In vivo blockade of T cell development reveals alternative pathways for generation of intraepithelial lymphocytes in mice

•We identify a novel CD8αα+ TCRαβ+ IEL subset.•Thy1.2 is a marker for a novel CD8αα+ TCRαβ+ IEL subset.•Thy1.2+ CD8αα+ TCRαβ+ IELs are generated from CD8α+ thymic precursors.

Intraepithelial lymphocytes (IELs) are resident cells localized within the intestinal epithelia and play an important role in regulating gut inflammations and host defense against pathogens. CD8α+ TCRαβ+ IELs are heterogeneous populations that are generated from T cell precursors including CD4− CD8α− double-negative (DN) cells and CD4+ CD8α+ double-positive (DP) cells. However, developmental pathways of TCRαβ+ IELs remained unclear. To gain insight into the mechanisms, we generated mice (Bcl11bΔDN2 mice) that lack thymic precursors (DN CD5+ TCRβ+ cells) for CD4− CD8αα+ TCRαβ+ IELs. Unexpectedly, we found that, in the absence of the precursors in thymi of Bcl11bΔDN2 mice, CD4− CD8αα+ TCRαβ+ IELs were still present in the intestine though the number was reduced. Adoptive transfer experiment showed that their precursors were highly enriched in CD8α+ TCRβ− thymocytes. The CD4− CD8αα+ TCRαβ+ IELs in Bcl11bΔDN2 mice are distinguished by Thy1.2 expression and are indeed present in WT mice. Taken together, our study reveal a novel developmental pathway for CD8αα+ TCRαβ+ IELs.