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Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.
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Carcinoma de Células Renais , Neoplasias Renais , Animais , Humanos , Actinas , Linfócitos T CD8-Positivos , Citoesqueleto , Semaforina-3A/genéticaRESUMO
Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.
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Antígenos CD , Apirase , Cadeias alfa de Integrinas , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Animais , Humanos , Camundongos , Antígenos CD/metabolismo , Antígenos CD/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Cadeias alfa de Integrinas/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.
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Proteínas Tirosina Fosfatases , Transdução de Sinais , Proteínas Tirosina Fosfatases/metabolismo , Antígenos CD28 , Receptores ImunológicosRESUMO
Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.
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Melanoma , Linfócitos T , Humanos , Células Apresentadoras de Antígenos , Melanoma/terapia , Imunoterapia , Imunoterapia AdotivaRESUMO
Group A Streptococcus (GAS) infection is associated with multiple clinical sequelae, including different subtypes of psoriasis. Such post-streptococcal disorders have been long known but are largely unexplained. CD1a is expressed at constitutively high levels by Langerhans cells and presents lipid antigens to T cells, but the potential relevance to GAS infection has not been studied. Here, we investigated whether GAS-responsive CD1a-restricted T cells contribute to the pathogenesis of psoriasis. Healthy individuals had high frequencies of circulating and cutaneous GAS-responsive CD4+ and CD8+ T cells with rapid effector functions, including the production of interleukin-22 (IL-22). Human skin and blood single-cell CITE-seq analyses of IL-22-producing T cells showed a type 17 signature with proliferative potential, whereas IFN-γ-producing T cells displayed cytotoxic T lymphocyte characteristics. Furthermore, individuals with psoriasis had significantly higher frequencies of circulating GAS-reactive T cells, enriched for markers of activation, cytolytic potential, and tissue association. In addition to responding to GAS, subsets of expanded GAS-reactive T cell clones/lines were found to be autoreactive, which included the recognition of the self-lipid antigen lysophosphatidylcholine. CD8+ T cell clones/lines produced cytolytic mediators and lysed infected CD1a-expressing cells. Furthermore, we established cutaneous models of GAS infection in a humanized CD1a transgenic mouse model and identified enhanced and prolonged local and systemic inflammation, with resolution through a psoriasis-like phenotype. Together, these findings link GAS infection to the CD1a pathway and show that GAS infection promotes the proliferation and activation of CD1a-autoreactive T cells, with relevance to post-streptococcal disease, including the pathogenesis and treatment of psoriasis.
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Linfócitos T CD8-Positivos , Psoríase , Humanos , Camundongos , Animais , Pele , Inflamação/patologia , Streptococcus pyogenes , Camundongos Transgênicos , LipídeosRESUMO
There is an increasing number of immune-checkpoint inhibitors being developed and approved for cancer immunotherapy. Most of the new therapies aim to reactivate tumour-infiltrating T cells, which are responsible for tumour killing. However, in many tumours, the most abundant infiltrating immune cells are macrophages and myeloid cells, which can be tumour-promoting as well as tumouricidal. CD200R was initially identified as a myeloid-restricted, inhibitory immune receptor, but was subsequently also found to be expressed within the lymphoid lineage. Using a mouse model humanised for CD200R and PD-1, we investigated the potential of a combination therapy comprising nivolumab, a clinically approved PD-1 blocking antibody, and OX108, a CD200R antagonist. We produced nivolumab as a murine IgG1 antibody and validated its binding activity in vitro as well as ex vivo. We then tested the combination therapy in the immunogenic colorectal cancer model MC38 as well as the PD-1 blockade-resistant lung cancer model LLC1, which is characterised by a large number of infiltrating myeloid cells, making it an attractive target for CD200R blockade. No significant improvement of overall survival was found in either model, compared to nivolumab mIgG1 monotherapy. There was a trend for more complete responses in the MC38 model, but investigation of the infiltrating immune cells failed to account for this. Importantly, MC38 cells expressed low levels of CD200, whereas LLC1 cells were CD200-negative. Further investigation of CD200R-blocking antibodies in tumours expressing high levels of CD200 could be warranted.
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Virus-based tumour vaccines offer many advantages compared to other antigen-delivering systems. They generate concerted innate and adaptive immune response, and robust CD8+ T cell responses. We engineered a non-replicating pseudotyped influenza virus (S-FLU) to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). Intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1-specific CD8+ T cell response in lungs and spleen that resulted in the regression of NY-ESO-1-expressing lung tumour and subcutaneous tumour, respectively. Combined administration with anti-PD-1 antibody, NY-ESO-1 S-FLU virus augmented the tumour protection by reducing the tumour metastasis. We propose that the antigen delivery through S-FLU is highly efficient in inducing antigen-specific CD8+ T cell response and protection against tumour development in combination with PD-1 blockade.
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Inibidores de Checkpoint Imunológico , Orthomyxoviridae , Masculino , Camundongos , Animais , Antígenos de Neoplasias , Proteínas de Membrana , Imunização , Anticorpos , Linfócitos T CD8-PositivosRESUMO
BACKGROUND: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Despite maximal treatment, median survival remains dismal at 14-24 months. Immunotherapies, such as checkpoint inhibition, have revolutionized management of some cancers but have little benefit for GBM patients. This is, in part, due to the low mutational and neoantigen burden in this immunogenically "cold" tumor. METHODS: U87MG and patient-derived cell lines were treated with 5-aza-2'-deoxycytidine (DAC) and underwent whole-exome and transcriptome sequencing. Cell lines were then subjected to cellular assays with neoantigen and cancer testis antigen (CTA) specific T cells. RESULTS: We demonstrate that DAC increases neoantigen and CTA mRNA expression through DNA hypomethylation. This results in increased neoantigen presentation by MHC class I in tumor cells, leading to increased neoantigen- and CTA-specific T-cell activation and killing of DAC-treated cancer cells. In addition, we show that patients have endogenous cancer-specific T cells in both tumor and blood, which show increased tumor-specific activation in the presence of DAC-treated cells. CONCLUSIONS: Our work shows that DAC increases GBM immunogenicity and consequent susceptibility to T-cell responses in vitro. Our results support a potential use of DAC as a sensitizing agent for immunotherapy.
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Glioblastoma , Adulto , Masculino , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Decitabina/farmacologia , Antígenos de Neoplasias/genética , Linfócitos T , Testículo , Linhagem Celular TumoralRESUMO
INTRODUCTION: The undiminished need for more effective cancer treatments stimulates the development of novel cancer immunotherapy candidates. The archetypical cancer immunotherapy would induce robust, targeted and long-lasting immune responses while simultaneously circumventing immunosuppression in the tumour microenvironment. For this purpose, we developed a novel immunomodulatory nanomedicine: PRECIOUS-01. As a PLGA-based nanocarrier, PRECIOUS-01 encapsulates a tumour antigen (NY-ESO-1) and an invariant natural killer T cell activator to target and augment specific antitumour immune responses in patients with NY-ESO-1-expressing advanced cancers. METHODS AND ANALYSIS: This open-label, first-in-human, phase I dose-escalation trial investigates the safety, tolerability and immune-modulatory activity of increasing doses of PRECIOUS-01 administered intravenously in subjects with advanced NY-ESO-1-expressing solid tumours. A total of 15 subjects will receive three intravenous infusions of PRECIOUS-01 at a 3-weekly interval in three dose-finding cohorts. The trial follows a 3+3 design for the dose-escalation steps to establish a maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D). Depending on the toxicity, the two highest dosing cohorts will be extended to delineate the immune-related parameters as a readout for pharmacodynamics. Subjects will be monitored for safety and the occurrence of dose-limiting toxicities. If the MTD is not reached in the planned dose-escalation cohorts, the RP2D will be based on the observed safety and immune-modulatory activity as a pharmacodynamic parameter supporting the RP2D. The preliminary efficacy will be evaluated as an exploratory endpoint using the best overall response rate, according to Response Evaluation Criteria in Solid Tumors V.1.1. ETHICS AND DISSEMINATION: The Dutch competent authority (CCMO) reviewed the trial application and the medical research ethics committee (CMO Arnhem-Nijmegen) approved the trial under registration number NL72876.000.20. The results will be disseminated via (inter)national conferences and submitted for publication to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04751786.
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Nanopartículas , Neoplasias , Antígenos de Neoplasias , Ensaios Clínicos Fase I como Assunto , Humanos , Imunidade , Dose Máxima Tolerável , Nanopartículas/efeitos adversos , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor-endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1-specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies. IMPLICATIONS: ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.
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Neoplasias da Mama/genética , Terapia de Imunossupressão/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Microambiente TumoralRESUMO
BACKGROUND: NY-ESO-1 is a tumor-specific, highly immunogenic, human germ cell antigen of the MAGE-1 family that is a promising vaccine and cell therapy candidate in clinical trial development. The mouse genome does not encode an NY-ESO-1 homolog thereby not subjecting transgenic T-cells to thymic tolerance mechanisms that might impair in-vivo studies. We hypothesized that an NY-ESO-1 T cell receptor (TCR) transgenic mouse would provide the unique opportunity to study avidity of TCR response against NY-ESO-1 for tumor vaccine and cellular therapy development against this clinically relevant and physiological human antigen. METHODS: To study in vitro and in vivo the requirements for shaping an effective T cell response against the clinically relevant NY-ESO-1, we generated a C57BL/6 HLA-A*0201 background TCR transgenic mouse encoding the 1G4 TCR specific for the human HLA-A2 restricted, NY-ESO-1157-165 SLLMWITQC (9C), initially identified in an NY-ESO-1 positive melanoma patient. RESULTS: The HLA-A*0201 restricted TCR was positively selected on both CD4+ and CD8+ cells. Mouse 1G4 T cells were not activated by endogenous autoimmune targets or a large library of non-cognate viral antigens. In contrast, their activation by HLA-A2 NY-ESO-1157-165 complexes was evident by proliferation, CD69 upregulation, interferon-γ production, and interleukin-2 production, and could be tuned using a twofold higher affinity altered peptide ligand, NY-ESO-1157-165V. NY-ESO-1157-165V recombinant vaccination of syngeneic mice adoptively transferred with m1G4 CD8+ T cells controlled tumor growth in vivo. 1G4 transgenic mice suppressed growth of syngeneic methylcholanthrene (MCA) induced HHD tumor cells expressing the full-length human NY-ESO-1 protein but not MCA HHD tumor cells lacking NY-ESO-1. CONCLUSIONS: The 1G4 TCR mouse model for the physiological human TCR against the clinically relevant antigen, NY-ESO-1, is a valuable tool with the potential to accelerate clinical development of NY-ESO-1-targeted T-cell and vaccine therapies.
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Antígeno HLA-A2/metabolismo , Proteínas de Neoplasias/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Receptores de Antígenos de Linfócitos T/genética , Timoma/tratamento farmacológico , Neoplasias do Timo/tratamento farmacológico , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Timoma/genética , Timoma/imunologia , Neoplasias do Timo/genética , Neoplasias do Timo/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPß binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPß, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPß binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.
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Arginina/metabolismo , Cromatina/metabolismo , Evasão da Resposta Imune/genética , Neoplasias/genética , Linfócitos T/metabolismo , Animais , HumanosRESUMO
Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.
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Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Portadores de Fármacos/farmacologia , Nanopartículas/uso terapêutico , Proteínas de Neoplasias/farmacologia , Fragmentos de Peptídeos/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Portadores de Fármacos/química , Humanos , Nanopartículas/química , Proteínas de Neoplasias/química , Fragmentos de Peptídeos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/químicaRESUMO
BACKGROUND: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. METHODS: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. FINDINGS: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. CONCLUSIONS: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders. FUNDING: Medical Research Council, UK.
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Deficiências de Ferro , Distúrbios do Metabolismo do Ferro , Animais , Hepcidinas/genética , Humanos , Imunidade Humoral , Ferro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Suínos , VacinaçãoRESUMO
Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
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Afinidade de Anticorpos , Arginase/química , Anticorpos de Cadeia Única/química , Regulação Alostérica , Cristalografia por Raios X , HumanosRESUMO
Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.
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BACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. RESULTS: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.
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Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias da Próstata/terapia , Hipofracionamento da Dose de Radiação , Microambiente Tumoral , Animais , Antígeno B7-H1/análise , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologiaRESUMO
Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8+ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.
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Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Epitopos/imunologia , Proteínas Mitocondriais/imunologia , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Neoplasias/terapiaRESUMO
Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.