Fig. 4

Immunophenotyping of CCK168 tumors in response to α-PD-1 and α-TGFβ therapy. a-k) CCK168 tumor cells were implanted sc into FVB mice according to Fig. 1b. After two drug doses on day 0 and 4, when some tumors began to show evidence of shrinkage, all tumors were harvested and analyzed by (a) immunohistochemistry or (b-k) multicolor flow cytometry. a Representative images of CD8a and CD45 immunohistochemistry for tumors from each of the four drug arms. Six to seven tumors were analyzed per drug arm per stain. Scale bar represents 50 uM. (b-k) Flow cytometry analysis shows increases in b CD45+ cells per live tumor cell, c CD8+ cytotoxic T cells per CD45+ immune cells and d increases in percentage of ICOS+ expressing CD8+ T cells. e α-PD-1 or α-TGFβ monotherapy elevates total CD4+ T cells with no additive effect. f α-TGFβ monotherapy neutralizes α-PD-1 induction of Tregs and, in combination therapy, reduces Treg levels to below baseline. g heterogeneous increase in CD4 + Th/CD4 + Treg ratio by α-TGFβ, h synergistic induction of CD8+/Treg ratios by α-PD-1 and α-TGFβ. The latter increased six to 40 fold in response to combinatorial therapy. (i) MHCII+CD11b + and (j) MHCII+CD11c + myeloid cells diminish as a percentage of total CD45 + cells following α-PD-1 or α-TGFβ therapy. k The ratio of mature T cells (CD4+ plus CD8+ cells) per CD11b myeloid cell (CD45 + Ly6G-CD11b + MHCII+) increases after combinatorial therapy. Flow cytometry data are representative of two to seven independent experiments for each cell type. * p < 0.05; ** p < 0.01; *** p < 0.001: Mann Whitney U test