Generation of BCR-ABL Reactive CD4+ T Helper Cells By Reprograming and Redifferentiation
Authors: Ueda Norihiro , Uemura Yasusi , Zhang Rhong , Kitayama Shuichi , Yasui Yutaka , Tatsumi Minako , Liu Tian-Yi , Kuzushima Kiyotaka , Kiyoi Hitoshi , Naoe Tomoki , Kaneko Shin 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder caused by BCR-ABL fusion protein that has constitutively active tyrosine kinase activity. Although the prognosis of the patient with CML in chronic phase has markedly improved by the advent of tyrosine kinase inhibiters, the management of the patients with CML in advanced phase remains to be the major challenge. Immunotherapy is considered to be one of the promising treatment strategies for refractory CML. BCR-ABL fusion region, b3a2 peptide, represents a neo-epitope that can induce CML-specific immune responses. The activation of b3a2 peptide-specific CD4+ T helper (Th) cells and their interaction with dendritic cells (DCs) can induce a robust cytotoxic T lymphocyte (CTL)-mediated anti-leukemic immunity through epitope spreading. However, current vaccination strategies cannot effectively induce the proliferation of antigen-specific Th cells in vivo, presumably due to the tumor-induced immunosuppressive milieu. In addition, ex vivo expansion of antigen-specific Th cells attenuates their effector functions by expansion-related cell senescence, and the procedure to establish antigen-specific Th cells for each patient's treatment is too complicated for the clinical application. The purpose of the present study is to establish a method to generate large amounts of functional b3a2-specific CD4+ Th cells enough for the treatment of the patients with refractory CML by using induced pluripotent stem cell (iPSC) technology.
First, we established b3a2-specific CD4+ Th clone from peripheral blood mononuclear cells of a healthy donor positive for HLA-DRB1*09:01 and HLA-A*24:02. The Th clone recognized b3a2 peptide in the context of HLA-DR9 and exhibited a Th1 profile. Second, we established iPSCs from the Th clone and differentiated them into T cell lineage by coculture with OP9 stromal cells expressing Notch ligand Delta-like 1. The iPSC-derived T cells (b3a2-iPS-T cells) expressed the same T cell antigen receptor (TCR) as the original Th clone but not CD4 molecule. Because CD4 acts as a co-receptor in the TCR-mediated Th responses, we transduced b3a2-iPS-T cells with CD4 gene. The CD4-expressing b3a2-iPS-T cells (CD4+ b3a2-iPS-T cells) recognized b3a2 peptide in the context of HLA-DR9 as the original Th clone. Moreover, CD4+ b3a2-iPS-T cells activated by b3a2 peptide induced DC maturation, as indicated by the upregulation of CD86 on DCs. In the additional presence of HLA-A24-restricted Wilms tumor 1 (WT1) peptide, the mature DCs stimulated primary expansion of WT1-specific CTLs. The CTLs exerted cytotoxicity against WT1 peptide-loaded HLA-A24 positive cell lines. These data suggest that the CD4+ b3a2-iPS-T cells have a potential to induce effective anti-leukemic immunity via DC maturation and subsequent CTL responses.
The current approach enable to provide large amounts of b3a2 specific CD4+ Th-like cells that would augment CTL-mediated anti-leukemic responses via DC maturation, which may contribute to the treatment of patients with refractory CML.

Disclosures
Kiyoi: Yakult Honsha Co.,Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Japan Blood Products Organization: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding. Naoe:Celgene K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; FUJIFILM Corporation: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding. Kaneko:AsTlym Co., Ltd: Other: founder, shareholder and scientific adviser.