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Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.
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Benzilisoquinolinas , Receptores de Antígenos Quiméricos , Animais , Benzilisoquinolinas/farmacologia , Camundongos , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Imunoterapia Adotiva/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Técnicas de Cultura de Células/métodosRESUMO
CD4+ T cells induced from human iPSCs (iCD4+ T cells) offer a therapeutic opportunity for overcoming immune pathologies arising from hematopoietic stem cell transplantation. However, most iCD4+ T cells are conventional helper T cells, which secrete inflammatory cytokines. We induced high-level expression of FOXP3, a master transcription factor of regulatory T cells, in iCD4+ T cells. Human iPSC-derived, FOXP3-induced CD4+ T (iCD4+ Treg-like) cells did not secrete inflammatory cytokines upon activation. Moreover, they showed demethylation of the Treg-specific demethylation region, suggesting successful conversion to immunosuppressive iCD4+ Treg-like cells. We further assessed these iCD4+ Treg-like cells for CAR-mediated immunosuppressive ability. HLA-A2 CAR-transduced iCD4+ Treg-like cells inhibited CD8+ cytotoxic T cell (CTL) division in a mixed lymphocyte reaction assay with A2+ allogeneic CTLs and suppressed xenogeneic graft-versus-host disease (GVHD) in NSG mice treated with A2+ human PBMCs. In most cases, these cells suppressed the xenogeneic GvHD progression as much as natural CD25+CD127- Tregs did.
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Doença Enxerto-Hospedeiro , Células-Tronco Pluripotentes Induzidas , Receptores de Antígenos Quiméricos , Linfócitos T Reguladores , Humanos , Doença Enxerto-Hospedeiro/imunologia , Animais , Linfócitos T Reguladores/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Camundongos , Fatores de Transcrição Forkhead/metabolismo , Xenoenxertos , Camundongos Endogâmicos NOD , Modelos Animais de Doenças , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismoRESUMO
In recent years, immunotherapy has become a standard cancer therapy, joining surgery, chemotherapy, and radiation therapy. This therapeutic approach involves the use of patient-derived antigen-specific T cells or genetically modified T cells engineered with chimeric antigen receptors (CAR) or T cell receptors (TCR) that specifically target cancer antigens. However, T cells require ex vivo stimulation for proliferation when used in therapy, and the resulting "exhaustion," which is characterized by a diminished proliferation capacity and anti-tumor activity, poses a significant challenge. As a solution, we reported "rejuvenated" CD8 + T cells that possess high proliferation capacity from induced pluripotent stem cells (iPSCs) in 2013. This review discusses the status and future developments in immunotherapy using iPSC-derived T cells, drawing insights from our research to overcome the exhaustion associated with antigen-specific T cell therapy.
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Introduction: Genetically modified human induced pluripotent stem cell (iPSC)-based regenerative medicine has substantial potential in the treatment of refractory human diseases. Thus, preclinical studies on the safety and efficacy of these products are essential. Non-human primate (NHP) models such as the rhesus macaque are highly similar to humans in terms of size, lifespan, and immune system, rendering them superior models. However, effective gene transduction in rhesus macaque iPSCs (Rh-iPSCs) remains challenging. In this study, we investigated the effective gene transduction into Rh-iPSCs and its effect on differentiation efficiency. Methods: We established a gene transduction method using the piggyBac transposon vector system. Gene transduced Rh-iPSCs were analyzed for undifferentiated markers. We did teratoma assay to check pluripotency. Gene transduced Rh-iPSCs were differentiated into hematopoietic stem and progenitor cells (HSPCs) and T-cell lineage cells. Additionally, gene transduced Rh-iPSCs were compared the differentiation efficiency with parental Rh-iPSCs. Results: We could establish a gene transduction method using the piggyBac transposon vector system, demonstrating high efficiency and stable transgene expression in Rh-iPSCs. These Rh-iPSCs maintained long-term gene expression while expressing undifferentiated markers. Teratoma assay indicated that these Rh-iPSCs had pluripotency. These Rh-iPSCs could differentiate into HPSCs and T cells that express transgenes. These Rh-iPSCs can differentiate into hematopoietic stem cells and T cells that express transgenes. No significant differences in efficiency of differentiation were observed between parental Rh-iPSCs and these Rh-iPSCs. Conclusions: These results indicate that the piggyBac transposon vector is an excellent gene transfer tool for rhesus macaque iPSCs and could contribute to the advancement of preclinical studies using rhesus macaque iPSCs.
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ABSTRACT: A major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14- CD235a-), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αß+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC-T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αß+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αß+ memory T cells to improve the outcome in patients with MM.
Assuntos
Antineoplásicos , Antígenos CD8 , Células-Tronco Pluripotentes Induzidas , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/terapia , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígeno de Maturação de Linfócitos B/metabolismo , Linfócitos T Citotóxicos , Antineoplásicos/metabolismoRESUMO
Immune rejection of allogeneic cell therapeutics remains a major problem for immuno-oncology and regenerative medicine. Allogeneic cell products so far have inferior persistence and efficacy when compared with autologous alternatives. Engineering of hypoimmune cells may greatly improve their therapeutic benefit. We present a new class of agonistic immune checkpoint engagers that protect human leukocyte antigen (HLA)-depleted induced pluripotent stem cell-derived endothelial cells (iECs) from innate immune cells. Engagers with agonistic functionality to their inhibitory receptors TIM3 and SIRPα effectively protect engineered iECs from natural killer (NK) cell and macrophage killing. The SIRPα engager can be combined with truncated CD64 to generate fully immune evasive iECs capable of escaping allogeneic cellular and immunoglobulin G (IgG) antibody-mediated rejection. Synthetic immune checkpoint engagers have high target specificity and lack retrograde signaling in the engineered cells. This modular design allows for the exploitation of more inhibitory immune pathways for immune evasion and could contribute to the advancement of allogeneic cell therapeutics.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Endoteliais/metabolismo , Antígenos HLA , Células Matadoras Naturais , Imunidade InataRESUMO
Allogeneic T cell platforms utilizing induced pluripotent stem cell (iPSC) technology exhibit significant promise for the facilitation of adoptive immunotherapies. While mature T cell receptor (TCR) signaling plays a crucial role in generating T cells from iPSCs, the introduction of exogenous mature TCR genes carries a potential risk of causing graft-versus-host disease (GvHD). In this study, we present the development of truncated TCRα and TCRß chains, termed mini-TCRs, which lack variable domains responsible for recognizing human leukocyte antigen (HLA)-peptide complexes. We successfully induced cytotoxic T lymphocytes (CTLs) from iPSCs by employing mini-TCRs. Combinations of TCRα and TCRß fragments were screened from mini-TCR libraries based on the surface localization of CD3 proteins and their ability to transduce T cell signaling. Consequently, mini-TCR-expressing iPSCs underwent physiological T cell development, progressing from the CD4 and CD8 double-positive stage to the CD8 single-positive stage. The resulting iPSC-derived CTLs exhibited comparable cytokine production and cytotoxicity in comparison to that of full-length TCR-expressing T lymphocytes when chimeric antigen receptors (CARs) were expressed. These findings demonstrate the potential of mini-TCR-carrying iPSCs as a versatile platform for CAR T cell therapy, offering a promising avenue for advancing adoptive immunotherapies.
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In recent years, cell-based immunotherapies, such as chimeric antigen receptor(CAR)-T cell therapy, have greatly advanced the treatment of some hematological malignancies, especially those resistant to other therapies. Nevertheless, there are significant obstacles to the clinical application of current autologous therapies, such as high cost, challenging large-scale manufacturing, and difficulty obtaining long-term therapeutic efficacy due to T cell exhaustion. Induced pluripotent stem(iPS)cells have the potential to solve these problems through their unlimited proliferative capacity and differentiation potency to every type of cell in a body. Furthermore, iPS cells can be genetically engineered and differentiated into various types of immune cells, providing an unlimited resource for the development of"off-the-shelf"cell therapies. Here, we review the clinical development status of regenerative immunotherapies using iPS cell-derived CD8 killer T cells and natural killer(NK)cells and outline regenerative immunotherapies using natural killer T(NKT)cells, γδ T cells, mucosal-associated invariant T(MAIT)cells, and macrophages.
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Células-Tronco Pluripotentes Induzidas , Neoplasias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Matadoras Naturais/metabolismo , Imunoterapia Adotiva , Imunoterapia , Linfócitos T CitotóxicosRESUMO
Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such "hu-NK mice" rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.
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Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Células Matadoras Naturais , Antígenos de Histocompatibilidade Classe I/metabolismo , Linfócitos T , Antígenos HLA/metabolismoRESUMO
The effectiveness of chimaeric antigen receptor (CAR) T-cell immunotherapies against solid tumours relies on the accumulation, proliferation and persistency of T cells at the tumour site. Here we show that the proliferation of CD8αß cytotoxic CAR T cells in solid tumours can be enhanced by deriving and expanding them from a single human induced-pluripotent-stem-cell clone bearing a CAR selected for efficient differentiation. We also show that the proliferation and persistency of the effector cells in the tumours can be further enhanced by genetically knocking out diacylglycerol kinase, which inhibits antigen-receptor signalling, and by transducing the cells with genes encoding for membrane-bound interleukin-15 (IL-15) and its receptor subunit IL-15Rα. In multiple tumour-bearing animal models, the engineered hiPSC-derived CAR T cells led to therapeutic outcomes similar to those of primary CD8 T cells bearing the same CAR. The optimization of effector CAR T cells derived from pluripotent stem cells may aid the development of long-lasting antigen-specific T-cell immunotherapies for the treatment of solid tumours.
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Células-Tronco Pluripotentes Induzidas , Neoplasias , Animais , Humanos , Receptores de Antígenos de Linfócitos T/genética , Células-Tronco Pluripotentes Induzidas/patologia , Linfócitos T CD8-Positivos , Neoplasias/terapia , Proliferação de CélulasRESUMO
Introduction: T cells induced from induced pluripotent stem cells(iPSCs) derived from antigen-specific T cells (T-iPS-T cells) are an attractive tool for T cell immunotherapy. The induction of cytotoxic T-iPS-T cells is well established in feeder-free condition for the aim of off-the-shelf production, however, the induction of helper T-iPS-T cells remains challenging. Methods: We analyzed T-iPS-T cells matured in 3D organoid culture at different steps in the culture process at the single-cell level. T-iPS-T cell datasets were merged with an available human thymocyte dataset based in single-cell RNA sequencing (scRNA-seq). Particularly, we searched for genes crucial for generation CD4+ T-iPS-T cells by comparing T-iPS-T cells established in 2D feeder-free or 3D organoid culture. Results: The scRNA-seq data indicated that T-iPS-T cells are similar to T cells transitioning to human thymocytes, with SELENOW, GIMAP4, 7, SATB1, SALMF1, IL7R, SYTL2, S100A11, STAT1, IFITM1, LZTFL1 and SOX4 identified as candidate genes for the 2D feeder-free induction of CD4+ T-iPS-T cells. Discussion: This study provides single cell transcriptome datasets of iPS-T cells and leads to further analysis for CD4+ T cell generation from T-iPSCs.
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Células-Tronco Pluripotentes Induzidas , Proteínas de Ligação à Região de Interação com a Matriz , Humanos , Técnicas de Cultura de Células , Diferenciação Celular , Genes Homeobox , Organoides , Fatores de Transcrição SOXC , Proteínas de Ligação ao GTPRESUMO
CAR-T therapy has shown excellent therapeutic efficacy in B-cell malignancy. Nevertheless, manufacturing stability, quality control, and CAR T-cell availability are still challenging because current CAR T-cell therapy is a personalized product derived from patient peripheral T-cells. However, allogeneic T-cells have emerged as a novel source to overcome this issue. Because induced pluripotent stem (iPS) cells are pluripotent stem cells derived from somatic cells and have in vitro self-renewal ability and pluripotency, they are expected to be a source of many regenerative medicinal products. Recently, it has become possible to generate CD8 killer T cells from iPS cells, and efforts have been made to generate CAR-CD8 killer T-cells from allogeneic iPS cells. This review discusses the induction of CD8 killer T-cells from iPS cells, efforts to improve the safety and certainty of the induction process for clinical use, and the utility of gene editing to reduce allogeneic antigenicity of iPS T-cells.
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Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/genética , Terapia Baseada em Transplante de Células e TecidosRESUMO
The efficacy of T-cell therapy depends on the maintenance of antigen specificity, memory phenotype, longterm viability, and proliferative capacity of T cells in vivo. Personalized autologous T-cell therapies pose a few manufacturing challenges, in terms of quality, and supply stability. Recently, it has become possible to derive CD8 killer T cells from induced pluripotent stem cells (iPSCs) and develop CAR-CD8 killer T cells from allogeneic iPSCs. This article reviews CD8 killer T-cell induction from iPSCs, attempts to enhance process safety and reliability, and discusses the use of gene-editing technology for reducing allogeneic antigenicity.
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Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Neoplasias , Humanos , Imunoterapia , Imunoterapia Adotiva , Reprodutibilidade dos TestesRESUMO
Recently, research has been conducted with chimeric antigen receptor (CAR)-T cells to improve efficacy against solid tumors. Humanized CAR improved the long-term survival of CAR-T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR was generated from a conventional nonhumanized antibody-NZ-1, the only anti-PDPN antibody for which a CAR was produced. In this study, we investigated other anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance the therapeutic potential of CAR-T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in mice that were significantly better than those in nonhumanized NZ-1 CAR-T cells.
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Neoplasias , Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Artificial thymic organoids (ATOs) allow the selective differentiation of chimeric antigen receptor (CAR)-transduced human iPSCs into CAR-T cells. In this issue of Cell Stem Cell, Wang et al. now use ATOs to produce human CD19+ CAR-T cells that mimic conventional CAR-T cells and effectively control the progression of human CD19+ leukemia in an animal model.
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Células-Tronco Pluripotentes Induzidas , Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Humanos , Imunoterapia Adotiva , Organoides , Receptores de Antígenos de Linfócitos TRESUMO
Persistence of HIV latently infected cells is a barrier to HIV cure. The "kick and kill" strategy for a cure includes clearance of the viral reservoir by HIV-specific cytotoxic T lymphocytes (CTLs). However, exhaustion and senescence of T cells accelerates during HIV infection, and does not fully recover, despite complete viral suppression under antiretroviral therapy. We previously established an induced pluripotent stem cell (iPSC) from a parental HIV-specific CTL clone and generated an iPSC-derived rejuvenated HIV-specific CTL clone (iPSC-CTL), which exhibited an early memory phenotype, high proliferation capacity and effector functions in vitro. Here, we assessed the antiviral efficacy of the HIV-specific iPSC-CTL by single- and multiple-round viral suppression assays (VSAs). The HIV-specific iPSC-CTL suppressed viral replication in an HLA-dependent manner with equivalent efficacy to the parental CTL clone in single-round VSA. In multiple-round VSA, however, the ability of the iPSC-CTL to suppress viral replication was longer than that of the parental CTL clone. These results indicate that HIV-specific iPSC-CTL can sustainably exert suppressive pressure on viral replication, suggesting a novel approach to facilitate clearance of the HIV reservoir via adoptive transfer of rejuvenated CTLs. IMPORTANCE Elimination of latently HIV-infected cells is required for HIV cure. In the "kick and kill" strategy proposed for a cure to HIV, the host immune system, including HIV-specific cytotoxic T lymphocytes (CTLs), play a central role in eliminating HIV antigen-expressing cells following reactivation by latency-reversing agents (LRAs). However, CTL dysfunction due to exhaustion and senescence in chronic HIV infection can be an obstacle to this strategy. Adoptive transfer with effective HIV-specific CTLs may be a solution of this problem. We previously generated an induced pluripotent stem cell (iPSC)-derived rejuvenated HIV-specific CTL clone (iPSC-CTL) with high functional and proliferative capacity. The present study demonstrates that iPSC-CTL can survive and suppress HIV replication in vitro longer than the parental CTL clone, indicating the potential of iPSC-CTL to sustainably exert suppressive pressure on viral replication. Adoptive transfer with rejuvenated HIV-specific CTLs in combination with LRAs may be a new intervention strategy for HIV cure/remission.
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Células-Tronco Pluripotentes Induzidas , Linfócitos T Citotóxicos , Antivirais/uso terapêutico , Células Cultivadas , Infecções por HIV/imunologia , Infecções por HIV/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Replicação Viral/imunologiaRESUMO
Tumor-infiltrating lymphocytes (TIL), which include tumor-specific T lymphocytes with frequency, are used for adoptive cell transfer therapy (ACT) in clinical practice. The optimization of TIL preparation has been investigated to reduce the senescence and increase the abundance of TIL, as both the quality and quantity of the transferred cells have great influence on the outcome of TIL-based ACT (TIL-ACT). Considering the effects of cell reprogramming on senescence, we expected that the anti-tumor effect could be enhanced by TIL regeneration. To confirm this hypothesis, we established tumor-specific TIL-derived iPS cells (TIL-iPSC) with human colorectal cancer specimens. T cells differentiated from TIL-iPSC (TIL-iPS-T) retained not only intrinsic T cell functions and tumor specificity, but also exhibited improved proliferation capacity and additional killing activity. Moreover, less differentiated profiles and prolonged persistency were seen in TIL-iPS-T compared with primary cells. Our findings imply that iPSC technology has great potential for TIL-ACT.
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Neoplasias Colorretais/terapia , Células-Tronco Pluripotentes Induzidas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias Colorretais/imunologia , Feminino , Humanos , Imunoterapia , Células-Tronco Pluripotentes Induzidas/citologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/citologia , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T/citologia , Linfócitos T/transplanteRESUMO
Adoptive immunotherapy has emerged as a powerful approach to cure cancer and chronic infections. Currently, the generation of a massive number of T cells that provide long-lasting immunity is challenged by exhaustion and differentiation-associated senescence, which inevitably arise during in vitro cloning and expansion. To circumvent these problems, several studies have proposed an induced pluripotent stem cell (iPSC)-mediated rejuvenation strategy to revitalize the exhausted/senescent T cell clones. Because iPSC-derived cytotoxic T lymphocytes (iPSC-CTLs) generated via commonly used monolayer systems have unfavorable, innate-like features such as aberrant natural killer (NK) activity and limited replication potential, we modified the redifferentiation culture to generate CD8αß+CD5+CCR7+CD45RA+CD56--adaptive iPSC-CTLs. The modified iPSC-CTLs exhibited early memory phenotype, including high replicative capacity and the ability to give rise to potent effector cells. In expansion culture with an optimized cytokine cocktail, iPSC-CTLs proliferated more than 1015-fold in a feeder-free condition. Our redifferentiation and expansion package of early memory iPSC-CTLs could supply memory and effector T cells for both autologous and allogeneic immunotherapies.
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Biomarcadores/metabolismo , Técnicas de Cultura de Células/métodos , Imunoterapia Adotiva/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Neoplasias/terapia , Linfócitos T Citotóxicos/citologia , Animais , Antígenos CD5/metabolismo , Antígeno CD56/deficiência , Antígenos CD8/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Reprogramação Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células K562 , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Receptores CCR7/metabolismo , Linfócitos T Citotóxicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Avoiding the immune rejection of transplanted T cells is central to the success of allogeneic cancer immunotherapies. One solution to protecting T-cell grafts from immune rejection involves the deletion of allogeneic factors and of factors that activate cytotoxic immune cells. Here we report the generation of hypoimmunogenic cancer-antigen-specific T cells derived from induced pluripotent stem cells (iPSCs) lacking ß2-microglobulin, the class-II major histocompatibility complex (MHC) transactivator and the natural killer (NK) cell-ligand poliovirus receptor CD155, and expressing single-chain MHC class-I antigen E. In mouse models of CD20-expressing leukaemia or lymphoma, differentiated T cells expressing a CD20 chimeric antigen receptor largely escaped recognition by NKG2A+ and DNAM-1+ NK cells and by CD8 and CD4 T cells in the allogeneic recipients while maintaining anti-tumour potency. Hypoimmunogenic iPSC-derived T cells may contribute to the creation of off-the-shelf T cell immunotherapies.
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Células-Tronco Pluripotentes Induzidas/citologia , Leucemia/terapia , Linfoma/terapia , Receptores Virais/genética , Linfócitos T/transplante , Microglobulina beta-2/genética , Animais , Antígenos de Diferenciação de Linfócitos T/metabolismo , Diferenciação Celular , Linhagem Celular , Técnicas de Inativação de Genes , Engenharia Genética , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Leucemia/imunologia , Linfoma/imunologia , Masculino , Camundongos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.