Controlled Major Histocompatibility Complex-T Cell Receptor Signaling Allows Efficient Generation of Functional, Antigen-Specific CD8⁺T Cells from Embryonic Stem Cells and Thymic Progenitors

Abstract

Generation of early T cells by coculturing stem cells on notch-ligand-expressing OP9 stromal cells (OP9-DL1) has been widely reported. However, further differentiation of these cells into mature, antigen-specific, functional T cells, without retroviral transduction of T cell receptors (TcRs), is yet to be achieved. In the thymic niche this differentiation is controlled by the interaction of developing TcRs with major histocompatibility (MHC) molecules on stromal cells. We hypothesized that by providing exogenous antigen-specific MHC=TcR signals, stem and progenitor cells could be engineered into functional, effector T cells specific for the same antigen. Here we demonstrate that both thymus-derived immature T cells (double positive [DP]: CD4 þ CD8 þ ) and mouse embryonic stem cells can be efficiently differentiated into antigen-specific CD8 þ T cells using either MHC tetramers or peptide-loaded stromal cells. DP cells, following MHC=TcR signaling, retained elevated recombination activating gene-1 levels, suggesting continuing TcR gene rearrangement. Both DP and embryonic stem-cell-derived CD8 þ T cells showed significant cytotoxic T lymphocytes activity against antigen-loaded target cells, indicating that these cells are functional. Such directed differentiation strategy could provide an efficient method for generating functional, antigen-specific T cells from stem cells for potential use in adoptive T cell therapy.

Generation of Functional, Antigen-Specific CD8+ Human T Cells from Cord Blood Stem Cells Using Exogenous Notch and Tetramer-TCR Signaling

“…20 Furthermore, an in vitro model of TCR revision in mature human CD8+ T cells, induced by the presence of anti-CD3 and IL-7, was recently described. 43 In particular, IL-7 has been shown to induce T cell survival and facilitate chromatin remodeling at the TCR loci in developing, immature T cells.…”

“…Recently, it has been demonstrated that treatment of mouse DP thymocytes with antigen-loaded H-2K b MHC class I tetramers, anti-CD28, anti-CD3, IL-2, and IL-7 results in RAG-1 upregulation, suggesting that TCR gene rearrangement is initiated or continued due to the presence of MHC tetramers [20]. Furthermore, an in vitro model of TCR revision in mature human CD81 T cells, induced by the presence of anti-CD3 and IL-7, was recently described [45].…”

“…19 Our group also demonstrated the ability of antigen-loaded MHC Class I tetramers to generate, from mouse DP cells or mouse embryonic stem cells, a population of CD8+ T cells specific for that particular antigen and capable of in vitro cytotoxic killing of target cells. 20 However, to date, direct in vitro generation of antigen-specific, functional human T cells from any stem cell population has not been achieved, except through stromal cell co-culture with HSCs retrovirally transduced with specific TCRs. 21,22 …”

Emerging strategies for spatiotemporal control of stem cell fate and morphogenesis

“…Understanding the co-culture signaling responses governing cell specification can instruct synthetic platforms to recapitulate native responses; for example, the maturation of stem cells to produce antigen specific T cells has been accomplished by providing major histocompatibility complex molecules from either stromal cells or as tetramers in solution [31]. Additionally, patterning approaches have demonstrated the ability to create complex co-culture platforms, and thereby may inform approaches to spatially control differentiation.…”

Development of Feeder-Free Culture Systems for Generation of ckit+sca1+ Progenitors from Mouse iPS Cells

“…In these studies iPS cells were differentiated into the hematopoietic lineage by co-culturing with a bone marrow stromal cell line (OP9) or its derivatives (e.g. notch-ligand transfected OP9 cells, OP9-DL1), which has been well established to support ES cell hematopoiesis and to generate lymphoid lineages [15, 16]. Despite significant advances, ultimate clinical application requires therapeutic cells to be free of stromal cell contamination, which is difficult to achieve through cell sorting techniques.…”