CD4 and CD8 TCRαβ Cells Are selected On MHC Expressed On Thymocyte Precursors in OP9-DL1 Cultures.

Abstract

Abstract 3670Poster Board III-606Human CD34+ haematopoietic precursor cells cultured on delta-like-ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells (TEC) which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected.Phenotypically mature CD27+ CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single positive (SP) TCRαβ cells were observed. Mature CD8 SP cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ+ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L and high levels of IL-2 and IL-4; CD8+ cells expressed EOMES, RUNX3 and high levels of granzyme, perforin and IFN-γ.Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells, whereas blocking MHC class I antibodies and CD8 antibodies inhibited generation of mature CD27+ CD8+ SP cells. The presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells.In conclusion, we have demonstrated positive selection of TCRαβ+ cells in the absence of TEC. The data suggest that positive selection is induced by interaction between T precursor cells without requirement for other haematologic or stromal elements. Disclosures:No relevant conflicts of interest to declare.