Semaphorin4D Inhibition Improves Response to Immune-Checkpoint Blockade via Attenuation of MDSC Recruitment and Function

Abstract

Tumor infiltration by immunosuppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs), causes resistance to immunotherapy. Semaphorin4D, originally characterized for its axonal guidance properties, also contributes to endothelial cell migration and survival and modulates global immune cytokine profiles and myeloid cell polarization within the tumor microenvironment. Here, we show how a therapeutic murine Sema4D mAb improves responses to immune-checkpoint blockade (ICB) in two murine carcinoma models. Treatment of tumor-bearing mice with Sema4D mAb abrogated Ly6Ghi PMN-MDSC recruitment through reducing MAPK-dependent chemokine production by tumor cells in Murine oral cancer-1 (MOC1) tumors. PMN-MDSC suppressive capacity was reduced through inhibition of Sema4D-driven arginase expression. These changes led to enhanced tumor infiltration by CD8+ TIL and activation of tumor-draining lymph node T lymphocytes in response to tumor antigen. Sema4D mAb in combination with either CTLA-4 or PD-1 blockade enhanced rejection of tumors or tumor growth delay, resulting in prolonged survival with either treatment. This function of Sema4D mAb provides a rationale for its evaluation in combination with ICB to treat tumors with immunosuppressive myeloid infiltration.

©2018 American Association for Cancer Research.

Figures

Figure 1.

Sema4D mAb plus ICB enhanced rejection of MOC1 tumors. A, Mice bearing MOC1 tumors were treated with Sema4D mAb and CTLA-4 mAb alone or in combination (n = 10 mice/group). Individual tumor growth curves are shown. The ratio of mice that rejected tumors is shown below the graph legends. Representative results from two independent experiments. B, Survival analysis of mice treated with Sema4D mAb and CTLA-4 mAb alone or in combination. C, Mice that rejected MOC1 tumors following combination Sema4D and CTLA-4 mAb treatments were challenged with MOC1 (3 × 106 cells/mouse) in the contralateral flank and followed for tumor engraftment. Control mice were untreated, wild-type B6 mice. Ratio of mice that rejected tumor engraftment is shown below the graph legend. D, Mice bearing MOC1 tumors were treated with Sema4D mAb and PD-1 mAb alone or in combination (n = 10 mice/group). Individual tumor growth curves are shown. Representative results from two independent experiments. E, Survival analysis of mice treated with Sema4D mAb and PD-1 mAb alone or in combination. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Sema4D mAb plus ICB enhanced rejection of LLC tumors. A, Mice bearing LLC tumors were treated with Sema4D mAb and CTLA-4 mAb alone or in combination (n = 10 mice/group). Individual tumor growth curves are shown. Ratio of mice that rejected tumors is shown below the graph legends. Representative results from two independent experiments. B, Survival analysis of mice treated with Sema4D mAb and CTLA-4 mAb alone or in combination. C, Mice bearing LLC tumors were treated with Sema4D mAb and PD-1 mAb alone or in combination (n = 10 mice/group). Individual tumor growth curves are shown. Representative results from two independent experiments. D, Survival analysis of mice treated with Sema4D mAb and PD-1 mAb alone or in combination. **, P < 0.01; ***, P < 0.001.

Figure 3.

Sema4D mAbs did not alter MOC1 tumor vascularity in vivo or directly alter MOC1 tumor cells in vitro. A, MOC1 tumors (n = 5) or oral mucosa from the same mice were digested into single-cell suspensions, and cellular subsets were analyzed for Sema4D and PlexinB1 expression by flow cytometry. Representative dot plot of a digested MOC1 tumor is shown. Expression levels on CD45.2−CD31− epithelial or tumor cells, CD45.2−CD31+ endothelial cells, CD45.2+CD11b+Ly6GhiLy6CintF4/80− myeloid cells, CD45.2+CD11b+ Ly6GlowLy6ChiF4/80− myeloid cells, CD45.2+CD11b+F4/80+ macrophages, and CD45.2+CD11b−CD3+ lymphocytes are quantified on the right. Negative refers to isotype control for Sema4D or PlexinB1 mAbs used for staining. B, MOC1 cells in vitro were analyzed for Sema4D and PlexinB1 expression by flow cytometry. Negative refers to isotype control for Sema4D or PlexinB1 mAbs used for staining. C, Real-time in vitro impedance of MOC1 cells exposed to recombinant Sema4D protein (10 μg/mL), Sema4D mAb (10 μg/mL), or isotype (10 μg/mL) alone or in combination was measured to assess changes in cell adhesion and proliferation. Cells were plated at time 0 in recombinant protein or antibody. D, MOC1 cells in vitro were assessed for induction of apoptosis via annexin V/7AAD flow cytometry after exposure to Sema4D mAb for 24 hours. E, MOC1 cells in vitro were assessed for changes in cell-surface expression of components of immunogenicity by flow cytometry after exposure to Sema4D mAb for 24 hours. All in vitro experiments were repeated at least twice, each with at least three technical replicates. F, Mice bearing MOC1 tumors were treated with Sema4D mAb or isotype control (n = 5/group). Tumors harvested 1 day after the last Sema4D mAb treatment were analyzed via flow cytometry for endothelial cell infiltration. G, Tumors from mice treated as in F were assessed for CD31 vessel density by CD31 immunofluorescence. Representative photomicrographs on left, with quantification by vessel count per high-power field (10 fields/condition) on right. MFI, mean fluorescent intensity; rSema4D, recombinant Sema4D. **, P < 0.01; ***, P < 0.001.

Figure 4.

Sema4D mAb inhibited recruitment of myeloid cells into MOC1 tumors through reduction of myeloid chemokines. A, Mice bearing MOC1 tumors were treated with Sema4D mAb or isotype control (n = 5/group) and whole tumor digests harvested 1 day after the last Sema4D mAb treatment were analyzed via flow cytometry for Ly6GhiLy6Cint myeloid cell or Ly6GlowLy6Chi myeloid cell infiltration. Representative dot plots on the left, quantified on the right. B, Mice bearing MOC1 tumors were treated as above (n = 5/group), tumor digests subjected to a percoll gradient, and leukocytes were analyzed for myeloid cell markers and intracellular Ki67 by flow cytometry. Representative dot plots on the left, quantified on the right. C, Mice bearing MOC1 tumors were treated as above (n = 5/group). Some tumor digests were subjected to a percoll gradient (to obtain leukocyte fraction). Whole tumor or leukocyte fraction digests were analyzed for myeloid chemokine expression via qRT-PCR. D, MOC1 cells in vitro were exposed to Sema4D mAb (10 μg/mL) or isotype for 24 hours and analyzed for myeloid chemokine expression by qRT-PCR. E, MOC1 cells in vitro were exposed to recombinant Sema4D protein (10 μg/mL), Sema4D mAb (10 μg/mL), or isotype alone or in combination for 1 hour and subjected to Western blot analysis. Band densitometry quantified below each blot. F, MOC1 cells in vitro were exposed to the MEK inhibitor U0126 (5 μmol/L) for 24 hours, and cell culture supernatant was analyzed for CXCL1 concentration by ELISA. All in vitro experiments were repeated at least twice. rSema4D, recombinant Sema4D. *, P < 0.05; ***, P < 0.001.

Figure 5.

Sema4D mAb inhibited the T-lymphocyte-suppressive capacity of Ly6GhiLy6Clow myeloid cells within MOC1 tumors. A, Mice bearing MOC1 tumors were treated with Sema4D mAb or isotype control (n = 5/group), tumor digests harvested 1 day after the last Sema4D mAb treatment were subjected to a percoll gradient, and Ly6GhiLy6Clow myeloid cells were sorted via positive magnetic selection. Myeloid cell–suppressive capacity was assessed in a T-lymphocyte proliferation assay. Representative overlaid CFSE histograms on the left, quantified on the right. B, MOC1 TILs were cocultured with MOC1 tumor cells in the presence or absence of Ly6GhiLy6Clow myeloid cells, and changes in MOC1 cell impedance were assessed in real time. Representative impedance plot on the left, and loss of impedance at the 24-hour time point qualified on the right. C, Mice bearing MOC1 tumors were treated Sema4D mAb or isotype control (n = 5/group), and whole tumor digests were analyzed for ARG1 expression via qRT-PCR. D, Mice bearing MOC1 tumors were treated as in C (n = 5/group), tumor digests were subjected to a percoll gradient, and tumor leukocytes were analyzed for lineage markers and intracellular phosphotranscription factors by flow cytometry. Representative dot plots and overlaid histograms are on the left, quantified on the right. E, Tumor Ly6GhiLy6Clow myeloid cells were sorted from untreated MOC1 tumors and exposed ex vivo to recombinant Sema4D protein (10 μg/mL), Sema4D mAb (10 μg/mL), or isotype alone or in combination for 12 hours. Ly6GhiLy6Clow myeloid cell–suppressive capacity was assessed in a T-lymphocyte proliferation assay (n = 5/group). Representative overlaid histograms on the left, quantified on the right. F, PMN-MDSCs as in E were assessed for ARG1 expression via qRT-PCR after exposure to recombinant Sema4D protein or antibody for 3 hours (n = 5/group). G, PMN-MDSCs as in E were assessed for intracellular phosphotranscription factors by flow cytometry after exposure to recombinant Sema4D protein or antibody for 1 hour (n = 5/group). MFI, mean fluorescent intensity; rSema4D, recombinant Sema4D. **, P < 0.01; ***, P < 0.001.

Figure 6.

Sema4D mAb enhanced CD8+ TIL infiltration and expansion and draining lymph node T-lymphocyte tumor antigen–specific responses. A, Mice bearing MOC1 tumors were treated with Sema4D mAb or isotype control (n = 5/group), and whole tumor digests harvested 1 day after the last Sema4D mAb treatment were analyzed via flow cytometry for CD3+CD8+ T-lymphocyte infiltration. Representative dot plots are on the left, quantified on the right. Inset bar graphs show expression of immune checkpoints on CD8+ TIL. B, Mice bearing MOC1 tumors were treated as in A (n = 5/group), tumor digests subjected to a percoll gradient, and leukocytes were analyzed for T-lymphocyte markers and intracellular Ki67 by flow cytometry. Representative dot plots on the left, quantified on the right. C, Mice bearing MOC1 tumors were treated as in A (n = 5/group), tumor-draining lymph nodes were harvested 1 day after the last Sema4D mAb treatment, and T lymphocytes were assessed for IFNγ concentration after stimulation. D, Mice bearing MOC1 tumors were treated as in A (n = 5/group) and analyzed via flow cytometry for expression of H-2Kb/Db and PD-L1 on CD45.2−CD31− tumor cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.