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The ecosystem of brain tumors is considered immunosuppressed, but our current knowledge may be incomplete. Here we analyzed clinical cell and tissue specimens derived from patients presenting with glioblastoma or nonmalignant intracranial disease to report that the cranial bone (CB) marrow, in juxtaposition to treatment-naive glioblastoma tumors, harbors active lymphoid populations at the time of initial diagnosis. Clinical and anatomical imaging, single-cell molecular and immune cell profiling and quantification of tumor reactivity identified CD8+ T cell clonotypes in the CB that were also found in the tumor. These were characterized by acute and durable antitumor response rooted in the entire T cell developmental spectrum. In contrast to distal bone marrow, the CB niche proximal to the tumor showed increased frequencies of tumor-reactive CD8+ effector types expressing the lymphoid egress marker S1PR1. In line with this, cranial enhancement of CXCR4 radiolabel may serve as a surrogate marker indicating focal association with improved progression-free survival. The data of this study advocate preservation and further exploitation of these cranioencephalic units for the clinical care of glioblastoma.
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H3K27M, a driver mutation with T and B cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient treated with an H3K27M peptide vaccine who subsequently entered complete remission. The vaccine robustly expanded class II human leukocyte antigen (HLA)-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their complementarity-determining region 3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ and HLA-DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.
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Glioma , Linfócitos T , Humanos , Antígenos HLA-DR , Vacinação , Glioma/genética , EpitoposRESUMO
Effective, unbiased, high-throughput methods to functionally identify both class II and class I HLA-presented T cell epitopes and their cognate T cell receptors (TCRs) are essential for and prerequisite to diagnostic and therapeutic applications, yet remain underdeveloped. Here, we present T-FINDER [T cell Functional Identification and (Neo)-antigen Discovery of Epitopes and Receptors], a system to rapidly deconvolute CD4 and CD8 TCRs and targets physiologically processed and presented by an individual's unmanipulated, complete human leukocyte antigen (HLA) haplotype. Combining a highly sensitive TCR signaling reporter with an antigen processing system to overcome previously undescribed limitations to target expression, T-FINDER both robustly identifies unknown peptide:HLA ligands from antigen libraries and rapidly screens and functionally validates the specificity of large TCR libraries against known or predicted targets. To demonstrate its capabilities, we apply the platform to multiple TCR-based applications, including diffuse midline glioma, celiac disease, and rheumatoid arthritis, providing unique biological insights and showcasing T-FINDER's potency and versatility.
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Antígenos de Histocompatibilidade Classe I , Receptores de Antígenos de Linfócitos T , Humanos , Ligantes , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos HLA , Antígenos de Histocompatibilidade Classe IIRESUMO
BACKGROUND: Neuroligin 4 X-linked (NLGN4X) harbors a human leukocyte antigen (HLA)-A*02-restricted tumor-associated antigen, overexpressed in human gliomas, that was found to induce specific cytotoxic T cell responses following multi-peptide vaccination in patients with newly diagnosed glioblastoma. METHODS: T cell receptor (TCR) discovery was performed using droplet-based single-cell TCR sequencing of NLGN4X-tetramer-sorted T cells postvaccination. The identified TCR was delivered to Jurkat T cells and primary human T cells (NLGN4X-TCR-T). Functional profiling of NLGN4X-TCR-T was performed by flow cytometry and cytotoxicity assays. Therapeutic efficacy of intracerebroventricular NLGN4X-TCR-T was assessed in NOD scid gamma (NSG) major histocompatibility complex (MHC) I/II knockout (KO) (NSG MHC I/II KO) mice bearing NLGN4X-expressing experimental gliomas. RESULTS: An HLA-A*02-restricted vaccine-induced T cell receptor specifically binding NLGN4X131-139 was applied for preclinical therapeutic use. Reactivity, cytotoxicity, and polyfunctionality of this NLGN4X-specific TCR are demonstrated in various cellular models. Intracerebroventricular administration of NLGN4X-TCR-T prolongs survival and leads to an objective response rate of 44.4% in experimental glioma-bearing NSG MHC I/II KO mice compared to 0.0% in control groups. CONCLUSION: NLGN4X-TCR-T demonstrate efficacy in a preclinical glioblastoma model. On a global scale, we provide the first evidence for the therapeutic retrieval of vaccine-induced human TCRs for the off-the-shelf treatment of glioblastoma patients.Keywords cell therapy | glioblastoma | T cell receptor | tumor antigen.
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Vacinas Anticâncer , Glioblastoma , Camundongos , Animais , Humanos , Glioblastoma/genética , Glioblastoma/terapia , Vacinas Anticâncer/uso terapêutico , Vacinas de Subunidades Antigênicas , Receptores de Antígenos de Linfócitos T , Linfócitos T , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular NeuronaisRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1011011.].
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Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms in several organisms that inhabit the shoreline suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.
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Fatores de Transcrição ARNTL , Relógios Circadianos , Isópodes , Animais , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Proteínas CLOCK/genética , Drosophila/metabolismo , Proteínas de Drosophila , Isópodes/genética , Isópodes/metabolismo , Mamíferos/metabolismo , NataçãoRESUMO
Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M+ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M+ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician's discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4+ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M+ diffuse midline glioma.
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Neoplasias Encefálicas , Glioma , Vacinas , Humanos , Adulto , Animais , Camundongos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Histonas/genética , Glioma/genética , Glioma/terapia , Mutação/genéticaRESUMO
BACKGROUND: Representative data describing serious infections in children aged ≥5 years and adults in Africa are limited. METHODS: We conducted population-based surveillance for pneumonia, meningitis, and septicemia in a demographic surveillance area in The Gambia between 12 May 2008 and 31 December 2015. We used standardized criteria to identify, diagnose, and investigate patients aged ≥5 years using conventional microbiology and radiology. RESULTS: We enrolled 1638 of 1657 eligible patients and investigated 1618. Suspected pneumonia, septicemia, or meningitis was diagnosed in 1392, 135, and 111 patients, respectively. Bacterial pathogens from sterile sites were isolated from 105 (7.5%) patients with suspected pneumonia, 11 (8.1%) with suspected septicemia, and 28 (25.2%) with suspected meningitis. Streptococcus pneumoniae (n = 84), Neisseria meningitidis (n = 16), and Staphylococcus aureus (n = 15) were the most common pathogens. Twenty-eight (1.7%) patients died in hospital and 40 (4.1%) died during the 4 months after discharge. Thirty postdischarge deaths occurred in patients aged ≥10 years with suspected pneumonia. The minimum annual incidence was 133 cases per 100 000 person-years for suspected pneumonia, 13 for meningitis, 11 for septicemia, 14 for culture-positive disease, and 46 for radiological pneumonia. At least 2.7% of all deaths in the surveillance area were due to suspected pneumonia, meningitis, or septicemia. CONCLUSIONS: Pneumonia, meningitis, and septicemia in children aged ≥5 years and adults in The Gambia are responsible for significant morbidity and mortality. Many deaths occur after hospital discharge and most cases are culture negative. Improvements in prevention, diagnosis, inpatient, and follow-up management are urgently needed.
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Meningites Bacterianas , Meningite , Pneumonia , Sepse , Criança , Humanos , Adulto , Lactente , Adolescente , Gâmbia/epidemiologia , Assistência ao Convalescente , Alta do Paciente , Meningites Bacterianas/epidemiologiaRESUMO
Background: Glioblastoma (GBM) is characterized by low numbers of glioma-infiltrating lymphocytes (GIL) with a dysfunctional phenotype. Whether this dysfunctional phenotype is fixed or can be reversed upon ex vivo culturing is poorly understood. The aim of this study was to assess T cell receptor (TCR)-dynamics and -specificities as well as determinants of in vitro GIL expansion by sequencing-based technologies and functional assays to explore the use of GIL for cell therapy. Methods: By means of flow cytometry, T cell functionality in GIL cultures was assessed from 9 GBM patients. TCR beta sequencing (TCRB-seq) was used for TCR repertoire profiling before and after in vitro expansion. Microarrays or RNA sequencing (RNA-seq) were performed from 6 micro-dissected GBM tissues and healthy brain RNA to assess the individual expression of GBM-associated antigens (GAA). GIL reactivity against in silico predicted tumor-associated antigens (TAA) and patient-individual GAA was assessed by ELISpot assay. Combined ex vivo single cell (sc)TCR-/RNA-seq and post-expansion TCRB-seq were used to evaluate transcriptional signatures that determine GIL expansion. Results: Human GIL regains cellular fitness upon in vitro expansion. Profound TCR dynamics were observed during in vitro expansion and only in one of six GIL cultures, reactivity against GAA was observed. Paired ex vivo scTCR/RNA-seq and TCRB-seq revealed predictive transcriptional signatures that determine GIL expansion. Conclusions: Profound TCR repertoire dynamics occur during GIL expansion. Ex vivo transcriptional T cell states determine expansion capacity in gliomas. Our observation has important implications for the use of GIL for cell therapy including genetic manipulation to maintain both antigen specificity and expansion capacity.
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The clock neurons of the fruit fly Drosophila melanogaster have become a useful model for expressing misfolded protein aggregates that accumulate in several human neurodegenerative diseases. One advantage of such an approach is that the behavioral effects can be readily quantified on circadian locomotor rhythms, sleep or activity levels via automated, highly reliable and objective procedures. Therefore, a rapid assay is required to visualize whether these neurons develop aggregates. Here we describe a modified immunoblot method, agarose gel electrophoresis (AGERA) that has been optimized for resolving aggregates from fly clock neurons.
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Proteínas de Drosophila , Drosophila melanogaster , Animais , Ritmo Circadiano/fisiologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Eletroforese em Gel de Ágar , Neurônios/metabolismoRESUMO
After solid-organ transplantation, reactivation of the cytomegalovirus (CMV) is often observed in seronegative patients and associated with a high risk of disease and mortality. CMV-specific T cells can prevent CMV reactivation. In a phase 1 trial, CMV-seronegative patients with end-stage renal disease listed for kidney transplantation were subjected to CMV phosphoprotein 65 (CMVpp65) peptide vaccination and further investigated for T-cell responses. To this end, CMV-specific CD8+ T cells were characterized by bulk T-cell-receptor (TCR) repertoire sequencing and combined single-cell RNA and TCR sequencing. In patients mounting an immune response to the vaccine, a common SYE(N)E TCR motif known to bind CMVpp65 was detected. CMV-peptide-vaccination-responder patients had TCR features distinct from those of non-responders. In a non-responder patient, a monoclonal inflammatory T-cell response was detected upon CMV reactivation. The identification of vaccine-induced CMV-reactive TCRs motifs might facilitate the development of cellular therapies for patients wait-listed for kidney transplantation.
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Infecções por Citomegalovirus/prevenção & controle , Falência Renal Crônica/terapia , Receptores de Antígenos de Linfócitos T/genética , Proteínas da Matriz Viral/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Ensaios Clínicos Fase I como Assunto , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/administração & dosagem , Vacinas contra Citomegalovirus/imunologia , Humanos , Falência Renal Crônica/imunologia , Transplante de Rim , Análise de Sequência de RNA , Imagem Individual de Molécula , Proteínas da Matriz Viral/imunologiaRESUMO
Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with evidence suggesting they represent a heterogeneous population. This study summarises the prognostic role of all proteins characterised in CAFs with immunohistochemistry in non-small cell lung cancer thus far. The functions of these proteins in cellular processes crucial to CAFs are also analysed. Five databases were searched to extract survival outcomes from published studies and statistical techniques, including a novel method, used to capture missing values from the literature. A total of 26 proteins were identified, 21 of which were combined into 7 common cellular processes key to CAFs. Quality assessments for sensitivity analyses were carried out for each study using the REMARK criteria whilst publication bias was assessed using funnel plots. Random effects models consistently identified the expression of podoplanin (Overall Survival (OS)/Disease-specific Survival (DSS), univariate analysis HR 2.25, 95% CIs 1.80-2.82) and α-SMA (OS/DSS, univariate analysis HR 2.11, 95% CIs 1.18-3.77) in CAFs as highly prognostic regardless of outcome measure or analysis method. Moreover, proteins involved in maintaining and generating the CAF phenotype (α-SMA, TGF-ß and p-Smad2) proved highly significant after sensitivity analysis (HR 2.74, 95% CIs 1.74-4.33) supporting attempts at targeting this pathway for therapeutic benefit.
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Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Actinas/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Fibroblastos/metabolismo , Heterogeneidade Genética , Humanos , Neoplasias Pulmonares/patologia , Fenótipo , Prognóstico , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
BACKGROUND: Meta-analyses of studies evaluating survival (time-to-event) outcomes are a powerful technique to assess the strength of evidence for a given disease or treatment. However, these studies rely on the adequate reporting of summary statistics in the source articles to facilitate further analysis. Unfortunately, many studies, especially within the field of prognostic research do not report such statistics, making secondary analyses challenging. Consequently, methods have been developed to infer missing statistics from the commonly published Kaplan-Meier (KM) plots but are liable to error especially when the published number at risk is not included. METHODS: We therefore developed a method using non-linear optimisation (nlopt) that only requires the KM plot and the commonly published P value to better estimate the underlying censoring pattern. We use this information to then calculate the natural logarithm of the hazard ratio (ln (HR)) and its variance (var) ln (HR), statistics important for meta-analyses. RESULTS: We compared this method to the Parmar method which also does not require the number at risk to be published. In a validation set consisting of 13 KM studies, a statistically significant improvement in calculating ln (HR) when using an exact P value was obtained (mean absolute error 0.014 vs 0.077, P = 0.003). Thus, when the true HR has a value of 1.5, inference of the HR using the proposed method would set limits between 1.49/1.52, an improvement of the 1.39/1.62 limits obtained using the Parmar method. We also used Monte Carlo simulations to establish recommendations for the number and positioning of points required for the method. CONCLUSION: The proposed non-linear optimisation method is an improvement on the existing method when only a KM plot and P value are included and as such will enhance the accuracy of meta-analyses performed for studies analysing time-to-event outcomes. The nlopt source code is available, as is a simple-to-use web implementation of the method.
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Projetos de Pesquisa , Humanos , Estimativa de Kaplan-Meier , Metanálise como Assunto , Prognóstico , Modelos de Riscos Proporcionais , Análise de SobrevidaRESUMO
Catabolism of the essential amino acid tryptophan is a key metabolic pathway contributing to the immunosuppressive tumor microenvironment and therefore a viable drug target for cancer immunotherapy. In addition to the rate-limiting enzyme indoleamine-2,3-dioxygenase-1 (IDO1), tryptophan catabolism via tryptophan-2,3-dioxygenase (TDO2) is a feature of many tumors, particularly malignant gliomas. The pathways regulating TDO2 in tumors are poorly understood; using unbiased promoter and gene expression analyses, we identify a distinct CCAAT/enhancer-binding protein ß (C/EBPß) binding site in the promoter of TDO2 essential for driving constitutive TDO2 expression in glioblastoma cells. Using The Cancer Genome Atlas (TCGA) data, we find that C/EBPß expression is correlated with TDO2, and both are enriched in malignant glioma of the mesenchymal subtype and associated with poor patient outcome. We determine that TDO2 expression is sustained mainly by the LAP isoform of CEBPB and interleukin-1ß, which activates TDO2 via C/EBPß in a mitogen-activated protein kinase (MAPK) kinase-dependent fashion. In summary, we provide evidence for a novel regulatory and therapeutically targetable pathway of immunosuppressive tryptophan degradation in a subtype of glioblastoma with a particularly poor prognosis.
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Biomarcadores Tumorais/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Glioblastoma/metabolismo , Regiões Promotoras Genéticas/genética , Triptofano Oxigenase/metabolismo , Biomarcadores Tumorais/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Carcinogênese , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Tolerância Imunológica , Interleucina-1beta/metabolismo , Terapia de Alvo Molecular , Transdução de Sinais , Triptofano Oxigenase/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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From 1980 to 1991, Kyriacou, Hall, and collaborators (K&H) reported that the Drosophila melanogaster courtship song has a 1-min cycle in the length of mean interpulse intervals (IPIs) that is modulated by circadian rhythm period mutations. In 2014, Stern failed to replicate these results using a fully automated method for detecting song pulses. Manual annotation of Stern's song records exposed a ~50% error rate in detection of IPIs, but the corrected data revealed period-dependent IPI cycles using a variety of statistical methods. In 2017, Stern et al. dismissed the sine/cosine method originally used by K&H to detect significant cycles, claiming that randomized songs showed as many significant values as real data using cosinor analysis. We first identify a simple mathematical error in Stern et al.'s cosinor implementation that invalidates their critique of the method. Stern et al. also concluded that although the manually corrected wild-type and perL mutant songs show similar periods to those observed by K&H, each song is usually not significantly rhythmic by the Lomb-Scargle (L-S) periodogram, so any genotypic effect simply reflects "noise." Here, we observe that L-S is extremely conservative compared with 3 other time-series analyses in assessing the significance of rhythmicity, both for conventional locomotor activity data collected in equally spaced time bins and for unequally spaced song records. Using randomization of locomotor and song data to generate confidence limits for L-S instead of the theoretically derived values, we find that L-S is now consistent with the other methods in determining significant rhythmicity in locomotor and song records and that it confirms period-dependent song cycles. We conclude that Stern and colleagues' failure to identify song cycles stems from the limitations of automated methods in accurately reflecting song parameters, combined with the use of an overly stringent method to discriminate rhythmicity in courtship songs.
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Ritmo Circadiano , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Comportamento Sexual Animal , Animais , Corte , Genótipo , Modelos Teóricos , Música , Mutação , Proteínas Circadianas Period/genética , Erro Científico ExperimentalRESUMO
The high-throughput discovery of T cell receptors (TCR) enables us to follow the longitudinal course of tumor disease, to monitor immunotherapy-related repertoire alterations, and exploit TCRs for therapeutic use. Here we discuss state of the art and future experimental and computational strategies to dissect the hypervariable world of TCRs and their corresponding antigens in the context of cancer immunotherapy.
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Mapeamento de Epitopos/métodos , Ensaios de Triagem em Larga Escala/métodos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Biologia Computacional/métodos , Mapeamento de Epitopos/instrumentação , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
The speed of T cell receptor (TCR) discovery has been revolutionized by barcode-based TCR sequencing approaches that allow the reconstitution of a T cell's paired alpha and beta TCR chain, and the process of TCR discovery promises to become ever faster and cheaper with the continuing development single cell analysis techniques. This technological progress has generated an urgent need to develop efficient TCR validation platforms for the rapid and safe clinical translation of TCRs into therapeutic agents. Whereas much attention has in the past focused on CD8-positive cytotoxic T cells recognizing MHC class I presented epitopes, the increasing demand to validate TCRs expressed on neoepitope-reactive CD4 T cells requires the implementation of large-scale T cell activation-based readout assays to complement existing multimer and cytotoxicity-based assays. Here, we present commonly used TCR validation assays, and include detailed guidance on TCR synthesis, delivery, and appropriate experimental control TCRs. We also comment on upcoming methods that hold promise for further speeding the process of TCR validation, hastening the translation of TCRs from the laboratory into the clinic.