‘Off-the-shelf’ immunotherapy with iPSC-derived rejuvenated cytotoxic T lymphocytes  Miki Ando, Hiromitsu Nakauchi  Experimental Hematology  Volume 47, Pages 2-12 (March 2017) DOI: 10.1016/j.exphem.2016.10.009 Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 1 Immunosuppressive state of effector CD8+ T cells in the tumor microenvironment. (A) How tumors “escape” the immune system. Reduction of MHC expression diminishes presentation of target antigens. The expression of Fas ligand on tumor cells induces apoptosis in CTLs. TGFβ and IL-10 drive the differentiation of CD4+ T cells toward the regulatory phenotype, which results in T-cell inhibition. The engagement of PD-1 with its ligand programmed cell death protein ligand 1 (PD-L1) inhibits the proliferation and effector function of T cells through the recruitment of SHP2, which inactivates TCR-mediated signaling. (B) T-cell exhaustion during chronic antigen exposure. The presentation of antigenic peptides in the context of MHC class I triggers the priming of naive CD8+ T cells and their differentiation into effector CD8+ T cells. After antigen clearance, effector CD8+ T cells differentiate into memory CD8+ T cells. In contrast, during continuous exposure to antigen, T cells gradually lose effector functions and proliferation capacity and finally are eliminated via apoptosis. The gain of cell surface inhibitory receptors such as PD-1 correlated with T-cell exhaustion reduces antitumor immune function. Experimental Hematology 2017 47, 2-12DOI: (10.1016/j.exphem.2016.10.009) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 2 The antigen recognition of T cells and CARs. (A) Antigens inside the cell are delivered to the cell surface as peptide fragments by the MHC and T cells interact with antigens through the TCR. The subsequent costimulatory signal is also necessary for optimal T-cell proliferation, differentiation, and survival. CD28 and 4-1BB interact with CD80 and 4-1BBL, respectively. (B) CARs react directly with native tumor antigens expressed on the surface of a tumor cell in a non-MHC-restricted manner. CARs are composed of antibody-binding domains fused to T-cell signaling domains. The target binding module of the single-chain antibody-variable fragment (scFv) are originated from a tumor-specific antibody. Experimental Hematology 2017 47, 2-12DOI: (10.1016/j.exphem.2016.10.009) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 3 Rejuvenation of antigen-specific CTLs for powerful adoptive immunotherapy. Reprogramming via iPSC technology generates T-iPSCs from antigen-specific CTLs. These T-iPSCs can redifferentiate into CTLs that retain ancestral antigen specificity and cytotoxicity. Rejuvenated CTLs have higher proliferative capacity and longer telomeres than do the original exhausted CTLs. Experimental Hematology 2017 47, 2-12DOI: (10.1016/j.exphem.2016.10.009) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 4 In vitro generation of rejuvenated antigen-specific CTLs. Antigen-specific CTL clones first are established from antigen-specific CTLs obtained from peripheral blood. These clones are reprogrammed into iPSCs (T-iPSCs) by transduction with vectors that contain the four Yamanaka reprogramming factors. For hematopoietic differentiation, T-iPSCs are cultured on C3H10T1/2 feeder cells for 2 weeks, yielding iPSC-derived sacs that contain many hematopoietic progenitor cells. Hematopoietic progenitor cells extracted from sacs are transferred onto Notch-ligand-expressing CH310T1/2 feeder cells. After 28 days of culture, T-lineage cells are harvested and stimulated, yielding huge numbers of rejuvenated antigen-specific CTLs that were generated from T-iPSCs. Experimental Hematology 2017 47, 2-12DOI: (10.1016/j.exphem.2016.10.009) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 5 Induction of apoptosis by activating iC9. In the intrinsic cell death pathway, activated BAX or BID translocates to the mitochondria, forming pores that allow cytochrome C to move from mitochondrial intermembrane space to cytosol. There, cytochrome C activates caspase-9, which in turn activates caspase-3 and leads to apoptosis. iC9 was made by the fusion of human caspase-9 to a modified FK506-binding protein (FKBP12), allowing conditional dimerization. When CID is added, iC9 is dimerized. The dimer activates caspase-3 directly, without mitochondrial poration, inducing apoptosis. Experimental Hematology 2017 47, 2-12DOI: (10.1016/j.exphem.2016.10.009) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 6 T-iPSCs provide a practical platform for gene therapy or engineered T-cell therapy. After reprogramming, T-iPSCs can be transduced with a suicide-gene-based safeguard system, transgenic TCRs, or CARs. Redifferentiation of T-iPSCs into which the suicide gene iC9 has been introduced yields rejCTLs that express iC9. Experimental Hematology 2017 47, 2-12DOI: (10.1016/j.exphem.2016.10.009) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions