Inhibiting myeloid-derived suppressor cell trafficking enhances T cell immunotherapy

Abstract

Recruitment of myeloid-derived suppressor cells (MDSCs) into tumors induces local immunosuppression in carcinomas. Here, we assessed whether SX-682, an orally bioavailable small-molecule inhibitor of CXCR1 and CXCR2, could block tumor MDSC recruitment and enhance T cell activation and antitumor immunity following multiple forms of immunotherapy. CXCR2+ neutrophilic MDSCs (PMN-MDSCs) were the most abundant myeloid cell subset within oral and lung syngeneic carcinomas. PMN-MDSCs demonstrated greater suppression of tumor-infiltrating lymphocyte killing of targets compared with macrophages. SX-682 significantly inhibited trafficking of PMN-MDSCs without altering CXCR2 ligand expression. Trafficking of CXCR1+ macrophages was unaltered, possibly due to coexpression of CSF1R. Reduced PMN-MDSC tumor infiltration correlated with enhanced accumulation of endogenous or adoptively transferred T cells. Accordingly, tumor growth inhibition or the rate of established tumor rejection following programed death-axis (PD-axis) immune checkpoint blockade or adoptive cell transfer of engineered T cells was enhanced in combination with SX-682. Despite CXCR1/2 expression on tumor cells, SX-682 appeared to have little direct antitumor effect on these carcinoma models. These data suggest that tumor-infiltrating CXCR2+ PMN-MDSCs may prevent optimal responses following both PD-axis immune checkpoint blockade and adoptive T cell transfer therapy. Abrogation of PMN-MDSC trafficking with SX-682 enhances T cell-based immunotherapeutic efficacy and may be of benefit to patients with MDSC-infiltrated cancers.

Keywords: Immunotherapy; Oncology.

Figure 1. Tumor-infiltrating CXCR2⁺Ly6G^hi myeloid cells suppress TIL function to a greater degree than CXCR1⁺ macrophages.

Day 20 tumors from wild-type (WT) B6 mice bearing MOC1 (A) or LLC (B) tumors were digested and assessed for infiltration of myeloid cells by flow cytometry. Representative dot plots of gating strategy on the left, with pie graphs of myeloid cell constituency on the right. Ly6G^hiLy6C^intF4/80^– myeloid cells or F4/80⁺ macrophages were sorted from MOC1 (C) or LLC (D) tumors and assessed for ability to suppress TIL (10:1 E/T) killing of parental tumor cells. Ly6G^hiLy6C^intF4/80^– myeloid cells or F4/80⁺ macrophages were plated at a 3:1 ratio to TILs. Representative impedance plots shown on the left, with quantification of percentage loss of cell index at 12 hours quantified on the right. CXCR1 and CXCR2 expression on MOC1 (E) and LLC (F) tumor-infiltrating immune cells was assessed via flow cytometry. Representative data from 1 of 2 independent assays with similar results shown. MFI, mean fluorescence intensity. *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA. n/s, nonsignificant.

Figure 2. SX-682 monotherapy abrogates CXCR2⁺ PMN-MDSC tumor infiltration.

WT B6 mice bearing MOC1 (A) or LLC (B) tumors were treated with SX-682 chow starting on either day 10 or day 20 after implantation and followed for tumor growth. Summary growth curves shown (n = 10/group). Day 25 tumors, spleens, and bone marrow harvested from MOC1 (C) or LLC (D) tumor-bearing mice treated with SX-682 chow beginning on day 10 or 20 after tumor implantation or control chow were assessed for infiltration/accumulation of PMN-MDSCs or Ly6G^loLy6C^hi myeloid cells by flow cytometry (n = 5/group). Representative dot plots on the left, with quantification of myeloid cells within each tissue compartment on the right. Representative data from 1 of 2 independent assays with similar results shown. n/s, nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA.

Figure 3. SX-682 treatment results in enhanced TIL infiltration and tumor cell PD-L1 expression.

Day 25 tumors from mice bearing MOC1 (A) or LLC (B) tumors were treated with SX-682 chow starting on either day 10 or day 20 after implantation, then digested and assessed for infiltration of TILs by flow cytometry. Representative dot plots of live CD45.2⁺ cells on the left with quantification of CD8⁺ and CD4⁺ TILs on the right. Inset is PD-1 and CD137 expression on TILs. From the same tumors, PD-L1 expression on MOC1 (C) or LLC (D) CD45.2^–CD31^–PDGFR^– tumor cells were assessed with flow cytometry. PD-L1 expression on day 25 CD45.2^–CD31^–PDGFR^– MOC1 (E) and LLC (F) tumor cells was assessed following treatment on day 10 with SX-682 with or without antibody depletion of CD8⁺ cells (clone YTS 169.4, 200 μg i.p. twice weekly). Representative data from 1 of 2 independent assays with similar results shown. n/s, nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA.

Figure 4. SX-682 enhances tumor control or rejection following PD-1 blockade.

WT B6 mice bearing established MOC1 (A) or LLC (B) tumors were treated with SX-682 or control chow and PD-1 mAb (200 μg i.p. twice weekly for a total of 4 injections) or isotype control IgG2a mAb, alone or in combination, starting on day 10 and followed for primary tumor growth (left) and survival (right). ***P < 0.001 by log-rank (Mantel-Cox) analysis. Mice bearing established MOC1 (C) or LLC (D) tumors (n = 8/group) were treated with a combination of SX-682 and PD-1 mAb with or without antibody depletion of CD8⁺ cells (clone YTS 169.4, 200 μg i.p. twice weekly). Representative data from 1 of 2 independent assays with similar results shown. ***P < 0.001 by Student’s t test between tumor volumes on final day of measurement.

Figure 5. SX-682 results in greater tumor infiltration of adoptively transferred T cells.

Day 15 tumors from RAG1^–/– mice bearing MOC1 (A) or LLC (B) tumors engineered to express SIINFEKL were digested and assessed for infiltration of PMN-MDSCs after treatment with SX-682 or control chow starting on day 7. Representative dot plots of live CD45.2⁺CD11b⁺F4/80^– cells on the left, with quantification on the right. RAG1^–/– mice bearing MOC1 (C) or LLC (D) SIINFEKL–expressing tumors were treated with SX-682 or control chow on day 7 and treated with a single adoptive transfer of 1 × 10⁶ OT-I T cells on day 10. Tumors were assessed for infiltration of Vα2⁺ OT-I T cells 12 hours later. Representative dot plots of live cells on the left, quantification on the right. mKate2-positive cells are SIINFEKL-positive tumor cells. FMO, fluorescence minus one. Representative data from 1 of 2 independent assays with similar results shown. **P < 0.01 by Student’s t test.

Figure 6. SX-682 enhances tumor control or rejection following adoptive transfer of antigen-specific T cells.

RAG1^–/– mice bearing MOC1 (A) or LLC (B) SIINFEKL–expressing tumors were treated with SX-682 or control chow on day 7 and treated with a single adoptive transfer of 1 × 10⁶ OT-I T cells (OT-1 ACT) on day 10. Mice were followed for primary tumor growth (left) and survival (right). Cumulative results from 2 independent experiments shown. *P < 0.05; **P < 0.01 by log-rank (Mantel-Cox) analysis.

Figure 7. SX-682 treatment effect is not due to direct alteration of tumor cell viability, invasive capacity, or immunogenicity.

(A) MOC1 or LLC cells were assessed for CXCR1 or CXCR2 expression by flow cytometry, compared to fibroblasts, endothelial, immune or epithelial cells from MOC1 or LLC tumors in vivo. Live CD45.2^–CD31^–PDGFR^– oral mucosal epithelial cells and lung epithelial cells were used as comparators for MOC1 and LLC, respectively. (B) CXCR1 and CXCR2 expression on day 25 CD45.2^–CD31^–PDGFR^– MOC1 or LLC tumor cells following SX-682 treatment beginning on day 10 or 20 after tumor implantation was assessed by flow cytometry. (C) MOC1 or LLC cells were plated in increasing doses of SX-682 and evaluated for alteration in viability via impedance analysis. (D) MOC1 or LLC cells were exposed to SX-682 (1 μM for 24 hours) and assessed for induction of apoptosis via flow cytometry. (E) Extracellular matrix invasion of MOC1 or LLC cells was assessed using 10% FBS (positive control) or CXCL1 (50 ng/ml) as the chemoattractant in the presence or absence of SX-682 (1 μM). (F) MOC1 or LLC cells expressing SIINFEKL were exposed to activated OT-I T cells in the presence or absence of SX-682 (cells plated in 1 μM) and T cell killing was assessed via impedance analysis. Representative impedance plots on the left, with quantification at 8 hours on the right. Representative data from 1 of at least 2 independent assays with similar results shown. *P < 0.05; ***P < 0.001 by Student’s t test.