anti cd3 anti cd28 dynabeads  (Thermo Fisher)


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    Dynabeads Human T Activator CD3 CD28 for T Cell Expansion and Activation
    Description:
    Dynabeads Human T Activator CD3 CD28 are for the activation and expansion of human T cells Advantages of Dynabeads Human T Activator CD3 CD28 • Activation of T cells without the need for feeder cells• Activated cells that retain in vivo like function• Maximum reproducibility without contamination by soluble antibodies or mitogens• From 100 to 1 000 fold T cell expansionAbout Dynabeads Human T Activator CD3 CD28Dynabeads Human T Activator CD3 CD28 offer a simple method for activation and expansion of T cells that does not require feeder cells antigen presenting cells or antigen The uniform 4 5 µm diameter inert superparamagnetic beads are similar in size to antigen presenting cells and are covalently coupled to anti CD3 and anti CD28 antibodies These two antibodies provide primary and co stimulatory signals optimized for efficient T cell activation and expansion Expansion of the T cell population can be stimulated using recombinant IL 2 and after activation or expansion the magnetic beads can be easily removed using a DynaMag magnet For expansion of antigen specific T cells from T cell clones or T cell lines we recommend using Dynabeads Human T Activator CD3 CD28 CD137 Starting samplesStart with mononuclear cells MNCs peripheral blood mononuclear cells PBMCs from either whole blood or buffy coat or T cell subsets including CD3 T cells CD4 or CD8 T cells or regulatory T cells Downstream applicationsThe activated T cells can be analyzed shortly after activation for transfection transduction or to study TCR signaling proteomics gene expression etc T cells can be left in culture to differentiate into T helper cell subsets or expansion of polyclonal antigen specific T cells Learn more about Dynabeads products• Find Dynabeads products for a whole range of applications • Find magnets for Dynabeads separations
    Catalog Number:
    11131D
    Price:
    None
    Category:
    Beads Microspheres
    Applications:
    Cell Activation & Expansion|Cell Analysis|Cell Isolation|Human Cell Activation & Expansion|Human Cell Isolation|Cell Isolation & Expansion
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    Structured Review

    Thermo Fisher anti cd3 anti cd28 dynabeads
    In vitro expanded CD8 + <t>CD28</t> − T cells suppress CD4 + T cells proliferation in a donor-specific manner. CD8 + T cells from donor A were stimulated with APCs from donor B plus γc cytokines for 9 days, followed by enrichment of the CD8 + CD28 − population as described in Materials and Methods . 5×10 4 CFSE labeled purified responder CD4 + T cells (R) from donor A were stimulated with 5×10 4 APCs from donor B (B- APC ) or from an indifferent donor (I- APC , HLA-A, B and DR fully mismatched with donor B), or 1×10 4 <t>anti-CD3/CD28</t> coated <t>Dynabeads</t> in triplicates in 96-well plates. The in vitro expanded CD8 + CD28 − T cells were added as putative suppressors (S) at S∶R ratios of 0.5∶1, 0.1∶1 and 0.02∶1 (with the cell number of “R” kept constant). Proliferation of the CD4 + cells was measured by CFSE dilution ( A ) or by 3 H thymidine uptake ( B ). Data shown are representative of three independent experiments. * P = 0.002; ** P = 0.009; *** P = 0.008.
    Dynabeads Human T Activator CD3 CD28 are for the activation and expansion of human T cells Advantages of Dynabeads Human T Activator CD3 CD28 • Activation of T cells without the need for feeder cells• Activated cells that retain in vivo like function• Maximum reproducibility without contamination by soluble antibodies or mitogens• From 100 to 1 000 fold T cell expansionAbout Dynabeads Human T Activator CD3 CD28Dynabeads Human T Activator CD3 CD28 offer a simple method for activation and expansion of T cells that does not require feeder cells antigen presenting cells or antigen The uniform 4 5 µm diameter inert superparamagnetic beads are similar in size to antigen presenting cells and are covalently coupled to anti CD3 and anti CD28 antibodies These two antibodies provide primary and co stimulatory signals optimized for efficient T cell activation and expansion Expansion of the T cell population can be stimulated using recombinant IL 2 and after activation or expansion the magnetic beads can be easily removed using a DynaMag magnet For expansion of antigen specific T cells from T cell clones or T cell lines we recommend using Dynabeads Human T Activator CD3 CD28 CD137 Starting samplesStart with mononuclear cells MNCs peripheral blood mononuclear cells PBMCs from either whole blood or buffy coat or T cell subsets including CD3 T cells CD4 or CD8 T cells or regulatory T cells Downstream applicationsThe activated T cells can be analyzed shortly after activation for transfection transduction or to study TCR signaling proteomics gene expression etc T cells can be left in culture to differentiate into T helper cell subsets or expansion of polyclonal antigen specific T cells Learn more about Dynabeads products• Find Dynabeads products for a whole range of applications • Find magnets for Dynabeads separations
    https://www.bioz.com/result/anti cd3 anti cd28 dynabeads/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
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    anti cd3 anti cd28 dynabeads - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Common Gamma Chain Cytokines Promote Rapid In Vitro Expansion of Allo-Specific Human CD8+ Suppressor T Cells"

    Article Title: Common Gamma Chain Cytokines Promote Rapid In Vitro Expansion of Allo-Specific Human CD8+ Suppressor T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028948

    In vitro expanded CD8 + CD28 − T cells suppress CD4 + T cells proliferation in a donor-specific manner. CD8 + T cells from donor A were stimulated with APCs from donor B plus γc cytokines for 9 days, followed by enrichment of the CD8 + CD28 − population as described in Materials and Methods . 5×10 4 CFSE labeled purified responder CD4 + T cells (R) from donor A were stimulated with 5×10 4 APCs from donor B (B- APC ) or from an indifferent donor (I- APC , HLA-A, B and DR fully mismatched with donor B), or 1×10 4 anti-CD3/CD28 coated Dynabeads in triplicates in 96-well plates. The in vitro expanded CD8 + CD28 − T cells were added as putative suppressors (S) at S∶R ratios of 0.5∶1, 0.1∶1 and 0.02∶1 (with the cell number of “R” kept constant). Proliferation of the CD4 + cells was measured by CFSE dilution ( A ) or by 3 H thymidine uptake ( B ). Data shown are representative of three independent experiments. * P = 0.002; ** P = 0.009; *** P = 0.008.
    Figure Legend Snippet: In vitro expanded CD8 + CD28 − T cells suppress CD4 + T cells proliferation in a donor-specific manner. CD8 + T cells from donor A were stimulated with APCs from donor B plus γc cytokines for 9 days, followed by enrichment of the CD8 + CD28 − population as described in Materials and Methods . 5×10 4 CFSE labeled purified responder CD4 + T cells (R) from donor A were stimulated with 5×10 4 APCs from donor B (B- APC ) or from an indifferent donor (I- APC , HLA-A, B and DR fully mismatched with donor B), or 1×10 4 anti-CD3/CD28 coated Dynabeads in triplicates in 96-well plates. The in vitro expanded CD8 + CD28 − T cells were added as putative suppressors (S) at S∶R ratios of 0.5∶1, 0.1∶1 and 0.02∶1 (with the cell number of “R” kept constant). Proliferation of the CD4 + cells was measured by CFSE dilution ( A ) or by 3 H thymidine uptake ( B ). Data shown are representative of three independent experiments. * P = 0.002; ** P = 0.009; *** P = 0.008.

    Techniques Used: In Vitro, Labeling, Purification

    2) Product Images from "Effects of wear particles of polyether-ether-ketone and cobalt-chromium-molybdenum on CD4- and CD8-T-cell responses"

    Article Title: Effects of wear particles of polyether-ether-ketone and cobalt-chromium-molybdenum on CD4- and CD8-T-cell responses

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23757

    CD4+ T-cell populations identified by immunostaining and flow cytometry in human peripheral blood ( A ) Raw data showing differences between PEEK and positive control groups in the induction of CD4+IL2+ and CD4+IL10+ T-cell responses. ( B) Th1, Th2, and Th17 cells are shown in the control group or the groups challenged with PEEK, and CoCrMo particles. The −Control group was unchallenged, resuspended in RPMI 1640 medium; the +Control group was treated with human T-activator CD3/28. Each data point represents the response of an individual participant; the bar indicates the mean value. * P
    Figure Legend Snippet: CD4+ T-cell populations identified by immunostaining and flow cytometry in human peripheral blood ( A ) Raw data showing differences between PEEK and positive control groups in the induction of CD4+IL2+ and CD4+IL10+ T-cell responses. ( B) Th1, Th2, and Th17 cells are shown in the control group or the groups challenged with PEEK, and CoCrMo particles. The −Control group was unchallenged, resuspended in RPMI 1640 medium; the +Control group was treated with human T-activator CD3/28. Each data point represents the response of an individual participant; the bar indicates the mean value. * P

    Techniques Used: Immunostaining, Flow Cytometry, Cytometry, Positive Control

    CD8+ T-cell populations identified by immunostaining and flow cytometry in human peripheral blood ( A ) Raw data showing differences between PEEK and CoCrMo particles in the induction of CD8+IL2+ and CD8+IL10+ T-cell responses. ( B ) CD8+IFNγ+/IL-2+/IL-10+/IL-17+ cells in the control groups or the groups challenged with PEEK, and CoCrMo particles. The −Control group was unchallenged, resuspended in RPMI 1640 medium; the +Control group was treated with human T-activator CD3/28. Each data point represents the response of an individual subject; the bar indicates the mean value. * P
    Figure Legend Snippet: CD8+ T-cell populations identified by immunostaining and flow cytometry in human peripheral blood ( A ) Raw data showing differences between PEEK and CoCrMo particles in the induction of CD8+IL2+ and CD8+IL10+ T-cell responses. ( B ) CD8+IFNγ+/IL-2+/IL-10+/IL-17+ cells in the control groups or the groups challenged with PEEK, and CoCrMo particles. The −Control group was unchallenged, resuspended in RPMI 1640 medium; the +Control group was treated with human T-activator CD3/28. Each data point represents the response of an individual subject; the bar indicates the mean value. * P

    Techniques Used: Immunostaining, Flow Cytometry, Cytometry

    3) Product Images from "Compromised Metabolic Reprogramming Is an Early Indicator of CD8+ T Cell Dysfunction during Chronic Mycobacterium tuberculosis Infection"

    Article Title: Compromised Metabolic Reprogramming Is an Early Indicator of CD8+ T Cell Dysfunction during Chronic Mycobacterium tuberculosis Infection

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.11.034

    Mtb Infection Suppresses Mitochondrial Respiration and Increases Glycolytic Dependency (A and B) Aerobic respiration within the mitochondria in the mammalian cell (A) and the cell mito stress test (CMST) profile generated during extracellular flux (XF) analysis (B). XF analysis was performed on purified lung CD8 + T cells during Mtb or BCG infection. (C) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) at baseline. (D–N) OCR measured during a CMST (D) with associated bioenergetic parameters (E) and corresponding phenogram after uncoupling with FCCP at D21 post-infection (F), D35 post-infection (G–J), and W12 post-infection (K–N). (O) CMST profile with/without anti-CD3/anti-CD28 activator beads. (P and Q) OCR and ECAR at baseline with/without activation (P) and phenogram before/after activation at baseline (Q), performed at D35 post-infection. (R) Glycolysis, measured by the change in ECAR pre- and post-injection with glucose. TB10.4-TET + cells are from Mtb -infected mice. A R, antimycin A and rotenone; F, carbonyl cyanide-4-[trifluoromethoxy] phenylhydrazone or FCCP; O, oligomycin;. Statistics, unless otherwise indicated, are relative to uninfected (UI). Data are representative of three independent experiments (n = 5 mice per group). “p ≤ 0.05, # p ≤ 0.01, ∞ p ≤ 0.005, and ∗ p ≤ 0.001 by one-way ANOVA. Error bars are mean ± SD.
    Figure Legend Snippet: Mtb Infection Suppresses Mitochondrial Respiration and Increases Glycolytic Dependency (A and B) Aerobic respiration within the mitochondria in the mammalian cell (A) and the cell mito stress test (CMST) profile generated during extracellular flux (XF) analysis (B). XF analysis was performed on purified lung CD8 + T cells during Mtb or BCG infection. (C) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) at baseline. (D–N) OCR measured during a CMST (D) with associated bioenergetic parameters (E) and corresponding phenogram after uncoupling with FCCP at D21 post-infection (F), D35 post-infection (G–J), and W12 post-infection (K–N). (O) CMST profile with/without anti-CD3/anti-CD28 activator beads. (P and Q) OCR and ECAR at baseline with/without activation (P) and phenogram before/after activation at baseline (Q), performed at D35 post-infection. (R) Glycolysis, measured by the change in ECAR pre- and post-injection with glucose. TB10.4-TET + cells are from Mtb -infected mice. A R, antimycin A and rotenone; F, carbonyl cyanide-4-[trifluoromethoxy] phenylhydrazone or FCCP; O, oligomycin;. Statistics, unless otherwise indicated, are relative to uninfected (UI). Data are representative of three independent experiments (n = 5 mice per group). “p ≤ 0.05, # p ≤ 0.01, ∞ p ≤ 0.005, and ∗ p ≤ 0.001 by one-way ANOVA. Error bars are mean ± SD.

    Techniques Used: Infection, Generated, Purification, Activation Assay, Injection, Mouse Assay

    4) Product Images from "Mechanism of splenic cell death and host mortality in a Plasmodium yoelii malaria model"

    Article Title: Mechanism of splenic cell death and host mortality in a Plasmodium yoelii malaria model

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10776-2

    Kinetic of immune cell populations and levels of cytokines and chemokines in the spleen of N67C-infected mice. Cell counts of red pulp macrophages (Mɸ) from uninfected (NI) and N67C-infected spleens days 2–6 p.i . (a) , of conventional dendritic cells (cDC) (b) , natural killer (NK) cells (c) , CD8 + T cells (d) , and CD4 + T cells (e), B lymphocytes (f) . Cells were counted using flow cytometry after staining with cell-specific markers. (g–l) Pro-inflammatory mediators in homogenates of spleens, measured using ELISA. (m) Representative flow cytometry plots of CD4 + CD44 hi and CD8 + CD44 hi splenic T cells of uninfected (NI) and N67C-infected mice expressing intracellular TNF-α and IFN-γ. Splenocytes were stimulated ex vivo with anti-CD3/CD28 in the presence of Brefeldin A before staining. dpi, day post infection. (n,o) Percentage of TNF-α or IFN-γ positive CD4+ and CD8+ splenic T cells day 2, 3 and 4 p.i ., after infection with N67C parasite. The results are expressed as mean ± SEM (3–5 mice) and are representative of two or three independent experiments. Kruskal-Wallis test, * p
    Figure Legend Snippet: Kinetic of immune cell populations and levels of cytokines and chemokines in the spleen of N67C-infected mice. Cell counts of red pulp macrophages (Mɸ) from uninfected (NI) and N67C-infected spleens days 2–6 p.i . (a) , of conventional dendritic cells (cDC) (b) , natural killer (NK) cells (c) , CD8 + T cells (d) , and CD4 + T cells (e), B lymphocytes (f) . Cells were counted using flow cytometry after staining with cell-specific markers. (g–l) Pro-inflammatory mediators in homogenates of spleens, measured using ELISA. (m) Representative flow cytometry plots of CD4 + CD44 hi and CD8 + CD44 hi splenic T cells of uninfected (NI) and N67C-infected mice expressing intracellular TNF-α and IFN-γ. Splenocytes were stimulated ex vivo with anti-CD3/CD28 in the presence of Brefeldin A before staining. dpi, day post infection. (n,o) Percentage of TNF-α or IFN-γ positive CD4+ and CD8+ splenic T cells day 2, 3 and 4 p.i ., after infection with N67C parasite. The results are expressed as mean ± SEM (3–5 mice) and are representative of two or three independent experiments. Kruskal-Wallis test, * p

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Ex Vivo

    5) Product Images from "Functional interrogation of primary human T cells via CRISPR genetic editing"

    Article Title: Functional interrogation of primary human T cells via CRISPR genetic editing

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

    doi: 10.4049/jimmunol.1701616

    Reduced apoptosis in CD95 -deleted Jurkat cells and primary human T cell subsets (A) Analysis of CD95-induced cell death in CD95 -deleted cells. Histogram overlays of Annexin V staining of CD95 + and CD95 − cells in activated versus non-activated Jurkat cells and primary CD4 + and CD8 + T cells. Jurkat or primary T cells were activated and transduced with lentivirus targeting the CD95 gene (gCD95) and primary T cells were expanded in IL-2, without selection, then reactivated with anti-CD95 crosslinking antibody or anti-CD3/anti-CD28, respectively, for 24 hours. Reactivated cells were analyzed for apoptosis by Annexin V staining. (B) T cell apoptosis after CD95 gene knockout. Cells were gated on either CD95 − or CD95 + 24 hours after reactivation as described in A.Data represent three independent experiments. Error bars represent SEM. ***p
    Figure Legend Snippet: Reduced apoptosis in CD95 -deleted Jurkat cells and primary human T cell subsets (A) Analysis of CD95-induced cell death in CD95 -deleted cells. Histogram overlays of Annexin V staining of CD95 + and CD95 − cells in activated versus non-activated Jurkat cells and primary CD4 + and CD8 + T cells. Jurkat or primary T cells were activated and transduced with lentivirus targeting the CD95 gene (gCD95) and primary T cells were expanded in IL-2, without selection, then reactivated with anti-CD95 crosslinking antibody or anti-CD3/anti-CD28, respectively, for 24 hours. Reactivated cells were analyzed for apoptosis by Annexin V staining. (B) T cell apoptosis after CD95 gene knockout. Cells were gated on either CD95 − or CD95 + 24 hours after reactivation as described in A.Data represent three independent experiments. Error bars represent SEM. ***p

    Techniques Used: Staining, Transduction, Selection, Gene Knockout

    Efficient and functional GARP expression using CRISPR-mediated promoter activation (A) GARP expression in Jurkat cells after CRISPR-mediated gene activation. Jurkat were first transduced with lentiviruses expressing dCas9 and p65 proteins, selected with hygromycin. Next the dCas9 and p65 expressing cells were transduced with lentiviruses expressing gRNAs targeting promoters regions of GARP (gGARP-g12) and selected with zeocin. GARP expression in Jurkat cells was analyzed after 11 days post transduction, compared to empty vector (control), after gating on GFP+ cells (marker for dCas9 expression). (B) LAP binding to GARP molecules on primary CD4 + T cells transduced with GARP-gRNA (g12) or empty vector (control). Primary CD4 + T cells were activated with anti-CD3/anti-CD28 beads, transduced with lentiviruses expressing dCas9 and p65 proteins and selected with hygromycin. After 2 weeks expansion, dCas9 and p65 expressing cells were then reactivated with anti-CD3/anti-CD28 beads and transduced with lentiviruses bearing the gRNAs targeting different promoter regions and selected with zeocin. Analysis was performed on T cells expanded for two weeks in culture post transduction and after gating on GFP+ T cells (as marker for dCas9+ population). (C) Percentage of GARP expression level in both Jurkat (as in Figure 5A) and primary CD4 + T cells(as in Figure 5B), transduced with 14 different gRNA clones targeting the GARP promoter in the range of −200 to 200 base pairs downstream and upstream of transcription start site (TSS). Background level of GARP expression from control was subtracted. Data represent three independent experiments. Error bars represent SEM. **p
    Figure Legend Snippet: Efficient and functional GARP expression using CRISPR-mediated promoter activation (A) GARP expression in Jurkat cells after CRISPR-mediated gene activation. Jurkat were first transduced with lentiviruses expressing dCas9 and p65 proteins, selected with hygromycin. Next the dCas9 and p65 expressing cells were transduced with lentiviruses expressing gRNAs targeting promoters regions of GARP (gGARP-g12) and selected with zeocin. GARP expression in Jurkat cells was analyzed after 11 days post transduction, compared to empty vector (control), after gating on GFP+ cells (marker for dCas9 expression). (B) LAP binding to GARP molecules on primary CD4 + T cells transduced with GARP-gRNA (g12) or empty vector (control). Primary CD4 + T cells were activated with anti-CD3/anti-CD28 beads, transduced with lentiviruses expressing dCas9 and p65 proteins and selected with hygromycin. After 2 weeks expansion, dCas9 and p65 expressing cells were then reactivated with anti-CD3/anti-CD28 beads and transduced with lentiviruses bearing the gRNAs targeting different promoter regions and selected with zeocin. Analysis was performed on T cells expanded for two weeks in culture post transduction and after gating on GFP+ T cells (as marker for dCas9+ population). (C) Percentage of GARP expression level in both Jurkat (as in Figure 5A) and primary CD4 + T cells(as in Figure 5B), transduced with 14 different gRNA clones targeting the GARP promoter in the range of −200 to 200 base pairs downstream and upstream of transcription start site (TSS). Background level of GARP expression from control was subtracted. Data represent three independent experiments. Error bars represent SEM. **p

    Techniques Used: Functional Assay, Expressing, CRISPR, Activation Assay, Transduction, Plasmid Preparation, Marker, Binding Assay, Clone Assay

    Suppression of Store-Operated Ca 2+ Entry (SOCE) and cytokine production in ORAI1-deleted human T cells (A) ORAI1 expression in Jurkat cells after CRISPR-mediated gene deletion. Jurkat cells were transduced with lentiviruses targeting the ORAI1 gene using two different gRNA clones (gORAI1-g1 or gORAI1-g2) and selected with puromycin for one week. ORAI1 protein expression was determined by anti-ORAI1 antibody staining. (B) Measurement of Ca 2+ influx in ORAI1 -deleted CD4 + T cells. CD4+ T cells were activated with anti-CD3/anti-CD28 beads and the next day transduced with gORAI1 lentiviruses. Puromycin was then added after 4 days post-activation and cells were selected and expanded for two weeks in culture. T cells were then loaded with Fura-2 and stimulated in 0 mM Ca 2+ Ringer solution with 1 μM thapsigargin (TG) to induce passive store depletion. Ca 2+ influx was measured after re-addition of 1 mM Ca 2+ to the extracellular buffer at 420 sec. T cells transduced with empty lentivectors (Control) was comparable to non-infected cells. (C) Cytokine expression in ORAI1 -deleted CD4 + T cells. Expression of TNF and IL-2 cytokines (top) by total CD4 + T cells, and IL-22 and IL-17 cytokines (bottom) by CD4 + CCR6 + T cells that were transduced with either empty-vector control or gORAI1-g1, expanded for 2 weeks after puromycin selection, and restimulated with PMA and Ionomycin for 4 hours in the presence of GolgiStop. Data is representative of three independent experiments. (D) Frequency of T cells expressing IFNγ, TNF, GM-CSF, IL-2, IL-17, and IL-22 analyzed from PMA and Ionomycin activation and intracellular staining as described in C. Data represent three independent experiments. Error bars represent SEM. ***p
    Figure Legend Snippet: Suppression of Store-Operated Ca 2+ Entry (SOCE) and cytokine production in ORAI1-deleted human T cells (A) ORAI1 expression in Jurkat cells after CRISPR-mediated gene deletion. Jurkat cells were transduced with lentiviruses targeting the ORAI1 gene using two different gRNA clones (gORAI1-g1 or gORAI1-g2) and selected with puromycin for one week. ORAI1 protein expression was determined by anti-ORAI1 antibody staining. (B) Measurement of Ca 2+ influx in ORAI1 -deleted CD4 + T cells. CD4+ T cells were activated with anti-CD3/anti-CD28 beads and the next day transduced with gORAI1 lentiviruses. Puromycin was then added after 4 days post-activation and cells were selected and expanded for two weeks in culture. T cells were then loaded with Fura-2 and stimulated in 0 mM Ca 2+ Ringer solution with 1 μM thapsigargin (TG) to induce passive store depletion. Ca 2+ influx was measured after re-addition of 1 mM Ca 2+ to the extracellular buffer at 420 sec. T cells transduced with empty lentivectors (Control) was comparable to non-infected cells. (C) Cytokine expression in ORAI1 -deleted CD4 + T cells. Expression of TNF and IL-2 cytokines (top) by total CD4 + T cells, and IL-22 and IL-17 cytokines (bottom) by CD4 + CCR6 + T cells that were transduced with either empty-vector control or gORAI1-g1, expanded for 2 weeks after puromycin selection, and restimulated with PMA and Ionomycin for 4 hours in the presence of GolgiStop. Data is representative of three independent experiments. (D) Frequency of T cells expressing IFNγ, TNF, GM-CSF, IL-2, IL-17, and IL-22 analyzed from PMA and Ionomycin activation and intracellular staining as described in C. Data represent three independent experiments. Error bars represent SEM. ***p

    Techniques Used: Expressing, CRISPR, Transduction, Clone Assay, Staining, Activation Assay, Size-exclusion Chromatography, Infection, Plasmid Preparation, Selection

    Functional CD127 and CD25 gene expression on primary CD4 + T cells using CRISPR-mediated promoter activation (A) Expression of CD127 (top left) and CD25 (bottom left) in primary CD4 + T cells transduced with gRNA targeting CD127 or CD25 promoter. Primary CD4 + T cells were activated with anti-CD3/anti-CD28 beads, transduced with lentiviruses expressing dCas9 and p65 proteins and selected with hygromycin. After 2 weeks expansion, dCas9 and p65 expressing cells were then reactivated with anti-CD3/anti-CD28 beads and transduced with lentiviruses targeting CD127 (gCD127) or CD25 (gCD25) promoters, selected with zeocin and expanded for 2 weeks. The histograms show expressions after gating on GFP + (gray histogram) and GFP − cells (empty histogram), GFP being the marker for dCas9 expression. Percentgate of CD127 (top right) or CD25 (bottom right) expression in primary CD4 + T cells transduced with CD25− or CD127− promoter targeting gRNAs, between GFP + or GFP − population (as in Figure 6A). Data represent three independent experiments. Error bars represent SEM. ***p
    Figure Legend Snippet: Functional CD127 and CD25 gene expression on primary CD4 + T cells using CRISPR-mediated promoter activation (A) Expression of CD127 (top left) and CD25 (bottom left) in primary CD4 + T cells transduced with gRNA targeting CD127 or CD25 promoter. Primary CD4 + T cells were activated with anti-CD3/anti-CD28 beads, transduced with lentiviruses expressing dCas9 and p65 proteins and selected with hygromycin. After 2 weeks expansion, dCas9 and p65 expressing cells were then reactivated with anti-CD3/anti-CD28 beads and transduced with lentiviruses targeting CD127 (gCD127) or CD25 (gCD25) promoters, selected with zeocin and expanded for 2 weeks. The histograms show expressions after gating on GFP + (gray histogram) and GFP − cells (empty histogram), GFP being the marker for dCas9 expression. Percentgate of CD127 (top right) or CD25 (bottom right) expression in primary CD4 + T cells transduced with CD25− or CD127− promoter targeting gRNAs, between GFP + or GFP − population (as in Figure 6A). Data represent three independent experiments. Error bars represent SEM. ***p

    Techniques Used: Functional Assay, Expressing, CRISPR, Activation Assay, Transduction, Marker

    CRISPR-mediated deletion of single or multiple genes in primary human T cells (A) CD4 and CD45 expression in purified human CD4 + T cells. CD4 + T cells were activated with anti-CD3/anti-CD28 beads and the next day transduced with entiviruses targeting CD45 gene (gCD45-g2), as described in methods. Cells were then expanded in IL-2 and stained at indicated time points with CD4 and CD45 antibodies. (B) Comparison of knockout efficiency using three different gRNAs targeting CD4 or CD45 gene loci in human CD4 + T cells with or without puromycin selection. Puromycin (0.4 μg/ml) was added at day 3 post-transduction to a portion of activated T cells infected with the lentiviruses. Cells were then stained for CD4 or CD45 expresssion at day 7 post-transduction (day 4 post-selection). Data represent three independent experiments with cells isolated from different donors. Error bars represent SEM. ***p
    Figure Legend Snippet: CRISPR-mediated deletion of single or multiple genes in primary human T cells (A) CD4 and CD45 expression in purified human CD4 + T cells. CD4 + T cells were activated with anti-CD3/anti-CD28 beads and the next day transduced with entiviruses targeting CD45 gene (gCD45-g2), as described in methods. Cells were then expanded in IL-2 and stained at indicated time points with CD4 and CD45 antibodies. (B) Comparison of knockout efficiency using three different gRNAs targeting CD4 or CD45 gene loci in human CD4 + T cells with or without puromycin selection. Puromycin (0.4 μg/ml) was added at day 3 post-transduction to a portion of activated T cells infected with the lentiviruses. Cells were then stained for CD4 or CD45 expresssion at day 7 post-transduction (day 4 post-selection). Data represent three independent experiments with cells isolated from different donors. Error bars represent SEM. ***p

    Techniques Used: CRISPR, Expressing, Purification, Transduction, Staining, Knock-Out, Selection, Infection, Isolation

    6) Product Images from "Human iPSC-Derived Neural Crest Stem Cells Exhibit Low Immunogenicity"

    Article Title: Human iPSC-Derived Neural Crest Stem Cells Exhibit Low Immunogenicity

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.12.015

    IPSC-NCSCs Are Not Immunosuppressive (A) Schematic outline of transwell assay format. (B) Representative flow cytometry histograms show T cell proliferation, detected by quantifying levels of CFSE by flow cytometry. Anti-CD3/anti-CD28-stimulated PBMCs were co-cultured with BM-MSCs, NIBSC8-MSCs, and NIBSC8-NCSCs, and reduction of T cell proliferation was measured by quantifying CFSE low cells (shown in pink) in total CD3 + T cells and CD3 + CD8 + T cells. (C) Graphs show relative total CD3 + T cell and CD3 + CD8 + T cell proliferation in a transwell assay format (tw) and without transwell inserts (w/o tw). Bars represent proliferation in PBMCs only, in stimulated (stim) PBMCs only, and in stim PBMCs in response to co-culture with BM-MSCs, NIBSC8-MSCs, and NIBSC8-NCSCs relative to stim PBMCs. Error bars represent ± SEM (w/o tw: n = 9 biological replicates in three separate experiments; with tw: n = 3 biological replicates in a single experiment). Ordinary one-way ANOVA with Bonferroni post-test was performed. Asterisks denote statistical significance in comparison with stim PBMCs: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.
    Figure Legend Snippet: IPSC-NCSCs Are Not Immunosuppressive (A) Schematic outline of transwell assay format. (B) Representative flow cytometry histograms show T cell proliferation, detected by quantifying levels of CFSE by flow cytometry. Anti-CD3/anti-CD28-stimulated PBMCs were co-cultured with BM-MSCs, NIBSC8-MSCs, and NIBSC8-NCSCs, and reduction of T cell proliferation was measured by quantifying CFSE low cells (shown in pink) in total CD3 + T cells and CD3 + CD8 + T cells. (C) Graphs show relative total CD3 + T cell and CD3 + CD8 + T cell proliferation in a transwell assay format (tw) and without transwell inserts (w/o tw). Bars represent proliferation in PBMCs only, in stimulated (stim) PBMCs only, and in stim PBMCs in response to co-culture with BM-MSCs, NIBSC8-MSCs, and NIBSC8-NCSCs relative to stim PBMCs. Error bars represent ± SEM (w/o tw: n = 9 biological replicates in three separate experiments; with tw: n = 3 biological replicates in a single experiment). Ordinary one-way ANOVA with Bonferroni post-test was performed. Asterisks denote statistical significance in comparison with stim PBMCs: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

    Techniques Used: Transwell Assay, Flow Cytometry, Cell Culture, Co-Culture Assay

    7) Product Images from "Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy"

    Article Title: Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161820

    Cell expansion with gamma-irradiated K562a cells and characterization of expanded cells. (A) Expansion fold based on total lymphocyte count after treatment with CD3/CD28 beads, CIK, Zometa (ZOL) or CIKZ methods. PBMCs (2 x 10 6 per well in a 6-well plate) were first treated with the four methods for 7 days, then 1 x 10 5 treated cells were mixed with K562a cells at a 1:200 ratio in a T75 flask and expanded for another 14 days. Fold expansion of total cells was determined on days 7 and 21. Data represent the calculated total cell expansion folds based on the actual expansion rates of total cells from Day 0 to Day 7 and Day 7 to Day 21. (B) Frequencies of αβ T, γδ T and NK cells on days 0 and 21. (C) Frequencies of CD8 + and CD4 + T cells on days 0 and 21. (D) Phenotypic analysis of CD8 + and CD4 + T cells. TN, TCM, TEM and TEFF: Naïve, central-memory, effector-memory, and effector CD8 + T cells, respectively. PD1: Immune exhaustion marker. Treg: CD4 + regulatory T cells. Data are displayed as Mean ± SD of total cell expansion fold at the end of the 21-day culture from four donors.
    Figure Legend Snippet: Cell expansion with gamma-irradiated K562a cells and characterization of expanded cells. (A) Expansion fold based on total lymphocyte count after treatment with CD3/CD28 beads, CIK, Zometa (ZOL) or CIKZ methods. PBMCs (2 x 10 6 per well in a 6-well plate) were first treated with the four methods for 7 days, then 1 x 10 5 treated cells were mixed with K562a cells at a 1:200 ratio in a T75 flask and expanded for another 14 days. Fold expansion of total cells was determined on days 7 and 21. Data represent the calculated total cell expansion folds based on the actual expansion rates of total cells from Day 0 to Day 7 and Day 7 to Day 21. (B) Frequencies of αβ T, γδ T and NK cells on days 0 and 21. (C) Frequencies of CD8 + and CD4 + T cells on days 0 and 21. (D) Phenotypic analysis of CD8 + and CD4 + T cells. TN, TCM, TEM and TEFF: Naïve, central-memory, effector-memory, and effector CD8 + T cells, respectively. PD1: Immune exhaustion marker. Treg: CD4 + regulatory T cells. Data are displayed as Mean ± SD of total cell expansion fold at the end of the 21-day culture from four donors.

    Techniques Used: Irradiation, Transmission Electron Microscopy, Marker

    Phenotyping of NKT cell populations and tumor-targeting efficiencies of expanded CIKZ cells. (A) Frequencies of the total CD3 + CD56 + NKT cells were examined by flow cytometry before and after cell expansion with CD3/CD28 beads, CIK, Zometa (ZOL) or CIKZ methods. (B) The detailed phenotypic analysis of CD3 + CD56 + T cells after gating with γδ T, CD4 + T and CD8 + T cell markers. (C) Cytolytic activities of the cells expanded with CIK, ZOL and CIKZ methods against Raji human malignant Burkitt lymphoma cell line. Effector and target cells were co-cultured at the indicated cell ratios for 3 hours before measuring cytolytic activities. (D) In vivo effects of CIKZ cells on Raji tumor growth. Tumor volumes and weights of in Raji tumor-bearing mice sacrificed 30 days after i.p. tumor inoculation. P value of tumor volume was found to be p
    Figure Legend Snippet: Phenotyping of NKT cell populations and tumor-targeting efficiencies of expanded CIKZ cells. (A) Frequencies of the total CD3 + CD56 + NKT cells were examined by flow cytometry before and after cell expansion with CD3/CD28 beads, CIK, Zometa (ZOL) or CIKZ methods. (B) The detailed phenotypic analysis of CD3 + CD56 + T cells after gating with γδ T, CD4 + T and CD8 + T cell markers. (C) Cytolytic activities of the cells expanded with CIK, ZOL and CIKZ methods against Raji human malignant Burkitt lymphoma cell line. Effector and target cells were co-cultured at the indicated cell ratios for 3 hours before measuring cytolytic activities. (D) In vivo effects of CIKZ cells on Raji tumor growth. Tumor volumes and weights of in Raji tumor-bearing mice sacrificed 30 days after i.p. tumor inoculation. P value of tumor volume was found to be p

    Techniques Used: Flow Cytometry, Cytometry, Cell Culture, In Vivo, Mouse Assay

    8) Product Images from "Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells"

    Article Title: Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells

    Journal: bioRxiv

    doi: 10.1101/2020.09.08.286914

    ASAP-seq and CITE-seq reveal coordinated and distinct changes in chromatin, RNA, and protein levels. a. Schematic of the experimental design. PBMCs were incubated with (stimulation) or without (control) multimeric a-CD3/CD28 for 16 hrs, followed by staining with the 227 antibody panel. An aliquot of the cells was withdrawn and subjected to CITE-seq, while the remaining cells were fixed and subjected to ASAP-seq. b,c. Reduced dimension representations using data integration methods and UMAP for ( b ) ASAP-seq and ( c ) CITE-seq for both control (left) and stimulated conditions (right). d. Correlation of surface marker fold changes (log 2 ) upon stimulation as detected by CITE-seq and ASAP-seq. Top upregulated markers are highlighted in red, and downregulated in blue. e. Schematic and summary of number and proportion of differential features (chromatin accessibility peaks, genes, and surface proteins) detected for T cells between the stimulation and control. f. Summary of changes in chromatin accessibility, gene expression and surface protein abundance for 84 expressed genes during T cell stimulation. g. Pearson correlation between the log 2 fold changes for each modality as shown in ( f ). h-j. UMAPs of single-cell chromatin accessibility, mRNA expression, and surface protein levels for both the control (top) and stimulation condition (bottom) shown on the reduced dimension space for ( h ) CD3, ( i ) CD69 and ( j ) PD-1.
    Figure Legend Snippet: ASAP-seq and CITE-seq reveal coordinated and distinct changes in chromatin, RNA, and protein levels. a. Schematic of the experimental design. PBMCs were incubated with (stimulation) or without (control) multimeric a-CD3/CD28 for 16 hrs, followed by staining with the 227 antibody panel. An aliquot of the cells was withdrawn and subjected to CITE-seq, while the remaining cells were fixed and subjected to ASAP-seq. b,c. Reduced dimension representations using data integration methods and UMAP for ( b ) ASAP-seq and ( c ) CITE-seq for both control (left) and stimulated conditions (right). d. Correlation of surface marker fold changes (log 2 ) upon stimulation as detected by CITE-seq and ASAP-seq. Top upregulated markers are highlighted in red, and downregulated in blue. e. Schematic and summary of number and proportion of differential features (chromatin accessibility peaks, genes, and surface proteins) detected for T cells between the stimulation and control. f. Summary of changes in chromatin accessibility, gene expression and surface protein abundance for 84 expressed genes during T cell stimulation. g. Pearson correlation between the log 2 fold changes for each modality as shown in ( f ). h-j. UMAPs of single-cell chromatin accessibility, mRNA expression, and surface protein levels for both the control (top) and stimulation condition (bottom) shown on the reduced dimension space for ( h ) CD3, ( i ) CD69 and ( j ) PD-1.

    Techniques Used: Incubation, Staining, Marker, Expressing, Cell Stimulation

    Related Articles

    Expressing:

    Article Title: Decreased sensitivity to 1,25-dihydroxyvitamin D3 in T cells from the rheumatoid joint
    Article Snippet: For experiments using isolated CD45RA + CD4+ naïve T cells, CD45RO + CD4+ memory T cells and CD14 + monocytes, cells were enriched by negative selection using cell separation reagents (StemCell Technologies and Biolegend). .. For 24 h post-stimulation analysis of gene expression, T cells were stimulated with anti-CD3/CD28 dynabeads (Life Technologies) at a ratio of 1 bead: 2 T cells in medium supplemented with 5% human AB serum (TCS Biosciences, Buckingham UK). ..

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Negative Control:

    Article Title: Effects of wear particles of polyether-ether-ketone and cobalt-chromium-molybdenum on CD4- and CD8-T-cell responses
    Article Snippet: The PBMCs were separated into five groups (two control groups and three experimental groups), and wear debris was added at a concentration of 20 particles/PBMC [ ]. .. The negative control was unchallenged, being resuspended in RPMI 1640 medium (Thermo Fisher Scientific), and the positive control group was treated with human T-activator CD3/28 (Thermo Fisher Scientific), which is known to activate human T-cells. .. The PBMCs were incubated at 37°C and 5% CO2 for 72 h, and then analyzed by flow cytometry as described below.

    Positive Control:

    Article Title: Effects of wear particles of polyether-ether-ketone and cobalt-chromium-molybdenum on CD4- and CD8-T-cell responses
    Article Snippet: The PBMCs were separated into five groups (two control groups and three experimental groups), and wear debris was added at a concentration of 20 particles/PBMC [ ]. .. The negative control was unchallenged, being resuspended in RPMI 1640 medium (Thermo Fisher Scientific), and the positive control group was treated with human T-activator CD3/28 (Thermo Fisher Scientific), which is known to activate human T-cells. .. The PBMCs were incubated at 37°C and 5% CO2 for 72 h, and then analyzed by flow cytometry as described below.

    Selection:

    Article Title: Ly6C defines a subset of memory-like CD27+ γδ T cells with inducible cancer-killing function
    Article Snippet: In brief, CD3+ T cells were enriched by negative selection on a LD column and QuadroMACS separator (Miltenyi Biotec), before positive selection of γδ T cells using MS columns with an OctoMacs separator (Miltenyi Biotec). .. To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio. .. Cells were kept in 96 U-well plates (Greiner Bio-One) in normoxic incubators at 37°C and propagated at a density of 4×104 cells per well.

    Cell Culture:

    Article Title: Ly6C defines a subset of memory-like CD27+ γδ T cells with inducible cancer-killing function
    Article Snippet: In brief, CD3+ T cells were enriched by negative selection on a LD column and QuadroMACS separator (Miltenyi Biotec), before positive selection of γδ T cells using MS columns with an OctoMacs separator (Miltenyi Biotec). .. To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio. .. Cells were kept in 96 U-well plates (Greiner Bio-One) in normoxic incubators at 37°C and propagated at a density of 4×104 cells per well.

    Activation Assay:

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Staining:

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Magnetic Beads:

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Incubation:

    Article Title: Antigen-specific culture of memory-like CD8 T cells for adoptive immunotherapy
    Article Snippet: Antigen-specific cells were enriched as previously described ( ) from freshly isolated lymph node and spleen cells (mouse) or overnight incubated PBMCs (human) after staining with dextramers according to manufacturer's instructions (Immudex). .. T cells were incubated mixed with peptide pulsed dendritic cells at a ratio of 2:1 or CD3/CD28 beads (Invitrogen) at a ratio of 1:1 and plated at a density of 10,000-20,000 T cells per well of round bottom 96-well plates in a volume of 150-200 μL per well. .. Fresh media containing the same concentration of cytokines and drugs was added to each well at half the volume initially plated after 3-4 days.

    Isolation:

    Article Title: PRMT5 regulates T cell interferon response and is a target for acute graft-versus-host disease
    Article Snippet: T cells were lysed in RIPA buffer, and Western blot was performed according to standard protocols. .. For time course experiments, T cells were isolated from mouse spleen or human T cells were isolated from healthy donor leukopaks and stimulated using CD3/CD28 Dynabeads according to the manufacturer’s protocol (Invitrogen, Thermo Fisher Scientific). .. Cells were lysed in RIPA buffer and immunoblotted using primary Abs against PRMT5 (Abcam), H3R8me2s, and H3R8me2a (EpiGentek polyclonal antibody, catalog A-3716-100). (See for a complete list of antibodies.)

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    Thermo Fisher cd3 cd28 beads
    HDACi exposure does not significantly increase expression of FOXP3 in human Tregs, and FOXP3 expression depends upon level of IL-2. (a) qPCR analysis of FOXP3 mRNA in fresh isolated (left) or expanded (middle) Tregs stimulated with <t>CD3/CD28</t> mAb-coated
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    HDACi exposure does not significantly increase expression of FOXP3 in human Tregs, and FOXP3 expression depends upon level of IL-2. (a) qPCR analysis of FOXP3 mRNA in fresh isolated (left) or expanded (middle) Tregs stimulated with CD3/CD28 mAb-coated

    Journal: Clinical immunology (Orlando, Fla.)

    Article Title: Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs 1

    doi: 10.1016/j.clim.2010.04.018

    Figure Lengend Snippet: HDACi exposure does not significantly increase expression of FOXP3 in human Tregs, and FOXP3 expression depends upon level of IL-2. (a) qPCR analysis of FOXP3 mRNA in fresh isolated (left) or expanded (middle) Tregs stimulated with CD3/CD28 mAb-coated

    Article Snippet: Tregs and Teffs were stimulated with CD3/CD28 beads (Dynabeads, Invitrogen) for 2, 4, 6, 21 or 24 h, 3 d or 5 d in the presence or absence of HDACi.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation

    FOXP3, CD25, CTLA4 expression and IL-2 production in CD4+ cells after stimulation with or without HDACi. Human PBMC were stimulated for 24 h with CD3/CD28 mAb-coated beads ± addition of HDACi (two experiments with valproic acid and bufexamac were

    Journal: Clinical immunology (Orlando, Fla.)

    Article Title: Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs 1

    doi: 10.1016/j.clim.2010.04.018

    Figure Lengend Snippet: FOXP3, CD25, CTLA4 expression and IL-2 production in CD4+ cells after stimulation with or without HDACi. Human PBMC were stimulated for 24 h with CD3/CD28 mAb-coated beads ± addition of HDACi (two experiments with valproic acid and bufexamac were

    Article Snippet: Tregs and Teffs were stimulated with CD3/CD28 beads (Dynabeads, Invitrogen) for 2, 4, 6, 21 or 24 h, 3 d or 5 d in the presence or absence of HDACi.

    Techniques: Expressing

    Serial expression of HDAC mRNA by human Tregs (black) or Teffs (grey) before and after CD3/CD28 stimulation. Freshly isolated CD4+CD25+CD127- Tregs and CD4+CD25- Teffs from the same donor were stimulated for the periods shown and HDAC gene expression

    Journal: Clinical immunology (Orlando, Fla.)

    Article Title: Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs 1

    doi: 10.1016/j.clim.2010.04.018

    Figure Lengend Snippet: Serial expression of HDAC mRNA by human Tregs (black) or Teffs (grey) before and after CD3/CD28 stimulation. Freshly isolated CD4+CD25+CD127- Tregs and CD4+CD25- Teffs from the same donor were stimulated for the periods shown and HDAC gene expression

    Article Snippet: Tregs and Teffs were stimulated with CD3/CD28 beads (Dynabeads, Invitrogen) for 2, 4, 6, 21 or 24 h, 3 d or 5 d in the presence or absence of HDACi.

    Techniques: Expressing, Isolation

    MUC1 peptides couple to anti-human DNGR-1 can induce CD8 + T cells in vitro. BDCA-3 + cells were isolated from PBMCs and incubated with the LLL peptide coupled to anti-human DNGR-1 or isotype control, in the presence of anti-CD40 and poly-IC, for 1 hour before incubating with autologous T cells for 7 days. The cells were then pulsed with peptide following a further 7 days incubation stimulated with autologous monocyte-derived DCs pulsed with peptide. (A) Seven days later, the cells were pulsed for 4 hour with LLL peptide and IFN-γ secretion by CD8 + T cells measured by IFN-γ Secretion Assay. An aliquot of anti-DNGR-1-peptide stimulated cells was incubated for 4 hour with 5 μg/mL PHA and 1 μg/mL anti-human CD3 (Control DNGR-1). Data shown are representative of four independent experiments. (B, C) Cells were also pulsed with (B) MCF-7 cells and (C) Capan-1 cells and IFN-γ production was measured by intracellular cytokine staining. An aliquot of either isotype control (Control Ig) or anti-DNGR-1 (Control DNGR-1) stimulated cells was incubated for 4 hour with PBMC:Dynabeads® human T-Expander CD3/CD28. All the cells were analysed after gating on live cells. (D, E) Expression of endogenous MUC1 by (D) Capan-1 cells and (E) MCF-7 cells was determined by HMFG2 binding (black-line histogram) as assessed by flow cytometry. (Filled histogram, isotype control.)

    Journal: European Journal of Immunology

    Article Title: Targeting DNGR-1 (CLEC9A) with antibody/MUC1 peptide conjugates as a vaccine for carcinomas

    doi: 10.1002/eji.201344076

    Figure Lengend Snippet: MUC1 peptides couple to anti-human DNGR-1 can induce CD8 + T cells in vitro. BDCA-3 + cells were isolated from PBMCs and incubated with the LLL peptide coupled to anti-human DNGR-1 or isotype control, in the presence of anti-CD40 and poly-IC, for 1 hour before incubating with autologous T cells for 7 days. The cells were then pulsed with peptide following a further 7 days incubation stimulated with autologous monocyte-derived DCs pulsed with peptide. (A) Seven days later, the cells were pulsed for 4 hour with LLL peptide and IFN-γ secretion by CD8 + T cells measured by IFN-γ Secretion Assay. An aliquot of anti-DNGR-1-peptide stimulated cells was incubated for 4 hour with 5 μg/mL PHA and 1 μg/mL anti-human CD3 (Control DNGR-1). Data shown are representative of four independent experiments. (B, C) Cells were also pulsed with (B) MCF-7 cells and (C) Capan-1 cells and IFN-γ production was measured by intracellular cytokine staining. An aliquot of either isotype control (Control Ig) or anti-DNGR-1 (Control DNGR-1) stimulated cells was incubated for 4 hour with PBMC:Dynabeads® human T-Expander CD3/CD28. All the cells were analysed after gating on live cells. (D, E) Expression of endogenous MUC1 by (D) Capan-1 cells and (E) MCF-7 cells was determined by HMFG2 binding (black-line histogram) as assessed by flow cytometry. (Filled histogram, isotype control.)

    Article Snippet: As a positive control for the assay, an aliquot of T cells was incubated with PBMC:Dynabeads® human T-Expander CD3/CD28 (Life Technologies) at a 1:1 ratio (cell:bead) instead of cancer cells.

    Techniques: In Vitro, Isolation, Incubation, Derivative Assay, Staining, Expressing, Binding Assay, Flow Cytometry, Cytometry

    miR-27a influences proliferation and IFN- γ production by CD4 + T cells in ex vivo experiments upon drug-mediated ICD. The CM from S-, AS- or C-transfected and treated HCT116 alone ( a ) or from co-cultures of the same transfected and pretreated cells cells with hu-iDCs ( b ) were added to naïve, freshly isolated CD4 + T cells either unstimulated or stimulated with anti-CD3/CD28 dynabeads to test their ability to proliferate. Samples were analyzed in triplicate and data are mean±S.D. and representative of three experiments. * P ⩽0.05 (two-tailed Student's t -test). ( c ) To analyze the production of IFN- γ , freshly isolated CD4 + T cells were cultured overnight in the presence or absence of anti-CD3/CD28 dynabeads and exposed to CMs from co-cultures as in ( b ). Samples were analyzed in triplicate and data are mean±S.D. and representative of three experiments. * P ⩽0.05; ** P ⩽0.01 (two-tailed Student's t -test)

    Journal: Cell Death & Disease

    Article Title: The miR-27a-calreticulin axis affects drug-induced immunogenic cell death in human colorectal cancer cells

    doi: 10.1038/cddis.2016.29

    Figure Lengend Snippet: miR-27a influences proliferation and IFN- γ production by CD4 + T cells in ex vivo experiments upon drug-mediated ICD. The CM from S-, AS- or C-transfected and treated HCT116 alone ( a ) or from co-cultures of the same transfected and pretreated cells cells with hu-iDCs ( b ) were added to naïve, freshly isolated CD4 + T cells either unstimulated or stimulated with anti-CD3/CD28 dynabeads to test their ability to proliferate. Samples were analyzed in triplicate and data are mean±S.D. and representative of three experiments. * P ⩽0.05 (two-tailed Student's t -test). ( c ) To analyze the production of IFN- γ , freshly isolated CD4 + T cells were cultured overnight in the presence or absence of anti-CD3/CD28 dynabeads and exposed to CMs from co-cultures as in ( b ). Samples were analyzed in triplicate and data are mean±S.D. and representative of three experiments. * P ⩽0.05; ** P ⩽0.01 (two-tailed Student's t -test)

    Article Snippet: Cells were stimulated for 3 days with an anti-CD3/CD28 Dynabeads (0.1 bead/cell) (Thermo Fisher) in the presence or absence of the CM obtained either from co-cultures, as reported above, or from HCT116 or RKO cells transfected with the miR-27a S, AS or C RNA and treated with MTX or OXP.

    Techniques: Ex Vivo, Transfection, Isolation, Two Tailed Test, Cell Culture

    TIGIT expressing CD8 + T cells have impaired cytokine responses. Representative flow cytometry plots gated on CD8 + T cells showing (A) TIGIT or (C) PD-1 expression against Ki-67 from a chronically HIV-infected individual. Compiled data of Ki-67 + CD8 + T cell frequency (%) separated into (B) TIGIT + and TIGIT - or (D) PD-1 + and PD-1 - ( n = 20). P values were calculated by Wilcoxon matched-pairs signed ranked test. Ex vivo PBMCs from chronically HIV-infected individuals were stimulated with HIV Gag peptide pool and assessed for cytokine production. (E) Representative flow cytometry plots gated on CD8 + T cells showing TIGIT expression and either IFN-γ, IL-2, or TNF-α content after no stimulation, stimulation with an HIV-1 Gag peptide pool, or a positive control stimulation with anti-CD3 + anti-CD28 Dynabeads. (F) Compiled data of IFN-γ, IL-2, or TNF-α CD8 + T cell frequency (%) from TIGIT + or TIGIT - CD8 + T cell compartments after HIV-1 Gag peptide pool stimulation (sample group includes; AS n = 4, EC n = 3, NC n = 3). P values were calculated by Wilcoxon matched-pairs signed ranked test. (G) Compiled data of TIGIT and PD-1 expression on HIV-1 Gag responding cells (sample group includes; AS n = 4, EC n = 3, NC n = 3). P values were calculated with repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test (*p

    Journal: PLoS Pathogens

    Article Title: TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection

    doi: 10.1371/journal.ppat.1005349

    Figure Lengend Snippet: TIGIT expressing CD8 + T cells have impaired cytokine responses. Representative flow cytometry plots gated on CD8 + T cells showing (A) TIGIT or (C) PD-1 expression against Ki-67 from a chronically HIV-infected individual. Compiled data of Ki-67 + CD8 + T cell frequency (%) separated into (B) TIGIT + and TIGIT - or (D) PD-1 + and PD-1 - ( n = 20). P values were calculated by Wilcoxon matched-pairs signed ranked test. Ex vivo PBMCs from chronically HIV-infected individuals were stimulated with HIV Gag peptide pool and assessed for cytokine production. (E) Representative flow cytometry plots gated on CD8 + T cells showing TIGIT expression and either IFN-γ, IL-2, or TNF-α content after no stimulation, stimulation with an HIV-1 Gag peptide pool, or a positive control stimulation with anti-CD3 + anti-CD28 Dynabeads. (F) Compiled data of IFN-γ, IL-2, or TNF-α CD8 + T cell frequency (%) from TIGIT + or TIGIT - CD8 + T cell compartments after HIV-1 Gag peptide pool stimulation (sample group includes; AS n = 4, EC n = 3, NC n = 3). P values were calculated by Wilcoxon matched-pairs signed ranked test. (G) Compiled data of TIGIT and PD-1 expression on HIV-1 Gag responding cells (sample group includes; AS n = 4, EC n = 3, NC n = 3). P values were calculated with repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test (*p

    Article Snippet: Functional assays In the intracellular cytokine stimulation assay studies, thawed cryopreserved PBMCs were stimulated for 12 hours in an incubator at 37°C with 5% CO2 with 5 μg/ml brefeldin A and 5 μg/ml monensin (Sigma-Aldrich) culture media, DMSO alone, pooled HIV-1 Gag peptides, or anti-CD3/CD28 dynabeads (Life Technologies) in the presence or absence of purified isotype IgG control, anti-TIGIT and/or anti-PD-L1 mAbs.

    Techniques: Expressing, Flow Cytometry, Cytometry, Infection, Ex Vivo, Positive Control

    Effect of in vitro blockade with anti-TIGIT/anti-PD-L1 mAbs on HIV-specific CD8 + T cell responses. Ex vivo PBMCs from chronically HIV-infected individuals were stimulated with HIV Gag peptide pool in the presence of mAb blocking antibodies. (A) Representative flow cytometry plots gated on CD8 + T cells, showing IFN-γ responses from an HIV-infected individual. No HIV-1 Gag stimulation with an isotype control, HIV-1 Gag stimulation with an isotype control, HIV-1 Gag stimulation with anti-TIGIT, HIV-1 Gag stimulation with anti-PD-L1, HIV-1 Gag stimulation with dual blockade (anti-TIGIT + anti-PD-L1) and a positive control (anti-CD3 + anti-CD28 Dynabeads). (B) Compiled data showing variation in the frequency (%) of IFN-γ in responses to HIV-1 Gag peptide pool with isotype control or mAb blockade; TIGIT blockade (left panel), PD-L1 blockade (middle panel), and dual blockade (right panel) ( n = 25). P values were calculated by Wilcoxon matched-pairs signed ranked test. (C) Representative flow cytometry plots gated on CD8 + T cells from HIV-infected individuals, showing intermediate and high CFSE dilution in response to HIV-1 Gag peptide pool stimulation in the presence of either an isotype control, anti-TIGIT mAb, anti-PD-L1 mAb, a combination of both anti-TIGIT and anti-PD-L1 mAbs or anti-CD3 + anti-CD28 Dynabeads as a positive control. (D) Graphs show compiled data showing variation in the frequency (%) of CFSE dim in responses to HIV-1 Gag peptide pool with either an isotype control or mAb blockade; TIGIT blockade (left panel), PD-L1 blockade (middle panel), and dual blockade (right panel) ( n = 24). P values were calculated by Wilcoxon matched-pairs signed ranked test.

    Journal: PLoS Pathogens

    Article Title: TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection

    doi: 10.1371/journal.ppat.1005349

    Figure Lengend Snippet: Effect of in vitro blockade with anti-TIGIT/anti-PD-L1 mAbs on HIV-specific CD8 + T cell responses. Ex vivo PBMCs from chronically HIV-infected individuals were stimulated with HIV Gag peptide pool in the presence of mAb blocking antibodies. (A) Representative flow cytometry plots gated on CD8 + T cells, showing IFN-γ responses from an HIV-infected individual. No HIV-1 Gag stimulation with an isotype control, HIV-1 Gag stimulation with an isotype control, HIV-1 Gag stimulation with anti-TIGIT, HIV-1 Gag stimulation with anti-PD-L1, HIV-1 Gag stimulation with dual blockade (anti-TIGIT + anti-PD-L1) and a positive control (anti-CD3 + anti-CD28 Dynabeads). (B) Compiled data showing variation in the frequency (%) of IFN-γ in responses to HIV-1 Gag peptide pool with isotype control or mAb blockade; TIGIT blockade (left panel), PD-L1 blockade (middle panel), and dual blockade (right panel) ( n = 25). P values were calculated by Wilcoxon matched-pairs signed ranked test. (C) Representative flow cytometry plots gated on CD8 + T cells from HIV-infected individuals, showing intermediate and high CFSE dilution in response to HIV-1 Gag peptide pool stimulation in the presence of either an isotype control, anti-TIGIT mAb, anti-PD-L1 mAb, a combination of both anti-TIGIT and anti-PD-L1 mAbs or anti-CD3 + anti-CD28 Dynabeads as a positive control. (D) Graphs show compiled data showing variation in the frequency (%) of CFSE dim in responses to HIV-1 Gag peptide pool with either an isotype control or mAb blockade; TIGIT blockade (left panel), PD-L1 blockade (middle panel), and dual blockade (right panel) ( n = 24). P values were calculated by Wilcoxon matched-pairs signed ranked test.

    Article Snippet: Functional assays In the intracellular cytokine stimulation assay studies, thawed cryopreserved PBMCs were stimulated for 12 hours in an incubator at 37°C with 5% CO2 with 5 μg/ml brefeldin A and 5 μg/ml monensin (Sigma-Aldrich) culture media, DMSO alone, pooled HIV-1 Gag peptides, or anti-CD3/CD28 dynabeads (Life Technologies) in the presence or absence of purified isotype IgG control, anti-TIGIT and/or anti-PD-L1 mAbs.

    Techniques: In Vitro, Ex Vivo, Infection, Blocking Assay, Flow Cytometry, Cytometry, Positive Control