Marked induction of CD4 +CD8 + T cells with multifunctional properties by coculturing CD2 + cells with xenogeneic stromal cells

A number of T cell subsets have been identified, and the in vitro characterization of these subsets largely depends on an appropriate induction system for each one. In previous studies, we characterized a unique T cell line, HOZOT, which possessed a CD4 +CD8 + double positive (DP) phenotype and multifunctional properties including cytotoxic, helper, and regulatory functions. Therefore, this T cell subset has been termed Tchreg cells. In this study, we established a new method of Tchreg cell induction, which consists of three steps and provides more efficient and reproducible results. By using a purified T cell population, the efficiency of Tchreg generation was profoundly increased, and the use of purified CD2 + cells rather than CD3 + cells was shown to be superior. One surprising finding was that at the initial step, stromal cell stimulation induced DP T cells more efficiently than dendritic cells or anti-T cell receptor stimulation, indicating a distinct antigen presenting cell (APC) ability of stromal cells as distinguished from conventional APCs. Even in subsequent steps, the presence of stromal cells was required to maintain the DP phenotype. In the second step, addition of stromal cell-conditioned (but not unconditioned) autologous CD14 + cells instead of interleukin-2 was beneficial for subsequent expansion of Tchreg cells. The improved three-step culture method described here represents a step forward in efforts to achieve clinical application and a good tool for elucidating how Tchreg cells, especially CD4 +CD8 + T cells, are generated. ► Tchreg cells are a unique T cell subset with CD4 +CD8 + phenotype and multifunctional properties. ► We developed the new Tchreg induction method consisting of three steps using purified CD2 + cells from cord blood. ► The improved culture method is more efficient and reproducible than the previous one. ► Co-culture with mouse stromal cells is required for the induction and maintenance of CD4 +CD8 + cells.