Precocious Differentiation of Somatic and Pluripotent Stem Cells Bearing Pre-Arranged TCR
Authors: Klemen Nicholas , Vizcardo Raul , Tran Linda , Restifo Nicholas P. 
Published: October 2019

Abstract
Adoptive cell transfer (ACT) of mutation-specific T lymphocytes may represent the future of immunotherapy for cancer. Current strategies utilize mature T cells isolated from tumors, but this approach may be hampered by limited persistence, advanced differentiation status, and exhaustion of transferred cells. Cellular rejuvenation is needed, and this could be accomplished through induced pluripotent stem cell (iPS) technology. IPS cells can be generated from various types of somatic cells using the four Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), and we have shown that iPS generated from T cells (T-iPS) retain their original TCR specificity upon re-differentiation into T cell lineage. With existing technology, tumor antigen-specific progenitor cells can be derived from T-iPS. These cells could have superior potential to engraft, proliferate, and persist for long periods of time. To explore this concept, we have generated a murine iPS cell line from our Pmel transgenic mouse model, in which T cells bear a pre-rearranged TCR specific for the melanoma antigen GP100. Differentiation in vitro employs the OP9-DL1 co-culture system. We characterize the DN1-DN4 stages by flow cytometry of T cell lineage differentiation markers, such as c-kit, CD3, CD4, CD8, CD25, TCRvb, and TCRvb13 (present in the Pmel transgene).
To study somatic stem cell differentiation, we used lineage negative bone marrow from femurs of wild type C57BL/6 (WT) and Pmel TCR transgenic (Pmel) mice. After 10 days of culture, cells were harvested and analyzed. Frequencies of cells in the DN1-DN4 stages for WT were 2%, 41%, 55%, and 2%; for Pmel they were 5%, 31%, 39%, and 25%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 2%. In contrast, CD3 expression in Pmel cells was 85% for cells in DN1/DN2 and 95% for cells in DN3/DN4.
To study pluripotent stem cell differentiation, we used WT embryonic stem cells (ES) and Pmel iPS. After 21 days of culture, cells were harvested and analyzed. The double positive compartment was 9% in WT ES and 30% in Pmel iPS; the double negative compartment was 60% and 37%. Frequencies of cells in DN1-DN4 stages for WT ES were 4%, 6%, 45%, and 45%; for Pmel they were 1%, 7%, 69%, and 23%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 13%. In contrast, CD3 expression in Pmel cells was 76% for cells in DN1/DN2 and 91% for cells in DN3/DN4.
These data show for the first time that in vitro differentiation of pluripotent stem-derived progenitor cells bearing a pre-rearranged TCR will undergo precocious expression of the CD3 molecule and enhanced progression to DP stage as compared to WT controls. The pattern is consistent with that previously seen during in vitro differentiation of somatic stem cells. It appears that the presence of a pre-rearranged TCR is driving precocious development of T cells in vitro, regardless of whether the source of progenitors is from somatic (bone marrow) or pluripotent (ES/iPS) stem cells. Our ultimate goal is to intricately characterize the nature of iPS-derived T cells, and study their antitumor efficiency in vitro and in vivo, in order to evaluate their suitability for clinical use.

Disclosures
No relevant conflicts of interest to declare.