Figure 4.
CD86 blockade reduces expression of inhibitory cell surface markers PD-1 and CTLA-4 on cHL-associated T cells. (A) Heat map illustrating expression of inhibitory signaling molecules of the NIVHAL cohort (n = 95 primary diagnosis samples). Each line represents 1 patient. (B) Change of inhibitory signaling molecules between primary (gray) and secondary (red) biopsies. Treatment response was evaluated by a board-certified pathologist and classified into HRSC clearance (light green, n = 4) or HRSC maintenance (red, n = 6). Statistical significance was calculated using 2-way ANOVA with Sidak multiple comparison correction. (C-D) Expression of indicated antigens on either CD14+ monocytes (left, CD86) or CD3+ T cells (middle left, CTLA-4; middle right, PD-1; right, CD28) measured by flow cytometry. cHL cell lines (L-540, C; L-428, D) and PBMCs were cocultured with either αCD86 antibody (mustard) or isotype control antibody (yellow). Data are mean ± SEM from 6 independent donors. Statistical significance was calculated using Wilcoxon signed rank test. (E) Summary of the treatment schedule used for humanized L-540 xenograft in vivo experiments. (F-G) BLI images (F) and fLuc-BLI–based quantification of tumor burden (G) of L-540 tumor-bearing mice injected with human PBMCs and treated with αCD86 antibody (mustard), isotype control antibody (yellow), or αPD-1 antibody (magenta). (H-I) Flow cytometric quantification of CTLA-4 (H) or PD-1 expression (I) in different organs on CD3+ T cells in different organs. There were 5 to 6 mice per group. Statistical significance was calculated using 2-way ANOVA with Tukey multiple comparison correction. (J-K) Representative histograms illustrating expression of CTLA-4 (J) or PD-1 (K) on CD3+ T cells in the spleen. (L) Overview of experimental scheme used to induce CD86 blockade in complex BMO. (M) Representative confocal images of cHL-PBMC-BMO cocultures. Yellow: mesenchymal tissue (CD271); violet: PBMC; turquoise: cHL tumor cells. Magnified images of the rectangle area are depicted at the bottom. (N-O) Expression of indicated antigens on CD3+ T cells (left, CTLA-4; middle PD-1; right CD28) in coculture with L-540 (N) or L-428 (O) cHL tumor cell lines measured by flow cytometry. cHL cell lines and BMO were cocultured either with αCD86 antibody or isotype control antibody. Data are mean ± SEM from n = 4-8 independent donors and BMO. Scale bar in panel M 100 µm (top), 30 µm (bottom). Statistical significance was calculated using Wilcoxon signed rank test. For all panels: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, P > .05.

CD86 blockade reduces expression of inhibitory cell surface markers PD-1 and CTLA-4 on cHL-associated T cells. (A) Heat map illustrating expression of inhibitory signaling molecules of the NIVHAL cohort (n = 95 primary diagnosis samples). Each line represents 1 patient. (B) Change of inhibitory signaling molecules between primary (gray) and secondary (red) biopsies. Treatment response was evaluated by a board-certified pathologist and classified into HRSC clearance (light green, n = 4) or HRSC maintenance (red, n = 6). Statistical significance was calculated using 2-way ANOVA with Sidak multiple comparison correction. (C-D) Expression of indicated antigens on either CD14+ monocytes (left, CD86) or CD3+ T cells (middle left, CTLA-4; middle right, PD-1; right, CD28) measured by flow cytometry. cHL cell lines (L-540, C; L-428, D) and PBMCs were cocultured with either αCD86 antibody (mustard) or isotype control antibody (yellow). Data are mean ± SEM from 6 independent donors. Statistical significance was calculated using Wilcoxon signed rank test. (E) Summary of the treatment schedule used for humanized L-540 xenograft in vivo experiments. (F-G) BLI images (F) and fLuc-BLI–based quantification of tumor burden (G) of L-540 tumor-bearing mice injected with human PBMCs and treated with αCD86 antibody (mustard), isotype control antibody (yellow), or αPD-1 antibody (magenta). (H-I) Flow cytometric quantification of CTLA-4 (H) or PD-1 expression (I) in different organs on CD3+ T cells in different organs. There were 5 to 6 mice per group. Statistical significance was calculated using 2-way ANOVA with Tukey multiple comparison correction. (J-K) Representative histograms illustrating expression of CTLA-4 (J) or PD-1 (K) on CD3+ T cells in the spleen. (L) Overview of experimental scheme used to induce CD86 blockade in complex BMO. (M) Representative confocal images of cHL-PBMC-BMO cocultures. Yellow: mesenchymal tissue (CD271); violet: PBMC; turquoise: cHL tumor cells. Magnified images of the rectangle area are depicted at the bottom. (N-O) Expression of indicated antigens on CD3+ T cells (left, CTLA-4; middle PD-1; right CD28) in coculture with L-540 (N) or L-428 (O) cHL tumor cell lines measured by flow cytometry. cHL cell lines and BMO were cocultured either with αCD86 antibody or isotype control antibody. Data are mean ± SEM from n = 4-8 independent donors and BMO. Scale bar in panel M 100 µm (top), 30 µm (bottom). Statistical significance was calculated using Wilcoxon signed rank test. For all panels: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, P > .05.

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