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Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunoglobulin-type lectins that mediate protein-carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33-related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec-5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec-5 suppresses T cell activation. In support of these findings, we found that Siglec-5 overexpression abrogates antigen receptor induced activation of NFAT and AP-1. Furthermore, we show that GBS ß-protein, a known bacterial ligand of Siglec-5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec-5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec-5 ligand(s), and that the presence of soluble Siglec-5 enhances tumor-cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec-5. Together, our data demonstrate that Siglec-5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggest that blockade of Siglec-5 could serve as a new strategy to enhance anti-tumor T cell functions.
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Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Proteínas de Checkpoint Imunológico , Lectinas , Linfócitos T , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Humanos , Proteínas de Checkpoint Imunológico/metabolismo , Imunoglobulinas , Lectinas/metabolismo , Ligantes , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Linfócitos T/metabolismo , TirosinaRESUMO
Adoptive transfer of tumor epitope-reactive T cells has emerged as a promising strategy to control tumor growth. However, chronically-stimulated T cells expanded for adoptive cell transfer are susceptible to cell death in an oxidative tumor microenvironment. Because oxidation of cell-surface thiols also alters protein functionality, we hypothesized that increasing the levels of thioredoxin (Trx), an antioxidant molecule facilitating reduction of proteins through cysteine thiol-disulfide exchange, in T cells will promote their sustained antitumor function. Using pre-melanosome protein (Pmel)-Trx1 transgenic mouse-derived splenic T cells, flow cytometry, and gene expression analysis, we observed here that higher Trx expression inversely correlated with reactive oxygen species and susceptibility to T-cell receptor restimulation or oxidation-mediated cell death. These Trx1-overexpressing T cells exhibited a cluster of differentiation 62Lhi (CD62Lhi) central memory-like phenotype with reduced glucose uptake (2-NBDGlo) and decreased effector function (interferon γlo). Furthermore, culturing tumor-reactive T cells in the presence of recombinant Trx increased the dependence of T cells on mitochondrial metabolism and improved tumor control. We conclude that strategies for increasing the antioxidant capacity of antitumor T cells modulate their immunometabolic phenotype leading to improved immunotherapeutic control of established tumors.
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Linfócitos T/metabolismo , Tiorredoxinas/metabolismo , Animais , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Transportador de Glucose Tipo 1/metabolismo , Selectina L/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Estresse Oxidativo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Tiorredoxinas/genética , Microambiente Tumoral , Antígeno gp100 de Melanoma/genética , Antígeno gp100 de Melanoma/metabolismoRESUMO
Therapeutic outcomes for adoptive cell transfer (ACT) therapy are constrained by the quality of the infused T cells. The rapid expansion necessary to obtain large numbers of cells results in a more terminally differentiated phenotype with decreased durability and functionality. N-acetyl cysteine (NAC) protects against activation-induced cell death (AICD) and improves anti-tumor efficacy of Pmel-1 T cells in vivo. Here, we show that these benefits of NAC can be extended to engineered T cells and significantly increases T-cell survival within the tumor microenvironment. The addition of NAC to the expansion protocol of human TIL13838I TCR-transduced T cells that are under evaluation in a Phase I clinical trial, demonstrated that findings in murine cells extend to human cells. Expansion of TIL13838I TCR-transduced T cells in NAC also increased their ability to kill target cells in vitro. Interestingly, NAC did not affect memory subsets, but diminished up-regulation of senescence (CD57) and exhaustion (PD-1) markers and significantly decreased expression of the transcription factors EOMES and Foxo1. Pharmacological inhibition of the PI3K/Akt pathway ablates the decrease in Foxo1 induced by NAC treatment of activated T cells. This suggests a model in which NAC through PI3K/Akt activation suppresses Foxo1 expression, thereby impacting its transcriptional targets EOMES, PD-1, and granzyme B. Taken together, our results indicate that NAC exerts pleiotropic effects that impact the quality of TCR-transduced T cells and suggest that the addition of NAC to current clinical protocols should be considered.
Assuntos
Acetilcisteína/farmacologia , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imunoterapia Adotiva , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Células Cultivadas , Sequestradores de Radicais Livres/farmacologia , Humanos , Ativação Linfocitária , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de SinaisRESUMO
Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.
Assuntos
Antígenos de Neoplasias/imunologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias Cutâneas/terapia , Subpopulações de Linfócitos T/transplante , Adulto , Idoso , Humanos , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/secundário , Subpopulações de Linfócitos T/imunologia , Transplante AutólogoRESUMO
The authors would like to make the following corrections to the published article.
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T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.
Assuntos
Transferência Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Animais , Ligação Competitiva/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Citometria de Fluxo , Células HEK293 , Células Hep G2 , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Células Jurkat , Camundongos , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismoRESUMO
Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.
Assuntos
Células Dendríticas/imunologia , Interferon gama/farmacologia , Linfócitos T/imunologia , Receptores Toll-Like/agonistas , Diferenciação Celular , HumanosRESUMO
The success in recent clinical trials using T cell receptor (TCR)-genetically engineered T cells to treat melanoma has encouraged the use of this approach toward other malignancies and viral infections. Although hepatitis C virus (HCV) infection is being treated with a new set of successful direct anti-viral agents, potential for virologic breakthrough or relapse by immune escape variants remains. Additionally, many HCV+ patients have HCV-associated disease, including hepatocellular carcinoma (HCC), which does not respond to these novel drugs. Further exploration of other approaches to address HCV infection and its associated disease are highly warranted. Here, we demonstrate the therapeutic potential of PBL-derived T cells genetically engineered with a high-affinity, HLA-A2-restricted, HCV NS3:1406-1415-reactive TCR. HCV1406 TCR-transduced T cells can recognize naturally processed antigen and elicit CD8-independent recognition of both peptide-loaded targets and HCV+ human HCC cell lines. Furthermore, these cells can mediate regression of established HCV+ HCC in vivo. Our results suggest that HCV TCR-engineered antigen-reactive T cells may be a plausible immunotherapy option to treat HCV-associated malignancies, such as HCC.
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Carcinoma Hepatocelular/terapia , Genes Codificadores dos Receptores de Linfócitos T/fisiologia , Hepatite C/complicações , Neoplasias Hepáticas/terapia , Linfócitos T/imunologia , Animais , Carcinoma Hepatocelular/etiologia , Linhagem Celular Tumoral , Engenharia Genética , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia , Neoplasias Hepáticas/etiologia , Camundongos , Proteínas não Estruturais Virais/genéticaRESUMO
Mouse CD8(+) T cells conditioned with interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8(+) T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8(+) T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8(+) T cells led to a tenfold-100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8(+) T cells genetically modified with a tyrosinase-specific T cell receptor (TCR) exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8(+) T cells for adoptive transfer and cancer therapy.
Assuntos
Transferência Adotiva , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/transplante , Interleucina-12/farmacologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias Cutâneas/terapia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Granzimas/biossíntese , Humanos , Interleucina-12/imunologia , Depleção Linfocítica , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Cutâneas/imunologiaRESUMO
ABSTRACT: CD19-specific chimeric antigen receptor (CAR) T cells have demonstrated impressive responses in patients with relapsed and refractory B cell malignancies. However, many patients relapse or fail to respond to CD19 CAR T cells, demonstrating the need to improve its efficacy and durability. Current protocols for generating CAR T cells involve T cell activation through CD3 stimulation to facilitate efficient CAR transfer followed by ex vivo expansion with exogenous cytokines to obtain adequate cell numbers for treatment. Both T cell activation and expansion inevitably lead to terminal differentiation and replicative senescence, which are suboptimal for therapy. Interleukin-7 (IL-7) was previously shown to allow for lentiviral transduction of T cells in the absence of activation. In these studies, we used IL-7 to generate CD19 CAR T cells without stimulating CD3. Nonactivated and IL-7 cultured (NICE) CD19 CAR T cells were enriched with the T memory stem cell population, retained novel markers of stemness, had lower expression of exhaustion markers, and increased proliferative potential. Furthermore, our findings are consistent with engraftment of NICE CD19 CAR T cells and demonstrate a superior therapeutic response in both intraperitoneal and subcutaneous in vivo B cell lymphoma models. These results suggest that NICE CD19 CAR T cells may improve outcomes for B cell malignancies and warrant clinical evaluation.
Assuntos
Interleucina-7 , Recidiva Local de Neoplasia , Receptores de Antígenos de Linfócitos T , Humanos , Linfócitos T , Células-Tronco , FenótipoRESUMO
Immunotherapy is a beneficial treatment approach for multiple cancers, however, current therapies are effective only in a small subset of patients. Adoptive cell transfer (ACT) is a facet of immunotherapy where T cells targeting the tumor cells are transferred to the patient with several primary forms, utilizing unmodified or modified T cells: tumor-infiltrating lymphocytes (TIL), genetically modified T cell receptor transduced T cells, and chimeric antigen receptor (CAR) transduced T cells. Many clinical trials are underway investigating the efficacy and safety of these different subsets of ACT, as well as trials that combine one of these subsets with another type of immunotherapy. The main challenges existing with ACT are improving clinical responses and decreasing adverse events. Current research focuses on identifying novel tumor targeting T cell receptors, improving safety and efficacy, and investigating ACT in combination with other immunotherapies.
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Adoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (TN) and memory stem cell populations (TSCM) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.
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Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Melanoma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Feminino , Células HEK293 , Humanos , Memória Imunológica/imunologia , Interleucina-7/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Transdução Genética/métodosRESUMO
T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells.
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BACKGROUND: Dendritic cells (DCs) enhance the quality of anti-tumor immune response in patients with cancer. Thus, we posit that DC-based immunotherapy, in conjunction with toll-like receptor (TLR)-3 agonist poly-ICLC, is a promising approach for harnessing immunity against metastatic or locally advanced unresectable pancreatic cancer (PC). METHODS: We generated autologous DCs from the peripheral blood of HLA-A2+ patients with PC. DCs were pulsed with three distinct A2-restricted peptides: 1) human telomerase reverse transcriptase (hTERT, TERT572Y), 2) carcinoembryonic antigen (CEA; Cap1-6D), and 3) survivin (SRV.A2). Patients received four intradermal injections of 1 × 107 peptide-pulsed DC vaccines every 2 weeks (Day 0, 14, 28, and 42). Concurrently, patients received intramuscular administration of Poly-ICLC at 30 µg/Kg on vaccination days (i.e., day 0, 14, 28, and 42), as well as on days 3, 17, 21, 31, 37, and 45. Our key objective was to assess safety and feasibility. The effect of DC vaccination on immune response was measured at each DC injection time point by enumerating the phenotype and function of patient T cells. RESULTS: Twelve patients underwent apheresis: nine patients with metastatic disease, and three patients with locally advanced unresectable disease. Vaccines were successfully manufactured from all individuals. We found that this treatment was well-tolerated, with the most common symptoms being fatigue and/or self-limiting flu-like symptoms. Among the eight patients who underwent imaging on day 56, four patients experienced stable disease while four patients had disease progression. The median overall survival was 7.7 months. One patient survived for 28 months post leukapheresis. MHC class I -tetramer analysis before and after vaccination revealed effective generation of antigen-specific T cells in three patients with stable disease. CONCLUSION: Vaccination with peptide-pulsed DCs in combination with poly-ICLC is safe and induces a measurable tumor specific T cell population in patients with advanced PC. TRIAL REGISTRATION: NCT01410968 ; Name of registry: clinicaltrials.gov; Date of registration: 08/04/2011).
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Vacinas Anticâncer/administração & dosagem , Carboximetilcelulose Sódica/análogos & derivados , Células Dendríticas/imunologia , Neoplasias Pancreáticas/terapia , Poli I-C/administração & dosagem , Polilisina/análogos & derivados , Vacinação/métodos , Idoso , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/farmacologia , Carboximetilcelulose Sódica/administração & dosagem , Carboximetilcelulose Sódica/farmacologia , Células Dendríticas/transplante , Feminino , Humanos , Imunoterapia Ativa/métodos , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Projetos Piloto , Poli I-C/farmacologia , Polilisina/administração & dosagem , Polilisina/farmacologia , Transplante AutólogoRESUMO
Although adoptive transfer of autologous tumor antigen-specific T-cell immunotherapy can produce remarkable clinical efficacy, most patients do not achieve durable complete responses. We hypothesized that reducing susceptibility of T cells to activation-induced cell death (AICD), which increases during the rapid in vitro expansion of therapeutic T cells before their infusion, might improve the persistence of adoptively transferred cells. Our investigations revealed that repetitive stimulation of the T-cell receptor (TCR) induced AICD, as a result of activating the DNA damage response pathway through ATM-mediated Ser15 phosphorylation of p53. Activation of this DNA damage response pathway also occurred upon antigen-specific restimulation in TCR-transduced TIL1383I T cells prepared for adoptive transfer to patients as part of a clinical trial. Notably, treatment with the antioxidant N-acetyl cysteine (NAC) significantly reduced upregulation of the DNA damage marker γH2AX, subsequent ATM activation, and cell death. In the Pmel mouse model of melanoma, the presence of NAC during ex vivo T-cell expansion improved the persistence of adoptively transferred cells, reduced tumor growth, and increased survival. Taken together, our results offer a preclinical proof of concept for the addition of NAC to current therapeutic T-cell expansion protocols, offering immediate potential to improve the quality and therapeutic efficacy of adoptive T-cell therapeutics infused into patients. Cancer Res; 76(20); 6006-16. ©2016 AACR.
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Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Imunoterapia Adotiva , Ativação Linfocitária , Neoplasias Experimentais/terapia , Linfócitos T/imunologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Dano ao DNA , Feminino , Humanos , Camundongos , Neoplasias Experimentais/imunologia , Fosforilação , Receptores de Antígenos de Linfócitos T/fisiologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Advancements in adoptive cell transfer therapy (ACT) has led to the use of T cells engineered with tumor specific T cell receptors, which after rapid expansion can be obtained in sufficient numbers for treating patients. However, due to massive proliferation these cells are close to replicative senescence, exhibit exhausted phenotype, and also display increased susceptibility to activation induced cell death. We have previously shown that tumor reactive T cells undergo caspase-independent cell death upon TCR restimulation with cognate antigen, which involves reactive oxygen species and c-jun N-terminal kinase. Herein, we show that a large fraction of the human melanoma epitope tyrosinase reactive TCR transduced T cells that exhibit effector memory (TEM) phenotype and undergo programmed necrosis, or necroptosis, upon TCR restimulation. As compared to the T central memory (TCM) subsets, the TEM subset displayed an increased expression of genes involved in necroptotic cell death, and a necrotic phenotype upon TCR restimulation as confirmed by electron microscopy. Higher expression of receptor-interacting kinases (RIPK) that mediate necroptosis was also observed in the TEM fraction. Further, the TEM cells were rescued from undergoing necroptosis when pretreated with necroptotic inhibitor NecroX2 before TCR restimulation. Importantly, NecroX2 pretreated tumor reactive T cells also exhibited better tumor control and increased in vivo persistence when adoptively-transferred to treat subcutaneously established murine melanoma B16-F10. Thus, it is likely that the outcome of ACT could be vastly improved by interfering with the necroptotic cell death pathway in activated tumor reactive T cells used in immunotherapy.
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Apoptose/imunologia , Linfócitos T CD8-Positivos/transplante , Imunoterapia Adotiva/métodos , Melanoma Experimental/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Selectina L/imunologia , Selectina L/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Necrose/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their inherent genomic instability. Accumulation of mutations can alter processing and presentation of antigens recognized by antibodies and T cells that can lead to immune escape variants. Use of an agent that can intrinsically combat rapidly mutating viral or cancer-associated antigens would be quite advantageous in developing effective immunity against such disease. We propose that T cells harboring cross-reactive TCRs could serve as a therapeutic agent in these instances. With the use of hepatitis C virus, known for its genomic instability as a model for mutated antigen recognition, we demonstrate cross-reactivity against immunogenic and mutagenic nonstructural protein 3:1406-1415 and nonstructural protein 3:1073-1081 epitopes in PBL-derived, TCR-gene-modified T cells. These single TCR-engineered T cells can CD8-independently recognize naturally occurring and epidemiologically relevant mutant variants. TCR-peptide MHC modeling data allow us to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how these substitutions impact the requirement of CD8 affinity enhancement for recognition. A better understanding of such TCRs' promiscuous behavior may allow for exploitation of these properties to develop novel, adoptive T cell-based therapies for viral infections and cancers exhibiting similar genomic instability.
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Epitopos de Linfócito T/imunologia , Instabilidade Genômica , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia , Receptores de Antígenos de Linfócitos T/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , Hepacivirus/genética , Hepatite C/etiologia , HumanosRESUMO
Mutations and inactivation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) are observed in 15%-25% of cases of human T cell acute lymphoblastic leukemia (T-ALL). Pten deletion induces myeloproliferative disorders (MPDs), acute myeloid leukemia (AML), and/or T-ALL in mice. Previous studies attributed Pten-loss-related hematopoietic defects and leukemogenesis to excessive activation of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. Although inhibition of this signal dramatically suppresses the growth of PTEN-null T-ALL cells in vitro, treatment with inhibitors of this pathway does not cause a complete remission in vivo. Here, we report that focal adhesion kinase (Fak), a protein substrate of Pten, also contributes to T-ALL development in Pten-null mice. Inactivation of the FAK signaling pathway by either genetic or pharmacologic methods significantly sensitizes both murine and human PTEN-null T-ALL cells to PI3K/AKT/mTOR inhibition when cultured in vitro on feeder layer cells or a matrix and in vivo.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Humanos , Leucemia/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacosRESUMO
Ex vivo-expanded CD8(+) T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced antitumor efficacy, relative to CD8(+) T cells with a central memory-like phenotype (TCM cells). Furthermore, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM cells possess relatively greater antioxidative capacity than TEM cells. Here, we report that TCM cells exhibit a relative increase compared with TEM cells in the expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity, and glycolytic enzyme levels. Notably, T-cell receptor-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, upregulated thiol levels and antioxidant genes. A comparison of antitumor CD8(+) T-cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth and that profiling this biomarker may have benefits to adoptive T-cell immunotherapy protocols.