Fig. 6.

Increased tumor growth by PD-1–deficient Treg cells. (A) C57BL/6 mice were inoculated with B16F0 melanoma cells in the right rear flank. Fifteen days after inoculation, T cells were prepared from tumors and draining inguinal lymph nodes and subjected to flow cytometry. Representative flow cytometry staining for PD-1 expressed by Treg cells (red), Tconv cells (blue), and CD8⁺ T cells (green) in TILs (Top) and Ki67 expressed by TIL Treg cells (red) from tumor and PD-1⁺ Treg cells (blue) and PD-1⁻ Treg cells (green) from draining lymph nodes (Bottom). (B) C57BL/6 mice were lympho-depleted by 6-Gy irradiation and then were transferred with spleen cells from CD4^CrePD1^floxedFDG mice and Treg cells from either FoxP3^IRES-Cre or FoxP3^IRES-CrePD1^floxed mice. After cell transfer, mice were injected s.c. with B16F0 cells. DT was administered intraperitoneally 3 d after cell transfer to deplete Treg cells from the CD4^CrePD1^floxedFDG transferred fraction. Tumor growth of B16 tumors was measured over 18 d. (C) Irradiated (6 Gy) CD45.2 B6 mice were transferred with CD45.2 CD4^CrePD1^floxedFDG spleen cells and PD-1–intact CD45.1 Treg cells. Mice were injected with B16 tumor cells and DT as in B, and anti–PD-1 or isotype-matched IgG mAb was administered on days 5, 10, and 15. Tumor growth of B16 tumors was measured over 18 d (Left). Tumor masses measured on day 18 are shown (Right). (D) Tumor-draining lymph nodes in anti–PD-1 mAb-treated or control mice were collected on day 18 posttransfer to assess transferred CD45.1⁺ Treg cells. Representative flow cytometry staining (Left) and percentage (Right) of proliferating (Ki67⁺) transferred CD45.1⁺ Treg cells from both groups. Data are representative of at least two independent experiments.