Fig. 5. TAMs establish ICB resistance through PD-L1-CD80-mediated CD4⁺ T cell suppression and T_reg expansion.

a, c Ex vivo T cell suppression by tumor-associated myeloid cells. CD11b⁺ cells were purified from ICB R, ICB NR, and C tumors on d27 by MACS and co-cultured for 72 h with pre-activated naive CD3⁺ splenocytes with and without 20 µg ml⁻¹ anti-PD-L1 (10 F.9G2; ICB R n = 4, ICB NR n = 4, C n = 4 animals). a CD4⁺ T cell proliferation after co-culture assessed by CFSE staining. b Frequency of CD80⁺ cells of CD8⁺ (left) and CD4⁺ (right) TILs (ICB R n = 3, ICB NR n = 6, C n = 8 animals). c CD80 expression on pre-activated naive CD4⁺ and CD8⁺ T cells before and after co-culture with tumor-associated myeloid cells from ICB R, ICB NR, and C. d Tumor growth (left) and response (right) of C57BL/6 J mice treated with 250 µg anti-PD-1 and 100 µg anti-CTLA-4, and as combinatory therapy with additional 200 µg anti-PD-L1 on d13, d16, and d19 post Gl261 inoculation (aPD-1 + aCTLA-4 n = 13, aPD-1 + aCTLA-4 + aPD-L1 n = 13 animals). e CIBERSORT analysis of a GBM expression dataset of PD-1 inhibitor-treated patients before therapy (R n = 4, NR n = 5 biologically independent samples)—two-sided WRST. f Mediators of ICB response (Z-transformed log2 fold change R/NR). Data are represented as mean ± SEM for a, b, c and e. For a, statistical significance was determined by one-way ANOVA in combination with Dunnett’s test (CD3⁺ cells + R, NR, or C CD11b⁺ cells vs. T cells only) and Sidak’s test for multiple comparison (CD3⁺ cells + R CD11b⁺ cells vs. CD3⁺ cells + NR CD11b⁺ cells) or two-tailed paired Student’s t-test (− PD-L1 vs. + PD-L1). Statistical significance was analyzed by one-way ANOVA with Tukey’s test for multiple comparison for b, by one-way ANOVA in combination with Dunnett’s test (CD3⁺ cells + R, NR, or C CD11b⁺ cells vs. T cells only) for c, by unpaired two-tailed Student’s t-test for d, and WRST with Benjamini–Hochberg correction for e. Source data are provided as a Source Data file.