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Immune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRß) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRß repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRß repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRß clonotypes was observed in responding tumours. Machine learning identified TCRß CDR3 signatures unique to each tumour model, and signatures associated with ICT response at various timepoints before or during ICT. Clonally expanded CD8+ T cells in responding tumours post ICT displayed effector T cell gene signatures and phenotype. An early burst of clonal expansion during ICT is associated with response, and we report unique dynamics in TCRß signatures associated with ICT response.
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Inhibidores de Puntos de Control Inmunológico , Linfocitos Infiltrantes de Tumor , Receptores de Antígenos de Linfocitos T alfa-beta , Animales , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Ratones , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Humanos , Ratones Endogámicos C57BL , FemeninoRESUMEN
While chemotherapy remains the first-line treatment for many cancers, it is still unclear what distinguishes responders from non-responders. Here, we characterize the chemotherapy-responsive tumor microenvironment in mice, using RNA sequencing on tumors before and after cyclophosphamide, and compare the gene expression profiles of responders with progressors. Responsive tumors have an inflammatory and highly immune infiltrated pre-treatment tumor microenvironment characterized by the enrichment of pathways associated with CD4+ T cells, interferons (IFNs), and tumor necrosis factor alpha (TNF-α). The same gene expression profile is associated with response to cyclophosphamide-based chemotherapy in patients with breast cancer. Finally, we demonstrate that tumors can be sensitized to cyclophosphamide and 5-FU chemotherapy by pre-treatment with recombinant TNF-α, IFNγ, and poly(I:C). Thus, a CD4+ T cell-inflamed pre-treatment tumor microenvironment is necessary for response to chemotherapy, and this state can be therapeutically attained by targeted immunotherapy.
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Neoplasias , Linfocitos T , Animales , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Microambiente Tumoral , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , Ciclofosfamida/metabolismo , Neoplasias/patología , Linfocitos T CD4-Positivos/metabolismoRESUMEN
Objectives: Immune checkpoint inhibitors have achieved clinical success in cancer treatment, but this treatment causes immune-related adverse events, including type 1 diabetes (T1D). Our aim was to test whether a JAK1/JAK2 inhibitor, effective at treating spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice, can prevent diabetes secondary to PD-L1 blockade. Methods: Anti-PD-L1 antibody was injected into NOD mice to induce diabetes, and JAK1/JAK2 inhibitor LN3103801 was administered by oral gavage to prevent diabetes. Flow cytometry was used to study T cells and beta cells. Mesothelioma cells were inoculated into BALB/c mice to induce a transplantable tumour model. Results: Anti-PD-L1-induced diabetes was associated with increased immune cell infiltration in the islets and upregulated MHC class I on islet cells. Anti-PD-L1 administration significantly increased islet T cell proliferation and islet-specific CD8+ T cell numbers in peripheral lymphoid organs. JAK1/JAK2 inhibitor treatment blocked IFNγ-mediated MHC class I upregulation on beta cells and T cell proliferation mediated by cytokines that use the common γ chain receptor. As a result, anti-PD-L1-induced diabetes was prevented by JAK1/JAK2 inhibitor administered before or after checkpoint inhibitor therapy. Diabetes was also reversed when the JAK1/JAK2 inhibitor was administered after the onset of anti-PD-L1-induced hyperglycaemia. Furthermore, JAK1/JAK2 inhibitor intervention after checkpoint inhibitors did not reverse or abrogate the antitumour effects in a transplantable tumour model. Conclusion: A JAK1/JAK2 inhibitor can prevent and reverse anti-PD-L1-induced diabetes by blocking IFNγ and γc cytokine activities. Our study provides preclinical validation of JAK1/JAK2 inhibitor use in checkpoint inhibitor-induced diabetes.
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Antibodies that target immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and the programmed cell death protein 1/ligand 1 (PD-1/PD-L1) are now a treatment option for multiple cancer types. However, as a monotherapy, objective responses only occur in a minority of patients. Chemotherapy is widely used in combination with immune checkpoint blockade (ICB). Although a variety of isolated immunostimulatory effects have been reported for several classes of chemotherapeutics, it is unclear which chemotherapeutics provide the most benefit when combined with ICB. We investigated 10 chemotherapies from the main canonical classes dosed at the clinically relevant maximum tolerated dose in combination with anti-CTLA-4/anti-PD-L1 ICB. We screened these chemo-immunotherapy combinations in two murine mesothelioma models from two different genetic backgrounds, and identified chemotherapies that produced additive, neutral or antagonistic effects when combined with ICB. Using flow cytometry and bulk RNAseq, we characterized the tumor immune milieu in additive chemo-immunotherapy combinations. 5-fluorouracil (5-FU) or cisplatin were additive when combined with ICB while vinorelbine and etoposide provided no additional benefit when combined with ICB. The combination of 5-FU with ICB augmented an inflammatory tumor microenvironment with markedly increased CD8+ T cell activation and upregulation of IFNγ, TNFα and IL-1ß signaling. The effective anti-tumor immune response of 5-FU chemo-immunotherapy was dependent on CD8+ T cells but was unaffected when TNFα or IL-1ß cytokine signaling pathways were blocked. Our study identified additive and non-additive chemotherapy/ICB combinations and suggests a possible role for increased inflammation in the tumor microenvironment as a basis for effective combination therapy.
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Inhibidores de Puntos de Control Inmunológico , Neoplasias , Animales , Linfocitos T CD8-positivos , Fluorouracilo/uso terapéutico , Humanos , Ratones , Neoplasias/terapia , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/uso terapéuticoRESUMEN
Interferon gamma (IFNγ) is a proinflammatory cytokine implicated in autoimmune diseases. However, deficiency or neutralization of IFNγ is ineffective in reducing disease. We characterize islet antigen-specific T cells in non-obese diabetic (NOD) mice lacking all three IFN receptor genes. Diabetes is minimally affected, but at 125 days of age, antigen-specific CD8+ T cells, quantified using major histocompatibility complex class I tetramers, are present in 10-fold greater numbers in Ifngr-mutant NOD mice. T cells from Ifngr-mutant mice have increased proliferative responses to interleukin-2 (IL-2). They also have reduced phosphorylated STAT1 and its target gene, suppressor of cytokine signaling 1 (SOCS-1). IFNγ controls the expansion of antigen-specific CD8+ T cells by mechanisms which include increased SOCS-1 expression that regulates IL-2 signaling. The expanded CD8+ T cells are likely to contribute to normal diabetes progression despite reduced inflammation in Ifngr-mutant mice.
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Diabetes Mellitus , Interleucina-2 , Animales , Autoantígenos , Linfocitos T CD8-positivos , Citocinas/metabolismo , Interferón gamma/metabolismo , Interferones/metabolismo , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos NOD , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Previous studies have indicated a synergistic effect between radiotherapy and immunotherapy. A better understanding of how this combination affects the immune system can help to clarify its role in the treatment of metastatic cancer. We performed T cell receptor (TCR) sequencing on 46 sequentially collected samples from 15 patients with stage IV non-small cell lung cancer, receiving stereotactic body radiotherapy combined with a programmed cell death ligand-1 (PD-L1) inhibitor. TCR repertoire diversity was assessed using Rényi diversity curves and the Shannon diversity index. TCR clones were tracked over time. We found decreasing or stable diversity in the best responders, and an increase in diversity at progression in patients with an initial response. Expansion of TCR clones was more often seen in responders. Several patients also developed new clones of high abundance. This seemed to be more related to radiotherapy than to immune checkpoint blockade. In summary, we observed similar dynamics in the TCR repertoire as have been described with immunotherapy alone. In addition, the occurrence of new unique clones of high abundance after radiotherapy may indicate that radiotherapy functions as a personalized cancer vaccine.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti-PD-1 and anti-CTLA-4) and are being targeted for vaccination-based therapies. However, ICB treatments show uneven responses between patients, and neoantigen vaccination efficiency has yet to be established. Here, we characterize neoantigen-specific CD8+ T cells in a tumor that is resistant to ICB and neoantigen vaccination. Leveraging the use of mass cytometry combined with multiplex major histocompatibility complex (MHC) class I tetramer staining, we screened and identified tumor neoantigen-specific CD8+ T cells in the Lewis Lung carcinoma (LLC) tumor model (mRiok1). We observed an expansion of mRiok1-specific CD8+ tumor-infiltrating lymphocytes (TILs) after ICB targeting PD-1 or CTLA-4 with no sign of tumor regression. The expanded neoantigen-specific CD8+ TILs remained phenotypically and functionally exhausted but displayed cytotoxic characteristics. When combining both ICB treatments, mRiok1-specific CD8+ TILs showed a stem-like phenotype and a higher capacity to produce cytokines, but tumors did not show signs of regression. Furthermore, combining both ICB treatments with neoantigen vaccination did not induce tumor regression either despite neoantigen-specific CD8+ TIL expansion. Overall, this work provides a model for studying neoantigens in an immunotherapy nonresponder model. We showed that a robust neoantigen-specific T-cell response in the LLC tumor model could fail in tumor response to ICB, which will have important implications in designing future immunotherapeutic strategies.
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Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Linfocitos T CD8-positivos/inmunología , Carcinoma Pulmonar de Lewis/inmunología , Resistencia a Antineoplásicos , Linfocitos Infiltrantes de Tumor/inmunología , Animales , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
Precise clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic and epigenetic manipulation of the immune response has become a promising immunotherapeutic approach toward combating tumorigenesis and tumor progression. CRISPR-based immunologic reprograming in cancer therapy comprises the locus-specific enhancement of host immunity, the improvement of tumor immunogenicity, and the suppression of tumor immunoevasion. To date, the ex vivo re-engineering of immune cells directed to inhibit the expression of immune checkpoints or to express synthetic immune receptors (chimeric antigen receptor therapy) has shown success in some settings, such as in the treatment of melanoma, lymphoma, liver, and lung cancer. However, advancements in nuclease-deactivated CRISPR-associated nuclease-9 (dCas9)-mediated transcriptional activation or repression and Cas13-directed gene suppression present novel avenues for the development of tumor immunotherapies. In this review, the basis for development, mechanism of action, and outcomes from recently published Cas9-based clinical trial (genetic editing) and dCas9/Cas13-based pre-clinical (epigenetic editing) data are discussed. Lastly, we review cancer immunotherapy-specific considerations and barriers surrounding use of these approaches in the clinic.
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The success of immunotherapy that targets inhibitory T cell receptors for the treatment of multiple cancers has seen the anti-tumor immune response re-emerge as a promising biomarker of response to therapy. Longitudinal characterization of T cells in the tumor microenvironment (TME) helps us understand how to promote effective anti-tumor immunity. However, serial analyses at the tumor site are rarely feasible in clinical practice. Malignant pleural effusions (MPE) associated with thoracic cancers are an abnormal accumulation of fluid in the pleural space that is routinely drained for patient symptom control. This fluid contains tumor cells and immune cells, including lymphocytes, macrophages and dendritic cells, providing a window into the local tumor microenvironment. Recurrent MPE is common, and provides an opportunity for longitudinal analysis of the tumor site in a clinical setting. Here, we review the phenotype of MPE-derived T cells, comparing them to tumor and blood T cells. We discuss the benefits and limitations of their use as potential dynamic biomarkers of response to therapy.
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Surgical resection of cancer remains the frontline therapy for millions of patients annually, but post-operative recurrence is common, with a relapse rate of around 45% for non-small cell lung cancer. The tumour draining lymph nodes (dLN) are resected at the time of surgery for staging purposes, and this cannot be a null event for patient survival and future response to immune checkpoint blockade treatment. This project investigates cancer surgery, lymphadenectomy, onset of metastatic disease, and response to immunotherapy in a novel model that closely reflects the clinical setting. In a murine metastatic lung cancer model, primary subcutaneous tumours were resected with associated dLNs remaining intact, completely resected or partially resected. Median survival after surgery was significantly shorter with complete dLN resection at the time of surgery (49 days (95%CI)) compared to when lymph nodes remained intact (> 88 days; p < 0.05). Survival was partially restored with incomplete lymph node resection and CD8 T cell dependent. Treatment with aCTLA4 whilst effective against the primary tumour was ineffective for metastatic lung disease. Conversely, aPD-1/aCD40 treatment was effective in both the primary and metastatic disease settings and restored the detrimental effects of complete dLN resection on survival. In this pre-clinical lung metastatic disease model that closely reflects the clinical setting, we observe decreased frequency of survival after complete lymphadenectomy, which was ameliorated with partial lymph node removal or with early administration of aPD-1/aCD40 therapy. These findings have direct relevance to surgical lymph node resection and adjuvant immunotherapy in lung cancer, and perhaps other cancer, patients.
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Linfocitos T CD8-positivos/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Escisión del Ganglio Linfático , Metástasis de la Neoplasia/inmunología , Animales , Quimioterapia Adyuvante/métodos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ratones , Metástasis de la Neoplasia/patología , Recurrencia Local de Neoplasia/patologíaRESUMEN
T-cell responses to insulin and its precursor proinsulin are central to islet autoimmunity in humans and non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes. Mice have two proinsulin genes proinsulin -1 and 2 that are differentially expressed, with predominant proinsulin-2 expression in the thymus and proinsulin-1 in islet beta-cells. In contrast to proinsulin-2, proinsulin-1 knockout NOD mice are protected from autoimmune diabetes. This indicates that proinsulin-1 epitopes in beta-cells maybe preferentially targeted by autoreactive T cells. To study the contribution of proinsulin-1 reactive T cells in autoimmune diabetes, we generated transgenic NOD mice with tetracycline-regulated expression of proinsulin-1 in antigen presenting cells (TIP-1 mice) with an aim to induce immune tolerance. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in in TIP-1 mice indicating immune tolerance. Moreover, T cells from TIP-1 mice expressing proinsulin-1 transferred diabetes at a significantly reduced frequency. However, proinsulin-1 expression in APCs had minimal impact on the immune responses to the downstream antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and did not prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, boosting immune tolerance to proinsulin-1 partially prevents islet-autoimmunity. This study further extends the previously established role of proinsulin-1 epitopes in autoimmune diabetes in NOD mice.
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Diabetes Mellitus Tipo 1/prevención & control , Tolerancia Inmunológica , Proinsulina/fisiología , Animales , Autoanticuerpos/análisis , Glucosa-6-Fosfatasa/fisiología , Insulina/inmunología , Ratones , Ratones Endogámicos NOD , Proinsulina/genética , Proinsulina/inmunologíaRESUMEN
Immune checkpoint therapy (ICT) results in durable responses in individuals with some cancers, but not all patients respond to treatment. ICT improves CD8+ cytotoxic T lymphocyte (CTL) function, but changes in tumor antigen-specific CTLs post-ICT that correlate with successful responses have not been well characterized. Here, we studied murine tumor models with dichotomous responses to ICT. We tracked tumor antigen-specific CTL frequencies and phenotype before and after ICT in responding and non-responding animals. Tumor antigen-specific CTLs increased within tumor and draining lymph nodes after ICT, and exhibited an effector memory-like phenotype, expressing IL-7R (CD127), KLRG1, T-bet, and granzyme B. Responding tumors exhibited higher infiltration of effector memory tumor antigen-specific CTLs, but lower frequencies of regulatory T cells compared to non-responders. Tumor antigen-specific CTLs persisted in responding animals and formed memory responses against tumor antigens. Our results suggest that increased effector memory tumor antigen-specific CTLs, in the presence of reduced immunosuppression within tumors is part of a successful ICT response. Temporal and nuanced analysis of T cell subsets provides a potential new source of immune based biomarkers for response to ICT.
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Linfocitos T CD8-positivos/inmunología , Inhibidores de Puntos de Control Inmunológico/inmunología , Memoria Inmunológica/inmunología , Animales , Antígenos de Neoplasias/inmunología , Biomarcadores/metabolismo , Línea Celular Tumoral , Granzimas/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Immunotherapies have revolutionized cancer treatment. In particular, immune checkpoint therapy (ICT) leads to durable responses in some patients with some cancers. However, the majority of treated patients do not respond. Understanding immune mechanisms that underlie responsiveness to ICT will help identify predictive biomarkers of response and develop treatments to convert non-responding patients to responding ones. ICT primarily acts at the level of adaptive immunity. The specificity of adaptive immune cells, such as T and B cells, is determined by antigen-specific receptors. T cell repertoires can be comprehensively profiled by high-throughput sequencing at the bulk and single-cell level. T cell receptor (TCR) sequencing allows for sensitive tracking of dynamic changes in antigen-specific T cells at the clonal level, giving unprecedented insight into the mechanisms by which ICT alters T cell responses. Here, we review how the repertoire influences response to ICT and conversely how ICT affects repertoire diversity. We will also explore how changes to the repertoire in different anatomical locations can better correlate and perhaps predict treatment outcome. We discuss the advantages and limitations of current metrics used to characterize and represent TCR repertoire diversity. Discovery of predictive biomarkers could lie in novel analysis approaches, such as network analysis of amino acids similarities between TCR sequences. Single-cell sequencing is a breakthrough technology that can link phenotype with specificity, identifying T cell clones that are crucial for successful ICT. The field of immuno-sequencing is rapidly developing and cross-disciplinary efforts are required to maximize the analysis, application, and validation of sequencing data. Unravelling the dynamic behavior of the TCR repertoire during ICT will be highly valuable for tracking and understanding anti-tumor immunity, biomarker discovery, and ultimately for the development of novel strategies to improve patient outcomes.
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Inhibidores de Puntos de Control Inmunológico/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Humanos , Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/terapiaRESUMEN
Immune checkpoint blockade (ICPB) is a powerfully effective cancer therapy in some patients. Tumor neo-antigens are likely main targets for attack but it is not clear which and how many tumor mutations in individual cancers are actually antigenic, with or without ICPB therapy and their role as neo-antigen vaccines or as predictors of ICPB responses. To examine this, we interrogated the immune response to tumor neo-antigens in a murine model in which the tumor is induced by a natural human carcinogen (i.e. asbestos) and mimics its human counterpart (i.e. mesothelioma). We identified and screened 33 candidate neo-antigens, and found T cell responses against one candidate in tumor-bearing animals, mutant UQCRC2. Interestingly, we found a high degree of inter-animal variation in the magnitude of neo-antigen responses in otherwise identical mice. ICPB therapy with Cytotoxic T-lymphocyte-associated protein (CTLA-4) and α-glucocorticoid-induced TNFR family related gene (GITR) in doses that induced tumor regression, increased the magnitude of responses and unmasked functional T cell responses against another neo-antigen, UNC45a. Importantly, the magnitude of the pre-treatment draining lymph node (dLN) response to UNC45a closely corresponded to ICPB therapy outcomes. Surprisingly however, boosting pre-treatment UNC45a-specific T cell numbers did not improve response rates to ICPB. These observations suggest a novel biomarker approach to the clinical prediction of ICPB response and have important implications for the development of neo-antigen vaccines.
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Vacunas contra el Cáncer , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias , Animales , Antígenos de Neoplasias/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ganglios Linfáticos , Ratones , Neoplasias/genética , Neoplasias/terapia , Linfocitos T CitotóxicosRESUMEN
Activation of naïve CD8+ T cells stimulates proliferation and differentiation into cytotoxic T-lymphocytes (CTLs). Adoptive T Cell Therapy (ACT) involves multiple rounds of ex vivo activation to generate enough CTLs for reinfusion into patients, but this drives differentiation into terminal effector T cells. Less differentiated CTL populations, such as stem cell memory T cells, are more ideal candidates for ACT because of increased self-renewal and persistent properties. Ex vivo targeting of T cell differentiation with epigenetic modifiers is a potential strategy to improve cytotoxic T-lymphocyte (CTL) generation for ACT. We established a pipeline to assess the effects of epigenetic modifiers on CD8+ T cell proliferation, differentiation, and efficacy in a preclinical melanoma model. Single treatment with epigenetic modifiers inhibited T cell proliferation in vitro, producing CD44hiCD62Lhi effector-like T cells rather than a stem cell memory T cell phenotype. Most epigenetic modifying agents had no significant effect on ACT efficacy with the notable exception of the bromodomain and extraterminal (BET)-inhibitor JQ1 which was associated with a decrease in efficacy compared to unmodified T cells. These findings reveal the complexity of epigenetic targeting of T cell differentiation, highlighting the need to precisely define the epigenetic targeting strategies to improve CTL generation for ACT.
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Proliferación Celular , Epigénesis Genética , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/terapia , Linfocitos T/efectos de los fármacos , Animales , Azepinas/farmacología , Benzodiazepinas/farmacología , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Indolizinas/farmacología , Ratones , Ratones Endogámicos C57BL , Sulfonas/farmacología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/fisiología , Triazoles/farmacologíaRESUMEN
CD8+ T cells play a central role in beta-cell destruction in type 1 diabetes. CD8+ T cells use two main effector pathways to kill target cells, perforin plus granzymes and FAS ligand (FASL). We and others have established that in non-obese diabetic (NOD) mice, perforin is the dominant effector molecule by which autoreactive CD8+ T cells kill beta cells. However, blocking FASL pharmacologically was shown to protect NOD mice from diabetes, indicating that FASL may have some role. FASL can engage with its receptor FAS on target cells either as membrane bound or soluble FASL. It has been shown that membrane-bound FASL is required to stimulate FAS-induced apoptosis in target cells, whereas excessive soluble FASL can induce NF-κB-dependent gene expression and inflammation. Because islet inflammation is a feature of autoimmune diabetes, we tested whether soluble FASL could be important in disease pathogenesis independent of its cell death function. We generated NOD mice deficient in soluble FASL, while maintaining expression of membrane-bound FASL due to a mutation in the FASL sequence required for cleavage by metalloproteinase. NOD mice lacking soluble FASL had normal numbers of lymphocytes in their spleen and thymus. Soluble FASL deficient NOD mice had similar islet inflammation as wild-type NOD mice and were not protected from diabetes. Our data indicate that soluble FASL is not required in development of autoimmune diabetes.
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Immunotherapies such as adoptive cell therapy (ACT) are promising treatments for solid cancers. However, relapsing disease remains a problem and the molecular mechanisms underlying resistance are poorly defined. We postulated that the deregulated epigenetic landscape in cancer cells could underpin the acquisition of resistance to immunotherapy. To address this question, two preclinical models of ACT were employed to study transcriptional and epigenetic regulatory processes within ACT-treated cancer cells. In these models ACT consistently causes robust tumor regression, but resistance develops and tumors relapse. We identified down-regulated expression of immunogenic antigens at the mRNA level correlated with escape from immune control. To determine whether this down-regulation was under epigenetic control, we treated escaped tumor cells with DNA demethylating agents, azacytidine (AZA) and decitabine (DEC). AZA or DEC treatment restored antigen expression in a proportion of the tumor population. To explore the importance of other epigenetic modifications we isolated tumor cells refractory to DNA demethylation and screened clones against a panel of 19 different epigenetic modifying agents (EMAs). The library of EMAs included inhibitors of a range of chromosomal and transcription regulatory protein complexes, however, when tested as single agents none restored further antigen expression. These findings suggest that tumor cells employ multiple epigenetic and genetic mechanisms to evade immune control, and a combinatorial approach employing several EMAs targeting transcription and genome stability may be required to overcome tumor resistance to immunotherapy.
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OBJECTIVE: Pleural effusion (PE) is a common feature of malignant pleural mesothelioma. These effusions typically contain lymphocytes and malignant cells. We postulated that the PE would be a source of lymphocytes for analysis of tumor immune milieu. The aim of this study was to compare the phenotype and T cell receptor usage of pleural effusion T cells with paired concurrently drawn peripheral blood lymphocytes. We used multi-parameter flow cytometry and high-throughput T cell receptor sequencing to analyse peripheral blood and pleural effusion mononuclear cells. RESULTS: Both CD8+ and CD4+ T cells from effusion showed increased expression of T cell inhibitory receptors PD-1, LAG-3 and Tim-3 compared to blood. Comprehensive T cell receptor sequencing on one of the patients showed a discordant distribution of clonotypes in the antigen-experienced (PD-1+) compartment between effusion and blood, suggesting an enrichment of antigen specific clonotypes in the effusion, with potential as an immunological response biomarker.
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Neoplasias Pulmonares/inmunología , Mesotelioma/inmunología , Derrame Pleural/inmunología , Receptores de Superficie Celular/metabolismo , Linfocitos T/inmunología , Anciano , Anciano de 80 o más Años , Humanos , Neoplasias Pulmonares/sangre , Mesotelioma/sangre , Mesotelioma Maligno , Persona de Mediana Edad , Derrame Pleural/sangre , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Mesothelioma is an aggressive asbestos induced cancer with extremely poor prognosis and limited treatment options. Immune checkpoint blockade (ICPB) has demonstrated effective therapy in melanoma and is now being applied to other cancers, including mesothelioma. However, the efficacy of ICPB and which immune checkpoint combinations constitute the best therapeutic option for mesothelioma have yet to be fully elucidated. Here, we used our well characterised mesothelioma tumour model to investigate the efficacy of different ICBP treatments to generate effective therapy for mesothelioma. We show that tumour resident regulatory T cell co-express high levels of CTLA-4, OX40 and GITR relative to T effector subsets and that these receptors are co-expressed on a large proportion of cells. Targeting any of CTLA-4, OX40 or GITR individually generated effective responses against mesothelioma. Furthermore, the combination of αCTLA-4 and αOX40 was synergistic, with an increase in complete tumour regressions from 20% to 80%. Other combinations did not synergise to enhance treatment outcomes. Finally, an early pattern in T cell response was predictive of response, with activation status and ICP receptor expression profile of T effector cells harvested from tumour and dLN correlating with response to immunotherapy. Taken together, these data demonstrate that combination ICPB can work synergistically to induce strong, durable immunity against mesothelioma in an animal model.