Abstract 2309: T-cell development from T cell-derived induced pluripotent stem cell

The ability to differentiate T cells of defined specificity and function from T-cell derived induced pluripotent stem cells (TiPSC) may be useful for the treatment of a range of pathologies, including cancer, infection and immune deficits. We recently reported that genetic engineering of TiPSC to express a Chimeric Antigen Receptor (CAR) is an effective strategy to combine the unlimited availability of TiPSC and facilitate reprogramming of the T cell specificity and functional potential. In vivo, CD19-retargeted human TiPS-derived CAR T cells (CARTiPSC-T) display therapeutic potency in a lymphoma model. Surprisingly, despite expression of the endogenous αβ TCR, the CARTiPSC-T cells possessed innate-like phenotype and function, most similar to γδ T cells. Although innate T cells have anti-tumor activity, they lack some vital features for therapeutic efficacy including long-term memory and in vivo persistence, which characterize mature CD8+ and CD4+ αβ TCR T cells. Additional research into the mechanisms underlying in vitro T cell differentiation of TiPSC is required to improve the development of mature TCRαβ T cells and facilitate the employment of their therapeutic potential. T cell lineage determination depends in part on the balance between Notch and TCR signaling, we are investigating their respective roles, as well as that of the CAR, in determining lineage commitment. We hypothesize that the combined CAR and early CD3/TCRαβ expression disrupts the TCR/Notch signaling balance, prohibiting mature TCRαβ T cell development. To study the effects of the different Notch ligands, TiPSC were differentiated on OP9 cells expressing one of four Notch ligands (DLL1, DLL4, Jagged-1 or Jagged-2). Preliminary data suggests that DLL4 is able to facilitate T cell development to CD4/CD8 double positive (DP) stage, however, this is hindered by CAR expression. To determine the role of early TCRαβ expression, elimination of TCRαβ expression was facilitated by targeted disruption of the TCRα constant region using the CRISPR/Cas9 system. Elimination of early TCRαβ expression showed improved development towards DP stage of TiPSC on OP9-DLL1. TiPSC are a valuable system for the study of human T cell differentiation. In addition, and most importantly they are further amenable to genetic engineering with TCRs or CARs, which may be useful for the generation of therapeutic “off-the-shelf”, antigen-specific T cells. Citation Format: Sjoukje J.C. van der Stegen, Maria Themeli, Justi