Galectin-9 regulates CD4+ T cell proliferation via intracellular HLA-DR binding in dendritic cells

To initiate T cell-mediated immunity, dendritic cells (DCs) present antigen to specific T cells through the establishment of an immune synapse. Despite its critical importance, the spatiotemporal dynamics of membrane receptor organization during synapse formation in DCs have been only partly characterized. Galectins, a family of ß-galactoside binding proteins, exert crucial roles in homeostasis and pathological processes but the molecular mechanisms underlying galectin function are largely unresolved. Here, we demonstrate that intracellular galectin-9 (gal9) is required for T cell activation. Murine and human DCs lacking gal9 showed impaired induction of CD4+, but not CD8+, T cell proliferation, suggesting a conserved function for gal9 in modulating DC–T cell interactions. Live-cell imaging further revealed that galectin-9-depleted DCs fail to establish stable immunological synapses with T cells, resulting in reduced T cell clustering and activation. Unbiased co-immunoprecipitation and mass spectrometry identified HLA-II as a gal9 binding partner in DCs, and we observed a marked reduction of HLA-II recruitment to the immune synapse as the underlying mechanism. Conditional gal9 knockout in DCs led to enhanced tumor growth, compared to their WT counterparts, underscoring galectin-9 role in T cell-dependent anti-tumor immunity. Collectively, this study provides the first detailed account of gal9-mediated HLA-II organization at the synaptic site of DCs, revealing a novel mechanism by which galectins orchestrate immune receptor positioning from within the cytoplasm to enhance CD4+ T cell activation. Overall design: Wildtype and galectin-9 knockdown dendritic cells