cd8 t cell depletion anti cd8 antibody  (Bio X Cell)

Journal: Journal for immunotherapy of cancer

Article Title: Glucocorticoid activation by HSD11B1 limits T cell-driven interferon signaling and response to PD-1 blockade in melanoma.

doi: 10.1136/jitc-2021-004150

Figure Lengend Snippet: Figure 3 HSD11B1 expression confers resistance to PD-1 blockade. (A) Overview of mouse melanoma cell lines and Hsd11b1 expression (3’mRNA-seq). (B) Kinetic of 11-DHCS to CS conversion in indicated cell lines assayed by CS-specific ELISA (n=3). Dashed line indicates input (100%) of 11-DHCS. Error bars, SD. (C) 11-DHCS to CS conversion (% of input 11- DHCS) in indicated cell lines at 40 min and 3 hours assayed by CS-specific ELISA (n=3). (D) GSEA plot for indicated gene set. Comparison of CM and LN transcriptomes (3’mRNA-seq). (E) In vitro cell growth of CM vs LN cells exposed to IFN-γ. Upper panel: Quantification of n=3. Lower panel: Representative images of stained tissue culture wells. (F) Tumor growth kinetics (left) and final tumor weight at day 12 (right) of CM and LN melanomas treated with αPD-1 or IgG control. (G) Heatmap showing proliferation-associated gene expression (3’mRNA-seq) in CM and LN melanomas from (F). (H, I) Correlation of Hsd11b1 expression with T cell (cytotoxic) marker genes (H) and myeloid cell marker genes (I) in CM melanomas treated with αPD-1 or IgG control. (J) Individual tumor growth curves and (K) tumor weight (at day 8) of CM melanomas ectopically expressing Hsd11b1 (pRP.Hsd11b1) vs CM controls (pRP) treated with αPD-1 or IgG control. (L, M) Intratumoral CD8+ T cells (L) and CD4+ T cells (M) assessed by immunofluorescence from multiple representative regions. Statistics: *p<0.05, **p<0.01, ***p<0.001. Two-sided unpaired t-tests (B, F, K–M), with logarithms (C, E). Correction for multiple comparison with Benjamini and Hochberg method (E). 11-DHCS, 11-dehydrocorticosterone; CM, cutaneous melanoma; CS, corticosterone; FDR, false discovery rate; GSEA, gene set enrichment analysis; IFN-γ, interferon-γ; LN, lymph node; (N)ES,(normalized) enrichment score; r, Pearson’s correlation coefficient.

Article Snippet: Immune depletion of CD8+ T cells Anti- CD8a depleting antibody (40 mg/kg, clone 2.43, BioXcell, #BE0061) was intraperitoneally injected every 4 days starting from day −1 prior CM cells transplantation until mice were euthanized.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Comparison, In Vitro, Staining, Control, Gene Expression, Marker, Immunofluorescence

Journal: Journal for immunotherapy of cancer

Article Title: Glucocorticoid activation by HSD11B1 limits T cell-driven interferon signaling and response to PD-1 blockade in melanoma.

doi: 10.1136/jitc-2021-004150

Figure Lengend Snippet: Figure 6 HSD11B1 inhibition augments IFN-γ production of CD8+ T cells under PD-1 blockade. (A) Effect of HSD11B1 inhibition on IFN-γ production by intratumoral (CM) CD8+ T cells. Gating strategy for flow cytometry. (B) Representative FACS blots showing frequencies of IFN-γ +CD8+ cells in CM melanomas treated as indicated. (C) Quantification of experiment described in (B). Two-sided unpaired t-test with logarithms. (D) Effect of HSD11B1 inhibition on IFN-γ production by Pmel-1 T cells in vitro. Gating strategy for flow cytometry. (E) Representative FACS blots (IFN-γ positivity) of gp100 activated Pmel-1 T cells treated as indicated. (F) Quantification of experiments (n=3) described in (D, E). 11-DHCS, DEXA (100 nM), CBX and 10j (10 µM). (G) Experimental outline of CD8+ T cell depletion in mice bearing CM melanomas and treatment conditions. (H, I) Frequencies of CD8+ T cells in (H) tumor and (I) tumor-draining LN assessed by flow cytometry. (J) Individual CM melanoma tumor growth curves and (K) tumor volume at day 8 after inoculation treated as indicated with or without antibody-mediated depletion of CD8+ cells. Statistics: *p<0.05, **p<0.01, ***p<0.001. Two-sided unpaired t-tests (for ratios with logarithms). 11- DHCS, 11-dehydrocorticosterone; CM, cutaneous melanoma; DEXA, dexamethasone; IFN-γ, interferon-γ; tdLN, tumor-draining lymph node.

Article Snippet: Immune depletion of CD8+ T cells Anti- CD8a depleting antibody (40 mg/kg, clone 2.43, BioXcell, #BE0061) was intraperitoneally injected every 4 days starting from day −1 prior CM cells transplantation until mice were euthanized.

Techniques: Inhibition, Flow Cytometry, In Vitro