Phagocytosis Checkpoint Blockade Overcomes Immunosuppression Triggered by Radiation Therapy and PD1 Inhibition

Abstract

<h3>Purpose/Objective(s)</h3> Trials combining radiation (RT) and anti-PD1 therapy have had largely disappointing results despite promising preclinical data. To better understand this lack of synergy, we used single-cell sequencing (scSeq) in a preclinical mouse model of RT ± anti-PD1 to dissect the dynamics of tumor infiltrating leukocytes (TILs) after treatment. We identified the phagocytic checkpoint SIRPα-CD47 on myeloid cells as a key immunosuppressive pathway and tested the impact of targeting this pathway on combined therapy efficacy. <h3>Materials/Methods</h3> Using a murine model of breast cancer (E0771) we tested the effect of RT (16 Gy x 1 using the SmART+ platform with image guidance) ± anti-PD1± anti-CD47. Tumor growth and Kaplan-Meier survival curves were compared for each treatment group. CD45⁺ TILs were isolated from treated tumors and analyzed by flow cytometry, scSeq, and cellular indexing of transcriptomes (CITEseq) to profile the immune microenvironment and its functional capacity. <h3>Results</h3> First we sequenced 94,503 individual TILs at various timepoints following RT, to date the largest collection of single-cell libraries of the post-RT immune compartment. Most baseline cells were CD11b⁺ macrophages and T cells which were rapidly eliminated by day 3 after RT. The immune compartment was subsequently repopulated by <i>de novo</i> T cells and macrophages at day 7. Several phagocytic checkpoints (SIRPα, LILRB3/4, SLAMF3/7, LRP1) were significantly upregulated on CD11b⁺ macrophages after RT. Given the importance of phagocytic checkpoints in mediating myeloid suppressive function, we tested whether SIRPα-CD47 blockade could enhance anti-PD1/RT activity by alleviating RT-mediated immunosuppression. While anti-PD1/RT and anti-CD47/RT delayed tumor growth versus RT alone, the addition of anti-CD47 to anti-PD1/RT showed the greatest reduction in tumor size and longest survival (P<0.001). To elucidate the mechanisms behind this, we sequenced 16,504 additional cells following RT ± anti-PD1 ± anti-CD47. We identified highly exhausted, M2-like macrophages (CD11b⁺Arg1⁺) in tumors treated with anti-PD1/RT, which were eliminated with the addition of anti-CD47. Moreover, several pro-inflammatory markers (Il1a, Nos2, Mif) and chemokines (CCL3, CXCL1-3) were upregulated with anti-PD1/RT but downregulated with the addition of anti-CD47 despite enhanced efficacy of the triple combination. Closer examination revealed that anti-PD1/RT was associated with neutrophil tumor infiltration that was abrogated with anti-CD47. Lastly addition of anti-CD47 to anti-PD1/RT resulted in dramatic increases in B cell tumor infiltration, which was absent with anti-PD1/RT alone. <h3>Conclusion</h3> SIRPα-CD47 blockade significantly enhances anti-tumor immunity and survival elicited by anti-PD1/RT likely by decreasing neutrophil infiltration and increasing B cell activity. Targeting phagocytic checkpoints may limit myeloid suppression induced by anti-PD1/RT by enhancing B and T cell interactions.