cd8 t cell exhaustion  (Sino Biological)

Journal: Febs Letters

Article Title: Function‐driven design of a surrogate interleukin‐2 receptor ligand

doi: 10.1002/1873-3468.70249

Figure Lengend Snippet: Proliferative effects of scDb‐2 and scDb‐2d on IL‐2Rβ•γc ‐expressing Ba/F3 cells and human PBMCs. (A) Proliferation of IL‐2 Rβ•γc‐expressing Ba/F3 cells treated with IL‐2, scDb‐2 and scDb‐2d (CCK‐8 assay). (B) Proliferation of activated PBMCs exposed to IL‐2, scDb‐2 and scDb‐2d (CCK‐8 assay). (C) Comparable expansion of total CD3+ T cells after 12‐day culture with 20 n m IL‐2 or scDb‐2d. Differential expansion of T cell subsets: (D) enhanced CD8+ T cell proliferation and (E) reduced Treg expansion with scDb‐2d versus IL‐2 (20 n m , 12 days). Data represent mean ± SD ( n = 3 technical replicates). (F) Exhaustion marker expression (TIM‐3, LAG‐3, PD‐1) on CD8+ T cells after 12‐day culture with 2.5 n m or 10 n m IL‐2/scDb‐2d. (G) Activation marker expression (CD27, CD69) on CD8+ T cells under same conditions. Data represent mean ± SEM ( n = 3 technical replicates). Statistical significance was determined by the two‐tailed unpaired t ‐test (GraphPad Prism): ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD8+ T cell exhaustion was evaluated through staining with PD‐1‐PE (#10377‐M140‐P; Sino Biological), LAG‐3‐PE (#369305; BioLegend), and TIM‐3‐FITC (#345021; BioLegend), while activation status was assessed using CD27‐FITC (#555440; BD Biosciences, San Jose, CA, USA) and CD69‐APC (#555533; BD Biosciences).

Techniques: Expressing, CCK-8 Assay, Marker, Activation Assay, Two Tailed Test

Journal: Gut Microbes

Article Title: Microbial metabolite butyrate promotes anti-PD-1 antitumor efficacy by modulating T cell receptor signaling of cytotoxic CD8 T cell

doi: 10.1080/19490976.2023.2249143

Figure Lengend Snippet: Serum butyric acid amount is correlated with anti-PD-1 efficacy in NSCLC patients. (a) Overview of experimental design. Targeted metabolomics (GC-MS/MS) analysis procedure. (b, c) the serum levels of acetic acid (AA), propionic acid (PA), isobutyric acid (IBA), butyric acid (BA), isovaleric acid (IVA), valeric acid (VA) and caproic acid (CA) in NSCLC patients were detected by GC-MS/MS ( n = 22). (d) Linear regression analysis between SCFAs (AA, PA, IBA, BA, IVA, VA and CA) levels and surface markers (PD-1 + , Tim-3 + , TIGIT + , CD28 + ) on T cells (CD4 + , CD8 + , Vδ1 + , and Vδ2 + ) in patients with advanced lung cancer ( n = 22). (e) experimental design. SCFAs levels in serum of patients with NSCLC were detected by GC-MS/MS before therapy with anti-PD-1 and chemotherapy. (f) CT scans showing changes of tumor size in patients with NSCLC who received intravenous anti-PD-1 and chemotherapy. (g) the serum SCFAs from patients with NSCLC were measured and compared between responders (complete response, partial response or stable disease; CR, PR or SD) and non-responders (progressive disease, PD or death). (h) the level of PD-1 on circulating CD8 + and Vδ2 + T cells in responders (N) versus non-responders (NR) of NSCLC (N, n = 11; NR, n = 11). The Pearson correlation was used in (d); two-tailed unpaired Student’s t -test (g and h). Data represented mean or mean±SD. * P < .05, ** P < .01, *** P < .001, **** P < .0001. n.s., not significant.

Article Snippet: Vehicle- or BA-pretreated CD8 + T cells were subjected to chromatin immunoprecipitation analysis using the Simple ChIP enzymatic chromatin IP Kit (CST, 9003) following the manufacturer’s instructions.

Techniques: Gas Chromatography-Mass Spectrometry, Two Tailed Test

Journal: Gut Microbes

Article Title: Microbial metabolite butyrate promotes anti-PD-1 antitumor efficacy by modulating T cell receptor signaling of cytotoxic CD8 T cell

doi: 10.1080/19490976.2023.2249143

Figure Lengend Snippet: Butyrate supplementation enhances the antitumor immune responses of anti-PD-1. (a) Overview of study design. (b, c) BA pretreated Vδ2 T cells (effector, E) were incubated with Jurkat cells (target, T) at the indicated ratios for 6 hours with or without anti-PD-1 treatment, and the percentages of dead cells out of total target cells identified as PI + were shown (healthy donors, n = 3 per group). (d) experiment approach. (e) images of B16-F0 tumors in mice at day 15 post-tumor implantation. (f) tumor weight in the mice in each group at the end of experiment ( n = 6 per group). (g-i) tumor growth (g and h) and survival curves (i) in the mice after treatment with IgG, butyrate, anti-PD-1 alone or the combination ( n = 6 per group). (j) Tissue pathology of the indicated organs was evaluated by H&E staining at the end of the experiment (days 15 after B16-F0 tumor implantation). (k-m) tumor-infiltrating IFN-γ + and TNF-α + T cells (CD8 + and CD4 + ) were analyzed by flow cytometry ( n = 6 per group). Two-tailed unpaired Student’s t -test used in (c, E: T = 15:1); one-way ANOVA with Tukey’s multiple comparisons test (f, g, l and m); log-rank (Mantel-Cox) test was used in (i). Data represented mean±SD. * P < .05, ** P < .01, *** P < .001, **** P < .0001. n.s., not significant.

Article Snippet: Vehicle- or BA-pretreated CD8 + T cells were subjected to chromatin immunoprecipitation analysis using the Simple ChIP enzymatic chromatin IP Kit (CST, 9003) following the manufacturer’s instructions.

Techniques: Incubation, Tumor Implantation, Staining, Flow Cytometry, Two Tailed Test

Journal: Gut Microbes

Article Title: Microbial metabolite butyrate promotes anti-PD-1 antitumor efficacy by modulating T cell receptor signaling of cytotoxic CD8 T cell

doi: 10.1080/19490976.2023.2249143

Figure Lengend Snippet: Microbial metabolites butyrate promotes anti-PD-1 immunotherapy efficacy through CD8 + T cell-dependent antitumor immunity. (a) experimental design: mice were treated with antibiotics, and inoculated with B16-F0 tumor cells followed by indicated therapy. (b-e) tumor growth was monitored. Mice were inoculated with B16-F0 in ABX pretreated mice, then received anti-PD-1, BA, or anti-PD-1 plus butyrate therapy ( n = 7 per group). Experiments were independently repeated three times (b-e). (f, g) serum IFN-γ and TNF-α levels in mice were measured by ELISA after indicated treatment (mice serum were collected on day 18), n = 7 per group. One-way ANOVA with Tukey’s multiple comparisons test (e-g). Data represented mean±SD. * P < .05, ** P < .01, *** P < .001, **** P < .0001. n.s., not significant.

Article Snippet: Vehicle- or BA-pretreated CD8 + T cells were subjected to chromatin immunoprecipitation analysis using the Simple ChIP enzymatic chromatin IP Kit (CST, 9003) following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Gut Microbes

Article Title: Microbial metabolite butyrate promotes anti-PD-1 antitumor efficacy by modulating T cell receptor signaling of cytotoxic CD8 T cell

doi: 10.1080/19490976.2023.2249143

Figure Lengend Snippet: Butyrate acts within activated T cells to enhance acetylation of the Pdcd1 and Cd28 locus. (a) mRNA levels of immune checkpoint receptors and co-stimulatory molecules in SCFAs (AA = 10 mM, PA = 1 mM and BA = 0.5 mM) pretreated CD8 + T cells were verified by Quantitative real-time PCR (qPCR), and their fold changes were shown in heat map (healthy donors, n = 3 per group). (b, c) the expression of PD-1 + and CD28 + on vehicle, AA, PA, BA-pretreated Vδ2 + and CD8 + T cells was analyzed by flow cytometry (Vδ2 + , n = 12; CD8 + , n = 5). (d-f) human cytotoxic T cells (CD8 + and Vδ2 + ) were treated with BA, A-485 (1 μM) alone or the combination for 48 hours followed by flow cytometry ( n = 3–7). (g) the levels of histone 3 lysine 9 acetylation (H3K9ac) and histone 3 lysine 27 acetylation (H3K27ac) in vehicle or BA-treated cytotoxic CD8 + T cells were determined by immunoblotting. (h) CD8 + T cells were treated with or without A485, BA or their combination for 48 hours, followed by western blot. (i-m) Overview of primers design (i). Graph showed ChIP-qPCR analysis of H3K27ac and H3K9ac at the promoter of Cd28 and Pdcd1 gene in vehicle or BA-pretreated CD8 + T cells. The levels of H3K9ac (j and k) and H3K27ac (l and m) were normalized to the input, n = 3 healthy donors. Primers 1–7 (qPCR primers for Cd28 and Pdcd1 1 to 7). Two-tailed unpaired Student’s t -test (b and c); one-way ANOVA with Tukey’s multiple comparisons test (e and f). Data represented mean±SD. * P < .05, ** P < .01, *** P < .001, **** P < .0001. n.s., not significant.

Article Snippet: Vehicle- or BA-pretreated CD8 + T cells were subjected to chromatin immunoprecipitation analysis using the Simple ChIP enzymatic chromatin IP Kit (CST, 9003) following the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Gut Microbes

Article Title: Microbial metabolite butyrate promotes anti-PD-1 antitumor efficacy by modulating T cell receptor signaling of cytotoxic CD8 T cell

doi: 10.1080/19490976.2023.2249143

Figure Lengend Snippet: Butyrate regulates the function of CD8 + T cells through T cell receptor signaling. (a) vehicle, AA (10 mM), PA (1 mM) or BA (0.5 mM)-pretreated human CD8 + T cells were activated with or without α-CD3/CD28 for 4 hours. The expression of various genes related to antitumor immunity in CD8 + T cells was measured by qPCR, and their fold changes were shown in heat map (healthy donors, n = 3 per group). (b-e) AA, PA, BA or control-pretreated CD8 + and Vδ2 + T cells were stimulated with α-CD3/CD28 for 4 hours, and the levels of IFN-γ + and TNF-α + in Vδ2 + and CD8 + T cells were detected by flow cytometry ( n = 3–5). (f) immunoblotting analysis the phosphorylation of protein from T cell receptor signaling pathway in vehicle or BA-pretreated CD8 + T cells upon α-CD3/CD28 activation. (g, h) BA or vehicle-pretreated CD8 + T cells were preloaded with fluo-3 AM in Ca 2+ free medium, then those cells were activated by α-CD3/CD28 or PMA/Ion with or without Ca 2+ supplementation. Ca 2+ fluorescence intensity (MFI) transformation was detected by flow cytometry. (i, j) BA-pretreated or vehicle Vδ2 + and CD8 + T cells were treated with BA, U73122 alone or BA+U73122 for another 1 hour, and then immunoblot analysis the expression of PLCγ1 and p-PLCγ1 from whole-cell lysate. (k-n) BA or vehicle-pretreated Vδ2 + and CD8 + T cells were treated with or without U73122 under the condition of α-CD3/CD28 activation for 4 hours. IFN-γ + and TNF-α + CD8 + or Vδ2 + T cells were measured by flow cytometry ( n = 6). Two-tailed unpaired Student’s t -test (c and e); one-way ANOVA with Tukey’s multiple comparisons test (l and n). Data represented mean±SD. * P < .05, ** P < .01, *** P < .001, **** P < .0001. n.s., not significant.

Article Snippet: Vehicle- or BA-pretreated CD8 + T cells were subjected to chromatin immunoprecipitation analysis using the Simple ChIP enzymatic chromatin IP Kit (CST, 9003) following the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Western Blot, Activation Assay, Fluorescence, Transformation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: An oral cancer vaccine using Bifidobacterium vector augments combination of anti-PD-1 and anti-CTLA-4 antibodies in mouse renal cell carcinoma model

doi: 10.1038/s41598-023-37234-6

Figure Lengend Snippet: Evaluation of the tumor infiltrating lymphocyte in experiment using anti-PD-1 antibody for immune checkpoint inhibitor. ( a ) Immunohistochemical staining for tumor-infiltrating T cells in Renca tumors. Immunohistochemical staining showed remarkably increased numbers of CD4 and CD8 T cells in tumor tissues in mice treated with combination therapy. ( b ) Tumor-infiltrating T cells after treatment in Renca model The population of tumor-infiltrating CD4 + T, CD8 + T, and CD107a + CD8 + T cells was analyzed by flow cytometry (n = 5). Each data point represents the average of the cell frequencies (bars, ± SE).

Article Snippet: Anti-mouse CD4 antibody (1:1,000, Abcam, Cambridge, UK), anti-mouse CD8 antibody (1:400, Cell Signaling Technology Japan, Tokyo, Japan), and Foxp3 antibody 236A/E7 (Mouse monoclonal anti-body; Abcam, Cambridge, UK) were used in the immunohistochemical staining.

Techniques: Immunohistochemical staining, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: An oral cancer vaccine using Bifidobacterium vector augments combination of anti-PD-1 and anti-CTLA-4 antibodies in mouse renal cell carcinoma model

doi: 10.1038/s41598-023-37234-6

Figure Lengend Snippet: Evaluation of the tumor infiltrating lymphocyte in experiment using anti-PD-1 and anti-CTLA-4 antibody for immune checkpoint inhibitor. ( a ) Immunohistochemical staining for tumor-infiltrating T cells in Renca tumors. Resected Renca tumors were immunohistochemically stained with anti-CD4 antibody, anti-CD8 antibody, or Foxp3 antibody. Representative immunohistochemical staining in each treatment group is shown (400 ×). ( b ) Tumor-infiltrating T cells after treatment in the Renca model. The population of tumor-infiltrating CD4T, CD8T, and CD107a + CD8 + T cells was analyzed by flow cytometry (n = 5). Each data point represents the average of the cell frequencies (bars, ± SE).

Article Snippet: Anti-mouse CD4 antibody (1:1,000, Abcam, Cambridge, UK), anti-mouse CD8 antibody (1:400, Cell Signaling Technology Japan, Tokyo, Japan), and Foxp3 antibody 236A/E7 (Mouse monoclonal anti-body; Abcam, Cambridge, UK) were used in the immunohistochemical staining.

Techniques: Immunohistochemical staining, Staining, Flow Cytometry