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Functionally Mature CD4 and CD8 TCRβ Cells Are Generated in OP9-DL1 Cultures from Human CD34⁺ Hematopoietic Cells¹

Stefanie Van Coppernolle,^* Greet Verstichel,^* Frank Timmermans,^* Imke Velghe,^* David Vermijlen, Magda De Smedt,^* Georges Leclercq,^* Jean Plum,^* Tom Taghon,^* Bart Vandekerckhove,^2* and Tessa Kerre^*

^*Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium; and Institute for Medical Immunology, ULB, Gosselies, Belgium

Human CD34⁺ hematopoietic 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 which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4⁺ and cytotoxic CD8⁺ TCRβ cells is not expected. Phenotypically mature CD27⁺CD1^– TCR as well as TCRβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRβ cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8β and one expressing CD8 dimers. TCRβ CD8 and TCR cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8β⁺ and CD4⁺ TCRβ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8β⁺ 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-. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8⁺ cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4⁺ or CD8⁺ T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grant G.0096.05 of the Fund for Scientific Research, Flanders (Fonds voor Wetenschappelijk Onderzoek Vlaanderen), Stichting tegen Kanker, and the Interuniversity Attraction Poles Program supported by the Belgian Science Policy. S.V.C. is supported by the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen. F.T., T.T., and T.K. are supported by the Fund for Scientific Research, Flanders (FWO Vlaanderen). I.V. is supported by the Interuniversity Attraction Poles Program.

² Address correspondence and reprint requests to Dr. Bart Vandekerckhove, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, University Hospital Ghent, Blok A, 4th floor, De Pintelaan 185, 9000 Ghent, Belgium. E-mail address: bart.vandekerckhove{at}ugent.be

³ Abbreviations used in this paper: HPC, hematopoietic precursor cell; CD4ISP, CD4 immature single positive; DL1, delta-like ligand 1; DN, double negative; DP, double positive; IEL, intraepithelial lymphocyte; MAIT, mucosal-associated invariant T; PNT, postnatal thymus; SP, single positive; TEC, thymic epithelial cell; NGFR, nerve growth factor receptor; GalCer, -galactosidase ceramide; Fw, forward; rev, reverse; DC, dendritic cell; nTreg, natural regulatory T cell.

⁴ The online version of this article contains supplemental material.