Disruption of CXCR2-mediated MDSC tumor trafficking enhances anti-PD1 efficacy

Abstract

Suppression of the host's immune system plays a major role in cancer progression. Tumor signaling of programmed death 1 (PD1) on T cells and expansion of myeloid-derived suppressor cells (MDSCs) are major mechanisms of tumor immune escape. We sought to target these pathways in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma of childhood. Murine RMS showed high surface expression of PD-L1, and anti-PD1 prevented tumor growth if initiated early after tumor inoculation; however, delayed anti-PD1 had limited benefit. RMS induced robust expansion of CXCR2(+)CD11b(+)Ly6G(hi) MDSCs, and CXCR2 deficiency prevented CD11b(+)Ly6G(hi) MDSC trafficking to the tumor. When tumor trafficking of MDSCs was inhibited by CXCR2 deficiency, or after anti-CXCR2 monoclonal antibody therapy, delayed anti-PD1 treatment induced significant antitumor effects. Thus, CXCR2(+)CD11b(+)Ly6G(hi) MDSCs mediate local immunosuppression, which limits the efficacy of checkpoint blockade in murine RMS. Human pediatric sarcomas also produce CXCR2 ligands, including CXCL8. Patients with metastatic pediatric sarcomas display elevated serum CXCR2 ligands, and elevated CXCL8 is associated with diminished survival in this population. We conclude that accumulation of MDSCs in the tumor bed limits the efficacy of checkpoint blockade in cancer. We also identify CXCR2 as a novel target for modulating tumor immune escape and present evidence that CXCR2(+)CD11b(+)Ly6G(hi) MDSCs are an important suppressive myeloid subset in pediatric sarcomas. These findings present a translatable strategy to improve the efficacy of checkpoint blockade by preventing trafficking of MDSCs to the tumor site.

Fig. 1.. Murine RMS is sensitive to anti-PD1 therapy.

Mice were orthotopically inoculated with M3-9-M RMS and control immunoglobulin G (No Tx), or anti-PD1 was administered intraperitoneally (200 μg per animal). (A and D) Survival and tumor size were monitored starting on day 0 (A) or on day 7 (D) and continued twice weekly until day 39. (B) Mice were inoculated with M3-9-M as above, and PD1 and Tim-3 expression on CD4 and CD8 T cells on day 20 is shown. NS, not significant. (C) Fluorescence-activated cell sorting (FACS) histograms showing surface expression of PD-L1 on M3-9-M and 76–9 RMS cells. (E and F) T cells from the spleen (E) and tumor (F) of mice from (A) were analyzed on day 12 for CD8⁺ T cell production of intracellular IFN-γ and TNF-α after stimulation using soluble anti-CD3/anti-CD28. (G and H) Mice were inoculated with 76–9 RMS and treated as in (A) and (D). Experiments consist of five mice per group and are representative of at least three separate experiments. Two-tailed unpaired t test or Kaplan-Meier survival analysis was used to calculate P values.

Fig. 2.. Treatment with anti-PD1 augments T cell reactivity to HY antigens expressed by M3-9-M.

(A) Reverse transcription polymerase chain reaction (RT-PCR) of male-specific UTY on M3-9-M and 76–9 RMS cell lines. NTC, no template control; MB49, a known HY-expressing cell line used as a positive control; HPRT, used as an endogenous housekeeping control gene. (B) M3-9-M RMS cells (1 × 10⁶) were injected into the gastrocnemius muscle of female (left) or male (right) wild-type (WT) B6 hosts, and cohorts were treated with anti-PD1 at 200 μg per animal from day 4 to day 22. Tumor growth curves of individual mice are shown. Kaplan-Meier survival curves (C) with log-rank statistics were used to compare the effects of anti-PD1 treatment in female (red) versus male (blue) hosts (n = 8 mice per group). (D) B6 female non-tumor-bearing mice or mice given M3-9-M or 76–9 tumor cells were treated with anti-PD1 starting at day 12. Splenocytes were harvested on day 25 and then stimulated with designated peptides overnight. IFN-γ–producing cells were enumerated using enzyme-linked immunospot (ELISpot). Five mice were used per group, and results shown are representative of two experiments. Two-tailed unpaired t test or Kaplan-Meier survival analysis was used to calculate P values.

Fig. 3.. Murine embryonal RMS predominantly expands GrMDSCs.

(A and B) Representative FACS plots (A) and absolute numbers (B) of MDSCs from blood on the days designated, and spleen and tumor (n = 5 per group) on day 20 after M3-9-M tumor inoculation. (C) RT-PCRwas performed to evaluate expression of arginase-1, iNOS in sorted populations of CD11b⁺Ly6G^hi, and CD11b⁺Ly6C^hi from the spleens of tumor-bearing mice versus BM of non-tumor-bearing mice. NTC, no template control. (D) Relative quantification of arginase-1 and iNOS transcripts in CD11b⁺Ly6G^hi and CD11b⁺Ly6C^hi subsets sorted from tumor-bearing mice. (E) Peripheral blood was collected from non-tumor-bearing and M3-9-M-bearing mice, and intracellular CD3ζ mean fluorescent intensity (MFI) was plotted for CD4⁺ and CD8⁺ T cells. (F) CD11b⁺Ly6G^hi and CD11b⁺Ly6C^hi subsets were purified from the spleens of tumor-bearing mice and placed in a proliferation assay with purified CellTrace Violet-stained B6 T cells at the designated MDSC/T cell ratios. Anti-CD3/CD28 beads were used at a 1:2 ratio with T cells to induce proliferation. Cells were harvested on day 5 and analyzed by FACS for Violet dilution. (G) CD11b⁺Ly6G^hi cells were purified and placed in a proliferation assay as in (F) with the addition of nor-NOHA (arginase inhibitor) and l-NMMA (iNOS inhibitor), each at 300 μM. (H) PD-L1 expression on gated populations of MoMDSCs (top) and GrMDSCs (bottom). Data are representative of at least two independent experiments. Two-tailed unpaired t test was used to calculate P values.

Fig. 4.. Tumor-induced CD11b⁺Ly6G^hi cells express CXCR2, and murine RMS produces CXCR2 ligands.

(A) Mice bearing M3-9-M or 76–9 tumors were analyzed for surface expression of CXCR2 and CCR2 on CD11b⁺Ly6G^hi and CD11b⁺Ly6C^hi subsets. (B) RT-PCR was performed to assess expression of CXCL1 and CXCL2 in M3-9-M and 76–9. NTC, no template control; HPRT, housekeeping control. (C) Protein analysis [enzyme-linked immunosorbent assay (ELISA)] of cell culture supernatants for CXCL1 and CXCL2. (D) ELISA analysis for CXCL1 (left panel) and CXCL2 (right panel) of serum obtained from blood samples taken from retro-orbital sinus of non–tumor-bearing and M3-9-M–bearing mice versus blood obtained directly from M3-9-M tumor puncture. (E) Peripheral blood CD11b⁺Ly6G^hi cells were electronically sorted from mice bearing M3-9-M tumors placed in the top chamber of a Transwell. M3-9-M was plated in the bottom chamber, and the absolute number of MDSCs migrating from the top to the bottom chamber after 8 hours was enumerated. Blocking antibodies for CXCL1/CXCL2 and CXCR2 (4 μg/ml) were added at the beginning of the experiment. Two-tailed unpaired t test was used to calculate P values.

Fig. 5.. CXCR2 is required for Ly6G^hi MDSCs homing to the tumor.

(A) WT B6 hosts were lethally irradiated and given syngeneic BM or CXCR2^−/− BM as described in Materials and Methods. Animals were inoculated orthotopically with 1 × 10⁶ M3-9-M (top) or 76–9 (bottom) on day 0. Tumors were harvested on day 22, and representative FACS plots gated on CD11b⁺ cells are shown. (B and C) Percentage and absolute numbers of designated subsets in M3-9-M (B) or 76–9 (C) tumors on day 22. (D) Tumor growth curves for M3-9-M and 76–9 in mice with WT BM or CXCR2^−/− BM. P values represent significance between the two groups at day 22. (E) Tissue sections of M3-9-M RMS from mice with WT or CXCR2^−/− marrow stained with fluorescently conjugated CD11b (top), Ly6G (middle), or merged images (bottom). All panels costained with 4′,6-diamidino-2-phenylindole (gray). Scale bars, 50 μm. Experiments represent n = 8 mice per group. Two-tailed unpaired t test was used to calculate P values.

Fig. 6.. Pediatric sarcoma tumors secrete CXCL1/CXCL8, and serumCXCL8 levels negatively correlate with survival in patients with pediatric sarcoma.

(A) CXCL8 and CXCL1 were measured in supernatant taken from in vitro–cultured human osteosarcoma (143b, MG63.2, and G292) and RMS (RH18) using ELISA. (B) CXCL1 and CXCL8 were measured by ELISA in serum samples collected from healthy donors (n = 22) and patients (n = 53) with pediatric sarcoma. (C) Serum CXCL8 is significantly higher in patients who died after enrollment on immunotherapy trials at NCI. Two-tailed unpaired t test was used to calculate P values.

Fig. 7.. PD1 checkpoint blockade is more effective in tumor-bearing hosts with CXCR2-deficient myeloid cells.

(A and B) Mice were inoculated with M3-9-M (A) or 76–9 (B) on day 0. Tumor growth in animals previously reconstituted with WT BM (left panel) or CXCR2^−/− BM (middle panel) as described in Materials and Methods, and treated with control mAb or anti-PD1 as noted in the legend. Kaplan-Meier survival curves are shown in the right panel and were evaluated using log-rank statistics. (C and D) Cohorts of mice with M3-9-M RMS tumors were analyzed for absolute number of tumor-infiltrating T cells and Ly6G⁺ MDSCs, and CD25 and OX40 expression on tumor-infiltrating CD8⁺ cells 48 hours after initiation of anti-PD1 therapy (WT hosts, n = 10 per group; CXCR2^−/− hosts, n = 7 per group). The ratio of T cell/MDSC was calculated. (E) Serum samples from cohorts of mice were analyzed for protein IFN-γ concentrations using ELISA. Two-tailed unpaired t test or Kaplan-Meier survival analysis was used to calculate P values.

Fig. 8.. The effectiveness of PD1 checkpoint blockade on established tumors is enhanced by anti-CXCR2 mAb therapy.

Mice were inoculated with M3-9-M RMS on day 0 as previously described. Cohorts were untreated and treated with anti-PD1 (200 μg per animal twice weekly starting on day 7 until day 35), anti-CXCR2 (200 μg per animal, days 6 and 10), or anti-CXCR2 plus anti-PD1. (A and B) Representative FACS plots (A) and cumulative data (B) from peripheral blood samples taken on day 15 from each group. (C and D) In a separate experiment, spleens (C) and RMS tumors (D) taken from untreated and treated mice were harvested and analyzed for MDSC infiltration. (E and F) Tumor growth curves and Kaplan-Meier survival curves for individual mice in (A). No Tx and anti-PD1 groups: n = 8 per group; anti-CXCR2 and anti- PD1 + anti-CXCR2 groups: n = 6 per group. Two-tailed unpaired t test or Kaplan-Meier survival analysis was used to calculate P values.