Discovery of Avian TCRVβ2⁺ T Cells Recognizing the Endogenous Ganglioside via a chCD1.1-Dependent Pathway

This study presents the discovery and first functional characterization of chicken lipid-reactive Natural Killer T (NKT)-like cells, resolving a long-standing knowledge gap in avian immunology. Using purified TCRVβ2⁺ lymphocytes, we demonstrate through rigorous experiments combining shRNA-mediated gene silencing, pharmacological pathway blockade, primary MACS-purified co-cultures, and intracellular cytokine flow cytometry that these cells are activated by CpG-ODN-stimulated antigen-presenting cells (APCs). Activation selectively depends on APC-driven glycosphingolipid (GSL) biosynthesis, with CpG-ODN significantly upregulating key biosynthetic enzymes (ST3GAL1 and ST3GAL5) in APCs. Silencing of enzymes involved in this pathway or chemical inhibition of GSL synthesis abrogated T cell activation. We identify GM3 as a potent, functional lipid ligand activating TCRVβ2⁺ lymphocytes. In parallel, IL-12 and IL-18 trigger IFN-γ production from TCRVβ2⁺ lymphocytes, crucially requiring live APC interactions. Upon activation of splenocytes with GM3, the cells expressed chIL-2, chIL-4, chIL-17, Granzyme A, and Perforin. By establishing functional, nonpeptide lipid-reactive T cells in chickens, these findings redefine the conceptual framework of avian innate-like T cell immunology. This discovery unlocks a highly translatable, universal target for lipid-adjuvanted poultry vaccines to enhance global animal health and food security.