Background Expression of CD47 on cancer cells inhibits phagocytosis by interacting with SIRPα on conventional dendritic cells (cDCs) thus preventing cancer cell antigen uptake and cross-presentation to T cells. As such, SIRPα is a myeloid checkpoint that limits the ability of cytotoxic therapies that induce an immunogenic cell death, such as radiation therapy (RT), to promote an in-situ vaccination and enhance tumor responses to immunotherapy. Based on this rationale, several agents targeting CD47 or SIRPα are in development and early clinical testing.

Methods and Results Although cDC1 are responsible for tumor antigen cross-presentation to CD8⁺ T cells, SIRPα expression is normally restricted to cDC2. Here we investigated the mechanisms whereby SIRPα-blockade promotes anti-tumor immune responses. We used MY1 (Boehringer-Ingelheim), a mouse anti-SIRPα antibody and two mouse models of subcutaneous luminal (TSA) and triple negative (AT3) breast cancer. MY1 monotherapy did not affect TSA or AT3 tumor growth. However, when used with RT, MY1 significantly improved control of irradiated and non-irradiated (abscopal) TSA tumors, as compared to RT alone (figure 1A, B). Improved tumor control was also seen in AT3-tumor bearing mice treated with RT+MY1, an effect that was abrogated by conditional deletion of cDCs in Zbtb46-DTR mice, or by depletion of CD8⁺ T cells. Having confirmed that MY1 promoted the development of anti-tumor immune responses in a cDC-dependent way when used with RT, we investigated cDC subsets in TSA tumors by cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing. This analysis resolved three cDC clusters: SIRPα⁺ cDC2, SIRPα^- cDC1 and a SIRPα⁺ cDC cluster expressing CCR7, CXCL16 and other markers of a previously described cDC3 subset, which was shown to trans-present IL15 and play a critical role for the survival and local expansion of effector CD8⁺ T cells in the tumor microenvironment¹ (figure 1C,D). In an independent experiment, multiparametric flow cytometry analysis confirmed that CCR7^high IL-15Rα⁺ CD103⁺DCs were significantly increased in RT+MY1-treated tumors (figure 1E), while cDC2 expressing high levels of activation and regulatory markers were depleted in MY1-treated tumors independently of RT.

Conclusions Overall, these results suggest a model whereby SIRPα signaling inhibits cDC functional differentiation into IL-15 trans-presenting cDC3 when exposed to dying cancer cells, rather than simply preventing cancer cell phagocytosis. We are currently testing if the CCR7⁺CXCL16⁺ DCs increased in RT+MY1-treated tumors promote the survival and local expansion of effector CD8 T cells via IL-15 trans-presentation, as previously described.¹

Reference

1. Di Pilato M, Kfuri-Rubens R, Pruessmann JN, Ozga AJ, Messemaker M, Cadilha BL, Sivakumar R, Cianciaruso C, Warner RD, Marangoni F, Carrizosa E, Lesch S, Billingsley J, Perez-Ramos D, Zavala F, Rheinbay E, Luster AD, Gerner MY, Kobold S, Pittet MJ, Mempel TR. CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment. Cell 2021;184(17):4512–4530 e4522.

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Abstract 487 Figure 1

RT+ MY1 improves local and systemic tumor control in TSA-bearing mice and promotes cDC differentiation into cDC3-like DC. BALB/c mice implanted with TSA cells on one or both flanks were treated with: isotype, MY1, RT+isotype and RT+MY1 (n=8 mice/group). (A) treatment with MY1 alone had no effect on tumor growth, however, combination with fractionated RT (3 consecutive doses of 8Gy) significantly improved tumor control, leading to complete regression that was durable in ≥50% mice. (B) RT+ MY1 combination also significantly delayed growth of a contralateral, non-irradiated model. Two-way Anova for statistical significance (*: p<0.05). (C) scRNAseq/CITEseq analysis of tumor infiltrating CD45+ cells from mice treated with isotype, MY1, RT+isotype and RT+MY1(n=3 per group) resolved 3 clusters of cDCs based on gene and protein expression. (D) SIRPα was expressed by CDC2 and by a second cluster with a cDC3 expression profile, while cDC1 were negative. Top panel: SIRP surface expression (CITEseq) and bottom panel: SIRPA RNA expression. (E) Flow cytometry analysis of tumor infiltrating cDCs revealed an increased frequency of highly activated CCR7+ CXCL16+ 115Ra+ cDCs. n=10 mice per group, mean±SD, groups are compared by Kruskal-Wallis test and Dunn’s multiple comparison for statistical significance