Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors

Abstract

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression.

Figure 1. Phenotypic analysis of CT26 and ID8-VEGF TIL

TIL isolated from CT26 and ID8-VEGF tumors were stained with antibodies to CD45, CD8, CD4, PD-1, CTLA-4, CD44, and CD62L as well as live/dead stain. Representative CT26 (A) and ID8-VEGF (B) tumor samples showing expression of PD-1 and CTLA-4 on CD8⁺CD45⁺ and CD4⁺CD45⁺ TIL with distinct cell populations, which are further analyzed for CD44 and CD62L. Summary data of CTLA-4 and PD-1 expression on T_EM (CD44^hiCD62L^lo) CD8⁺ and CD4⁺ TIL. Data represent mean ± SD of n=6 mice per group and are representative of 4 independent analyses.

Figure 2. Co-expression of PD-1 and CTLA-4 correlates with more severe dysfunction of CT26 and ID8-VEGF antigen-specific CD8⁺ T cells

A) PD-1⁺CTLA-4⁺ and PD-1⁺CTLA-4⁻ CD8⁺ TIL populations from either CT26 (i) or ID8-VEGF (ii) tumors were sorted, labeled with CFSE, and stimulated with AH-1 or FR peptide, respectively for 3 days in the presence of irradiated splenic CD45.1⁻ APCs. CD8⁺ T cell proliferation was determined by dilution of CFSE; numbers indicate the percentage of CFSE^lo (AH-1 or FR reactive cells). B) Frequency of CT26 (i) or ID8-VEGF (ii)-specific PD-1⁺CTLA-4⁻ and PD1⁺CTLA-4⁺ CD8⁺ T cells showing cytokine and degranulation after peptide stimulation (n=6). PD-1⁺CTLA-4⁻ and PD1⁺CTLA-4⁺ CD8⁺ TILs cells were further analyzed for differential phenotype and expression of other inhibitory receptors. C) PD-1⁺CTLA-4⁺ or PD-1⁺CTLA-4⁻ CD8⁺ TILs from CT26 (i) or ID8-VEGF (ii) were stimulated with AH-1 or FR peptide, respectively and cultured with blocking antibodies for 3 days. Proliferation was determined by ³[H]-thymidine incorporation. Statistical significance was determined by Student’s t-test.

Figure 3. In vivo PD-1 and CTLA-4 blockade promotes CT26 and ID8-VEGF tumor rejection in mice

Regression and survival (<200 mm²) of CT26 (A) and ID8-VEGF (B) inoculated mice following treatment as described in the Methods section. Figures represent one of the 5 (CT26) and 3 (ID8-VEGF) independent experiments.

Figure 4. Therapeutic adoptive transfer of in vitro α-PD-1 and α-CTLA-4 pre-treated TILs cause regression of CT26 tumors in mice

A) Tumor regression in mice transferred with in vitro expanded CT26 antigen-specific CD8⁺CTLA-4⁺PD-1⁺ CT26 TILs. (B) The percent of IFN-γ⁺ and Ki67⁺ of in vitro pre-treated CD8⁺ T cells just before adoptive transfer (left panel) and the TILs recovered from tumor one week after the final transfer (right panel) are shown.

Figure 5. In vivo PD-1 and CTLA-4 blockade increases TIL activation and CT26 antigen-specific inflammatory cytokine production

Summary data showing percentage of Ki-67⁺ CD8⁺ TILs (A) and mean fluorescence intensity (MFI) of granzyme B⁺ CD8⁺ TILs (B) from mice treated with various antibodies, as indicated. C) Flow cytometry analysis showing MFI of pT-bet, pEomes and pS6K expression by CD8⁺ TIL from treated mice. Representative and summary data showing IFN-γ and TNF-α production by CD8⁺CD45⁺ TIL from antibody (D) as well as GVAX ± antibody (E) treated mice 13–15 days following tumor inoculation. Statistical significance was determined by Student’s t-test.

Figure 6. PD-1 and CTLA-4 blockade enhance lymphocyte infiltration and reduce the frequency of Tregs in CT26 tumors

A) CD45⁺ leukocyte infiltration, and CD8⁺ and CD4⁺ TIL as percent of total CD45⁺ cells 13–15 days after tumor inoculation. B) Representative data showing the percentage of Tregs and expression of GITR among Tregs. C) Percentage of CD45⁺ TILs of Tregs, and the ratios of CD8⁺ or CD4⁺ effector T cells to Tregs in the tumors of treated mice with indicated blocking antibodies. Values are shown for mice analyzed independently and the sum of 3 independent experiments with 6–12 mice per group. Statistical significance was determined by Student’s t-test.

Figure 7. PD-1 and CTLA-4 pathway blockade reduces Treg-mediated suppression of CD8⁺ T cells in vitro and secretion of regulatory cytokines TGF-β and IL-10

A) Representative expression of PD-1 and CTLA-4 by intra-tumoral and splenic Tregs in the same mouse. B) ICOS and folate receptor 4 (FR4) expression and MFI in PD-1^hi and PD-1^lo Tregs. C) TIL Tregs (± αPD-1) were co-cultured with CFSE-labeled CD8⁺ T cells and DCs (pretreated) in the presence of αCD3. Dividing cells were quantified after 4-days of culture by gating on the CFSE^lo population. D) [³H]-thymidine incorporation into effector T cells in the presence or absence of TIL Tregs. Tregs were either in direct contact with the effector cells or were separated by a transwell membrane. The whole tumor leucocytes were isolated and cultured ex vivo for 72 hours in the presence of AH-1 peptide and PD-1 and CTLA-4 blocking antibodies as indicated. After culture, supernatants were analyzed for secretion of TGF-β (E), IL-10 (F), and IFN-γ (G). Results are from 3 representative experiments. Bar graphs show mean ± SD.