Fig. 6
Enhanced suppressive function of PD-1-expressing tumor-infiltrating Treg. a Enhanced suppression of CD8+ T cells by PD-1-expressing tumor-infiltrating Treg. At 3 weeks after intravenous injection of TC-1 cells, Treg were isolated from the spleen (SP) and lung of mice with TC-1 cell-induced tumors. SP Treg and tumor-infiltrating Treg expressed low and high levels of PD-1, respectively. CellTrace Violet (CTV)-labeled CD8+ T cells were stimulated in vitro with CD3/CD28 Dynabeads for 72 h in the absence or presence of each Treg population. CTV dilution in proliferating CD8+ T cells is indicated in each histogram. Histograms represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. b Contact-dependent Treg-mediated suppression of CD8+ T proliferation. CTV-labeled CD8+ T cells were stimulated in vitro with CD3/CD28 Dynabeads and cocultured with tumor-infiltrating Treg for 72 h in the absence or presence of a transwell membrane. c Homeostatic proliferation of donor Ly5.1+CD8+ T cells in the spleen isolated from Rag2−/− mice at 7 d after adoptive cell transfer. Representative plot (left) and absolute number (right) of donor Ly5.1+CD8+ T cells in the spleen. d PD-1-mediated suppressive activity of tumor-infiltrating Treg isolated from the lungs of tumor-bearing mice 2 weeks after intravenous injection of TC-1 cells. At this time point, Treg expressed intermediate levels of PD-1. CTV-labeled CD8+ T cells were stimulated as shown in (a). Before co-culture of CD8+ T cells with tumor-infiltrating Treg, the latter were pre-incubated with an anti-PD-1 antibody or its isotype as control. CTV dilution in proliferating CD8+ T cells is shown in the histograms, which represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. (e) Representative immunofluorescence images of mouse lung tumor samples. Antibodies against Foxp3, CD8, and PD-1 were used to label and examine the interaction between Treg and CD8+ T cells expressing PD-1. Data are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test)