Rewiring the dendritic cell: Regulatory T cell axis sensitizes prostate cancer to immunotherapy

Introduction: The prostate cancer (PCa) tumor microenvironment (TME) is immunosuppressive and resistant to immune checkpoint blockade (ICB). Based on multi-omic single cell correlatives of response to neoadjuvant Fc-enhanced (FcE) alphaCTLA-4 in high-risk localized PCa (NCT04301414), we explored how modulating the balance between regulatory T cells (Tregs) and dendritic cells (DCs) in the PCa TME affects sensitivity to ICB. Methods: We leveraged three single cell transcriptomic atlases of PCa to assess how Tregs and DC frequencies are dynamically regulated during PCa progression. To investigate responses to Treg depletion (via FcE-alphaCTLA-4) alongside DC expansion (via Flt3L-Ig) and stimulation (via STING agonist ADU-S100) in PCa, we employed the castration-sensitive MycCaP PCa model and 45-parameter spectral flow cytometry. To investigate novel direct mechanisms of DC stimulation by FcE-alphaCTLA-4 via Fcgamma receptor (FcgammaR) engagement, we optimized reductionist in vitro assays involving bone marrow-derived DCs (BMDCs) from wild-type (WT) mice or mice deficient in activating FcgammaRs ( Fcer1g -/- ) or inhibitory FcgammaRIIb ( Fcgr2b -/- ). To validate this effect in vivo we established a tumor antigen-specific T cell priming assay with both flow cytometry and ex vivo 2-photon live cell imaging readouts. Results: In three independent single cell transcriptomic PCa atlases, we find cDC2 frequencies are significantly decreased in advanced PCa while effector Tregs ( TNFRSF9 + ) increase in proportion. Hypothesizing that excessive Treg function and DC insufficiency drive ICB resistance in PCa, we find in vivo DC expansion (Flt3L-Ig) prior to hormonal therapy, Treg depletion (FcE-alphaCTLA-4), and in situ DC stimulation (ADU-S100) drives robust control of established MycCaP tumors, with 87% of animals tumor-free 40-days post-treatment (27/31). Using 45-parameter spectral flow cytometry we find FcE-alphaCTLA-4 drives DC activation prior to Treg depletion in the TME, supporting a novel direct mechanism of DC modulation by FcE-alphaCTLA-4. In vitro , we find cell-associated FcE-alphaCTLA-4 is sufficient to drive BMDC maturation in an FcgammaR-dependent manner. In vivo , FcE-alphaCTLA-4 augments DC-mediated T cell priming in an FcgammaR-dependent manner. Ongoing experiments are investigating how FcE-alphaCTLA-4 shapes DC-T cell interactions via FcgammaR engagement and how DC/Treg axis modulation can be optimized to improve long-term curative response rates in the MycCaP model. Conclusions: DC insufficiency and effector Treg differentiation are hallmarks of advanced PCa. Enhancing DC number and function concomitant with Treg depletion can drive effective antitumor immunity in a pre-clinical PCa model. Direct DC modulation via FcgammaR engagement represents a novel mechanism of response to CTLA-4-targeted ICB. These findings will inform ongoing translation of FcE-alphaCTLA-4 agents and development of novel immunotherapy strategies for PCa.