The immune checkpoint B7x promotes tumor-infiltrating tregs and resistance to anti-CTLA-4 therapy.

Tumor cells inhibit the anti-tumor immune response by expressing immunomodulatory factors and recruiting immunosuppressive cells to their microenvironment. Regulatory T cells (Tregs) are a population of CD4+ T cells that are commonly found in tumors, promoting a tolerogenic microenvironment and aiding in tumor immune evasion. The immune checkpoint ligand B7x is widely expressed in a broad variety of tumor types and is often associated with greater Treg infiltration, but the mechanism by which B7x promotes tumor-infiltrating Tregs is unknown. Here, we recapitulate the B7x-mediated increase in Tregs in murine tumor models and show that B7x promotes conversion of conventional CD4+ T cells into potently suppressive Tregs. We found that B7x induces global transcriptomic changes in Tregs, driving these cells to adopt an activated and suppressive phenotype. Mechanistically, B7x increased Foxp3 transcriptional expression by modulating the Akt/Foxo signaling pathway. Further, we found that expression of B7x by tumor cells reduced the efficacy of anti-CTLA-4 in syngeneic murine models in a Treg-dependent manner, but this resistance phenotype was overcome by combination anti-B7x and anti-CTLA-4 treatment. Put together, B7x mediates a novel Treg-promoting pathway within tumors and is a promising candidate for combination immunotherapy. In vivo: Foxp3-GFP-DTR mice (strain C57BL6) were engrafted with MC38-B7x or MC38-Control tumors, and tumors were enzymatically dissociated with collagenase-dispase-DNase solution. 10,000 to 20,000 tumor-infiltrating Tregs, defined as CD45+ CD4+ GFP+ cells, were flow-sorted on the BD FACSAria directly into RNA lysis buffer. In vitro: splenic CD4+ T cells from Foxp3-GFP-DTR mice were differentiated into iTregs with either B7x-Ig or Control-Ig, and subsequently up to 10,000 GFP+ CD4+ cells were sorted directly intro RNA lysis buffer.