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

Purpose/Objective(s)

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.

Materials/Methods

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.

Results

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 de novo 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.

Conclusion

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.