RESUMEN
BACKGROUND: γ-Secretase inhibitors (GSIs) increase B cell maturation antigen (BCMA) density on malignant plasma cells and enhance antitumour activity of BCMA chimeric antigen receptor (CAR) T cells in preclinical models. We aimed to evaluate the safety and identify the recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat (LY3039478) for individuals with relapsed or refractory multiple myeloma. METHODS: We conducted a phase 1, first-in-human trial combining crenigacestat with BCMA CAR T-cells at a single cancer centre in Seattle, WA, USA. We included individuals aged 21 years or older with relapsed or refractory multiple myeloma, previous autologous stem-cell transplant or persistent disease after more than four cycles of induction therapy, and Eastern Cooperative Oncology Group performance status of 0-2, regardless of previous BCMA-targeted therapy. To assess the effect of the GSI on BCMA surface density on bone marrow plasma cells, participants received GSI during a pretreatment run-in, consisting of three doses administered 48 h apart. BCMA CAR T cells were infused at doses of 50â×â106 CAR T cells, 150â×â106 CAR T cells, 300â×â106 CAR T cells, and 450â×â106 CAR T cells (total cell dose), in combination with the 25 mg crenigacestat dosed three times a week for up to nine doses. The primary endpoints were the safety and recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat, an oral GSI. This study is registered with ClinicalTrials.gov, NCT03502577, and has met accrual goals. FINDINGS: 19 participants were enrolled between June 1, 2018, and March 1, 2021, and one participant did not proceed with BCMA CAR T-cell infusion. 18 participants (eight [44%] men and ten [56%] women) with multiple myeloma received treatment between July 11, 2018, and April 14, 2021, with a median follow up of 36 months (95% CI 26 to not reached). The most common non-haematological adverse events of grade 3 or higher were hypophosphataemia in 14 (78%) participants, fatigue in 11 (61%), hypocalcaemia in nine (50%), and hypertension in seven (39%). Two deaths reported outside of the 28-day adverse event collection window were related to treatment. Participants were treated at doses up to 450â×â106 CAR+ cells, and the recommended phase 2 dose was not reached. INTERPRETATIONS: Combining a GSI with BCMA CAR T cells appears to be well tolerated, and crenigacestat increases target antigen density. Deep responses were observed among heavily pretreated participants with multiple myeloma who had previously received BCMA-targeted therapy and those who were naive to previous BCMA-targeted therapy. Further study of GSIs given with BCMA-targeted therapeutics is warranted in clinical trials. FUNDING: Juno Therapeutics-a Bristol Myers Squibb company and the National Institutes of Health.
Asunto(s)
Mieloma Múltiple , Receptores Quiméricos de Antígenos , Masculino , Humanos , Femenino , Mieloma Múltiple/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/uso terapéutico , Antígeno de Maduración de Linfocitos B , Inmunoterapia Adoptiva/efectos adversos , Linfocitos TRESUMEN
X-linked agammaglobulinemia (XLA) is an immune disorder caused by mutations in Bruton's tyrosine kinase (BTK). BTK is expressed in B and myeloid cells, and its deficiency results in a lack of mature B cells and protective antibodies. We previously reported a lentivirus (LV) BTK replacement therapy that restored B cell development and function in Btk and Tec double knockout mice (a phenocopy of human XLA). In this study, with the goal of optimizing both the level and lineage specificity of BTK expression, we generated LV incorporating the proximal human BTK promoter. Hematopoietic stem cells from Btk -/- Tec -/- mice transduced with this vector rescued lineage-specific expression and restored B cell function in Btk -/- Tec -/- recipients. Next, we tested addition of candidate enhancers and/or ubiquitous chromatin opening elements (UCOEs), as well as codon optimization to improve BTK expression. An Eµ enhancer improved B cell rescue, but increased immunoglobulin G (IgG) autoantibodies. Addition of the UCOE avoided autoantibody generation while improving B cell development and function and reducing vector silencing. An optimized vector containing a truncated UCOE upstream of the BTK promoter and codon-optimized BTK cDNA resulted in stable, lineage-regulated BTK expression that mirrored endogenous BTK, making it a strong candidate for XLA therapy.
RESUMEN
A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection.
RESUMEN
Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.
Asunto(s)
Desoxirribonucleasas/metabolismo , Dependovirus/metabolismo , Células Madre Hematopoyéticas/metabolismo , Receptores CCR5/metabolismo , Adulto , Antígenos CD34/metabolismo , Complejo CD3/metabolismo , Células Cultivadas , Reparación del ADN , Sitios Genéticos , Terapia Genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Edición de ARN/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Transplantation of genetically modified hematopoietic stem cells (HSCs) is a promising therapeutic strategy for genetic diseases, HIV, and cancer. However, a barrier for clinical HSC gene therapy is the limited efficiency of gene delivery via lentiviral vectors (LVs) into HSCs. We show here that rapamycin, an allosteric inhibitor of the mammalian target of rapamycin complexes, facilitates highly efficient lentiviral transduction of mouse and human HSCs and dramatically enhances marking frequency in long-term engrafting cells in mice. Mechanistically, rapamycin enhanced postbinding endocytic events, leading to increased levels of LV cytoplasmic entry, reverse transcription, and genomic integration. Despite increasing LV copy number, rapamycin did not significantly alter LV integration site profile or chromosomal distribution in mouse HSCs. Rapamycin also enhanced in situ transduction of mouse HSCs via direct intraosseous infusion. Collectively, rapamycin strongly augments LV transduction of HSCs in vitro and in vivo and may prove useful for therapeutic gene delivery.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Lentivirus/efectos de los fármacos , Lentivirus/genética , Sirolimus/farmacología , Transducción Genética/métodos , Animales , Vectores Genéticos/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/virología , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.
Asunto(s)
Linfocitos B/inmunología , Enfermedad de Chagas/inmunología , Glicoproteínas/metabolismo , Interleucina-17/inmunología , Neuraminidasa/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/inmunología , Animales , Linfocitos B/parasitología , Proliferación Celular , Células Cultivadas , Enfermedad de Chagas/genética , Glicoproteínas/genética , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuraminidasa/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/parasitología , Células Th17/inmunología , Células Th17/parasitología , Activación Transcripcional/inmunologíaRESUMEN
The immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to life-threatening hematopoietic cell dysfunction. We used WAS protein (WASp)-deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression. Transplantation of stem cells transduced with MND-huWASp LV resulted in sustained, endogenous levels of WASp in all hematopoietic lineages, progressive selection for WASp+ T, natural killer T and B cells, rescue of T-cell proliferation and cytokine production, and substantial restoration of marginal zone (MZ) B cells. In contrast, WS1.6-huWASp LV recipients exhibited subendogenous WASp expression in all cell types with only partial selection of WASp+ T cells and limited correction in MZ B-cell numbers. In parallel, WS1.6-huWASp LV recipients exhibited an altered B-cell compartment, including higher numbers of λ-light-chain+ naive B cells, development of self-reactive CD11c+FAS+ B cells, and evidence for spontaneous germinal center (GC) responses. These observations correlated with B-cell hyperactivity and increased titers of immunoglobulin (Ig)G2c autoantibodies, suggesting that partial gene correction may predispose toward autoimmunity. Our findings identify the advantages and disadvantages associated with each vector and suggest further clinical development of the MND-huWASp LV for a future clinical trial for WAS.
Asunto(s)
Linaje de la Célula/genética , Terapia Genética/métodos , Células Madre Hematopoyéticas/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animales , Células Cultivadas , Regulación de la Expresión Génica , Vectores Genéticos , Células Madre Hematopoyéticas/fisiología , Humanos , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Resultado del Tratamiento , Regulación hacia Arriba/genética , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/fisiologíaRESUMEN
Patients with the immunodeficiency Wiskott-Aldrich syndrome (WAS) frequently develop systemic autoimmunity. Here, we demonstrate that mutation of the WAS gene results in B cells that are hyperresponsive to B cell receptor and Toll-like receptor (TLR) signals in vitro, thereby promoting a B cell-intrinsic break in tolerance. Whereas this defect leads to autoantibody production in WAS protein-deficient (WASp(-/-)) mice without overt disease, chimeric mice in which only the B cell lineage lacks WASp exhibit severe autoimmunity characterized by spontaneous germinal center formation, class-switched autoantibodies, renal histopathology, and early mortality. Both T cell help and B cell-intrinsic TLR engagement play important roles in promoting disease in this model, as depletion with anti-CD4 antibodies or generation of chimeric mice with B cells deficient in both WASp and MyD88 prevented development of autoimmune disease. These data highlight the potentially harmful role for cell-intrinsic loss of B cell tolerance in the setting of normal T cell function, and may explain why WAS patients with mixed chimerism after stem cell transplantation often develop severe humoral autoimmunity.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos B/inmunología , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Animales , Anticuerpos Antinucleares/inmunología , Autoanticuerpos/inmunología , Linfocitos B/fisiología , Linfocitos T CD4-Positivos/inmunología , Quimera , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores Toll-Like/inmunología , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin ß (Igß) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC). In murine primary cells, EµB29 and EµBtkp LV-mediated high-level expression in immature and mature B cells compared with all other lineages. Expression increased with B cell maturation and was maintained in peripheral subsets. Expression in T and myeloid cells was much lower in percentage and intensity. Similarly, both EµB29 and EµBtkp LV exhibited high-level activity in human primary B cells. In contrast to EµB29, Btkp and EµBtkp LV also exhibited modest activity in myeloid cells, consistent with the expression profile of endogenous Bruton's tyrosine kinase (Btk). Notably, EµB29 and EµBtkp activity was superior in all expression models to an alternative, B-lineage targeted vector containing the EµS.CD19 enhancer/promoter. In summary, EµB29 and EµBtkp LV comprise efficient delivery platforms for gene expression in B-lineage cells.
Asunto(s)
Linfocitos B/metabolismo , Linfocitos B/patología , Terapia Genética , Vectores Genéticos/genética , Lentivirus/genética , Proteínas Tirosina Quinasas , Agammaglobulinemia Tirosina Quinasa , Agammaglobulinemia/genética , Agammaglobulinemia/terapia , Animales , Linfocitos B/inmunología , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Orden Génico , Genes Reporteros/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Vectores Genéticos/administración & dosificación , Células HEK293 , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Células Mieloides/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Type 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing ß-islet cells in the pancreas. The genetic and environmental mechanisms promoting the development of this disease remain poorly understood. We have explored the cellular requirements for T1D development in DO11.10xRIPmOVA (DORmO) mice, which carry a TCR transgene specific for an MHC class II-restricted epitope from OVA and express membrane-bound OVA in the pancreas under the control of the rat insulin promoter. We found that DORmO.RAG2(-/-) mice do not develop insulitis and are completely protected from diabetes, demonstrating that endogenous lymphocyte receptor rearrangement is required for disease development. Diabetes in DORmO mice is preceded by the development of OVA-specific autoantibodies and is delayed in B cell-deficient DORmO.JhD(-/-) mice, demonstrating that B cells contribute to disease progression. In addition, transfer of CD8(+) T cells from diabetic animals into DORmO.RAG2(-/-) mice promoted insulitis by OVA-specific CD4(+) T cells. Finally, although diabetes develops in DORmO mice in the presence of a significant population of Foxp3(+) OVA-specific regulatory T cells, boosting regulatory T cell numbers by injecting IL-2 immune complexes dampens autoantibody production and prevents development of insulitis and overt diabetes. These results help define the events leading to diabetes in DORmO mice and provide new insights into the cellular interactions required for disease development in an Ag-specific model of T1D.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Diabetes Mellitus Tipo 1/inmunología , Activación de Linfocitos/inmunología , Traslado Adoptivo , Animales , Linfocitos B/citología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Antígenos de Histocompatibilidad Clase II/genética , Inmunohistoquímica , Insulina/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Ovalbúmina/inmunología , Regiones Promotoras Genéticas , Ratas , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA, we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice, a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells, treated mice showed significant, albeit incomplete, rescue of mature B cells in the bone marrow, peripheral blood, spleen, and peritoneal cavity, and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression, viral integration, and partial functional responses, consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA.
Asunto(s)
Agammaglobulinemia/fisiopatología , Agammaglobulinemia/terapia , Linfocitos B/fisiología , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Terapia Genética , Lentivirus/genética , Recuperación de la Función/fisiología , Agammaglobulinemia Tirosina Quinasa , Animales , Linfocitos B/citología , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Antígenos CD79/genética , Línea Celular , Linaje de la Célula , Modelos Animales de Enfermedad , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Terapia Genética/efectos adversos , Vectores Genéticos/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos/genética , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/uso terapéuticoRESUMEN
CD4(+)Foxp3(+) regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4(+)CD103(hi) phenotype. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4(+)Foxp3(+) T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Inflamación/etiología , Inflamación/inmunología , Autotolerancia/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Antígenos CD/metabolismo , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Inflamación/patología , Cadenas alfa de Integrinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores CCR4/metabolismo , Piel/inmunología , Piel/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
The pathogenesis of multiple sclerosis involves a breakdown in T cell tolerance to myelin proteins like myelin basic protein (MBP). Most MBP-specific T cells are eliminated by central tolerance in adult mice, however, the developmentally regulated expression of MBP allows MBP-specific thymocytes in young mice to escape negative selection. It is not known how these T cells that encounter MBP for the first time in the periphery are regulated. We show that naive MBP-specific T cells transferred into T cell-deficient mice induce severe autoimmunity. Regulatory T cells prevent disease, however, suppression of the newly transferred MBP-specific T cells is abrogated by activating APCs in vivo. Without APC activation, MBP-specific T cells persist in the periphery of protected mice but do not become anergic, raising the question of how long-term tolerance can be maintained if APCs presenting endogenous MBP become activated. Our results demonstrate that regulatory T cells induce naive MBP-specific T cells responding to nonactivated APCs to differentiate into a unique, tolerized state with the ability to produce IL-10 and TGF-beta1 in response to activated, but not nonactivated, APCs presenting MBP. This tolerant response depends on continuous activity of regulatory T cells because, in their absence, these uniquely tolerized MBP-specific T cells can again induce autoimmunity.
Asunto(s)
Tolerancia Inmunológica/inmunología , Proteína Básica de Mielina/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Autoinmunidad/inmunología , Proliferación Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Citocinas/biosíntesis , Factores de Transcripción Forkhead/metabolismo , Transfusión de Linfocitos , Ratones , Ratones Transgénicos , Proteína Básica de Mielina/deficiencia , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T Reguladores/citología , Células TH1/inmunología , Células TH1/metabolismo , Factores de TiempoRESUMEN
CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell-mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor alpha chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica/inmunología , Inmunoterapia Adoptiva , Interleucina-15/farmacología , Neoplasias/terapia , Animales , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/citología , Proliferación Celular , Proteína Ligando Fas , Humanos , Memoria Inmunológica/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Interleucina-15 , Receptores de Interleucina-2/metabolismo , Factores de Necrosis Tumoral/metabolismoRESUMEN
CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon gamma. This split tolerance was accompanied by inhibition of Ca(2+) flux, ERK1/2, and Jun kinase phosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.