PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors

Abstract

Vaccination with irradiated B16 melanoma cells expressing either GM-CSF (Gvax) or Flt3-ligand (Fvax) combined with antibody blockade of the negative T-cell costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) promotes rejection of preimplanted tumors. Despite CTLA-4 blockade, T-cell proliferation and cytokine production can be inhibited by the interaction of programmed death-1 (PD-1) with its ligands PD-L1 and PD-L2 or by the interaction of PD-L1 with B7-1. Here, we show that the combination of CTLA-4 and PD-1 blockade is more than twice as effective as either alone in promoting the rejection of B16 melanomas in conjunction with Fvax. Adding alphaPD-L1 to this regimen results in rejection of 65% of preimplanted tumors vs. 10% with CTLA-4 blockade alone. Combination PD-1 and CTLA-4 blockade increases effector T-cell (Teff) infiltration, resulting in highly advantageous Teff-to-regulatory T-cell ratios with the tumor. The fraction of tumor-infiltrating Teffs expressing CTLA-4 and PD-1 increases, reflecting the proliferation and accumulation of cells that would otherwise be anergized. Combination blockade also synergistically increases Teff-to-myeloid-derived suppressor cell ratios within B16 melanomas. IFN-gamma production increases in both the tumor and vaccine draining lymph nodes, as does the frequency of IFN-gamma/TNF-alpha double-producing CD8(+) T cells within the tumor. These results suggest that combination blockade of the PD-1/PD-L1- and CTLA-4-negative costimulatory pathways allows tumor-specific T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby shifting the tumor microenvironment from suppressive to inflammatory.

Fig. 1.

Survival of mice challenged with 5 × 10⁴ B16-BL6 cells and vaccinated on days 3, 6, and 9 with 1 × 10⁶ Gvax (A) or Fvax (B) intradermally and the indicated antibody or combination i.p.. Lack of survival was defined as death or tumor size >1,000 mm³. Each curve represents three to four independent experiments of 5–15 mice per group.

Fig. 2.

Lymphocyte infiltration of B16 melanoma. Mice were challenged with 1.5 × 10⁵ B16-BL6 cells; treated with Fvax and the indicated antibody on days 3, 6, and 9; and killed on day 16. Percentages of CD45⁺ TILs of CD8⁺ T cells (A), CD4⁺ Teffs (B), and Tregs (C) are shown. The ratios of CD8⁺ T cells to Tregs (D) and CD4⁺ Teffs to Tregs (E) are shown. Values shown are for individually analyzed mice and are the sum of four to seven independent experiments with 5–15 mice per group. To determine statistical significance between samples, t tests were performed. *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001.

Fig. 3.

CTLA-4 and PD-1 expression by TILs. Mice challenged with 1.5 × 10⁵ B16-BL6 cells and treated on days 3, 6, and 9 were killed on day 16. TILs were fixed and stained for lymphocyte lineage and activation markers using the FoxP3 fixation kit. CTLA-4 and PD-1 were stained for surface and intracellular expression. Percentages of CD8⁺ T cells (A) and CD4⁺ Teffs (B) expressing PD-1, CTLA-4, or both are shown. Values shown are for individually analyzed mice and are the sum of three to six independent experiments with 5–15 mice per group. To determine statistical significance between samples, t tests were performed. *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001.

Fig. 4.

Ratios of Teffs to MDSCs within tumors. Mice challenged with 1.5 × 10⁵ B16-BL6 cells and treated on days 3, 6, and 9, were killed on day 16. TILs were fixed and stained for lymphocyte and myeloid lineage and activation markers using the FoxP3 fixation kit. Arginase-1 was stained using the 8C9 clone from Santa Cruz. The ratios of CD8⁺ T cells to CD11b⁺arginase-1⁺ MDSCs (A) and CD4⁺ Teffs to CD11b⁺arginase-1⁺ cells (B) within CD45⁺ melanoma TILs are shown for mice receiving the indicated therapy. Values shown are for individually analyzed mice and are the sum of three independent experiments with 5–15 mice per group. To determine statistical significance between samples, t tests were performed. *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001.

Fig. 5.

Changes in cytokine production in response to coinhibitory blockade. Mice challenged with 2.5 × 10⁵ B16-Ova cells and treated on days 6, 9, and 12 were killed on day 14. T cells were purified from tumor-draining lymph nodes and vaccine-draining lymph nodes, stained with antibodies, and sorted by flow cytometry into CD4⁺ and CD8⁺ subsets. Cytokine production was measured after 36 h using the TH1/TH2/TH17 CBA Kit and is shown for 2 × 10⁵ CD8 T cells restimulated on 1 × 10⁵ Ova 257–264 peptide-pulsed DCs (A) and for 2.5 × 10⁵ CD4 T cells restimulated on 1 × 10⁵ Ova 323–339 peptide-pulsed DCs (B). Data are shown for three to four independent experiments with five pooled mice per group. (C and D) TILs were purified from 5–10 pooled tumors per group and enriched using the Miltenyi T-cell purification kit. A total of 2 × 10⁶ TILs were restimulated with 7.5 × 10⁵ DCs [1:1 mix of Ova(257) and Ova(323) pulsed DCs] per well for 8 h in the presence of GolgiPlug. Cells were fixed using the FoxP3 kit and analyzed by flow cytometry for lymphocyte markers and intracellular IFN-γ and TNF-α production for CD8 cells (C) and CD4 Teffs (D) and also for Ki67 expression in CD8 cells (E) and CD4 Teffs (F). TIL data are from four independent experiments. All means shown are ± SEM.