Fig. 7.

Treg derived CD27 and PD-1 signaling limits tumor infiltration of CD8⁺ T cells and CTL effector functions. Mixed BM chimeric mice were generated by the reconstitution of lethally irradiated mice with equal numbers of FoxP3.LuciDTR-5 (CD45.1) cells and CD27 − / − BM cells. Eight weeks upon reconstitution, mice were injected s.c. with MC38 tumor cells. Seven and nine days after tumor inoculation, mice were treated either with DT i.v. or with αPD-1 i.v. or with a combination of both. As for the control, some mice were left untreated. Tumors were removed 15 days after inoculation. Tumors were digested and tumor-infiltrating lymphocytes (TILs) were enriched using CD45 beads. Total numbers of all TILs (A), of WT CD8⁺ TIL (B), and CD8⁺ T cells/ Treg TIL ratios (C) were determined by flow cytometric analysis. TILs were restimulated with PMA + Ionomycin for 4.5 h and IFNγ (D), TNFα (E), and Granzyme B (F) production of wt CD8 + TILs were determined using flow cytometry. Data of 2 independent experiments are shown. Horizontal bars represent mean ± SD (n = 4; DT, n = 5; untreated, n = 3). Statistical significance was determined with one-way ANOVA with Bonferroni’s multiple comparisons correction *p < 0.05, **p $\le$ 0.01, ***p $\le$ 0.001