Immunosuppressive Effects of Multiple Myeloma Are Overcome by PD-L1 Blockade  William H.D. Hallett, Weiqing Jing, William R. Drobyski, Bryon D. Johnson  Biology of Blood and Marrow Transplantation  Volume 17, Issue 8, Pages 1133-1145 (August 2011) DOI: 10.1016/j.bbmt.2011.03.011 Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 CD138+ plasma cells in myeloma patients express PD-L1. (A) Freshly isolated human bone marrow aspirates from patients with multiple myeloma were analyzed for expression of CD138+ myeloma cells; a representative example is shown. (B) PD-L1 expression on CD138+ cells (thick black line) and CD138− cells (thin black line) and isotype control staining (gray fill) on the gated CD138+ cells in panel A are compared. The PD-L1 expression is representative of more than 5 samples. (C) PD-L1 expression on CD138+ cells from a multiple myeloma patient (black line) are compared with PD-L1 expression on CD138+ cells from a normal healthy adult (gray fill). Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Murine 5T33 myeloma cells express high levels of PD-L1, and T cells from myeloma-bearing mice express elevated levels of PD-1. (A) Nontumor-bearing and moribund tumor-bearing mice were analyzed for the presence of CD138+PD-L1+ 5T33 myeloma cells in their spleens. The percentage of CD138+ cells from naïve mice and tumor-bearing mice is shown. PD-1 expression on (B) CD8 and (C) CD4 T cells from the same mice in panel A is shown. Average percentages of (D) CD138+ cells, (E) PD-1+ CD8 T cells, and (F) PD-1+ CD4 T cells were calculated. Each bar represents the average of more than 10 mice, and statistical comparisons were determined using the t test. Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 PD-1+ T cells are localized to the same tissue sites as myeloma. A single representative mouse with an advanced tumor-burden was euthanized and (A) blood, (B) bone marrow, (C) an inguinal lymph node, and (D) the spleen were harvested and analyzed for expression of tumor cells (PD-L1+ and CD138+) and PD-1 expression on CD8 T cells. The data are representative of more than 3 experiments. Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 PD-1+ CD8 T cells have an effector phenotype. (A) Percentages of splenic CD8 T cells with a naïve (CD44lowCD62L+), central memory (CD44hiCD62L+) and effector memory (CD44hiCD62L−) phenotype are shown for a nontumor-bearing mouse and a mouse with advanced myeloma. (B) CD8 T cells from a myeloma-bearing mouse were separated based on their expression of PD-1, and the PD-1− and PD-1+ cells were analyzed for CD44 and CD62L expression. Data are representative of more than 10 mice. Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 PD-1+ CD8 T cells in myeloma-bearing mice are undergoing apoptosis. (A) The apoptotic profiles of CD8 cells from a nontumor-bearing mouse, and (B) PD-1− and (C) PD-1+ CD8 T cells from a tumor-bearing mouse, are depicted. Percentages of healthy (annexin V−, propidium iodide−), early apoptotic (annexin V+, propidium iodide−), and necrotic (annexin V+, propidium iodide+) cells were determined using fluorescent-minus-one (FMO) controls. Data are representative of 2 individual experiments. Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 PD-1+ CD8 T cells do not produce pro-inflammatory cytokines and express the exhausted T cell marker Tim3. PD-1+ and PD-1− CD8+ T cells from tumor-bearing mice and CD8+ T cells from nontumor-bearing mice were sorted and cocultured for 72 hours in either media (No Stim) or in media with plate-bound anti-CD3 antibodies (Anti-CD3). Culture supernatants were harvested and the production of (A) IL-2, (B) TNF-α, (C) IFN-γ, and (D) IL-10 were measured by Bio-Plex. The cytokine data is representative of 2 independent experiments. (E) CD8+ T cells from nontumor-bearing mice and tumor-bearing mice were analyzed for the expression of PD-1 and Tim3. (F) The percent of PD-1+Tim3+ CD8 T cells was quantitated. The data are from 3 mice and are representative of 2 independent experiments. Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 7 Myeloma-bearing mice have elevated PD-1 expression on NK cells, and elevated numbers of regulatory T cells and myeloid-derived suppressor cells. (A) Percentages of PD-1+ NK cells (NKp46+CD3−) from naïve (nontumor-bearing) and tumor-bearing mice are shown in the left and center panels, and are quantitated in the right panel. (B) The percentages of regulatory T cells (CD4+Foxp3+) from naïve (left panel) and tumor-bearing (middle panel) mice are shown, and are quantitated in the right panel. (C) The percentages of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) from naïve (left panel) and tumor-bearing (middle panel) mice are shown, and are quantitated in the right panel. Quantitated data are combined from more than 5 mice per group, and the data are representative of more than 3 experiments. Statistical comparisons were performed using the t test. Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 8 PD-L1 blockade improves the efficacy of posttransplant vaccine-based immunotherapy for myeloma, but it has no effect in non-transplanted mice. (A) Experimental design for transplanted tumor-bearing mice. (B) Survival curves are shown for transplanted mice treated with vaccines and anti-PD-L1 mAb or control antibodies. Specifically, mice were injected with 2×106 5T33 cells i.v., lethally irradiated (1100 cGy TBI) 7 days later, and then rescued with 2.5×106 BM cells plus 2×107 splenocytes. The transplanted mice were then either given control antibody alone (rIgG), anti-PD-L1 mAb alone (anti-PD-L1), an empty vector vaccine plus control antibody (Empty Vector Vaccine + rIgG), an empty vector vaccine plus anti-PD-L1 (Empty Vector Vaccine + anti-PD-L1), 5T33-CD80/137L vaccine cells plus control antibody (CD80/CD137L Vaccine + rIgG), or 5T33-CD80/137L vaccine cells plus anti-PD-L1 (CD80/CD137L Vaccine + anti-PD-L1). The asterisks represent P < 0.05 as determined by the log-rank test. Data are combined from two independent experiments, and are representative of four experiments. (C) Experimental design for non-transplanted tumor-bearing mice. (D) Survival curves are shown for non-transplanted mice injected with 2×106 5T33 cells i.v., and either given no further treatment (No Tx) or treated with 5T33-CD80/137L vaccine cells plus anti-PD-L1 at the indicated time points. Data are combined from 2 experiments. Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 9 Peripheral blood CD4 and CD8 T cells in multiple myeloma patients express PD-1 after autologous HSCT. (A) PD-1 expression was analyzed on CD8 and CD4 T cells in a myeloma patient before transplantation. Representative expression of PD-1 on peripheral blood CD8 and CD4 T cells, as well as pretransplant bone marrow resident CD8 and CD4 T cells in myeloma patients is shown. (B) Expression of PD-1 on CD8 T cells or (C) CD4 T cells before transplantation (PreT) and 30, 60, and 100 days after autologous hematopoietic stem cell transplantation. The PD-1 expression profiles in panels B and C represent data from 37 patients. (D) PD-1 expression on CD8 T cells from tumor-free C57BL KaLwRij mice before transplantation and at 7-day intervals following syngeneic transplantation of 2.5 × 106 BM and 2 × 107 splenocytes are shown. Data are from 3 to 4 mice per group. (E) A/J mice were treated with a lethal dose of TBI on day −1. On day 0, the mice were injected with 107 syngeneic BM cells supplemented with 6 × 106 T cells. Spleens were collected on days 7, 14, 21, 28, and 45 after HSCT, and the expression of PD-1 on CD8 T cells was analyzed. The data is combined from 2 to 3 experiments, with 6 to 9 total mice per group. Results were compared using the Student t test (∗P < .05, ∗∗P < .01, compared with naïve nontransplanted control mice). Biology of Blood and Marrow Transplantation 2011 17, 1133-1145DOI: (10.1016/j.bbmt.2011.03.011) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions