Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer

Abstract

The tumor microenvironment mediates induction of the immunosuppressive programmed cell death-1 (PD-1) pathway, and targeted interventions against this pathway can help restore antitumor immunity. To gain insight into these responses, we studied the interaction between PD-1 expressed on T cells and its ligands (PD-1:PD-L1, PD-1:PD-L2, and PD-L1:B7.1), expressed on other cells in the tumor microenvironment, using a syngeneic orthotopic mouse model of epithelial ovarian cancer (ID8). Exhaustion of tumor-infiltrating lymphocytes (TIL) correlated with expression of PD-1 ligands by tumor cells and tumor-derived myeloid cells, including tumor-associated macrophages (TAM), dendritic cells, and myeloid-derived suppressor cells (MDSC). When combined with GVAX or FVAX vaccination (consisting of irradiated ID8 cells expressing granulocyte macrophage colony-stimulating factor or FLT3 ligand) and costimulation by agonistic α-4-1BB or TLR 9 ligand, antibody-mediated blockade of PD-1 or PD-L1 triggered rejection of ID8 tumors in 75% of tumor-bearing mice. This therapeutic effect was associated with increased proliferation and function of tumor antigen-specific effector CD8(+) T cells, inhibition of suppressive regulatory T cells (Treg) and MDSC, upregulation of effector T-cell signaling molecules, and generation of T memory precursor cells. Overall, PD-1/PD-L1 blockade enhanced the amplitude of tumor immunity by reprogramming suppressive and stimulatory signals that yielded more powerful cancer control.

Figure 1. Accumulation of myeloid and T cell populations in ID8 tumor

(a) Six to 8 weeks-old mice (n=12) were inoculated i.p. with 5 × 10⁶ ID8 tumor cells. The survival (i) and scheme (ii) are shown. (b) T (CD3⁺, CD8⁺, and CD4⁺) cells, Treg (CD4⁺CD25⁺Foxp3⁺) cells, and MDSC (CD11b⁺Gr1⁺) from tumor, ascites, spleen of ID8 tumor-bearing mice were isolated and counted (using 0.4% trypan blue stain) from 6 mice during early (4–5 weeks after tumor inoculation) or rest of the 6 mice during advanced (7–8 weeks after tumor inoculation) tumors. All cell subtypes were CD45⁺ gated. Bars represent mean ± SEM.

Figure 2. Expression of PD-L1, PD-L2 and PD-1 in ID8 mice

Mice were inoculated i.p. with 5×10⁶ ID8 tumor cells (n=12), and their tumor, ascites, spleen, liver, lung and blood were harvested from half of the mice at early and the other half at advanced tumor. Tumor cells (EpCAM⁺), macrophages (CD45⁺CD11B⁺F4/80⁺), DCs (CD45⁺CD11c⁺) and MDSCs (CD45⁺CD11B⁺Gr1⁺) were isolated from the tumor, ascites and spleen. Histograms show PD-L1 (a) and PD-L2 (b) expression by ID-8 tumor cells as well as macrophages, DCs and MDSCs from tumor, ascites and spleen of ID-8 tumor-bearing mice. (c) PD-1 expression on CD8⁺ T cells from tumor, ascites, spleen, blood, liver and lung of ID-8 tumor-bearing mice. Results are from one of the 3 experiments.

Figure 3. PD-1 or PD-L1 blockade causes regression of ID8 tumor

Mice were inoculated i.p. with 5 × 10⁶ ID8 tumor cells (n=12). These mice were injected either therapeutically (starting at 30 days after tumor inoculation) or prophylactically (starting at 20 days after tumor inoculation) 5 times i.p. with αPD-1 (200ug), αPD-L1 (200ug), or αPD-L2 (200ug) blocking antibodies on alternate days either alone or in combination as indicated. The scheme and survival of therapeutic (i) and prophylactic models (ii) are shown. Results are from one of the 3 representative experiments.

Figure 4. PD-1 or PD-L1 blockade increases immune activation of TILs in ID8 tumor

One week following completion of blockade treatment, the TILs were harvested from regressing tumors and stained with various markers. Percentages of CD8⁺ and CD4⁺ TIL (CD45⁺) infiltration of total leucocytes (a) and the ratio of CD8⁺ T cells to Tregs (b) are shown in treated versus untreated groups. (c) Blocking PD-1/PD-L1 interaction reduced Treg-mediated suppression of CD8⁺ T cells in vitro. CFSE-labeled CD8⁺ T cells were co-cultured with syngeneic, αCD3-loaded DCs with or without Tregs and αPD-L1 or αCTLA-4 as indicated. CD8⁺ T cells and stimulator APCs were obtained from naive B6 mice. Tregs were obtained from ID8-tumor bearing mice. Treg-mediated suppression of proliferation of naïve CD8+ T cells was noticed. Results from one of 3 experiments are shown. (d) The ratio of CD8⁺ T cells to MDSCs are shown. (e) CD8⁺ TILs from αPD-L1 treated mice were stained with arginase-1 (8C9 clone from Santa Cruz) and analyzed by flow cytometry. The CD11b⁺arginase-1⁺ MDSCs within CD45⁺ TILs are shown. (f) Tumor-dervived MDSCs were plated at 1×10⁶/well in 24-well plates and stimulated with equal amount of tumor supernatants (from ID8 cells). Following stimulation, cells were added with αPD-L1 and then arginase I was analyzed after 24 h following washing with PBS and lysis buffer. (g) Percentage of Ki67⁺ and Granzyme B⁺ as well as MFI of pT-bet, pEomes, pS6, and pAkt expression by CD8⁺ TILs are shown. The results are the sum of three independent experiments with 8–10 mice per group.

Figure 5. Synergistic effect of PD-1 blockade, GVAX or FVAX vaccination, and α4-1BB costimulation on ovarian ID8 tumor rejection

(a) Three weeks after ID8 tumor inoculation, the mice were given i.p with 2 × 10⁶ irradiated (150Gy) GVAX (top) or FVAX (bottom) once. A week later mice were injected 5 times i.p. with αPD-1 (200ug), αPD-L1 (200ug), αPD-L2 (200ug) on alternate days or a single dose 200ug of α-4-1BB either alone or in combination as indicated. Results are from one of the 3 experiments.

Figure 6. Synergistic effect of PD-1 blockade, GVAX vaccination, and α4-1BB costimulation on immune activation

TILs from regressing tumors of ID8 mice were harvested a week following completion of treatment and stained for various markers. Percentages of CD8⁺ and CD4⁺ TIL (CD45⁺) infiltration of total leucocytes are shown in treated versus untreated groups. The ratio of CD8⁺ T cells to Tregs and MDSC are also shown. Percentages of Ki67⁺CD8⁺ and MFI of Granzyme B⁺CD8⁺ TIL (CD45⁺) infiltration of total leukocytes are shown in treated versus untreated groups. Mean intensity of pT-bet, pEomes, and pS6 expression by CD8⁺ TILs is shown. Results from one of the 3 experiments are shown. Bars represent mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001.

Figure 7. In vivo PD-L1 blockade increases ovarian tumor antigen (FR-α) specific inflammatory cytokine production by ID8 TILs

(a) Representative data and summary showing percentage of IFN-γ production by CD8⁺ TILs from treated mice as indicated. Following treatment with GVAX or FVAX and/or in vivo αPD-L1 and α-4-1BB antibody treatment, the TILs were harvested and stimulated ex vivo with FR-α peptide for 5 h at 37°C in the presence of brefeldin-A. For intracellular staining, permeabilized cells were stained for IFN-γ or phosphorylated form of transcription factors (T-bet, eomes and S6 kinase). (b) A week after completion of treatment, 2 × 10⁵ CD8⁺ T cells from 5–10 pooled tumor draining mediastinal lymph nodes were re-stimulated with 1 × 10⁵ FR-α peptide pulsed DCs for 36–48 h. Cytokine (IFN-γ and TNF-α) production from culture supernatant was measured following in vitro culture using cytokine bead array (CBA) kit. (c) Flow cytometry analysis showing percentage of phosphorylated (p) T-bet, pEomes and pS6K expression by CD8⁺ TIL from treated mice. All analyses were performed using CD8⁺CD45⁺ TIL from treated mice. N.S. not significant.