T Cell Receptor Is Required for Differentiation, but Not Maintenance, of Intestinal CD4⁺ Intraepithelial Lymphocytes

The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. However, the role of T cell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4⁺CD8αα⁺ IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4⁺ T cells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4⁺CD8αα⁺ IEL differentiation. However, TCR ablation on differentiated CD4⁺CD8αα⁺ IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4⁺CD8αα⁺ IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4⁺ T cells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling.