In Vitro Generation of Mature T Lymphocytes From Human Ips Cells and Genetic Analysis of TCR Gene Rearrangements

Abstract 2984

T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens via T-cell receptors (TCRs) in HLA-restricted and antigen-specific manner. Adoptive cell transfer therapy (ACT), the administration of ex vivo-activated and -expanded autologous tumor-reactive T lymphocytes, is currently one of the effective methods for immunotherapy, especially for treatment of metastatic solid tumors including melanoma. However, the successful applications of this method are currently limited for tumor therapies. To broaden the range of the application of ACT, we endeavored to develop easier method to obtain cells that carry antigen-specific TCR genes. For the purpose, generation of induced pluripotent stem (iPS) cells from an antigen-reactive single T lymphocyte is attractive and rewarding way. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features may enable us to induce an unlimited number of T lymphocytes, especially high proliferative naïve / central memory-type T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro.

Peripheral T lymphocytes were isolated from healthy volunteers. Then reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) were transduced into fresh or frozen / thawed T lymphocytes. T lymphocyte-derived iPS-like colonies were observed within 3 weeks and they were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface marker expression, alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm that the iPS cells were generated from T lymphocyte. The TCR gene rearrangements encoded in an iPS colony were clonal for all iPS lines, indicating that each iPS colony was derived from a single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process.

Next, we tried to re-differentiate T lymphocyte derived-iPS (T-iPS) cells into T cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). T-cell differentiation was evidenced by the expression of T-cell markers, such as CD5, CD7, CD27, CD4, CD8, TCR α β and CD3. We obtained 33.5 ± 17.9% CD4⁺ CD8⁺ double positive (DP) cells, 6.51 ± 5.40% CD4⁺ CD8⁻ single positive (SP) cells and 3.80 ± 1.28% CD4⁻ CD8⁺ SP cells. They could be activated via TCR stimulation, and produce cytokines as functionally matured T lymphocytes do. The re-differentiation efficiency of T-iPS cells was higher than those of other pluripotent stem cells, such as embryonic stem (ES) cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. Transcribed TCR mRNA sequences in re-differentiated T cells were analyzed, and they were revealed to be identical to that engraved in the pre-differentiated T-iPS cells genome in CD4⁺ CD8⁺ DP phase. However, fully matured CD4⁺ CD8⁻ or CD4⁻ CD8⁺ SP phase cells had several TCRA gene rearrangement patterns distinct from the original T-iPS cell's. On the other hand, TCRB gene maintained identity with the original. The variance of the sequences, especially antigen-recognition site sequences, indicated that the antigen-specificity in the original T lymphocyte might be converted during DP to SP transition process in vitro.

These data indicate that functionally matured T cells were generated by re-differentiating T-iPS cells in vitro, and that re-assemble of TCRA genes could take place during SP T cell maturation process. In order to fulfill the T-iPS-mediated immunotherapy, we need to overcome the obstacle of further TCRA gene rearrangements. We think the solution lies in refinement of the re-differentiation method for controlling the expression of RAG1 and RAG2 recombinases or for inhibiting their activities.