NOTCH Activation at the Definitive Mesoderm Stage Facilitates Efficient Generation of T Cells with High Proliferation Potential from Human Pluripotent Stem Cells

Adoptive T cell therapies show promise in the treatment of several types of blood cancers. Developing off-the-shelf T cell products will further advance immunotherapies to the clinic and broaden their application. Human pluripotent stem cells (hPSCs) offer the potential to serve as a versatile and scalable source of T cells for immunotherapies, which could be coupled with genetic engineering technologies to meet specific clinical needs. However, production and expansion of T cells from hPSCs remains inefficient. In order to improve T cell production from hPSCs it is essential to identify cell subsets that are highly enriched in T cell progenitors, and those stages of development at which NOTCH activation induces the most potent T cells. Previously, we have developed both OP9-based and chemically defined systems for hematopoietic differentiation from iPSCs (Vodyanik et al., 2006 and Uenishi et. al, 2014). In these differentiation systems, hPSCs undergo stepwise progression towards APLNR⁺PDGFRa⁺ mesoderm with hemangioblast colony forming cells (HB-CFCs) that reflect primitive hematopoiesis (day 3 of differentiation), KDR^brightPDGFRa^low/- hematovascular mesodermal progenitors (HVMP) with definitive hematopoietic potential, VE-cadherin (VEC)⁺CD43^-CD73^- HE with definitive hematopoietic potential (day 4-5 of differentiation) and CD43⁺ hematopoietic progenitors, including CD235⁺CD41⁺ erythromegakaryocytic progenitors (E-MkP) and CD235a/41a^-CD45^+/- multipotent hematopoietic progenitors (MHP) that have lin^-CD34⁺CD90⁺CD38^-CD45RA^- hematopoietic stem progenitor cells (HSPC) phenotype (days 6-8 of differentiation). To assess the stage at which NOTCH activation induces the most potent T cells, we isolated the aforementioned blood forming populations and cultured them in T cell conditions on OP9-DLL4. We found that Day 3 APLNR⁺PDGFRa⁺ primitive posterior mesodermal cells did not produce T cells, while all downstream subsets except CD235a⁺CD41a⁺CD45^- cells do produce T cells when cultured on OP9-DLL4. As determined by limiting dilution assay, the highest frequency of T cell precursors was detected from day 4 HVMP (1 in 14 HVMP). The frequency of T cell precursors in day 5 HE and day 8 HPs was 1 in 16 HEs and 1 in 20 MHPs, respectively. In addition, we found that T cells generated from HVMPs have the capacity to proliferate for 8 weeks, in comparison to HEs and MHPs subsets, which could only be expanded for 4-5 weeks. T cell differentiation from hPSCs proceeded through a CD5⁺CD7⁺ progenitor stage that eventually transitions into CD8⁺CD4⁺ double positive cells (~90%), CD3⁺TCRa/b⁺ and CD3⁺TCRg/d⁺ cells. To confirm T cell development, we analyzed the genomic DNA of the hematopoietic cells from OP9-DLL4 cultures for the presence of T cell receptor (TCR) rearrangements. This analysis demonstrated the presence of multiple PCR products of random V-J and D-J rearrangements at the β locus and V-J rearrangements at the γ locus, indicative of a polyclonal T lineage repertoire. In vitro generated T-cells were functionally active and proliferated upon stimulation with PMA and IL-2. Upon activation, the cells express CD25⁺CD69⁺ (~73%) markers, cytokines (IFN-γ ~87%, TNFa~22%, IL2 ~34.5%) and cytolytic proteins (Perforin~37%). We also demonstrated that CD5⁺CD7⁺ T cell progenitors can be genetically modified to express CD19 CARs and eventually differentiate into CAR T cells with significant cytotoxic effect on Raji cells. Overall, our studies may aid in establishing protocols for the efficient off-the shelf production and expansion of PSC-derived CAR T cells for treating hematologic malignancies.