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

ABSTRACT 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.