The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Here, we demonstrate that a subset of TAMs that express folate receptor β (FRβ) possess an immunosuppressive M2-like profile. In syngeneic tumor mouse models, chimeric antigen receptor (CAR)-T cell-mediated selective elimination of FRβ+ TAMs in the TME results in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRβ-specific CAR-T cells also improves the effectiveness of tumor-directed anti-mesothelin CAR-T cells, while simultaneous co-administration of both CAR products does not. These results highlight the pro-tumor role of FRβ+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

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The detection of tumor-specific T cells in solid tumors is integral to interrogate endogenous antitumor responses and to advance downstream therapeutic applications. Multiple biomarkers are reported to identify endogenous tumor-specific tumor-infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39; however, a direct comparison of these molecules has yet to be performed. We evaluated these biomarkers in primary human ovarian tumor samples using single-cell mass cytometry to compare their relative phenotypic profiles, and examined their response to autologous tumor cells ex vivo. PD-1 + , CD103 + , and CD39 + TILs all contain a CD137 + cell subset, while CD137 + TILs highly co-express the aforementioned markers. CD137 + TILs exhibit the highest expression of cytotoxic effector molecules compared to PD-1 + , CD103 + , or CD39 + TILs. Removal of CD137 + cells from PD-1 + , CD103 + , or CD39 + TILs diminish their IFN-γ secretion in response to autologous tumor cell stimulation, while CD137 + TILs maintain high HLAdependent IFN-γ secretion. CD137 + TILs exhibited an exhausted phenotype but with CD28 co-expression, suggesting possible receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1 + , CD103 + , and CD39 + TILs are mainly derived from a subset of CD137-expressing TILs, implicating CD137 as a more selective biomarker for naturally occurring tumor-specific TILs.