 1, 5, or 25 μg of recombinant MBP fusion proteins per ml, consisting of the whole RAP-1 protein (closed circles), the N terminal fragment of CT-1 (CT-1N) consisting of aa 386 to 448 (closed triangles), the C-terminal fragment of CT-1 (CT-1C) consisting of aa 418 to 480 (open triangles), or control MBP protein (open circles). Cells were radiolabeled for 6 h with [ 3 H]thymidine, harvested, and counted. The results are presented as the mean ± range of variation around the mean of duplicate cultures. Background proliferative responses of cells in medium plus IL-2 for the clones were as follows: 2216.2B2, 15,305 ± 639 cpm; 2216.1G8, 12,652 ± 2,376 cpm; 2234.1E3, 12,635 ± 775 cpm; and 2216.1H4, 16,353 ± 3,758 cpm.

    Journal: Infection and Immunity

    Article Title: Helper T-Cell Epitopes Encoded by the Babesia bigemina rap-1 Gene Family in the Constant and Variant Domains Are Conserved among Parasite Strains

    doi:

    Figure Lengend Snippet: B. bigemina RAP-1 CT-1-specific T cells respond to aa 418 to 480. Three CT-1-specific Th cell clones (A to C) and one RAP-1-specific Th cell clone specific for an epitope within aa 144 to 187 (D) were cultured in a 3-day proliferation assay with autologous APC, 2 U of recombinant human IL-2 per ml, and 1, 5, or 25 μg of recombinant MBP fusion proteins per ml, consisting of the whole RAP-1 protein (closed circles), the N terminal fragment of CT-1 (CT-1N) consisting of aa 386 to 448 (closed triangles), the C-terminal fragment of CT-1 (CT-1C) consisting of aa 418 to 480 (open triangles), or control MBP protein (open circles). Cells were radiolabeled for 6 h with [ 3 H]thymidine, harvested, and counted. The results are presented as the mean ± range of variation around the mean of duplicate cultures. Background proliferative responses of cells in medium plus IL-2 for the clones were as follows: 2216.2B2, 15,305 ± 639 cpm; 2216.1G8, 12,652 ± 2,376 cpm; 2234.1E3, 12,635 ± 775 cpm; and 2216.1H4, 16,353 ± 3,758 cpm.

    Article Snippet: In some experiments with T-cell clones, 1 or 2 U of recombinant human IL-2 (Boehringer Mannheim, Indianapolis, Ind.) per ml was added to all assay wells to amplify proliferation.

    Techniques: Clone Assay, Cell Culture, Proliferation Assay, Recombinant