Extrathymic T cell differentiation in vitro from human CD34(+) stem cells

Although it is well established that T cells are derived from CD34(+) stem cells in vivo, and that T cells can develop in the absence of a functioning thymus, it has not proven possible thus far to generate human T cells in v itro from CD34(+) cells in the absence of any thymic influence. We now pres ent a limiting dilution cloning culture system that supports the differenti ation of highly purified human CD34(+) cells to CD3(+) T cells in vitro in the complete absence of any thymic components. The culture system features the use of a serum-free medium supplemented with a cocktail of cytokines in cluding flt-3 Ligand, interleukin-3 (IL-3), stem cell factor (SCF), and IL- 2. CD4(+) T cell clones capable of mitogen-stimulated proliferation and res ponse to IL-2, and expressing a varied TCR-V beta repertoire were obtained under these conditions. This culture system therefore supports human T lymp hopoiesis in the absence of any thymic influence and may prove useful for t he evaluation of extrathymic T cell differentiation in vitro.