B7-H4 Binds Galectin-9 Glycosylation-Dependently and Attenuates Galectin-9-Mediated CD28/AKT Activation and T Cell Death

B7-H4, a member of the B7 family, is broadly expressed on various cancer cells and has been implicated in negative immune regulation, particularly in suppressing anti-tumor immunity. However, the receptor for B7-H4 and the molecular mechanisms underlying its immunoinhibitory effects remain poorly understood. In this study, using peritoneal immune cells from mice adoptively transferred with OVA-expressing tumor cells and OVA-specific OT-1 T cells, we identified Galectin-9 (Gal-9) as a binding partner for B7-H4 and elucidated its role in modulating T cell responses through this interaction. We demonstrated that glycosylation in the IgC domain of B7-H4 is required for its binding to Gal-9; while the N-terminal carbohydrate recognition domain (N-CRD) of Gal-9, including the R65 residue in the N-CRD, is essential for this interaction. Additionally, we found that other B7 family members (B7.1, B7.2, B7-H2, and B7-DC) and immune cell surface receptors (CD28, 2B4, CD226, and SLAMF1) also bind to Gal-9 at comparable levels to B7-H4 and T cell immunoglobulin mucin receptor 3 (TIM-3). In vitro functional assays revealed that B7-H4 inhibits Gal-9-induced activation of CD28 downstream signaling and reduces Gal-9-mediated T cell death. In vivo, Gal-9 deficiency in mice resulted in a significant reduction in the proportion of splenic CD4+ T cells, whereas B7-H4 deficiency exhibited no observable phenotype. Furthermore, B7-H4 and Gal-9 double-knockout mice displayed no additional phenotype differences compared to Gal-9 single-knockout mice. Notably, tumor growth following tumor cell challenge was unaffected in all three knockout models (Gal-9 single-, B7-H4 single-, or double-knockout). Collectively, these findings suggest that B7-H4, other B7 family members, Gal-9, and T cell surface immune receptors form a complex regulatory network that modulates T cell activity and anti-tumor responses, although no single member exerts a major effect. This study provides a detailed molecular characterization of the interaction between B7-H4 and Gal-9 and identifies other previously unknown Gal-9 binding partners, offering valuable insights into the intricate regulatory network involving these molecules.