Rejuvenated iPSC-derived GD2-directed CART Cells Harbor Robust Cytotoxicity Against Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is exceptionally aggressive, with limited treatment options. Disialoganglioside (GD2) is highly expressed on SCLC and is considered a good target for chimeric antigen receptor (CAR) T cells (CART). Although GD2-directed CARTs (GD2-CART) exhibit cytotoxicity against various GD2-expressing tumors, they lack significant cytotoxicity against SCLC. To enhance cytotoxicity of GD2-CARTs against SCLC, we introduced GD2-CAR into induced pluripotent stem cells (iPSC)-derived rejuvenated cytotoxic T lymphocytes (GD2-CARrejT). GD2-CARrejTs acted much more strongly against SCLC cells than did GD2-CARTs both in vitro and in vivo. Single-cell RNA sequencing elucidated that levels of expression of TIGIT were significantly lower and levels of expression of genes associated with cytotoxicity were significantly higher in GD2-CARrejTs than those in GD2-CARTs. Dual blockade of TIGIT and programmed death-1 (PD-1) increased the cytotoxicity of GD2-CARTs to some extent, suggesting that low TIGIT and PD-1 expression by GD2-CARrejTs is a major factor required for robust cytotoxicity against SCLC. Not only for robust cytotoxicity but also for availability as "off-the-shelf" T-cell therapy, iPSC-derived GD2-CARrejTs are a promising novel treatment for SCLC. SIGNIFICANCE: This research introduces iPSC-derived rejuvenated GD2-CARTs (GD2-CARrejT) as a novel approach to combat SCLC. Compared with conventional GD2-CARTs, GD2-CARrejTs with reduced TIGIT and PD-1 expression demonstrate robust cytotoxicity against SCLC and would be a promising therapy for SCLC.

Successful management of acute graft-versus-host disease with ibrutinib during cord blood transplantation for germline DDX41-mutated acute myeloid leukemia.

Background: Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) with significant morbidity and mortality, and efficacy of currently available therapeutics are limited. Acute and chronic GVHD are similar in that both are initiated by antigen presenting cells and activation of alloreactive B-cells and T-cells, subsequently leading to inflammation, tissue damage, and organ failure. One difference is that acute GVHD is mostly attributed to T-cell activation and cytokine release, whereas B-cells are the key players in chronic GVHD. Ibrutinib is an irreversible inhibitor of the Bruton's tyrosine kinase (BTK), which is part of B-cell receptor signaling. Ibrutinib is currently used for treating chronic GVHD, but its efficacy towards acute GVHD is unknown. Besides BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK), which is predominantly expressed in T-cells and a crucial enzyme for activating the downstream pathway of TCR signaling. ITK activates PLCγ2 and facilitates signaling through NF-κB, NFAT, and MAPK, leading to activation and proliferation of T-cells and enhanced cytokine production. Therefore, the TCR signaling pathway is indispensable for development of acute GVHD, and ITK inhibition by ibrutinib would be a rational therapeutic approach. Case presentation: A 56-year-old male acute myeloid leukemia patient with Myeloid neoplasms with germline DEAD-box RNA helicase 41 (DDX41) mutation underwent cord blood transplantation and developed severe gastrointestinal (GI) acute GVHD which was refractory to steroids and mesenchymal stem cell therapy. While acute GVHD accommodated by multiple life-threatening GI bleeding events persisted, chronic cutaneous GVHD developed, and ibrutinib 420 mg/day was initiated from day 147 of transplant. Although ibrutinib was commenced targeting the chronic GVHD, unexpected and abrupt remission of acute GVHD along with remission of chronic GVHD was observed. Conclusion: Ibrutinib is a promising therapeutic for treating acute GVHD, and further studies are warranted.

An unconventional regulatory circuitry involving Aurora B controls anaphase onset and error-free chromosome segregation in trypanosomes.

Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the interface between chromosomes and spindle microtubules and as such are under tight control by complex regulatory circuitry. As part of the chromosomal passenger complex (CPC), the Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint, a feedback control system that delays the onset of anaphase by inhibiting the anaphase-promoting complex/cyclosome. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, whose kinetochores are composed of a unique set of structural and regulatory proteins. Kinetoplastids do not have a canonical spindle checkpoint and it remains unclear how their kinetochores are regulated to ensure the fidelity and timing of chromosome segregation. Here, we show in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness, that inhibition of Aurora B using an analogue-sensitive approach arrests cells in metaphase, with a reduction in properly bi-oriented kinetochores. Aurora B phosphorylates several kinetochore proteins in vitro, including the N-terminal region of the divergent Bub1-like protein KKT14. Depletion of KKT14 partially overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 protein results in a prominent delay in the metaphase-to-anaphase transition. Finally, we demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes massive chromosome mis-segregation in anaphase. Our results indicate that the CPC and KKT14 are involved in an unconventional pathway controlling mitotic exit and error-free chromosome segregation in trypanosomes.

Polatuzumab vedotin pharmacokinetics in a hemodialysis patient with diffuse large B-cell lymphoma.

PURPOSE: Chemotherapy for the hemodialysis (HD) patient is a challenging situation because it requires special considerations including dose modifications and timing of drug administration in relation with HD sessions. Polaltuzumab vedotin (PV), an antibody-drug conjugate in which monomethyl auristatin E (MMAE) is linked to an anti-CD79b monoclonal antibody, is an extremely promising therapeutic for treating diffuse large B cell lymphoma (DLBCL), but the pharmacokinetics are unknown in HD patients. METHODS: We carried out pharmacokinetic studies of PV when administered at 1.2 mg/kg to a DLBCL patient on HD, and compared the results with that of non-HD patients. PV was administered in conjunction with bendamustine and rituximab. RESULTS: Serum concentration-time curves of both antibodyconjugated and unconjugated MMAE in the presented HD patient were similar compared to that of non-HD patients. We also demonstrate that elimination of both antibody-conjugated and unconjugated MMAE through HD is limited. PV administration at 1.2 mg/kg to an HD patient was also clinically feasible, and no signs of peripheral neuropathy were observed. CONCLUSIONS: PV therapy may be a relatively safe treatment method for DLBCL patients on HD.

Establishment of ganglioside GD2-expressing extranodal NK/T-cell lymphoma cell line with scRNA-seq analysis.

Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKL), is characterized by Epstein-Barr virus infection and poor prognosis. We established a novel cell line, ENKL-J1, from bone marrow cells of an ENKL patient. We found that ENKL-J1 cells express the ganglioside GD2 (GD2) and that GD2-directed chimeric antigen receptor T cells exhibit cytotoxicity against ENKL-J1 cells, indicating that GD2 would be a suitable target of GD2-expressing ENKL cells. Targeted next-generation sequencing revealed TP53 and TET2 variants in ENKL-J1 cells. Furthermore, single-cell RNA sequencing in ENKL-J1 cells showed high gene-expression levels in the oncogenic signaling pathways JAK-STAT, NF-κB, and MAPK. Genes related to multidrug resistance (ABCC1), tumor suppression (ATG5, CRYBG1, FOXO3, TP53, MGA), anti-apoptosis (BCL2, BCL2L1), immune checkpoints (CD274, CD47), and epigenetic regulation (DDX3X, EZH2, HDAC2/3) also were expressed at high levels. The molecular targeting agents eprenetapopt, tazemetostat, and vorinostat efficiently induced apoptosis in ENKL-J1 cells in vitro. Furthermore, GD2-directed chimeric antigen receptor T cells showed cytotoxicity against ENKL-J1 cells in vivo. These findings not only contribute to understanding the molecular and genomic characteristics of ENKL; they also suggest new treatment options for patients with advanced or relapsed ENKL.

iPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer.

Functionally rejuvenated human papilloma virus-specific cytotoxic T lymphocytes (HPV-rejTs) generated from induced pluripotent stem cells robustly suppress cervical cancer. However, autologous rejT generation is time consuming, leading to difficulty in treating patients with advanced cancer. Although use of allogeneic HPV-rejTs can obviate this, the major obstacle is rejection by the patient immune system. To overcome this, we develop HLA-A24&-E dual integrated HPV-rejTs after erasing HLA class I antigens. These rejTs effectively suppress recipient immune rejection while maintaining more robust cytotoxicity than original cytotoxic T lymphocytes. Single-cell RNA sequencing performed to gain deeper insights reveal that HPV-rejTs are highly enriched with tissue resident memory T cells, which enhance cytotoxicity against cervical cancer through TGFßR signaling, with increased CD103 expression. Genes associated with the immunological synapse also are upregulated, suggesting that these features promote stronger activation of T cell receptor (TCR) and increased TCR-mediated target cell death. We believe that our work will contribute to feasible "off-the-shelf" T cell therapy with robust anti-cervical cancer effects.

Successful production of offspring derived from mouse zygotes vitrified with carboxylated ε-poly-L-lysine and polyvinyl alcohol without serum.

The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.

Targeted protein degradation using deGradFP in Trypanosoma brucei.

Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available in Trypanosoma brucei, an evolutionarily divergent eukaryote that causes human African trypanosomiasis. Here, we have adapted deGradFP (degrade green fluorescent protein [GFP]), a protein degradation system based on the SCF E3 ubiquitin ligase complex and anti-GFP nanobody, in T. brucei. As a proof of principle, we targeted a kinetoplastid kinetochore protein (KKT3) that constitutively localizes at kinetochores in the nucleus. Induction of deGradFP in a cell line that had both alleles of KKT3 tagged with yellow fluorescent protein (YFP) caused a more severe growth defect than RNAi in procyclic (insect form) cells. deGradFP also worked on a cytoplasmic protein (COPII subunit, SEC31). Given the ease in making GFP fusion cell lines in T. brucei, deGradFP can serve as a powerful tool to rapidly deplete proteins of interest, especially those with low turnover rates.

Divergent polo boxes in KKT2 bind KKT1 to initiate the kinetochore assembly cascade in Trypanosoma brucei.

Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 interact directly with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains that initiate kinetochore assembly in T. brucei.

Stage-specific transcription activator ESB1 regulates monoallelic antigen expression in Trypanosoma brucei.

Variant surface glycoprotein (VSG) coats bloodstream form Trypanosoma brucei parasites, and monoallelic VSG expression underpins the antigenic variation necessary for pathogenicity. One of thousands of VSG genes is transcribed by RNA polymerase I in a singular nuclear structure called the expression site body (ESB), but how monoallelic VSG transcription is achieved remains unclear. Using a localization screen of 153 proteins we found one, ESB-specific protein 1 (ESB1), that localized only to the ESB and is expressed only in VSG-expressing life cycle stages. ESB1 associates with DNA near the active VSG promoter and is necessary for VSG expression, with overexpression activating inactive VSG promoters. Mechanistically, ESB1 is necessary for recruitment of a subset of ESB components, including RNA polymerase I, revealing that the ESB has separately assembled subdomains. Because many trypanosomatid parasites have divergent ESB1 orthologues yet do not undergo antigenic variation, ESB1 probably represents an important class of transcription regulators.

Plasticity in centromere organization and kinetochore composition: Lessons from diversity.

Kinetochores are the macromolecular protein complexes that govern chromosome movement by binding spindle microtubules during mitosis and meiosis. Centromeres are the specific chromosomal regions that serve as the platform on which kinetochores assemble. Despite their essentiality for proper chromosome segregation, the size and organization of centromeres vary dramatically between species, while different compositions of kinetochores are found among eukaryotes. Here we discuss recent progress in understanding centromeres and kinetochores in non-traditional model eukaryotes. We specifically focus on select lineages (holocentric insects, early diverging fungi, and kinetoplastids) that lack CENP-A, a centromere-specific histone H3 variant that is critical for kinetochore specification and assembly in many eukaryotes. We also highlight some organisms that might have hitherto unknown types of kinetochore proteins.

Dual-antigen targeted iPSC-derived chimeric antigen receptor-T cell therapy for refractory lymphoma.

We generated dual-antigen receptor (DR) T cells from induced pluripotent stem cells (iPSCs) to mitigate tumor antigen escape. These cells were engineered to express a chimeric antigen receptor (CAR) for the antigen cell surface latent membrane protein 1 (LMP1; LMP1-CAR) and a T cell receptor directed to cell surface latent membrane protein 2 (LMP2), in association with human leucocyte antigen A24, to treat therapy-refractory Epstein-Barr virus-associated lymphomas. We introduced LMP1-CAR into iPSCs derived from LMP2-specific cytotoxic T lymphocytes (CTLs) to generate rejuvenated CTLs (rejTs) active against LMP1 and LMP2, or DRrejTs. All DRrejT-treated mice survived >100 days. Furthermore, DRrejTs rejected follow-up inocula of lymphoma cells, demonstrating that DRrejTs persisted long-term. We also demonstrated that DRrejTs targeting CD19 and LMP2 antigens exhibited a robust tumor suppressive effect and conferred a clear survival advantage. Co-operative antitumor effect and in vivo persistence, with unlimited availability of DRrejT therapy, will provide powerful and sustainable T cell immunotherapy.

Trigenic ADH5/ALDH2/ADGRV1 mutations in myelodysplasia with Usher syndrome.

Trio-next generation sequencing is useful to identify undiagnosed inherited diseases. We have attended a patient with trigenic ADH5/ALDH2/ADGRV1 pathogenic variants, which caused two distinct diseases, myelodysplastic syndrome and Usher syndrome. Whole genome sequencing of peripheral blood from the patient and his parents were applied to identify disease-causing genes. Sanger sequencing was performed to validate the identified ADH5/ALDH2/ADGRV1 variants. Our results identified disease-associated variants in ADGRV1 (disease inheritance autosomal recessive) and in ADH5 (disease inheritance also autosomal recessive) and a variant in ALDH2 (disease inheritance autosomal dominant). Although the variants identified in ADH5 and ALDH2 have been reported, their co-existence in association with disease-causing variation in a third gene has not. They broaden the spectrum of ADGRV1 in Usher syndrome. Findings on next generation sequencing guided rapid and accurate diagnosis, resulting in patient-tailored therapeutic intervention.

iPSC-Derived Neoantigen-Specific CTL Therapy for Ewing Sarcoma.

The prognosis of Ewing sarcoma caused by EWS/FLI1 fusion is poor, especially after metastasis. Although therapy with CTLs targeted against altered EWS/FLI1 sequences at the gene break/fusion site may be effective, CTLs generated from peripheral blood are often exhausted because of continuous exposure to tumor antigens. We addressed this by generating induced pluripotent stem cell (iPSC)-derived functionally rejuvenated CTLs (rejT) directed against the neoantigen encoded by the EWS/FLI1 fusion gene. In this study, we examined the antitumor effects of EWS/FLI1-rejTs against Ewing sarcoma. The altered amino acid sequence at the break/fusion point of EWS/FLI1, when presented as a neoantigen, evokes an immune response that targets EWS/FLI1 + sarcoma. Although the frequency of generated EWS/FLI1-specific CTLs was only 0.003%, we successfully established CTL clones from a healthy donor. We established iPSCs from a EWS/FLI1-specific CTL clone and redifferentiated them into EWS/FLI1-specific rejTs. To evaluate cytotoxicity, we cocultured EWS/FLI1-rejTs with Ewing sarcoma cell lines. EWS/FLI1-rejTs rapidly and continuously suppressed the proliferation of Ewing sarcoma for >40 hours. Using a Ewing sarcoma xenograft mouse model, we verified the antitumor effect of EWS/FLI1-rejTs via imaging, and EWS/FLI1-rejTs conferred a statistically significant survival advantage. "Off-the-shelf" therapy is less destructive and disruptive than chemotherapy, and radiation is always desirable, particularly in adolescents, whom Ewing sarcoma most often affects. Thus, EWS/FLI1-rejTs targeting a Ewing sarcoma neoantigen could be a promising new therapeutic tool.

SCLC-J1, a novel small cell lung cancer cell line.

Small cell lung cancer (SCLC) is a type of high-grade neuroendocrine carcinoma. It initially responds to chemotherapy but rapidly becomes chemoresistant and it is highly proliferative. The prognosis in SCLC is poor. We have established a novel SCLC cell line, SCLC-J1, from a malignant pleural effusion in a patient with advanced SCLC. SCLC-J1 cells express ganglioside GD2, CD276, and Delta-like protein 3. RB1 is lost. These features of the new SCLC cell line may be useful in understanding the cellular and molecular biology of SCLC and in designing better treatment.

Kinetoplastid kinetochore proteins KKT2 and KKT3 have unique centromere localization domains.

The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.

Sustainable Tumor-Suppressive Effect of iPSC-Derived Rejuvenated T Cells Targeting Cervical Cancers.

Immunotherapy utilizing induced pluripotent stem cell (iPSC) technology has great potential. Functionally rejuvenated cytotoxic T lymphocytes (CTLs) can survive long-term as young memory T cells in vivo, with continuous tumor eradication. Banking of iPSCs as an unlimited "off-the-shelf" source of therapeutic T cells may be feasible. To generate safer iPSCs, we reprogrammed human papilloma virus type 16 (HPV16) E6-specific CTLs by Sendai virus vector without cotransduction of SV40 large T antigen. The iPSCs efficiently differentiated into HPV16-specific rejuvenated CTLs that demonstrated robust cytotoxicity against cervical cancer. The tumor-suppressive effect of rejuvenated CTLs was stronger and more persistent than that of original peripheral blood CTLs. These rejuvenated HPV16-specific CTLs provide a sustained tumor-suppressive effect even for epithelial cancers and constitute promising immunotherapy for cervical cancer.

Preoperative denosumab treatment with curettage may be a risk factor for recurrence of giant cell tumor of bone.

PURPOSE: Giant cell tumor of bone (GCTB) is a local aggressive bone tumor, histologically classified as intermediate malignancy. Recently, the RANKL inhibitor, denosumab, was developed as a novel and effective treatment option for GCTB. Since the risk of preoperative use of denosumab with curettage had been previously reported, this study aimed to investigate the relationship between recurrences and clinicopathological features associated with adjuvant denosumab treatment in GCTB. METHODS: A total of 87 GCTB cases were treated at our institution. We reviewed 66 patients with conventional-type GCTB occurring in the extremities and analyzed 78 surgical treatments, including curettages and resections, with clinicopathological features and denosumab treatment. RESULTS: GCTB lesions, including 66 primary and 12 recurring, underwent surgical treatment like curettage and resection. Recurrence-free survivals in 78 GCTB surgeries were 78.7% in 3 years and 71.9% in 5 years. In the resected cases of GCTBs, there was no recurrence either with or without denosumab. In curettage cases, 3-year recurrence-free survivals were 0.0% (n = 3) in preoperative treatment of denosumab, 66.7% (n = 6) in postoperative treatment, and 76.6% (n = 43) in no treatment. Interestingly, three preoperative treatment cases demonstrated low MIB-1 index despite 100% recurrence. The other clinicopathological factors did not contribute much to the risk of recurrence in curettage cases. CONCLUSION: Our findings revealed the use of denosumab in GCTB, prior to curettage, to possibly increase the risk of local recurrence. Together with previous reports, our finding might provide information for beneficial treatment of GCTB.

Characterization of unconventional kinetochore kinases KKT10 and KKT19 in Trypanosoma brucei.

The kinetochore is a macromolecular protein complex that drives chromosome segregation in eukaryotes. Unlike most eukaryotes that have canonical kinetochore proteins, evolutionarily divergent kinetoplastids, such as Trypanosoma brucei, have unconventional kinetochore proteins. T. brucei also lacks a canonical spindle checkpoint system, and it therefore remains unknown how mitotic progression is regulated in this organism. Here, we characterized, in the procyclic form of T. brucei, two paralogous kinetochore proteins with a CLK-like kinase domain, KKT10 and KKT19, which localize at kinetochores in metaphase but disappear at the onset of anaphase. We found that these proteins are functionally redundant. Double knockdown of KKT10 and KKT19 led to a significant delay in the metaphase to anaphase transition. We also found that phosphorylation of two kinetochore proteins, KKT4 and KKT7, depended on KKT10 and KKT19 in vivo Finally, we showed that the N-terminal part of KKT7 directly interacts with KKT10 and that kinetochore localization of KKT10 depends not only on KKT7 but also on the KKT8 complex. Our results reveal that kinetochore localization of KKT10 and KKT19 is tightly controlled to regulate the metaphase to anaphase transition in T. bruceiThis article has an associated First Person interview with the first author of the paper.

Long-term eradication of extranodal natural killer/T-cell lymphoma, nasal type, by induced pluripotent stem cell-derived Epstein-Barr virus-specific rejuvenated T cells in vivo.

Functionally rejuvenated induced pluripotent stem cell (iPSC)-derived antigen-specific cytotoxic T lymphocytes (CTL) are expected to be a potent immunotherapy for tumors. When L-asparaginase-containing standard chemotherapy fails in extranodal natural killer/T-cell lymphoma, nasal type (ENKL), no effective salvage therapy exists. The clinical course then is miserable. We demonstrate prolonged and robust eradication of ENKL in vivo by Epstein-Barr virus-specific iPSC-derived antigen-specific CTL, with iPSC-derived antigen-specific CTL persisting as central memory T cells in the mouse spleen for at least six months. The anti-tumor response is so strong that any concomitant effect of the programmed cell death 1 (PD-1) blockade is unclear. These results suggest that long-term persistent Epstein-Barr virus-specific iPSC-derived antigen-specific CTL contribute to a continuous anti-tumor effect and offer an effective salvage therapy for relapsed and refractory ENKL.