Fig. 5. Chemokine-mediated recruitment of T_regs in the tumor micro-environment supported by CD8⁺ T cells in vivo.

(A and B) B16 cells were engrafted into Rag2^−/− (subcutaneously) followed, 24 hours later, by injection of 3 × 10⁶ CD8⁺ T cells (intravenously) (if indicated), and on day 10 after tumor inoculation, 1 × 10⁶ T_regs (CD4⁺ CD25⁺) were given (intravenously). PTX T_regs were treated with pertussis toxin for 1.5 hours in vitro before injection, and C021 T_regs were treated with the CCR4 antagonist C021 for 2 hours before injection. Forty-eight hours after T_reg injection, tumor and spleen were analyzed for the number of infiltrating T_regs (CD3⁺, CD4⁺,FoxP3⁺). T_regs could only be detected in the tumor site if nontreated and given in combination with pre-engrafted CD8⁺ T cells (A), whereas chemokine receptor inhibitory treatment did not alter homing to the spleen (B). Shown are means ± SEM of n = 6 out of two independent experiments; significance was tested using two-sided Mann-Whitney U test (**P = 0.0024; ****P < 0.0001). (C) Naïve (CD62L⁺) CD8 T cells were cultured for 6 hours or 7 days ± 6 hours or assayed ex vivo. After activation, mRNA expression levels of CCL22 were assessed by qRT-PCR, normalized to 18S RNA, and were relative to the ex vivo expression level of CCL22 (*) (left panel). Tumor-infiltrating CD8 T cells were sorted out of B16 tumors 12 days after engraftment and analyzed either ex vivo or after activation for 6 hours (right panel). Shown are means ± SEM of n = 3, with numbers indicating the fold change compared to ex vivo naïve or tumor-infiltrating CD8⁺ T cells.