Fig. 3

CTLA-4 and PD-1 blockade each potentiate intra-tumoral TLR9 activation through distinct mechanisms. (A) C57BL/6 J mice were injected with 1.5 × 10⁵ B16-Ova melanoma cells on the right and left flanks. The right flank tumor was then injected with 30μg of the TLR9 agonist ODN1826 or PBS in 50ul on days 3, 6 and 9 with or without concordant injection of 100μg anti-CTLA-4 9H10 i.p. or 250μg of anti-PD-1 RMP1–14 i.p. or 10μg of either antibody intra-tumorally. Mice were euthanized on day 14, tumors treated with Collagenase H (Sigma) and DNase (Roche) to produce single cell suspensions, and tumor infiltrating lymphocytes enriched via separation over a Histopaque 1119 (Sigma) density gradient (n = 1 experiment with 8 mice / group). Cells were fixed with the FoxP3 fixation kit (Thermo), stained with antibodies and analyzed by flow cytometry. (a) The ratios of intra-tumoral CD8 T cells versus FoxP3+ Treg and (b) versus CD11b⁺GR-1⁺ MDSC were determined. (c) For intra-tumoral CD8 T cells, the percentage expressing Granzyme B and (d) Ki67 was also measured. Statistical significance was calculated using the student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001