The Transcription Factors Thpok And Runx3 Identify Two Distinct CD4−CD8− Double Negative T Cell Populations And Respectively Define Their Commitment In The Helper Or Cytotoxic Fate

Abstract

Abstract In the small intestine epithelium, the major TCRab population is the double negative (DNT) cells lacking the expression of either CD4 or CD8. In contrast to the conventional selected cells, the DNT cells undergo an agonist selection, which leads to their functional education and capacity to migrate to intestine. DNT cells also possess the capacity to reach the liver, where we observed a heterogeneity of the DNT cells populations, based on expression of ThPOK and Runx3. In the gut, the latter is only expressed by the DNT cells. GSEA analysis and cytokines intra-cellular staining have shown that ThPOK+ DNT cells and Runx3 DNT cells are strongly divergent in their biological function. ThPOK+ DNT cells signature is enriched in genes expressed by CD4 T helper cells with a strong production of the inflammatory cytokine Il-17 whereas Runx3+ DNT cells signature is enriched in gene upregulated in CD8 T cytotoxic cells. In the thymus, based on the expression of these two transcription factors, we also found distinct DNT populations. Adoptive transfer, as well as hanging drop fetal thymus culture have demonstrated that solely Runx3+ DNT cells in the thymus are the precursors of the Runx3+ DNT cells in the gut. Furthermore, we demonstrated in the thymus both DNT cells populations are selected upon an agonist interaction with a self-antigen presented by developing thymocytes, for the development of ThPOK+ DNT cells, and TECs for Runx3+ DNT cells. RNA-seq experiment of thymic DNT populations revealed that the agonist selection process results in their differential functional education since T helper pathways are upregulated in ThPOK+ DNT cells whereas pathways involved in function and differentiation of cytotoxic cells are upregulated in Runx3+ DNT cells.