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Promise of Therapeutically Relevant T Cells

“Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+ T Cells” and “Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation”

Antigen-specific T cells have been proposed as a potentially important therapeutic cell type in the prevention or treatment of cancer or viral infections and, while cytotoxic T lymphocytes (CTLs) have been used in a clinical setting (Sensi and Anichini), there remains problems with the generation of sufficient numbers of fully functional cells for wider therapeutic use. Now, two groups (Hiroshi Kawamoto at the RIKEN Research Center for Allergy and Immunology, Yokohama, Japan and Shin Kaneko and Hiromitsu Nakauchi at the Center for Stem Cell Biology and Regenerative Medicine at The University of Tokyo, Japan) have proposed that induced pluripotent stem cell (iPSC) production from T cells and subsequent redifferentiation back into T cells could be an answer to this problem. In these two studies they describe the generation of iPSCs from a cancer-epitope specific T cell (Vizcardo, Masuda and Yamada et al) and an HIV type 1-epitope specific T cell (Nishimura et al) which both give rise to fully functional epitope-specific mature T cell progeny.

In the first study, Vizcardo, Masuda and Yamada et al, began by comparing reprogramming of CD8+ T cells isolated from human cord blood and adult peripheral blood (CD8+-iPSCs), CD34+ cord blood cells (hCB-iPSCs) and CD3+ T cells (hT-iPSCs). After activation by anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), cells were reprogrammed using Sendai viruses (Fusaki et al) carrying KLF4, SOX2, OCT4, c-MYC and SV40 large T Antigen (Park et al). Interestingly, in comparison to hESCs and hCB-iPSCs, hCD8-iPSCs produced a larger proportion of T Cell Receptor b (TCRb)+CD3+ cells from the CD4+CD8+ double-positive fraction under T cell conducive differentiation conditions.

Next, generation of iPSCs from antigen-specific T cells was analysed; specifically for the melanoma epitope MART-1. To this end, JKF6 lymphocytes originally derived from a melanoma patient (Yang et al) which specifically recognize a complex of the MART-1-peptide were transduced as before, generating 2 clones which were highly characterised and found to be similar to hESCs and retained the same rearranged configuration of TCRa and b chain genes as original JKF6 cells (‘‘MART-1-iPSCs’’).  When differentiated as described before, 70% of CD4+CD8+ double positive cells generated from MART-1-iPSCs expressed a TCR pattern specific to the MART-1 epitope. After stimulation with anti-CD3 mAb, CD8+CD3+ MART-1+ cells increased by 300-fold over the 6 weeks, with the disappearance of CD4+ cells.   These cells were functionally mature, as demonstrated by the appropriate release of IFNg production following CD3/CD28 stimulation, and also in response to the MART-1-peptide.

In the second study Nishimura et al, started by transducing activated CD3+ T cells from peripheral blood mononuclear cells (PBMCs) with OCT3/4, SOX2, KLF4, and c-MYC using retroviruses. Alongside this, PBMCs from an HLA-A24-positive patient with a chronic HIV-1 infection were collected and CD8+ CTL clones specific for an antigenic peptide were established from the HIV-1 Nef protein (Altfield et al). However, reprogramming using retroviral transduction with OCT3/4, SOX2, KLF4, c-MYC, NANOG, and LIN28A failed, but switching to Sendai viruses encoding for OCT3/4, SOX2, KLF4, and c-MYC, the miR- 302 target sequence, and SV40 large T antigen allowed for the production of ESC-like colonies by day 40. Resultant iPSCs also retained specific TCRa and TCRb gene rearrangements, suggesting that the generated iPSCs originated from a reprogrammed T cell.

A two stage co-culture mediated haematopoietic differentiation protocol was then used to generate CD45+, CD38+, CD7+, CD45RA+, CD3+, and TCRab+ T lineage cells. These cells were then cultured on OP9-DL1 stimulated with a-CD3/28 beads or PHA and then subsequently co-cultured on irradiated HLA-A24-negative PMBCs in the presence of IL-7 and IL-15, required for the generation of memory phenotype mature CD8+ T cells. By day 14, CD8+ T cells were generated which stained positive for A24/Nef by cytometric analysis suggesting that these cells recognized the same epitope on the same HLA. These cells could be expanded greatly in vitro, an essential facet if they are to be used therapeutically, did not lose expression of central memory T cell markers such as CCR7, CD27, and CD28 and had longer telomeres than the original T cell clone. Finally, the cytotoxic nature of these cells was qualified initially through the appearance of CD107a (a marker of degranulation CD8+ lymphocytes) in response to a-CD3/28 beads or a wild type Nef-peptide and also the detection of IFN-g in response to a wild type Nef-peptide.

Overall, these two studies show it is feasible to generate sufficient quantities of good quality antigen-specific T cells through iPSC-mediated reprogramming and redifferentiation. Next, these studies will no doubt move to in vivo studies where the efficacy of protection against cancer and HIV-1 can be tested as part of the next stage in moving these findings into a therapeutic context.

During the writing of this article, another study has reported similar findings (“Expansion of Functional Human Mucosal-Associated Invariant T Cells via Reprogramming to Pluripotency and Redifferentiation” Wakao et al) which demonstrate the reprogramming and differentiation of mucosal-associated invariant T (MAIT) cells which and protect against Mycobacterium abscessus infection.

References

  • Altfeld, M. et al. (2006). HLA Alleles Associated with Delayed Progression to AIDS Contribute Strongly to the Initial CD8(+) T Cell Response against HIV-1. PLoS Med. 3, e403.
  • Fusaki, N. et al (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 85, 348–362.
  • Park, I.H. et al (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146.
  • Sensi, M., and Anichini, A. (2006). Unique tumor antigens: evidence for immune control of genome integrity and immunogenic targets for T cell-mediated patient-specific immunotherapy. Clin. Cancer Res. 12, 5023–5032.
  • Yang, S. et al (2011). The shedding of CD62L (L-selectin) regulates the acquisition of lytic activity in human tumor reactive T lymphocytes. PLoS ONE 6, e22560.

Study originally appeared in Cell.

Stem Cell Correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.