In Vitro Generation of Human Pluripotent Stem Cell-Derived T Cells for Immunotherapy
Authors: Montel-Hagen Amélie , Seet Christopher S. , Li Suwen , Chick Brent , Chang Patrick , Zhu Yuhua , He Chongbin , Lopez Shawn , Crooks Gay M. 
Published: April 2021

Abstract
Adoptive cell therapy using T cells engineered to express antigen-specific T cell receptors (TCR-T) or chimeric antigen receptors (CAR-T) offer targeted and potentially curative treatments for malignancy. Current approaches rely on the genetic modification and expansion of mature circulating T-cells. Such processes are limited to autologous T cells due to the risk of graft-versus-host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. Furthermore, prolonged ex-vivo expansion of T cells may reduce in vivo efficacy and harvesting sufficient T cells from lymphopenic patients is challenging.
Direct in vitro differentiation of engineered T cells from human pluripotent stem cells (HSPCs) may overcome these problems by providing an unlimited source of cells that can be genetically edited, permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells.
We have developed an in vitro Artificial Thymic Organoid (ATO) system that induces highly efficient and reproducible production of mature naïve T cells from human hematopoietic stem cells and progenitor cells (HSPC). Here, we report the preclinical development of a modified ATO system that supports highly efficient in vitro differentiation and positive selection of naive human T cells from at least 5 different lines of human pluripotent stem cells (PSC), including Embryonic stem cells (ESC) and induced Pluripotent Stem Cells (iPSC). T cell differentiation from PSC was very similar phenotypically to that from HSPC. As in normal human thymopoiesis, the first evidence for T cell commitment was expression of CD7 and CD5, followed by the CD3-CD8lo "ISP8" stage, then CD4+CD8+ "DP" stage and finally production of CD3+CD8+CD4- "CD8SP" and Cd3+CD4+CD8- "CD4SP". As is typical with both monolayer cultures and ATOs (and opposite to normal thymus), CD8SP predominated over CD4SP. Surprisingly, differentiation occurred more rapidly from PSC than with HSPC. As with HSPC-ATOs, CD8SP from PSC ATOs showed a mature naïve conventional T cell phenotype i.e. CD3+TCRab+CD4- CD45RA+CD62L+CD27+ and exhibited a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation.
The differentiation in ATOs of an ESC line that expresses an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell yield with an enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. TCR-engineered T cells produced from PSC in ATOs displayed a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. The TCR engineered T cells underwent polyfunctional cytokine release, and proliferation in response to artificial APCs.
Moreover, the differentiation in ATOs of an ESC line that expresses a CD19-specific 2nd generation (CD28/CD3zeta) CAR construct resulted in the production of CD5+CD7+ CD45RA+ CAR T cells. As reported previously, the ESC-derived CAR T cells did not express CD4, CD8 or CD3; however, they responded to PMA/ionomycin and underwent specific cytokine release and degranulation in response to target cells expressing CD19.
PSC-derivedATOs thus present a highly efficient platform for the generation of clinically relevant mature naïve and potentially non-alloreactive TCR and CAR engineered T cells for adoptive immunotherapy.

Disclosures
Montel-Hagen: Kite Pharma: Research Funding. Seet: Kite Pharma: Research Funding. Crooks: Kite Pharma: Research Funding.