CD93+ Myeloid Cells Suppress T-Cell-Mediated Anti-Tumor Immunity

Background Bispecific T cell engager (TCE) therapy demonstrated promising efficacy in B-cell non-Hodgkin's lymphoma (B-NHL), but acquired resistance usually occurs. The mechanism of resistance of TCE immunotherapy is still not clear. Three different mechanisms have been hypothesized to result in TCE resistance. Many studies have shown the loss of antigen and the intrinsic T cell dysfunction may be responsible for TCE resistance. However, very limited studies suggested that the suppressive tumor microenvironment may contribute to TCE resistance in in-vitro assays and mouse models. No evidence has been shown in human patients yet. Methods The immune microenvironment of TCE-nonresponse or untreated B-NHL patients was characterized by single-cell RNA sequencing, high-dimensional flow cytometry, and immunohistochemistry analyses. The cellular signaling that govern immune modulation were determined by tumor cell-immune cell co-culture assays with the tyrosine kinase inhibitor screening. Humanized B-NHL mouse models were established to determine the efficacy of TCE and combination treatments. Results We found immunosuppressive tumor-associated myeloid cells (TAMs) were increased in tumor microenvironment of relapse patients post-TCE therapy. Depletion of TAMs synergistically enhanced TCE anti-tumor efficacy in a humanized mouse tumor model. By screening a kinase inhibitors library of 533 small molecule compounds in our co-culture system, we discovered the kinase inhibitors could boost T cell cytotoxicity against B cell lymphoma cells which was impaired by TAMs and decrease the IL-4-induced CD163 expression on TAMs. BTK inhibitors were the most effective drug in both up-regulated the relative cell death and downregulated relative CD163 MFI of macrophages with statistical significance among those screened tyrosine kinase inhibitors. According to KEGG analysis, the mitogen-activated protein kinase (MAPK) signaling pathway was the top enriched pathway downregulated in BTKi-treated or BTK-knockout macrophages, and a C-type lectin transmembrane protein, CD93, was one of the intriguing genes, which was downregulated by kinase inhibitors involved MAPK and PKC signaling. Moreover, we indeed identified BTK signaling and its downstream molecule CD93 in TAMs promoted T cell suppression. TCE-engaged cytotoxic T cells recruited monocytes and macrophages from the circulation to tumor via a chemokine receptor, CCR5, that in turn promote CD93 expression through the activation of BTK signaling. TCE-engaged T cells also secreted IGFBP7 that binds and activates CD93 on TAMs. Both BTK signaling and its downstream molecule CD93 in TAMs could contribute to T cell suppression. BTK inhibition or CD93 blockade dramatically increased TCE anti-tumor efficacy. Furthermore, we found CD93 is critical for the immunosuppressive function of TAMs in various solid tumors. Conclusion In summary, our results suggest that the immunosuppressive tumor microenvironment is a major cause of TCE resistance for B-NHL patients and a novel myeloid checkpoint, CD93, might be a promising target for cancer immunotherapy. Keywords B cell lymphoma, immunotherapy, bispecific antibody, tumor-associated myeloid cells, immunosuppression, BTK, CD93