Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Key role of NOS2 and the canonical cGMP-mediated NO signaling pathway in the MDSC-promoted Th17 differentiation of TILs from OvCa patients and human naive and memory CD4 + Th cells. (A) Induction of IL-17A, Rorc (encoding RORγt), FoxP3 (indicating de novo differentiation of FoxP3 + T reg cells from naive precursors), and T-bet gene expression in anti-CD3/CD28–expanded naive CD4 + T cells by tumor–isolated MDSCs (mean ± SD from six patients), as compared with control CD11b + cells (mean ± SD from three healthy donors). (B) IL-17A production levels and percentages of IL-17A + cells (mean ± SD from n donors), and representative intracellular staining (IL-17A vs. IFN-γ, right) in naive CD4 + T cells ( n = 6 healthy donors) or OvCa-infiltrating CD4 + TILs ( n = 3 patients) stimulated with anti-CD3/CD28 antibodies in the presence of cancer-isolated MDSCs or control CD11b + cells. (C) IL-17A production by naive versus memory CD4 + T cells (mean ± SD from n = 4 healthy donors) stimulated with either anti-CD3/CD28 antibodies or TNF-matured allogeneic DCs in the presence of MDSCs or control CD11b + cells. (D and E) Percentage of IL-17A + or IFN-γ + CD4 + T cells (D) and IL-17A secretion (E) in anti-CD3/CD28/MDSC–expanded naive CD4 + T cells (D) and CD4 + TILs (E) in the presence of specific inhibitors of NOS (L-NMMA), COX2 (celecoxib), IDO-, ARG-, or IL-10. The data (mean ± SD) from one representative experiment (performed in replicates: D, triplicate cultures; E, quadruplicate cultures). The results were confirmed in three independent experiments using different patients/healthy donors. (F) Representative staining of IL-17A + or IFN-γ + CD4 + T cells in co-cultures of anti-CD3/CD28–expanded CD4 + TILs and MDSCs in the presence of specific inhibitors of COX2 (celecoxib) and NOS (L-NMMA). The results were confirmed in three independent experiments using different patients. (G) Relative gene expression of IL-17A and Rorc, induced in anti-CD3/CD28–expanded naive CD4 + T cells cultured in the presence of MDSCs in the presence of a specific inhibitor of NOS (L-NMMA). The graphs present the mean ± SD from one representative experiment (triplicate cultures) of two (using different patients/healthy donors). (H) Relative gene expression of NOS2 and IL-17A in 7 d ex vivo anti-CD3/CD28–expanded cultures of OvCa ascites cells from 10 OvCa patients ( n = 10; r 2 = 0.7692; P < 0.001). (I) IL-17A production in anti-CD3/CD28–expanded cultures of naive or memory CD4 + T cells in the presence of MDSCs, with or without specific inhibitors of NOS2 (1400W) or cGMP (ODQ). Data (mean ± SD) from one representative experiment (triplicate cultures). The results were confirmed in three independent experiments using different patients/healthy donors. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: Expressing, Isolation, Staining, Cell Culture, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Exogenous NO supports the cytokine-driven induction of Th17 function in memory Th cells and promotes the de novo induction of Th17 cells from naive precursors. (A) NO 2 − levels (mean ± SD from four patients) in co-cultures of CD4 + TILs with tumor-isolated MDSCs (as compared with blood CD11b + cells) and in the presence of NOS inhibitors ADMA and l -NMMA. (B) Expression of IL-1β, IL-6, TGF-β 1 (spontaneous expression, left), and IL-23 (stimulation with CD40L, right) in MDSCs. Data (mean ± SD) from three experiments involving MDSCs from three different patients. (C) Induction of IL-17A or IFN-γ production by anti-CD3/CD28–stimulated bulk CD4 + T cells from healthy donors, cultured in the absence or presence of Th1 (200 U/ml rhIL-12, 200 ng/ml αIL-4-Ab), Th17 (20 ng/ml rhIL-1β, 50 ng/ml rhIL-6, 10 ng/ml rhIL-23), and T reg cell (5 ng/ml TGF-β 1 , 10 nM 9-cis retinoic acid) -driving cytokines, and physiological concentrations of exogenous NO donor (DETA-NONOate). IL-17A was undetectable in Th1- and T reg cell–driving conditions. Percentage of FoxP3 + cells in control cultures were as follows: (−), 10.4%; Th1, 12.7%; Th17, 5.2%; and T reg cell, 41.2%. The graphs present the mean ± SD from one representative experiment (triplicate cultures) of two (healthy donors). (D) Suppression of CD4 + T cells differentiating in Th1-, T reg -, and Th17-driving conditions by high concentrations (>100 µM) of DETA-NONOate. Proliferation of CFSE-labeled anti-CD3/CD28–stimulated bulk CD4 + T cells cultured in the absence or presence of Th1, Th17, and T reg cell–driving cytokines and supplemented with increasing concentrations of DETA-NONOate. The graph presents the mean ± SD from one representative experiment (triplicate cultures) of two (different healthy donors). (E and F) Relative gene expression of IL-17A (log scale) and Rorc (log scale), and secretion of IL-17A by naive (E) or naive and memory (F) CD4 + T cells, expanded with anti-CD3/CD28 antibodies in the absence or presence of Th17-driving cytokines and NO donor (DETA-NONOate). The graphs present the mean ± SD from one representative experiment (E, triplicate cultures; F, quadruplicate cultures). The results were confirmed in three independent experiments using cells of different healthy donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: Isolation, Expressing, Cell Culture, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Endogenous T cell–expressed NOS2 and T cell-produced NO are required for de novo Th17 differentiation from naive precursors and induction of the Th17 phenotype in memory cells. (A) Comparative induction of NOS2 (left) and IL-17A (right) gene expression in naive and memory CD4 + T cells (mean ± SD from three healthy donors) stimulated with anti-CD3/CD28 antibodies in the absence or presence of Th17-driving cytokines. (B) Dose-dependent induction of NOS2 gene expression in naive CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the presence of increasing concentrations of NO donor (DETA-NONOate) and Th17-driving cytokines. The graph presents the mean ± SD from one representative experiment (performed with triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (C) Dose-dependent induction of NOS2 gene expression in bulk CD4 + T cells, stimulated with anti-CD3/CD28 antibodies and Th17-driving cytokines (high: 20 ng/ml IL-1β, 50 ng/ml IL-6, 10 ng/ml IL-23; low: 25× dilution). The graph presents the mean ± SD from one representative experiment (triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (D) Induction of intracellular NOS2 protein (left and middle, representative data; right, mean ± SD from n = 3 healthy donors) in CD3 + -gated bulk CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the presence of Th17-driving cytokines and NO donor (DETA-NONOate). Bar, 10 µm. Data in the right panel is represented as the fold change of the mean fluorescence intensity (MFI) over the isotype control. (E) Induction of intracellular NO (DAF-FM staining; representative experiment, left; mean ± SD from n = 3 healthy donors, right) in CD3 + -gated bulk CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the presence of Th17-driving cytokines and NO donor (DETA-NONOate), or the presence of general NOS inhibitor (L-NMMA) or NOS2-specific inhibitor (1400W). Data in the right panel is expressed as the fold increase of DAF-FM MFI over CD4 + T cells cultured in the absence of Th17-driving cytokines and NO donor. When not otherwise indicated, statistically significant differences compared with the absence of NO inhibitors are shown. (F) IL-17A secretion by naive CD4 + T cells stimulated with Th17-driving cytokines in the presence of a general NOS inhibitor (L-NMMA) or NOS2-specific inhibitor (1400W). The graph presents the mean ± SD from one representative experiment (quadruplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (G) Induction of NOS2 (left, mean ± SD from four healthy donors) gene expression correlated with the IL-17A production (right, mean ± SD from three healthy donors) in bulk CD4 + T cells by the individual Th17-inducing factors IL-1β, IL-6, IL-23, and/or TGF-β 1 . *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: Produced, Expressing, Fluorescence, Staining, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Endogenous NOS2 and persistent cGMP signaling are required for the NO-assisted de novo induction of Th17 cells and for the stability of human in vivo-developed Th17 cells from cancer patients. (A) Relative gene expression of IL-17A, IL-17F, and IL-23R in bulk CD4 + T cells, expanded with anti-CD3/CD28 in the absence or presence of Th17-driving cytokines and general NOS inhibitor (L-NMMA). The graphs present the mean ± SD from a representative experiment (triplicate cultures) of two (using different patients/healthy donors), that both yielded similar results. (B) Regulation of (left) surface IL-23R expression on naive and memory CD4 + T cells (mean ± SD from four healthy donors) activated with anti-CD3/CD28 in the presence of NOS inhibitor (L-NMMA) or NO donor (DETA-NONOate). (right) Relative gene expression of IL-23R and IL-2R (right) in naive CD4 + T cells in the presence of increasing concentrations of NO donor and Th17-driving cytokines. Statistically significant differences compared with the absence of DETA-NONOate and Th17-driving cytokines are indicated. The graphs present the mean ± SD from a representative experiment (performed with triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (C) IL-17A production by naive CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the absence or presence of cGMP inhibitor (ODQ, left) or supplemented with cGMP analogue (Br-cGMP, right) in the absence or presence of Th17-driving cytokines. The graphs present the mean ± SD from a representative experiment (triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (D) IL-17A (left) or IFN-γ (right) production by OvCa-isolated CD4 + TILs (mean ± SD from five patients), expanded with anti-CD3/CD28 antibodies and restimulated in the absence or presence of NOS inhibitor (L-NMMA) or cGMP inhibitor (ODQ) for 48 h (statistically significant differences compared with the absence of inhibitors are indicated). (E) IL-17A production by in vitro–generated Th17 cells (generated in 8 d cultures of CD4 + T cells stimulated with anti-CD3/CD28 in the presence of Th17-driving cytokines), pretreated or not for 48 h with NOS inhibitor (L-NMMA) or cGMP inhibitor (ODQ). The data are shown as mean ± SD from 4 healthy donors. Statistically significant differences compared with condition in the absence of inhibitors are indicated. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: In Vivo, Expressing, Isolation, In Vitro, Generated

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Hepatic MTH1+CD3+ T cells correlated with disease activity in AIH. (A) Representative confocal staining of CD3 (red), MTH1 (green), and DAPI (for nuclei in blue) (magnification ×400) in the liver of patients with AIH. Scale bar, 20 μm. (B) Number of MTH1+CD3+ T cells in portal areas was positively correlated with degree of hepatic inflammation but showed no clear link with fibrosis stages in AIH. (C) Number of MTH1+CD3+ T cells in portal areas had a significant positive correlation with levels of serum ALT and AST in patients with AIH. (D) Number of MTH1+CD3+ T cells in portal areas was positively correlated with levels of serum ALP and GGT. Bars reflect the mean ± SEM. Abbreviations: DAPI, 4 0, 6‐diamidino‐2‐phenylindole; hpf, high‐power field.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: Activity Assay, Staining

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib significantly inhibited T‐cell proliferation in human T cells in vitro . Isolated human CD3+ T cells were cultured with/without anti‐CD3/CD28 beads for 72 hours. Representative western blot analyses of MTH1 72 hours after anti‐CD3/CD28 beads stimulation. (B,C) Isolated T cells were activated with anti‐CD3/CD28 beads with or without 2 μM karonudib for 72 hours. The percentage of CD25+ and CD69+ T cells was determined on day 3 by flow cytometry. (D) Analysis of T‐cell number treated with karonudib for 72 hours after anti‐CD3/CD28 beads stimulation. (E) Statistical analysis of the T‐cell proliferation assay treated with/without 2 μM karonudib for 72 hours from HCs and patients with AIH who were treatment naive. (F) Representative western blot analyses of P53, P21, P27, CDK2, and cyclin E 72 hours after anti‐CD3/CD28 beads stimulation. The GAPDH blot was used as a loading control. Data are from one experimental representative of at least three independent experiments and represent triplicate wells. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Cell Culture, Western Blot, Flow Cytometry, Proliferation Assay, Control

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib rendered activated T cells more susceptible to DNA damage in vitro . (A) Representative fields corresponding to each treatment were photographed. Isolated human T cells were activated with/without anti‐CD3/CD28 beads in the presence of DMSO or 2 μM karonudib for 72 hours. The alkaline comet assay was conducted and nucleoids were visualized by epifluorescence microscopy using a fluorescein isothiocyanate filter. (B) Quantification of comet tail moment. Values represent mean ± SEM from three independent experiments (100 comets per experiment). (C) Levels of cleaved‐PARP and phosphorylated histone H2AX (γ‐H2AX) were determined by immunoblot analysis. The GAPDH blot was used as a loading control. (D,E) Intracellular flow cytometry assessment of annexin V and propidium iodide expression in anti‐CD3/CD28 beads‐stimulated CD4+ and CD8+ T lymphocytes treated with 2 μM karonudib or DMSO after 72 hours. Data are from one experimental representative of at least three independent experiments. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Alkaline Single Cell Gel Electrophoresis, Epifluorescence Microscopy, Western Blot, Control, Flow Cytometry, Expressing

Journal: Frontiers in Aging

Article Title: Obesity and Fatty Acids Promote Mitochondrial Translocation of STAT3 Through ROS-Dependent Mechanisms

doi: 10.3389/fragi.2022.924003

Figure Lengend Snippet: Reagent or resource.

Article Snippet: Dynabeads Human T-Activator CD3⁄CD28 for T Cell Activation , GIBCO life technologies , Cat# 11132D.

Techniques: Confocal Microscopy, Recombinant, Software, Activation Assay, Isolation

Journal: eLife

Article Title: Umbilical cord blood-derived ILC1-like cells constitute a novel precursor for mature KIR+NKG2A- NK cells

doi: 10.7554/elife.55232

Figure Lengend Snippet: Figure 1. ILC1-like cells have a unique gene signature distinct from NK cells. (a) CB mononuclear cells (MNCs) were enriched prior to sorting via biotin- labelled antibodies (anti-CD3/CD19/CD14/CD66b) and sorted for ILC1-like cells, CD56dim, and CD56bright NK cells (see Figure 1—figure supplement 1 for sorting strategy). RNA sequencing was done on the Illumina platform. The heat map indicates the top 100 differentially expressed genes between ILC1-like cells and CD56bright NK cells including CD56dim NK cells. (b) A two-dimensional principle component analyses based on the top 2000 Figure 1 continued on next page

Article Snippet: In brief, ~10–20 107 cells were stained with the biotinylated antibodies anti-CD3 (OKT3, 3.2 ml/10 107 cells), anti-CD14 (63D3, 4.8 ml/10 107), anti-CD19 (HIB19, 4.8 ml/10 107), and anti-CD66b (G10F5, 2.4 ml/10 107) from BioLegend for 15 min on ice.

Techniques: RNA Sequencing

Journal: eLife

Article Title: Umbilical cord blood-derived ILC1-like cells constitute a novel precursor for mature KIR+NKG2A- NK cells

doi: 10.7554/elife.55232

Figure Lengend Snippet: Figure 4. ILC1-like cells possess high NKP potential without previous epigenetic priming for NK cell receptor expression. (a) Scheme of the experimental set-up. CB MNCS were freshly isolated, enriched using biotinylated antibodies (anti-CD3, CD14, CD19, CD66b), and sorted for CD5+ILC1- like cells, CD5-ILC1-like cells, CD56bright NK cells, and ILC2. One day prior to sorting, OP9-DL1 cells were plated in 96-well flat-bottom plates. Cells were either supplemented with IL-7 and FLt3L for the T cell condition or IL-2, IL-7, and IL-15 for the NK cell condition and cultured for 8 days with medium change at day 5. (b) Exemplary dot plots and quantification of CD94 expression together with either NKG2A or KIR2DL2/L3/S2 expression after Figure 4 continued on next page

Article Snippet: In brief, ~10–20 107 cells were stained with the biotinylated antibodies anti-CD3 (OKT3, 3.2 ml/10 107 cells), anti-CD14 (63D3, 4.8 ml/10 107), anti-CD19 (HIB19, 4.8 ml/10 107), and anti-CD66b (G10F5, 2.4 ml/10 107) from BioLegend for 15 min on ice.

Techniques: Expressing, Isolation, Cell Culture

Journal: Journal for immunotherapy of cancer

Article Title: PARP inhibitors enhance antitumor immune responses by triggering pyroptosis via TNF-caspase 8-GSDMD/E axis in ovarian cancer.

doi: 10.1136/jitc-2024-009032

Figure Lengend Snippet: Figure 5 Deficiency of GSDME blunts the immune response triggered by PAPR inhibitor in vivo. (A–L) Immunocompetent C57BL/6 mice were transplanted with wild type (WT) or Gsdme-deficient ID8 cells and challenged with niraparib for about 4 weeks. Tumors were harvested and subjected to bulk T-cell receptor (TCR) sequencing and evaluation of immune status. The frequency of T-cell clonotypes in the top 25% of TCR repertoires was shown using pie charts ((A) n=4) and quantified (B). The clonal expansion was evaluated using clonality (C). The TCR diversity was calculated by normalized Shannon diversity entropy (D). The proportion of dendritic cell differentiation (CD11c+MHCIIHigh) and maturation (CD11c+MHCIIHighCD86High) in tumors (E), lymph nodes (F), and spleens (G) was determined by flow cytometry. The proportion of CD3+ T cells and NK cells among CD45+ immune cells in tumors was determined by flow cytometry (H). The production of IFN-γ in tumor-infiltrated CD4/8+ T and NK cells was examined by flow cytometry (I). The expression of granzyme B in tumor-infiltrated CD8+ T and NK cells was evaluated by mean fluorescence intensity (J). Representative images of GSDME, CD4, CD8, and GZMB IHC staining in tumor sections (K) and numbers of indicated immune cells in a ×20 field of a microscope (L). Scale bars: 200 µm. WT or Gsdme-deficient ID8 cells were intrabursally transplanted into C57BL/6 mice and received niraparib and/or anti-PD-1 treatment. Representative bioluminescent images of mice bearing WT and Gsdme-KO ID8 tumors at the endpoint (M). The tumor weight was quantified in each mouse treated with niraparib and/or anti-PD-1 antibodies ((N) n=7). Mean values±SEM. *P<0.05, **p<0.01, and ***p<0.001 by Student’s t-test in (B–J, L, and N).

Article Snippet: Subsequently, these isolated T cells were stimulated with anti- CD3 (BioXCell, BE0002) and anti- CD28 antibodies (BioXCell, BE0328) in the presence or absence of olaparib/niraparib.

Techniques: In Vivo, Sequencing, Cell Differentiation, Flow Cytometry, Expressing, Fluorescence, Immunohistochemistry, Microscopy

Journal: Oncoimmunology

Article Title: Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer

doi: 10.1080/2162402X.2016.1145333

Figure Lengend Snippet: Combining a modulator of infiltrating-myeloid cells and an inhibitor of PD-1/PD-L1 axis increases T cell proliferation and T cell producing IFNγ. Myeloid cells CD11b+ were isolated from tumors using BD FACSAria II (BD Biosciences). T cells were isolated from spleen of naive mice using mouse pan T cell isolation Kit (Miltenyei). CFSE-labeled T cells were stimulated with CD3/CD28 beads ratio 1:1 (Life Technologies). Stimulated T cells were cultured with CD11b+ (at a ratio CD11b:T cells of 1:1) and incubated for 72 h at 37°C. (A) Representative histograms obtained by FACS analysis showing the fluorescence intensity of CFSE-T cells gated on CD8 + . (B) The percentage of proliferating CD8 + cells from the different treated groups is shown. (C) IFNγ secretion in the supernatant of the co-culture is measured 72 h following incubation at 37°C using Luminex Technology. Experiments were repeated twice (Kruskal–Wallis test, * p < 0.05).

Article Snippet: T cells were labeled with CellTrace™ CFSE Cell Proliferation Kit (Life Technologies) and activated by Dynabeads® Mouse T-Activator CD3/CD28 (Life Technologies) at a bead-to-cell ratio of 1:1.

Techniques: Isolation, Cell Isolation, Labeling, Cell Culture, Incubation, Fluorescence, Co-Culture Assay, Luminex