Expression data from T cells in vivo

Induced pluripotent stem cells (iPSC) might provide an unlimited source of T cells for a variety of clinical applications, including restoration of T cell immunity following stem cell transplantation and generation of minimally differentiated antigen-specific T cells for adoptive immunotherapy. However, current methods of differentiating T cells from iPSC using OP9/DLL1, an extrinsic source of Notch signaling, result in the accumulation of unconventional T cells either lacking CD8 co-receptors or expressing CD8αα homodimers. We sought to explore the conditions that cause iPSC to give rise to unconventional T cells, and to test whether alternative culture conditions that convey physiologic signals for selection at various stages of in vitro differentiation could allow for the generation of conventional T cells. Here, we demonstrate that inappropriate signals for selection are a major driver of the unconventional T cell fate, and that a non-substrate bound thymic culture system can provide the requisite signals required for selection and survival of conventional T cells expressing the CD8αβ heterodimer. This culture system is capable of generating large numbers of both polyclonal and antigen-specific T cells that recapitulate the phenotypic, functional, and gene expression profiles of bona fide naïve CD8αβ+ T cells. These data represent an important proof-of-concept that therapeutically useful mature T cells can be generated from iPSC entirely in vitro, a finding with major implications for the use of reprogramming technology for cellular therapy. Samples were injected into 6 week old littermate Rag1-/- female recipients with 2e7 recombinant vaccinia with hgp100. Mice were sacrificed 25 days later and spleens were recovered. Live, congenically marked cells were recovered by FACS.