Programmed death receptor-1/programmed death receptor ligand-1 blockade after transient lymphodepletion to treat myeloma

Abstract

Early phase clinical trials targeting the programmed death receptor-1/ligand-1 (PD-1/PD-L1) pathway to overcome tumor-mediated immunosuppression have reported promising results for a variety of cancers. This pathway appears to play an important role in the failure of immune reactivity to malignant plasma cells in multiple myeloma patients, as the tumor cells express relatively high levels of PD-L1, and T cells show increased PD-1 expression. In the current study, we demonstrate that PD-1/PD-L1 blockade with a PD-L1-specific Ab elicits rejection of a murine myeloma when combined with lymphodepleting irradiation. This particular combined approach by itself has not previously been shown to be efficacious in other tumor models. The antitumor effect of lymphodepletion/anti-PD-L1 therapy was most robust when tumor Ag-experienced T cells were present either through cell transfer or survival after nonmyeloablative irradiation. In vivo depletion of CD4 or CD8 T cells completely eliminated antitumor efficacy of the lymphodepletion/anti-PD-L1 therapy, indicating that both T cell subsets are necessary for tumor rejection. Elimination of myeloma by T cells occurs relatively quickly as tumor cells in the bone marrow were nearly nondetectable by 5 d after the first anti-PD-L1 treatment, suggesting that antimyeloma reactivity is primarily mediated by preactivated T cells, rather than newly generated myeloma-reactive T cells. Anti-PD-L1 plus lymphodepletion failed to improve survival in two solid tumor models, but demonstrated significant efficacy in two hematologic malignancy models. In summary, our results support the clinical testing of lymphodepletion and PD-1/PD-L1 blockade as a novel approach for improving the survival of patients with multiple myeloma.

Figure 1. Increased PD-1 expression on CD8 T cells in the spleens and bone marrow of myeloma-bearing mice occurs preferentially on T cells with the potential for tumor antigen reactivity

A. KaLwRij mice were inoculated with 2×10⁶ 5T33 cells i.v. Myeloma-bearing (MB) mice became moribund and were euthanized between days 28-40 after inoculation. Splenocytes and femoral bone marrows were harvested and the CD8 T cells analyzed by flow cytometry for PD-1 expression. At the same time points, naïve (non-myeloma-bearing) mice were analyzed as controls. CD8 T cells were gated as CD3⁺CD8⁺7AAD⁻. PD-1 percentage was based on isotype controls. Data are combined from 4 independent experiments; n=13 (MB) and 4 (naive) mice. B. and C. (KaLwRij × B6.SJL) F1 mice were sublethally irradiated (500 cGy) and given 1.5×10⁶ purified OT-1 CD8 splenocytes i.v. Some mice were inoculated 1-2 weeks later with 2×10⁶ 5T33 cells i.v. Myeloma-bearing (MB) mice were euthanized when they became moribund, between days 28-35 after inoculation; naive (non-myeloma-bearing) control mice were euthanized at the same time points. Splenocytes and femoral bone marrow cells were analyzed by flow cytometry to determine PD-1 expression levels on host (CD45.1⁺) versus OT-1 (CD45.1⁻) CD8 T cells. CD8 T cells were gated as CD3⁺CD8⁺7AAD⁻. B. Representative flow cytometry dot/contour plots and histograms depicting PD-1 expression on host and transferred OT-1 CD8 T cells from MB and naïve mice. C. Percentages of PD-1⁺ host and OT-1 CD8 T cells in individual mice are shown. Each group contains of 2-6 mice from 2 independent experiments, and the mean +/− SEM for each group is shown. OT-1 cells could only be found in the bone marrow of 2/5 MB mice and 5/6 naive mice.

Figure 2. Sublethal-irradiation and anti-PD-L1 administration facilitate the rejection of myeloma

A. Experimental design: Myeloma-bearing KaLwRij (B) or (KaLwRij × B6.SJL) F1 (C) mice received either 500 cGy or no irradiation (0 cGy) seven days after tumor cell inoculation. Treatment with anti-PD-L1 or control IgG (200 μg i.p.) was initiated 5 days later, and specifically given 12, 14, 19, 21, 26 and 28 days after tumor inoculation. Some mice were euthanized at day 17 for use in IFN-γ ELISPOT assays (D & E). B & C. Survival curves from showing the combined data from 3 (B) or 4 (C) independent experiments; n=12-15 mice per experimental group. D & E. CD8 T cells were isolated from spleens and BM 17 days after tumor inoculation or 10 days after irradiation in naïve mice treated with anti-PD-L1 or control IgG. The CD8 T cells were assayed in IFN-γ ELISPOT assays with tumor cell stimulators to determine tumor-reactive IFN-γ secreting cell frequencies. Anti-PD-L1 or control IgG (10 μg/mL) was added to the assays in vitro. The graphs are representative of 2 independent experiments in which the CD8 T cells for each group were pooled from 5 individual mice. ***p<0.001; **p<0.01.

Figure 3. The anti-myeloma effect induced by sublethal irradiation and anti-PD-L1 occurs relatively quickly

Myeloma-bearing mice were treated with 500 cGy irradiation on day 8 after 5T33GFP inoculation, and anti-PD-L1 or control IgG was administered on days 13, 15, 20, 22, 27 and 29. Mice were euthanized on days 18 and 32 and spleens and BM were harvested. The tissues were analyzed by flow cytometry to detect the presence of 5T33GPF. A. Representative flow cytometry dot/contour plots depicting GFP expression (tumor cells) on the y-axis and CD8 on the x-axis. B. The percentages of GFP+ 5T33 myeloma cells in the tissues of individual experimental mice are shown. Spleens were not analyzed on day 18 due to the inability to detect myeloma at this time point. The data is representative of 2 separate experiments; n=7-10 mice per group at each time point. Groups were compared using the Mann Whitney test.

Figure 4. The time course of irradiation-induced lymphodepletion and the effect of irradiation and anti-PD-L1 treatment on PD-1 and FoxP3 expression

A. Experimental design: Myeloma-bearing (KaLwRij × B6.SJL) F1 mice were euthanized on the indicated days during treatment with irradiation and anti-PD-L1 or control IgG. Splenocytes and bone marrow cells were harvested. B. Absolute numbers of splenocytes and bone marrow (femurs and tibias) are shown. C. Absolute numbers (solid lines) and percentages (dotted lines) of the indicated splenocyte immune cell populations. T cells were gated as 7AAD⁻CD3⁺, B cells were gated as 7AAD⁻CD3⁻CD19⁺, DC were gated as 7AAD⁻CD3⁻CD11c⁺, and −NK cells were gated as 7AAD⁻CD3⁻NK1.1⁺. The effect of treatment at specific time points was compared using the Student's T test (*p<0.05). D. percentages of splenic PD-1⁺ CD4 and CD8 T cells. E. Percentages of splenic Foxp3⁺ CD4 T cells. The data are the combined results of 1-3 separate experiments; n=4-14 mice per group at each time point.

Figure 5. The anti-myeloma effect of sublethal irradiation and anti-PDL1 is dependent upon both CD4 and CD8 T cells

A. Experimental design: Myeloma-bearing KaLwRij mice received in vivo depleting antibodies (250 μg i.p. of anti-CD4, anti-CD8, or anti-NK1.1) on days 6, 9, 12, 15, and 20 after 5T33 inoculation. The mice were irradiated (500 cGy) and treated with anti-PDL1 blocking antibody as illustrated. Peripheral blood from some mice was obtained after administration of depleting antibodies to verify depletion. In each case, >95% of the targeted cell population was depleted (data not shown). B. The survival curves depict the combined results of 3 independent experiments; n=12-13 mice per experimental group.

Figure 6. ACT with T cells from syngeneic myeloma-bearing donor mice provides superior anti-myeloma immunity in combination with anti-PD-L1 treatment

A. Experimental design: Myeloma-bearing (KaLwRij × B6.SJL) F1 mice were lethally-irradiated (1100 cGy) seven days after 5T33 inoculation (some mice were irradiated on day 8). One day following irradiation, the mice were intravenously injected with 5-10×10⁶ bone marrow cells from naïve mice and 6×10⁶ purified T cells from myeloma-bearing (MB) or naïve donors. Experimental groups were treated with control IgG or anti-PD-L1 blocking antibody as depicted. B. The survival curves compare experimental groups of mice given T cells from MB versus naïve donors. Data for control IgG mice are from 1 experiment; n=5-6. Data for anti-PD-L1 mice are combined from 4 independent experiments; n=19-20 mice per experimental group.

Figure 7. Sublethal irradiation and anti-PD-L1 is also effective in the treatment of other hematologic malignancies

A. PD-L1 expression on cultured 5T33, B16F10, AGN2a, A20, EL4, and C1498 cells is depicted in flow cytometry histograms. Solid curves represent isotype controls. B. Survival curves are shown for mice inoculated with the indicated tumors. The experimental design followed that depicted in Figure 2A. Briefly, approximately one week after inoculation mice received 0 or 500 cGy irradiation and then were treated on six days with anti-PD-L1 or control IgG. Survival curves are from 1-3 independent experiments; n=8-20 mice per experimental group.