Asunto(s)
Epítopos de Linfocito T/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoterapia/métodos , Neoplasias/terapia , Animales , Línea Celular Tumoral , Biología Computacional/métodos , Internet , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Neoplasias/genética , Neoplasias/inmunología , Programas InformáticosRESUMEN
Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide-MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103-397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4-18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0-43%, n = 6). Thus, frameshift indels are equally represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.
Asunto(s)
Antígenos de Neoplasias/inmunología , Carcinoma de Células Renales/inmunología , Neoplasias Renales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos T/inmunología , Antígenos de Neoplasias/genética , Carcinoma de Células Renales/genética , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Mutación del Sistema de Lectura , Humanos , Neoplasias Renales/genética , Mutación PuntualRESUMEN
Human endogenous retroviruses (HERV) form a substantial part of the human genome, but mostly remain transcriptionally silent under strict epigenetic regulation, yet can potentially be reactivated by malignant transformation or epigenetic therapies. Here, we evaluate the potential for T cell recognition of HERV elements in myeloid malignancies by mapping transcribed HERV genes and generating a library of 1169 potential antigenic HERV-derived peptides predicted for presentation by 4 HLA class I molecules. Using DNA barcode-labeled MHC-I multimers, we find CD8+ T cell populations recognizing 29 HERV-derived peptides representing 18 different HERV loci, of which HERVH-5, HERVW-1, and HERVE-3 have more profound responses; such HERV-specific T cells are present in 17 of the 34 patients, but less frequently in healthy donors. Transcriptomic analyses reveal enhanced transcription of the HERVs in patients; meanwhile DNA-demethylating therapy causes a small and heterogeneous enhancement in HERV transcription without altering T cell recognition. Our study thus uncovers T cell recognition of HERVs in myeloid malignancies, thereby implicating HERVs as potential targets for immunotherapeutic therapies.
Asunto(s)
Retrovirus Endógenos/genética , Neoplasias Hematológicas/virología , Linfocitos T/metabolismo , Linfocitos T/virología , Linfocitos T CD8-positivos , Epigénesis Genética , Epítopos de Linfocito T , Perfilación de la Expresión Génica , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Inmunoterapia , Monitorización Inmunológica , Células Mieloides , NeoplasiasRESUMEN
Narcolepsy Type 1 (NT1) is a neurological sleep disorder, characterized by the loss of hypocretin/orexin signaling in the brain. Genetic, epidemiological and experimental data support the hypothesis that NT1 is a T-cell-mediated autoimmune disease targeting the hypocretin producing neurons. While autoreactive CD4+ T cells have been detected in patients, CD8+ T cells have only been examined to a minor extent. Here we detect CD8+ T cells specific toward narcolepsy-relevant peptides presented primarily by NT1-associated HLA types in the blood of 20 patients with NT1 as well as in 52 healthy controls, using peptide-MHC-I multimers labeled with DNA barcodes. In healthy controls carrying the disease-predisposing HLA-DQB1*06:02 allele, the frequency of autoreactive CD8+ T cells was lower as compared with both NT1 patients and HLA-DQB1*06:02-negative healthy individuals. These findings suggest that a certain level of CD8+ T-cell reactivity combined with HLA-DQB1*06:02 expression is important for NT1 development.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Cadenas beta de HLA-DQ/genética , Narcolepsia/inmunología , Orexinas/inmunología , Péptidos/inmunología , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Narcolepsia/genética , Neuronas/metabolismo , Orexinas/metabolismoRESUMEN
Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.
Asunto(s)
Antígenos/genética , Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Genes MHC Clase I/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Inmunoensayo/métodos , Células Cultivadas , Código de Barras del ADN Taxonómico , Genes MHC Clase I/inmunología , Humanos , Péptidos/genética , Péptidos/inmunología , Multimerización de Proteína/genética , Multimerización de Proteína/inmunología , Coloración y Etiquetado/métodosRESUMEN
BACKGROUND: A substantial proportion of cancer cases present with a metastatic tumor and require further testing to determine the primary site; many of these are never fully diagnosed and remain cancer of unknown primary origin (CUP). It has been previously demonstrated that the somatic point mutations detected in a tumor can be used to identify its site of origin with limited accuracy. We hypothesized that higher accuracy could be achieved by a classification algorithm based on the following feature sets: 1) the number of nonsynonymous point mutations in a set of 232 specific cancer-associated genes, 2) frequencies of the 96 classes of single-nucleotide substitution determined by the flanking bases, and 3) copy number profiles, if available. METHODS: We used publicly available somatic mutation data from the COSMIC database to train random forest classifiers to distinguish among those tissues of origin for which sufficient data was available. We selected feature sets using cross-validation and then derived two final classifiers (with or without copy number profiles) using 80 % of the available tumors. We evaluated the accuracy using the remaining 20 %. For further validation, we assessed accuracy of the without-copy-number classifier on three independent data sets: 1669 newly available public tumors of various types, a cohort of 91 breast metastases, and a set of 24 specimens from 9 lung cancer patients subjected to multiregion sequencing. RESULTS: The cross-validation accuracy was highest when all three types of information were used. On the left-out COSMIC data not used for training, we achieved a classification accuracy of 85 % across 6 primary sites (with copy numbers), and 69 % across 10 primary sites (without copy numbers). Importantly, a derived confidence score could distinguish tumors that could be identified with 95 % accuracy (32 %/75 % of tumors with/without copy numbers) from those that were less certain. Accuracy in the independent data sets was 46 %, 53 % and 89 % respectively, similar to the accuracy expected from the training data. CONCLUSIONS: Identification of primary site from point mutation and/or copy number data may be accurate enough to aid clinical diagnosis of cancers of unknown primary origin.