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Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.

Introduction: Loss-of-function mutations in Runt-related transcription factor 1 (RUNX1) are commonly found in both germline and somatic hematopoietic malignancies and confer particularly poor prognosis in AML. However, it remains unclear how RUNX1 functions during hematopoietic and leukemic development, particularly because RUNX1 mutations alone are not sufficient to cause myeloid malignancy and some models show that RUNX1 mutations confer hematopoietic stem cell defects. Recently, mouse models have shown that RUNX1-deficient neutrophils upregulate NFκB activity, and hematopoietic stem and progenitor cells (HSPCs) with overactive inflammatory pathways gain competitive advantage under chronic inflammation. Thus, we hypothesized that while RUNX1 mutations impair normal HSPC function, inflammation may select for or rescue RUNX1 mutant HSPCs. Methods: To interrogate the effect of RUNX1 loss in human CD34+ HSPCs, we disrupted the RUNX1 locus using CRISPR/Cas9 and AAV6-mediated homology directed repair. Importantly, by using an AAV6 vector that carries arms of homology flanking a fluorescent reporter expression cassette, we are able to track and isolate cells edited at the RUNX1 locus for in vitro and in vivo functional analyses and for molecular characterization using RNA-seq and ATAC-seq. Results: First, we used this system to evaluate the functional consequences of RUNX1 knockout (KO) in human CD34+ HSPCs. Loss of RUNX1 caused early erythroid-megakaryocytic differentiation arrest and skewing toward monocytic differentiation. RUNX1 KO cells demonstrated decreased proliferation, cell cycle arrest, and reduction in serial replating potential in vitro. In competitive transplantation experiments in NSG mice, RUNX1 KO engraftment decreased over time in both primary and secondary transplant, revealing a competitive disadvantage. Second, ATAC-seq peak motif analysis showed that PU.1 and NFκB motifs are more accessible upon RUNX1 KO whereas GATA, TAL1, and RUNX motifs were less accessible. Similarly, gene set enrichment analysis of transcriptional data confirmed the broad upregulation of NFκB-mediated inflammatory programs; downregulation of GATA1-dependent heme metabolism and platelet development pathways; and downregulation of MYC- and E2F-dependent cell cycle programs. These observations imply that RUNX1 directs cell fate decisions by recruiting and activating lineage-specific hematopoietic transcription factors and augmenting stem cell proliferation programs. We next sought to determine which cytokines are sufficient to drive RUNX1 KO cell expansion. RUNX1 KO cells not only expanded preferentially in NSG mice expressing human SCF, GM-CSF, and IL-3 (NSGS mice), but also were no longer defective in competitive transplants in these mice. Further, treatment with IL-3 was sufficient to significantly expand RUNX1 KO cells in vitro. Flow cytometry revealed that the IL-3 receptor CD123 is upregulated in RUNX1 KO cells compared to control. Similarly, RUNX1-mutant AML patient samples express higher levels of CD123 than RUNX1-wildtype AML patient samples. Finally, evaluation of publicly available RUNX1 ChIP-seq of bone marrow CD34+ HSPCs revealed that RUNX1 directly binds the promoter of CD123. Ongoing efforts are aimed at determining whether targeting CD123 and IL-3 signaling may be a viable therapeutic approach for the prevention or treatment of RUNX1-mutant myeloid malignancies. Conclusion: In summary, we established a RUNX1-deficient human HSPC model not only to evaluate the role of RUNX1 in hematopoiesis, but also to characterize intrinsic and extrinsic factors involved in RUNX1-deficient clonal expansion and leukemic transformation. We show that RUNX1 KO causes monocytic skew at the expense of erythro-megakaryocytic potential and severely limits HSC engraftment and expansion in vivo. Molecular profiling reveals that these effects are associated with dysregulation of both transcription factor activity and cytokine signaling. However, exposure to IL-3 rescues RUNX1-deficient cell proliferative defects in vitro and competitive engraftment defects in vivo. This hypersensitivity to IL-3 signaling is mediated in part by increased expression of the IL-3 receptor CD123. These findings reveal how RUNX1 mutations may initially behave in a deleterious manner but can ultimately confer an advantage to HSPCs under certain environmental conditions. Disclosures Majeti: CD47 Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Kodikaz Therapeutic Solutions Inc: Membership on an entity's Board of Directors or advisory committees.

Although loss-of-function RUNX1 mutations are commonly found in hematopoietic malignancies, how RUNX1 functions during hematopoietic and leukemic development is unclear. Using a CRISPR/AAV6 system to target the RUNX1 locus in human CD34+ hematopoietic stem and progenitor cells (HSPCs), we show that RUNX1 deficiency causes monocytic skew at the expense of erythro-megakaryocytic potential and stem cell activity, including a severe in vivo stem cell competitive defect. RNA-seq and ATAC-seq review that these effects are mediated by broad upregulation of PU.1 and NFKB transcriptional programs; downregulation of GATA1- and TAL1-dependent erythro-megakaryocytic differentiation; and downregulation of cell cycle programs mediated by MYC and E2F. Treatment with IL-3 rescues RUNX1-deficient cell proliferative and stem cell defects. Together, these results show that RUNX1 controls transcription factor activity and cytokine signaling, and loss of RUNX1 causes monocytic skewing and hypersensitivity to IL-3-dependent expansion. We are currently studying how the IL-3 selects for inflammation-sensitive RUNX1 deficient cells and whether targeting IL-3 signaling may be a viable therapeutic in the prevention or treatment of RUNX1 mutant malignancies.

Introduction: Mutations in additional sex combs like 1 (ASXL1) are recurrently found in myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but can also be found in pre-malignant states such as clonal hematopoiesis of indeterminate potential (CHIP). However, the mechanisms by which these mutations contribute to disease initiation and progression remain unresolved. Initial observations using murine models suggested a loss of function of the ASXL1 protein as relevant to disease. However more recently, several studies have suggested that recurrent mutations of ASXL1 result in a change-of function caused by a truncated ASXL1 protein. Most of these studies have been performed in a murine system and have used over-expression of truncated ASXL1. Methods: To interrogate the functional relevance of recurrent mutations in ASXL1 in human HSPCs, we generated a novel CRISPR/Cas9-mediated model that allows for the introduction of truncations or complete knockout of ASXL1 into CD34+ cord blood cells. Importantly, this is the first model which efficiently introduces these mutations into the native ASXL1 locus in human cord-blood and bone-marrow derived HSPCs while simultaneously introducing a fluorescent marker corresponding to the genotype, so individual cells can be tracked in vitro and in vivo. Results: Using this system, we demonstrate that truncated, but not deleted, ASXL1 shows a proliferative advantage, decreased ability to differentiate along the megakaryocyte and erythroid lineages, as well as increased serial replating in vitro. Interestingly, truncation of ASXL1 alone did not result in a myeloid differentiation block in vitro, in line with observations of mutant HSPCs in CHIP. In vivo, CD34+ cells harboring truncation of ASXL1 exhibited myeloid skewing and a proliferative advantage in competitive engraftment studies using a xenograft NSGS mouse model (both p Conclusion: In summary, our work demonstrates the utilization of a novel CRISPR/Cas9 model of human ASXL1-mutant HSPCs that closely resembles the phenotype seen in individuals with ASXL1 mutant CHIP and patients with myeloid malignancies carrying mutant ASXL1. Additionally, our model allows us to directly compare the deletion of ASXL1 with the truncation, adding to the evidence that truncated ASXL1 phenocopies pre-malignant and malignant hematopoiesis with recurrent ASXL1 mutations. Finally, we provide the first model for the spontaneous progression of pre-malignant human CHIP to myeloid malignancy, which should enable further studies of leukemogenesis and potentially disease prevention. Disclosures Majeti: FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy.

Hematopoietic stem cell (HSC) differentiation is accompanied by a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) to meet the increasing energy demand during proliferation and differentiation. However, the role of mitochondrial metabolism in HSC differentiation goes beyond ATP production. Metabolites generated during mitochondrial metabolism may impact in HSC fate decisions through stable epigenetic modifications. Despite some progress in understanding mitochondrial communication during HSC development, their role in human hematopoiesis remains largely elusive, where the lack of appropriate model systems poses a major obstacle. Reticular Dysgenesis (RD), a rare and particularly severe form of severe combined immunodeficiency (SCID), offers an attractive model for studying the role of mitochondrial metabolism in hematopoiesis. RD is an autosomal recessive disease caused by biallelic mutations of the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), which serves as the substrate for the ATP synthase. In addition to defective lymphocyte development typical of classic SCID, RD patients also suffer from impaired myeloid development, suggestive of a global defect in hematopoiesis. In a human induced pluripotent stem cell (iPSC) model for RD, hematopoietic stem and progenitor cells (HSPCs) recapitulate a profound maturation arrest of the myeloid lineage, increased oxidative stress and an energy-depleted metabolite and transcriptional profile. We hypothesize that AK2 defects drive hematopoietic cell fate decisions through changes in metabolites that regulate the activities of DNA/histone modifying enzymes and result in stable epigenetic modifications. Methods: Since iPSCs are not suitable to model the epigenetic characteristics of definitive hematopoiesis, we developed a novel model system in which we deleted AK2 in primary human HSCs using CRISPR/Cas9 gene editing technique. We found a highly effective single-guide RNA (sgRNA) targeting the catalytic LID domain of the AK2 gene to introduce directed DNA double stranded breaks (DSBs), and use a homologous recombination (HR)-mediated dual reporter system to track and isolate cells with biallelic AK2 disruption. Results: Our single-color GFP reporter system consistently produces a >60% GFP+ population of AK2-targeted CD34+ umbilical cord blood (UCB) cells. With dual GFP/BFP reporters, we were able to achieve 6% GFP/BFP double positive cells with confirmed biallelic AK2 knock-out. Since HR events on one allele are biologically linked to CRISPR/Cas9 mediated DSBs on the other, we assessed insertion and/or deletion (INDEL) frequency and protein expression in a single reporter (GFP+) population of AK2-targeted UCBs. We detected an INDEL frequency of over 90% on the non-HR alleles along with nearly absent AK2 protein expression by Western Blot. These results indicated that the highly efficient single-color reporter system with >60% targeting efficiency is sufficient to achieve an AK2 biallelic knock-out population in primary HSCs. in vitro myeloid differentiation of these AK2-targeted HSCs recapitulates the RD phenotype with impaired neutrophil but preserved monocyte development. Conclusion: This novel disease model for RD will now allow us to examine the cellular and molecular impact of perturbations in metabolism on human HSC development. We will investigate the effect on differentiation potential, metabolite profile, transcriptome and epigenome in vitro as well as in a xenograft mouse model. Elucidating how metabolism governs differentiation and self-renewal of human HSCs will not only advance our basic understanding of many blood and immune diseases, but has important translational implications for improving the use of HSCs in hematopoietic stem cell transplantation, gene and cell therapy. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine. To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in "Hit & Run," we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis. First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p Next, we investigated the effects of TET2 disruption on hematopoietic colony formation in methylcellulose. Both methods resulted in increased numbers of TET2-disrupted colonies compared to control (Hit & Run, n=4 independent experiments, p Next, we transplanted control or TET2-disrupted Hit & Run CD34+ cells into NSG mice. Primary transplantation at 4 months showed no statistical differences in either engraftment rate (human CD45+) or differentiation (T/ B/ Myeloid cells), although the frequency of TET2 indels increased gradually in CD33+ cells. Intriguingly, 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells (lymphoid=22.1%, myeloid=73.0%, Mann-Whitney U, p=0.0485) and a particular increase in a CMML-like CD33highCD14+CD16- population. Furthermore, preliminary data from tertiary transplantation (8 weeks after transplantation) indicates persistent myeloid skewing in the bone marrow in some mice and expansion of TET2-mutant cells, suggesting a CMML-like disease. Finally, we used in vivo competition studies to determine if TET2-disrupted HSPCs are selectively targeted by azacitidine or ascorbate treatment compared to controls. NSG mice were intrafemorally transplanted with a one-to-one ratio of control and TET2-disrupted HSPCs, and 4 months later, these mice were treated with azacitidine (2.5mg/kg/dose, i.p. daily on days 1-5 of a 14-day cycle for 2 cycles) or ascorbate (4g/kg/dose, i.p. twice daily for a month). In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p=0.036), but did not eradicate established TET2 pre-leukemia in all evaluable mice. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p=0.14). Our data show that TET2 disruption in primary human HSPCs blocked erythroid differentiation, increased colony formation and replating, and caused myeloid skewing and a CMML-like disease in vivo after an extended period of time. In this model, azacitidine or ascorbate treatment slowed expansion of TET2-mutant human pre-leukemic clones raising the intriguing possibility of preventing CHIP progression to de novo AML. Disclosures No relevant conflicts of interest to declare.

Summary Aging is linked to functional deterioration and hematological diseases. The hematopoietic system is maintained by hematopoietic stem cells (HSCs), and dysfunction within the HSC compartment is thought to be a key mechanism underlying age-related hematopoietic perturbations. Using single-cell transplantation assays with five blood-lineage analysis, we previously identified myeloid-restricted repopulating progenitors (MyRPs) within the phenotypic HSC compartment in young mice. Here, we determined the age-related functional changes to the HSC compartment using over 400 single-cell transplantation assays. Notably, MyRP frequency increased dramatically with age, while multipotent HSCs expanded modestly within the bone marrow. We also identified a subset of functional cells that were myeloid restricted in primary recipients but displayed multipotent (five blood-lineage) output in secondary recipients. We have termed this cell type latent-HSCs, which appear exclusive to the aged HSC compartment. These results question the traditional dogma of HSC aging and our current approaches to assay and define HSCs., Graphical Abstract, Highlights • Single-cell transplantation reveals dramatic age-related changes in HSC composition • MyRPs/MySCs increase with age as a frequency of whole BM cells and the HSC compartment • Latent-HSCs were identified exclusively in the aged bone marrow • Latent-HSCs have restricted potential in primary, but not secondary, transplants, Yamamoto et al. explore age-related changes to HSC function through large-scale clonal analysis using single-cell transplantation. They find large increases in myeloid-restricted repopulating progenitors (MyRPs) as well as a population of MyRPs that display a broader differentiation capacity only in secondary transplants, suggesting additional mechanisms contributing to hematopoietic aging.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) using cord blood or haploidentical donor is a promising alternative option for patients who can not find an HLA-matched donor. However, HLA-mismatch allo-HSCT may be complicated by graft-versus-host disease (GVHD), a major cause of non-relapse mortality mediated by alloreactive T cells. Infusion of anti-thymocyte globulin (ATG) is used both as a treatment for and as a prophylactic against GVHD but ATG, like other immunosuppressive regimens, reacts against cells without distinguishing between donor and recipient cells. ATG is polyclonal and causes many side effects such as allergic reaction and increasing susceptibility to infection due to repression of T cells. Our ultimate goal is to develop a novel therapeutic approach to GVHD with anti-HLA monoclonal antibody specifically targeting the mismatched HLA molecule of donor origin to transiently ablate alloreactive T cells. To generate allele-specific anti-HLA antibody (ASHmAb), we established a rapid and efficient strategy using two different HLA-transgenic mouse strains, HLA-A24 and HLA-A2 and immunized them with HLA-A* 02:01 and HLA-A* 24:02 tetramers loaded with cytomegalovirus PP65 peptide, respectively. Antibodies were screened for HLA allele-specific complement-dependent cytotoxicity in vitro. We successfully established three killing ASHmAbs (kASHmAbs) against HLA-A* 02:01, -A* 02:01/–A* 03:01, and -A* 23:01/-A* 24:02. In vivo cytotoxicity and specificity of a kASHmAb against HLA-A* 02:01 (A* 02:01-kASHmAb) was examined in detail using a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγnull (NOG) mice were injected with either HLA-A2 or HLA-A26 positive human peripheral blood mononuclear cells (PBMCs) from healthy donors (i.e. HLA-A2 mice or HLA-A26 mice, respectively). Administration of A* 02:01-kASHmAb promoted the survival of HLA-A2 mice to 100%, with a mean survival of more than 6 months. In contrast, administration of A* 02:01-kASHmAb to nonreactive HLA was not effective; all HLA-A26 mice died within 2 months (p Next, we examined the cytotoxicity of the kASHmAb on the human graft in bone marrow using humanized NOG mice. Sublethally irradiated NOG mice were reconstituted with CD34 positive hematopoietic stem/progenitor cells from HLA-A2 positive cord blood. These humanized mice showed about 70-90% human T, B and myeloid cells in the peripheral blood. When these mice were administrated with A* 02:01-kASHmAb for five consecutive days (60 mg/day), mature human blood cells disappeared immediately from the mouse peripheral blood. Interestingly, human PBMCs were detectable again in the mouse blood in 2-4 weeks after the last kASHmAb administration, suggesting kASHmAb may have preferential cytotoxic effect on mature PBMCs sparing hematopoietic stem progenitor cells. The mechanism of this preferential killing is not clear, but an optimal dose of kASHmAb may be safely administered to GVHD patients without permanently ablating the graft and necessitating repeated transplantation. This study is the first reported instance of using the cytotoxic effect of HLA antibodies against GVHD. Unlike ATG, kASHmAb specifically killed donor cells and contributed to the survival of the recipients. While this novel therapeutic approach requires a panel of kASHmAbs to different HLA alleles, this may open a new door for treatment of severe GVHD. Furthermore, optimization of the kASHmAb administration protocol may contribute to tolerance induction. Disclosures: No relevant conflicts of interest to declare.

Abstract 4544 [Introduction] Hematopoietic cell transplantation (HCT) is a promising strategy for the treatment of both hematologic malignancies and non-malignant hematologic dyscrasias. However, HCT is sometimes complicated by infectious diseases, especially those caused by virus. Although the most important defense mechanism to control viral replication is inducing virus-specific cytotoxic T-lymphocytes (CTLs), the details of CTL development are not fully elucidated in human transplantation. In 1978, Zinkernagel, et al., clarified in murine hematopoietic stem cell transplantation models that MHC-restricted T cells are not governed by donor HLA expressed in bone marrow stem and progenitor cells. Instead, MHC restriction is acquired by interaction with recipient thymic epithelial cells. For this reason, virus-specific T cells are not induced by the mismatched HLA. Due to the increasing number of cord blood and haplo-identical transplantations, many transplantations are being performed under HLA-mismatched conditions. However, the full repercussions of this HLA-mismatch on viral immunity are still unknown. [Clinical case / Methods] For 4 years we followed a 35-year-old patient (HLA-A*24:02, A*33:03) with acute myeloid leukaemia of subtype t(8;21)(q22;q22);RUNX1(AML1)-RUNX1T1(ETO) of the 2008 WHO classification who underwent cord blood (HLA-A*02:01, A*24:02) transplantation following myeloablative conditioning regimen in first complete molecular remission. We analyzed the cytomegalovirus-specific CD8(+) T cells (CMV-CTLs) through HLA-A*02:01 tetramer (HLA-mismatched) and HLA-A*24:02 tetramer (HLA-matched) by flow cytometer. T cell receptor repertoire of these tetramer-positive CMV-CTLs were analyzed by CTL cloning and CDR3 sequencing. [Results / Discussion] The patient had a short period of CMV antigenemia but not CMV disease which was well-controlled by drip infusion of the anti-viral reagent ganciclovir after transplantation. On day 41 after transplantation, only A24-restricted (HLA-matched) tetramer-positive CMV-CTLs were present. By day 195, however, A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs also appeared. We considered that the donor-derived dendritic cells (DCs) induced A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs from naïve T cells, which are originally contained in cord blood inoculum. These mismatched donor HLA restricted memory T cells existed for a long time with a wide-ranging repertoire after transplantation, however, they were considered to have a limited functional role in infection immunity because lungs and intestinal tracts epithelial cells do not express HLA-A2. Usually, cord blood with 0, 1, or 2 loci mismatches is applicable for transplantation. Since HLA-A and B loci are checked only on a serological level, there are some possibilities in which every HLA-A and B loci are mismatched in the genetic level. In such a case, induction of virus-specific CTLs from donor naïve T cells might be impaired and result in uncontrollable viral infections. The findings of this case will be helpful to understand the impact of HLA-mismatch on viral immunity. Disclosures: No relevant conflicts of interest to declare.

Summary The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy., Graphical Abstract, Highlights • iPSC-derived rejuvenated CTLs are effective against EBV-induced tumors in vivo • Rejuvenated CTLs are implemented with an inducible caspase-9 (iC9)-based suicide system • Upon induction, the iC9 system efficiently leads to apoptosis in rejuvenated CTLs • The iC9-based system provides a safeguard for future iPSC-mediated cell therapy, In this article, Nakauchi and colleagues show that iPSC-derived rejuvenated CTLs (rejCTLs) implemented with an inducible caspase-9 (iC9)-based suicide system are effective against EBV-induced tumors in vivo. Upon induction, the iC9-based system efficiently and safely leads to apoptosis in these rejCTLs in vitro and in vivo and provides a safeguard for future iPSC-mediated cell therapy.