Identification of Galectin-9 (Gal-9) as a B7-H4 binding partner and characterization of their glycosylation-dependent interaction that modulates T cell signaling within a multi-ligand/receptor network.

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, its receptor and the mechanisms underlying its immunosuppression remain poorly understood. Here, we identify Galectin-9 (Gal-9) as a binding partner of B7-H4 and investigate its role in modulating T cell responses. We show that glycosylation within the IgC domain of B7-H4 is required for Gal-9 binding, while the N-terminal carbohydrate recognition domain (N-CRD) of Gal-9-specifically residue R65-is essential for its binding with B7-H4. In addition, several 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 levels comparable to those observed with B7-H4 or 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 an increased proportion of splenic CD4+ T cells, whereas B7-H4 deficiency produced no detectable phenotype. Moreover, B7-H4 and Gal-9 double-knockout mice showed no additive phenotype compared with Gal-9 single-knockout mice, and tumor growth following tumor cell challenge was unaffected in all three knockout models. Collectively, these findings indicate that B7-H4, Gal-9, other B7 family members, and T cell surface immune receptors form a complex regulatory network that modulates T cell activity and anti-tumor responses, with no single component exerting a dominant effect. This study provides a detailed molecular characterization of the B7-H4-Gal-9 interaction and uncovers additional Gal-9 binding partners, offering insights into the finely tuned immune regulation mediated by the B7 family.