Proximal signaling through the innate immune inhibitory receptor signal regulatory protein alpha (SIRPα) in primary macrophages
Sabrin Mishel1,2, Jayne Danska1,2,3
1Department of Immunology, University of Toronto, Toronto, ON, Canada; 2Program in Genetics and Genomic Biology, Hospital for Sick Children, Toronto, ON, Canada; 3Department of Medical Biophysics and Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada
SIRPα is an innate immune inhibitory receptor expressed on myeloid cells. When SIRPα engages the widely expressed ligand CD47, the interaction inhibits macrophage phagocytosis of the detected cell. Drugs that target the CD47-SIRPα axis are under intensive investigation for cancer immunotherapy. We previously showed that treatment with a SIRPα-Fc fusion protein in the setting of patient-derived acute leukemia xenografts resulted in macrophage-dependent phagocytosis and removal. SIRPα-Fc (TTI-621) is currently in clinical trails for blood and other cancers. Despite the promise of CD47-directed therapies, little is known about how SIRPα conveys inhibitory signals in macrophages. SIRPα is known to restrain phagocytosis resulting from Fcγ receptor (FcγR) engagement; however, the mechanism of SIRPα inhibition of this activating receptor remains not well defined. We established a flow cytometry platform to evaluate proximal signalling effects of FcγR with and without SIRPα engagement in primary mouse macrophages using antibodies specific for phospho epitopes of intracellular kinases. We assessed the kinetics and downstream targets of FcγR-mediated activation and the inhibitory effects of SIRPα engagement on these FcγR-dependent phospho-epitopes and calcium flux. Our results indicate that SIRPα signalling reduced FcγR-mediated increases in cellular pBTK, pPLCγ2, and pERK but not pFAK. Furthermore, SIRPα inhibited FcγR-induced calcium flux, an early signal required for phagosome maturation. To further define the specificity of SIRPα inhibitory effects on FcγR activation pathways we also present evidence using kinase-specific inhibitors. Characterizing the specificity and kinetics of proximal inhibitory signalling will be useful to identify potential additive effects of small molecule kinase inhibitors in combination with the checkpoint inhibitor SIRPα-Fc for future combinatorial treatment of several cancers.