Blockade of programmed death ligand 1 enhances the therapeutic efficacy of combination immunotherapy against melanoma

Abstract

Inhibition of antitumor T cell responses can be mediated by the productive interaction between the programmed death-1 (PD-1) receptor on T cells and its ligand PD-L1. PD-L1 is highly expressed on both murine bone marrow-derived dendritic cells (DCs) and B16 melanoma. In this study, in vitro blockade of PD-L1 interaction on DCs led to enhanced IFN-gamma production and cytotoxicity by Ag-specific T cells. In vivo, the systemic administration of anti-PD-L1 Ab plus melanoma peptide-pulsed DCs resulted in a higher number of melanoma peptide-specific CD8(+) T cells, but this combination was insufficient to delay the growth of established B16 melanoma. Although the addition of 600 rad of total body irradiation delayed tumor growth, further adoptive transfer of Ag-specific CD8(+) T cells was needed to achieve tumor regression and long-term survival of the treated mice. Lymphopenic mice treated with anti-PD-L1 Ab demonstrated increased activation and persistence of adoptively transferred T cells, including a higher number of CD8(+) T cells infiltrating the tumor mass. Together, these studies support the blocking of PD-L1 signaling as a means to enhance combined immunotherapy approaches against melanoma.

Figure 1

Peptide-loaded DC pre-treated with anti-PD-L1 antibody enhance stimulation of a specific T cell response. (A) After 5 days of in vitro culture, bone marrow-derived DC were stained with anti-IA^b and PD-L1 antibodies and analyzed by flow cytometry. Filled histogram = anti-PD-L1, open histogram = isotype control. (B) DC were pulsed with OVA_257–264 or gp100_25–33 peptide and treated with either 10 µg/ml NrIgG or anti-PD-L1 antibody for 24 hours. These DC were then co-cultured with OT-I splenocytes at a 1:10 ratio for 48 hours. (C) DC were pulsed with OVA_323–337 peptide and treated with either 10 µg/ml NrIgG or anti-PD-L1 antibody for 24 hours. These DC were co-cultured with OT-II splenocytes at a 1:10 ratio for 48 hours. Supernatants were collected and tested in an IFN-gamma ELISA assay. One of 2 representative experiments is shown. *indicates p<0.001, **indicates p<0.01.

Figure 2

Stimulation with anti-PD-L1 antibody-treated DC enhances T cell cytotoxicity. Purified pmel T cells were stimulated in vitro for 5 days with gp100-pulsed DC pre-treated with NrIgG or anti-PD-L1 antibody. Cell cytotoxicity against gp100-coated EL4 target cells was measured in a 5 hour ⁵¹Cr release assay. *indicates p<0.01.

Figure 3

Immunization with OVA peptide-pulsed DC in combination with anti-PD-L1 antibody treatment leads to enhanced CD8⁺ T cell response. C57BL/6 mice (n=4) were immunized s.c. three times with DC pulsed with OVA_257–264 peptide at one week intervals. Mice also received NrIgG or anti-PD-L1 antibody every 3–4 days. Splenocytes were collected one week after the final peptide-pulsed DC immunization. (A) The percentage of CD8⁺ and OVA tetramer⁺ cells was measured by flow cytometry. * indicates p<0.01. (B) Splenocytes alone or co-cultured with 10 µg/ml OVA_257–264 peptide in vitro for 48 hours. Supernatants were collected and IFN-gamma was measured by an ELISA assay. This experiment was repeated twice with similar results. ** indicates p<0.05.

Figure 4

Expression of PD-L1 on B16 melanoma cells. B16 cells were stained with anti-PD-L1 antibody. Expression was measured by flow cytometry. Open histogram = Isotype control; filled histogram: PD-L1 expression.

Figure 5

Adoptive transfer of pmel T cells enhances the anti-B16 tumor response in tumor-bearing mice treated with anti-PD-L1 antibody and peptide-pulsed DC. C57BL/6 (n=6 per group) mice were injected s.c. with B16 cells. On day 3, mice received 600 rad of TBI. On day 4, mice received 1×10⁷ pmel T cells i.v. Beginning on day 4 and continuing every 3–4 days until day 60, mice received i.p. injections of NrIgG or anti-PD-L1 antibody. Mice also received s.c. injections of DC pulsed with gp100 peptide on days 4 and 11 after B16 injection. Tumor sizes were measured twice each week. (A) Tumor growth. (B) Survival. The results are representative of two similar experiments. * indicates p<0.05 compared to pmel + DC-gp100 + NrIgG treated mice.

Figure 6

Pmel T cells persist in mice treated with anti-PD-L1 antibody. Ly5.2 (CD45.1⁺) mice (n=20) were treated with 600 rad of TBI. On day 3, mice received i.v. 1×10⁷ pmel T cells. Beginning on day 3 and continuing every 3–4 days, mice received i.p. injections of either NrIgG or anti-PD-L1 antibody. Spleens (n=4) were collected at each time point and (A) the percentage of CD45.2⁺ pmel T cells was measured by flow cytometry. Splenocytes were restimulated in vitro with gp100-peptide pulsed DC for 48 hours. (B) Supernatants were collected and IFN-gamma was measured by an ELISA assay. (C) ³H-thymidine was added for an additional 18 hours. Cells were harvested and the amount of thymidine incorporation was measured. These experiments were repeated two times. * indicates p<0.01, **indicates p<0.05.

Figure 7

Higher numbers of pmel T cells infiltrate B16 tumor in mice treated with anti-PD-L1 antibody. Ly5.2 mice (n=10) were injected s.c. with B16 tumor cells. On day 11, mice received 1×10⁷ pmel T cells i.v. Mice also received i.p. injections of either NrIgG or anti-PD-L1 antibody on days 11, 14, and 17. Spleens and tumors were collected on day 19 and the percentage of CD45.2⁺ pmel T cells was measured. * indicates p<0.05 compared to NrIgG treated mice. This experiment was repeated two times with similar results.

Figure 8

Adoptive transfer of OVA-immune T cells enhances the anti-tumor response in M05-bearing mice treated with anti-PD-L1 antibody and peptide-pulsed DC. (A) Expression of PD-L1 on the surface of M05 cells. Filled histogram: anti-PD-L1, open histogram: NrIgG. (B) C57BL/6 (n=6 per group) mice were injected s.c. with M05 cells. On day 3, mice received 600 rad of TBI. On day 4, mice received 1×10⁷ T cells i.v. that were isolated from mice immunized four times with DC-OVA_SIINFEKL. Beginning on day 4 and continuing every 3–4 days until day 60, mice received i.p. injections of either NrIgG or anti-PD-L1 antibody. Mice also received DC pulsed with OVA_SIINFEKL peptide s.c. on days 4 and 11 after B16 injection. Tumors were measured twice each week. The results are representative of two experiments. * indicates p<0.05 compared to T cells + DC-OVA + NrIgG treated mice.