RESUMO
Acute myeloid leukemia (AML) has not benefited from innovative immunotherapies, mainly because of the lack of actionable immune targets. Using an original proteogenomic approach, we analyzed the major histocompatibility complex class I (MHC class I)-associated immunopeptidome of 19 primary AML samples and identified 58 tumor-specific antigens (TSAs). These TSAs bore no mutations and derived mainly (86%) from supposedly non-coding genomic regions. Two AML-specific aberrations were instrumental in the biogenesis of TSAs, intron retention, and epigenetic changes. Indeed, 48% of TSAs resulted from intron retention and translation, and their RNA expression correlated with mutations of epigenetic modifiers (e.g., DNMT3A). AML TSA-coding transcripts were highly shared among patients and were expressed in both blasts and leukemic stem cells. In AML patients, the predicted number of TSAs correlated with spontaneous expansion of cognate T cell receptor clonotypes, accumulation of activated cytotoxic T cells, immunoediting, and improved survival. These TSAs represent attractive targets for AML immunotherapy.
Assuntos
Epitopos/genética , Antígenos de Histocompatibilidade Classe I/genética , Leucemia Mieloide Aguda/genética , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular , Epigênese Genética/genética , Epigênese Genética/imunologia , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação/genética , Mutação/imunologia , Células-Tronco Neoplásicas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Colorectal cancer is the second leading cause of cancer death worldwide, and the incidence of this disease is expected to increase as global socioeconomic changes occur. Immune checkpoint inhibition therapy is effective in treating a minority of colorectal cancer tumors; however, microsatellite stable tumors do not respond well to this treatment. Emerging cancer immunotherapeutic strategies aim to activate a cytotoxic T cell response against tumor-specific antigens, presented exclusively at the cell surface of cancer cells. These antigens are rare and are most effectively identified with a mass spectrometry-based approach, which allows the direct sampling and sequencing of these peptides. Although the few tumor-specific antigens identified to date are derived from coding regions of the genome, recent findings indicate that a large proportion of tumor-specific antigens originate from allegedly noncoding regions. Here, we employed a novel proteogenomic approach to identify tumor antigens in a collection of colorectal cancer-derived cell lines and biopsy samples consisting of matched tumor and normal adjacent tissue. The generation of personalized cancer databases paired with mass spectrometry analyses permitted the identification of more than 30,000 unique MHC I-associated peptides. We identified 19 tumor-specific antigens in both microsatellite stable and unstable tumors, over two-thirds of which were derived from noncoding regions. Many of these peptides were derived from source genes known to be involved in colorectal cancer progression, suggesting that antigens from these genes could have therapeutic potential in a wide range of tumors. These findings could benefit the development of T cell-based vaccines, in which T cells are primed against these antigens to target and eradicate tumors. Such a vaccine could be used in tandem with existing immune checkpoint inhibition therapies, to bridge the gap in treatment efficacy across subtypes of colorectal cancer with varying prognoses. Data are available via ProteomeXchange with identifier PXD028309.
Assuntos
Neoplasias Colorretais , Instabilidade de Microssatélites , Antígenos de Neoplasias/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Inibidores de Checkpoint Imunológico , Imunoterapia/métodos , Peptídeos/genéticaRESUMO
Proteomic diversity in biological samples can be characterized by mass spectrometry (MS)-based proteomics using customized protein databases generated from sets of transcripts previously detected by RNA-seq. This diversity has only been increased by the recent discovery that many translated alternative open reading frames rest unannotated at unsuspected locations of mRNAs and ncRNAs. These novel protein products, termed alternative proteins, have been left out of all previous custom database generation tools. Consequently, genetic variations that impact alternative open reading frames and variant peptides from their translated proteins are not detectable with current computational workflows. To fill this gap, we present OpenCustomDB, a bioinformatics tool that uses sample-specific RNaseq data to identify genomic variants in canonical and alternative open reading frames, allowing for more than one coding region per transcript. In a test reanalysis of a cohort of 16 patients with acute myeloid leukemia, 5666 peptides from alternative proteins were detected, including 201 variant peptides. We also observed that a significant fraction of peptide-spectrum matches previously assigned to peptides from canonical proteins got better scores when reassigned to peptides from alternative proteins. Custom protein libraries that include sample-specific sequence variations of all possible open reading frames are promising contributions to the development of proteomics and precision medicine. The raw and processed proteomics data presented in this study can be found in PRIDE repository with accession number PXD029240.
Assuntos
Proteínas , Proteômica , Humanos , Proteômica/métodos , Bases de Dados de Proteínas , Fases de Leitura Aberta , Proteínas/genética , Peptídeos/genética , Peptídeos/análiseRESUMO
T cell development depends on sequential interactions of thymocytes with cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells. PSMB11 is a catalytic proteasomal subunit present exclusively in cTECs. Because proteasomes regulate transcriptional activity, we asked whether PSMB11 might affect gene expression in cTECs. We report that PSMB11 regulates the expression of 850 cTEC genes that modulate lymphostromal interactions primarily via the WNT signaling pathway. cTECs from Psmb11 -/- mice 1) acquire features of medullary thymic epithelial cells and 2) retain CD8 thymocytes in the thymic cortex, thereby impairing phase 2 of positive selection, 3) perturbing CD8 T cell development, and 4) causing dramatic oxidative stress leading to apoptosis of CD8 thymocytes. Deletion of Psmb11 also causes major oxidative stress in CD4 thymocytes. However, CD4 thymocytes do not undergo apoptosis because, unlike CD8 thymocytes, they upregulate expression of chaperones and inhibitors of apoptosis. We conclude that PSMB11 has pervasive effects on both CD4 and CD8 thymocytes via regulation of gene expression in cTECs.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células Epiteliais/citologia , Complexo de Endopeptidases do Proteassoma/genética , Timócitos/citologia , Animais , Apoptose , Diferenciação Celular , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/imunologia , Timo/imunologia , Via de Sinalização WntRESUMO
Defining the repertoire of peptides presented by the major histocompatibility complex class I (MHC I) is a key step toward the identification of relevant antigens for cancer immunotherapy. However, the identification of cancer-specific antigens is a significant analytical challenge in view of their low abundance and low mutational load found in most primary cancer specimens. Here, we describe the application of isobaric peptide labeling with tandem mass tag (TMT) to improve the detection of the MHC I peptides. Isobaric peptide labeling was found to promote the formation of multiply charged ions and to enhance the formation of b-type fragment ions, thus resulting in a 50% improvement of MHC I peptide identification. The gain in sensitivity obtained using TMT labeling enabled the detection of low-abundance MHC I peptides including tumor-specific antigens (TSAs) and minor histocompatibility antigens (MiHAs). We further demonstrate the application of this approach to quantify MiHAs presented by B-cell lymphocytes and determined their expression levels by LC-MS/MS using both synchronous precursor selection (SPS) and high-field asymmetric waveform ion mobility spectrometry (FAIMS).
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Sondas Moleculares/química , Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos/imunologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoprecipitação , Espectrometria de Mobilidade Iônica , Camundongos , Camundongos Endogâmicos NOD , Peptídeos/química , Succinimidas/química , Transplante HeterólogoRESUMO
Significant technological advances in both affinity chromatography and mass spectrometry have facilitated the identification of peptides associated with the major histocompatibility complex class I (MHC I) molecules, and enabled a greater understanding of the dynamic nature of the immunopeptidome of normal and neoplastic cells. While the isolation of MHC I-associated peptides (MIPs) typically used mild acid elution (MAE) or immunoprecipitation (IP), limited information currently exists regarding their respective analytical merits. Here, a comparison of these approaches for the isolation of two different B-cell lymphoblast cell models is presented, and it is reported on the recovery, reproducibility, scalability, and complementarity of identification from each method. Both approaches yielded reproducible datasets for peptide extracts obtained from 2 to 100 million cells, with 2016 to 5093 MIPs, respectively. The IP typically provides up to 6.4-fold increase in MIPs compared to the MAE. The comprehensiveness of these immunopeptidome analyses is extended using personalized genomic database of B-cell lymphoblasts, and it is discovered that 0.4% of their respective MIP repertoire harbored nonsynonymous single nucleotide variations (also known as minor histocompatibility antigens, MiHAs).
Assuntos
Ácidos/química , Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Imunoprecipitação/métodos , Fragmentos de Peptídeos/isolamento & purificação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Adulto , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Células Cultivadas , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
By regulating protein degradation, constitutive proteasomes (CPs) control practically all cellular functions. In addition to CPs, vertebrates express immunoproteasomes (IPs). The major nonredundant role ascribed to IPs is their enhanced ability to generate antigenic peptides. We report that CPs and IPs differentially regulate the expression of >8000 transcripts in maturing mouse dendritic cells (DCs) via regulation of signaling pathways such as IFN regulatory factors, STATs, and NF-κB. IPs regulate the transcription of many mRNAs and maturation of a few of them. Moreover, even when engineered to present optimal amounts of antigenic peptide, IP-deficient DCs are inefficient for in vivo T cell priming. Our study shows that the role of IPs in DCs is not limited to Ag processing and reveals a major nonredundant role for IPs in transcription regulation. The dramatic effect of IPs on the transcriptional landscape could explain the various immune and nonimmune phenotypes observed in vertebrates with IP deficiency or mutations.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Transcriptoma/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/genéticaRESUMO
In a context where injection of antigen (Ag)-specific T cells probably represents the future of leukemia immunotherapy, identification of optimal target Ags is crucial. We therefore sought to discover a reliable marker for selection of the most potent Ags. To this end, (1) we immunized mice against 8 individual Ags: 4 minor histocompatibility Ags (miHAs) and 4 leukemia-associated Ags (LAAs) that were overexpressed on leukemic relative to normal thymocytes; (2) we assessed their ability to reject EL4 leukemic cells; and (3) we correlated the properties of our Ags (and their cognate T cells) with their ability to induce protective antileukemic responses. Overall, individual miHAs instigated more potent antileukemic responses than LAAs. Three features had no influence on the ability of primed T cells to reject leukemic cells: (1) MHC-peptide affinity; (2) the stability of MHC-peptide complexes; and (3) epitope density at the surface of leukemic cells, as assessed using mass spectrometry. The cardinal feature of successful Ags is that they were recognized by high-avidity CD8 T cells that proliferated extensively in vivo. Our work suggests that in vitro evaluation of functional avidity represents the best criterion for selection of Ags, which should be prioritized in clinical trials of leukemia immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva , Antígenos de Histocompatibilidade Menor/imunologia , Peptídeos/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/prevenção & controle , Animais , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/transplante , Proliferação de Células , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Expressão Gênica , Imunização , Complexo Principal de Histocompatibilidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/genética , Peptídeos/administração & dosagem , Peptídeos/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Timócitos/efeitos dos fármacos , Timócitos/imunologia , Timócitos/patologiaRESUMO
Epithelial ovarian cancer (EOC) has not significantly benefited from advances in immunotherapy, mainly because of the lack of well-defined actionable antigen targets. Using proteogenomic analyses of primary EOC tumors, we previously identified 91 aberrantly expressed tumor-specific antigens (TSAs) originating from unmutated genomic sequences. Most of these TSAs derive from non-exonic regions, and their expression results from cancer-specific epigenetic changes. The present study aimed to evaluate the immunogenicity of 48 TSAs selected according to two criteria: presentation by highly prevalent HLA allotypes and expression in a significant fraction of EOC tumors. Using targeted mass spectrometry analyses, we found that pulsing with synthetic TSA peptides leads to a high-level presentation on dendritic cells. TSA abundance correlated with the predicted binding affinity to the HLA allotype. We stimulated naïve CD8 T cells from healthy blood donors with TSA-pulsed dendritic cells and assessed their expansion with two assays: MHC-peptide tetramer staining and TCR Vß CDR3 sequencing. We report that these TSAs can expand sizeable populations of CD8 T cells and, therefore, represent attractive targets for EOC immunotherapy.
Assuntos
Antígenos de Neoplasias , Neoplasias Ovarianas , Humanos , Feminino , Antígenos de Neoplasias/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/genética , Células Dendríticas/imunologia , Carcinoma Epitelial do Ovário/imunologia , Carcinoma Epitelial do Ovário/genética , Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodosRESUMO
Hormone receptor-positive breast cancer (HR+) is immunologically cold and has not benefited from advances in immunotherapy. In contrast, subsets of triple-negative breast cancer (TNBC) display high leukocytic infiltration and respond to checkpoint blockade. CD8+ T cells, the main effectors of anticancer responses, recognize MHC I-associated peptides (MAPs). Our work aimed to characterize the repertoire of MAPs presented by HR+ and TNBC tumors. Using mass spectrometry, we identified 57,094 unique MAPs in 26 primary breast cancer samples. MAP source genes highly overlapped between both subtypes. We identified 25 tumor-specific antigens (TSAs) mainly deriving from aberrantly expressed regions. TSAs were most frequently identified in TNBC samples and were more shared among The Cancer Genome Atlas (TCGA) database TNBC than HR+ samples. In the TNBC cohort, the predicted number of TSAs positively correlated with leukocytic infiltration and overall survival, supporting their immunogenicity in vivo. We detected 49 tumor-associated antigens (TAAs), some of which derived from cancer-associated fibroblasts. Functional expansion of specific T cell assays confirmed the in vitro immunogenicity of several TSAs and TAAs. Our study identified attractive targets for cancer immunotherapy in both breast cancer subtypes. The higher prevalence of TSAs in TNBC tumors provides a rationale for their responsiveness to checkpoint blockade.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Antígenos de Neoplasias/genética , Imunoterapia/métodos , Linfócitos T CD8-Positivos/patologiaRESUMO
The hypomethylating agent 5-azacytidine (AZA) is the first-line treatment for AML patients unfit for intensive chemotherapy. The effect of AZA results in part from T-cell cytotoxic responses against MHC-I-associated peptides (MAPs) deriving from hypermethylated genomic regions such as cancer-testis antigens (CTAs), or endogenous retroelements (EREs). However, evidence supporting higher ERE MAPs presentation after AZA treatment is lacking. Therefore, using proteogenomics, we examined the impact of AZA on the repertoire of MAPs and their source transcripts. AZA-treated AML upregulated both CTA and ERE transcripts, but only CTA MAPs were presented at greater levels. Upregulated ERE transcripts triggered innate immune responses against double-stranded RNAs but were degraded by autophagy, and not processed into MAPs. Autophagy resulted from the formation of protein aggregates caused by AZA-dependent inhibition of DNMT2. Autophagy inhibition had an additive effect with AZA on AML cell proliferation and survival, increased ERE levels, increased pro-inflammatory responses, and generated immunogenic tumor-specific ERE-derived MAPs. Finally, autophagy was associated with a lower abundance of CD8+ T-cell markers in AML patients expressing high levels of EREs. This work demonstrates that AZA-induced EREs are degraded by autophagy and shows that inhibiting autophagy can improve the immune recognition of AML blasts in treated patients.
Assuntos
Antimetabólitos Antineoplásicos , Autofagia , Azacitidina , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Azacitidina/farmacologia , Autofagia/efeitos dos fármacos , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Proliferação de Células , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologiaRESUMO
Circadian clocks regulate many important aspects of physiology, and their disturbance leads to various medical conditions. Circadian variations have been found in immune system variables, including daily rhythms in circulating WBC numbers and serum concentration of cytokines. However, control of immune functional responses by the circadian clock has remained relatively unexplored. In this study, we show that mouse lymph nodes exhibit rhythmic clock gene expression. T cells from lymph nodes collected over 24 h show a circadian variation in proliferation after stimulation via the TCR, which is blunted in Clock gene mutant mice. The tyrosine kinase ZAP70, which is just downstream of the TCR in the T cell activation pathway and crucial for T cell function, exhibits rhythmic protein expression. Lastly, mice immunized with OVA peptide-loaded dendritic cells in the day show a stronger specific T cell response than mice immunized at night. These data reveal circadian control of the Ag-specific immune response and a novel regulatory mode of T cell proliferation, and may provide clues for more efficient vaccination strategies.
Assuntos
Ritmo Circadiano/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD28/fisiologia , Proteínas CLOCK/administração & dosagem , Proteínas CLOCK/biossíntese , Proteínas CLOCK/imunologia , Proliferação de Células , Células Cultivadas , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/metabolismo , Fatores de Tempo , Regulação para Cima/imunologiaRESUMO
Innate CD8 T cells are found in mutant mouse models, but whether they are produced in a normal thymus remains controversial. Using the RAG2p-GFP mouse model, we found that â¼10% of TCRαß(+) CD4(-)CD8(+) thymocytes were innate polyclonal T cells (GFP(+)CD44(hi)). Relative to conventional T cells, innate CD8 thymocytes displayed increased cell surface amounts of B7-H1, CD2, CD5, CD38, IL-2Rß, and IL-4Rα and downmodulation of TCRß. Moreover, they overexpressed several transcripts, including T-bet, Id3, Klf2, and, most of all, Eomes. Innate CD8 thymocytes were positively selected, mainly by nonhematopoietic MHCIa(+) cells. They rapidly produced high levels of IFN-γ upon stimulation and readily proliferated in response to IL-2 and IL-4. Furthermore, low numbers of innate CD8 thymocytes were sufficient to help conventional CD8 T cells expand and secrete cytokine following Ag recognition. This helper effect depended on CD44-mediated interactions between innate and conventional CD8 T cells. We concluded that innate TCRαß(+) CD8 T cells represent a sizeable proportion of normal thymocytes whose development and function differ in many ways from those of conventional CD8 T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Imunidade Inata/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismoRESUMO
MHC-I-associated peptides deriving from non-coding genomic regions and mutations can generate tumor-specific antigens, including neoantigens. Quantifying tumor-specific antigens' RNA expression in malignant and benign tissues is critical for discriminating actionable targets. We present BamQuery, a tool attributing an exhaustive RNA expression to MHC-I-associated peptides of any origin from bulk and single-cell RNA-sequencing data. We show that many cryptic and mutated tumor-specific antigens can derive from multiple discrete genomic regions, abundantly expressed in normal tissues. BamQuery can also be used to predict MHC-I-associated peptides immunogenicity and identify actionable tumor-specific antigens de novo.
Assuntos
Neoplasias , Proteogenômica , Humanos , Antígenos de Neoplasias/genética , Antígenos de Histocompatibilidade Classe I , Neoplasias/genética , Peptídeos/genética , RNARESUMO
Self/non-self discrimination is a fundamental requirement of life. Endogenous peptides presented by major histocompatibility complex class I (MHC I) molecules represent the essence of self for CD8 T lymphocytes. These MHC I peptides (MIPs) are collectively referred to as the immunopeptidome. From a systems-level perspective, very little is known about the origin, composition and plasticity of the immunopeptidome. Here, we show that the immunopeptidome, and therefore the nature of the immune self, is plastic and moulded by cellular metabolic activity. By using a quantitative high-throughput mass spectrometry-based approach, we found that altering cellular metabolism via the inhibition of the mammalian target of rapamycin results in dynamic changes in the cell surface MIPs landscape. Moreover, we provide systems-level evidence that the immunopeptidome projects at the cell surface a representation of biochemical networks and metabolic events regulated at multiple levels inside the cell. Our findings open up new perspectives in systems immunology and predictive biology. Indeed, predicting variations in the immunopeptidome in response to cell-intrinsic and -extrinsic factors could be relevant to the rational design of immunotherapeutic interventions.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Imunidade , Complexo Principal de Histocompatibilidade/genética , Redes e Vias Metabólicas/genética , Proteômica , Transdução de Sinais/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/imunologia , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Antígenos de Histocompatibilidade Classe I/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Espectrometria de Massas , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/imunologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/genética , Peptídeos/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Biologia de Sistemas/métodos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Espectrometria de Massas em TandemRESUMO
Proteasome-mediated proteolysis plays a crucial role in many basic cellular processes. In addition to constitutive proteasomes (CPs), which are found in all eukaryotes, jawed vertebrates also express immunoproteasomes (IPs). Evidence suggests that the key role of IPs may hinge on their impact on the repertoire of peptides associated to major histocompatibility complex (MHC) I molecules. Using a label-free quantitative proteomics approach, we identified 417 peptides presented by MHC I molecules on primary mouse dendritic cells (DCs). By comparing MHC I-associated peptides (MIPs) eluted from primary DCs and thymocytes, we found that the MIP repertoire concealed a cell type-specific signature correlating with cell function. Notably, mass spectrometry analyses of DCs expressing or not IP subunits MECL1 and LMP7 showed that IPs substantially increase the abundance and diversity of MIPs. Bioinformatic analyses provided evidence that proteasomes harboring LMP7 and MECL1 have specific cleavage preferences and recognize unstructured protein regions. Moreover, while differences in MIP repertoire cannot be attributed to potential effects of IPs on gene transcription, IP subunits deficiency altered mRNA levels of a set of genes controlling DC function. Regulated genes segregated in clusters that were enriched in chromosomes 4 and 8. Our peptidomic studies performed on untransfected primary cells provide a detailed account of the MHC I-associated immune self. This work uncovers the dramatic impact of IP subunits MECL1 and LMP7 on the MIP repertoire and their non-redundant influence on expression of immune-related genes.
Assuntos
Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/química , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Western Blotting , Cromatografia Líquida , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Espectrometria de Massas em TandemRESUMO
Previous reports showed that mouse vaccination with pluripotent stem cells (PSCs) induces durable anti-tumor immune responses via T cell recognition of some elusive oncofetal epitopes. We characterize the MHC I-associated peptide (MAP) repertoire of human induced PSCs (iPSCs) using proteogenomics. Our analyses reveal a set of 46 pluripotency-associated MAPs (paMAPs) absent from the transcriptome of normal tissues and adult stem cells but expressed in PSCs and multiple adult cancers. These paMAPs derive from coding and allegedly non-coding (48%) transcripts involved in pluripotency maintenance, and their expression in The Cancer Genome Atlas samples correlates with source gene hypomethylation and genomic aberrations common across cancer types. We find that several of these paMAPs were immunogenic. However, paMAP expression in tumors coincides with activation of pathways instrumental in immune evasion (WNT, TGF-ß, and CDK4/6). We propose that currently available inhibitors of these pathways could synergize with immune targeting of paMAPs for the treatment of poorly differentiated cancers.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias , Células-Tronco Pluripotentes , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Peptídeos/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
The factors controlling memory T (Tm)-cell longevity are still poorly defined, and their identification is pivotal to the design of a vaccine conferring long-term protection against infection. Tm cells have the ability to survive in the absence of the T-cell receptor (TCR)-MHC interaction. This does not exclude a possible role for TCR-intrinsic ligand-independent constitutive signaling in Tm-cell homeostasis. Using a unique TCR tetracycline-inducible expression system, we show that the ablation of TCR expression, which abrogates any possible signaling via the TCR, did not influence the survival and self-renewal of antigen-specific CD8(+) Tm cells even when they have to compete with endogenous T cells for survival factors. Moreover, CD8(+) Tm-cell functionality was not altered even on prolonged maintenance in the absence of TCR-MHC interactions. Furthermore, our results show that a subset of CD4(+) Tm cells can survive in the absence of TCR expression in nonlymphopenic hosts.
Assuntos
Memória Imunológica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Sobrevivência Celular/imunologia , Expressão Gênica/efeitos dos fármacos , Camundongos , Subpopulações de Linfócitos T , Linfócitos T/transplante , Tetraciclina/farmacologiaRESUMO
Combining RNA sequencing, ribosome profiling, and mass spectrometry, we elucidate the contribution of non-canonical translation to the proteome and major histocompatibility complex (MHC) class I immunopeptidome. Remarkably, of 14,498 proteins identified in three human B cell lymphomas, 2,503 are non-canonical proteins. Of these, 28% are novel isoforms and 72% are cryptic proteins encoded by ostensibly non-coding regions (60%) or frameshifted canonical genes (12%). Cryptic proteins are translated as efficiently as canonical proteins, have more predicted disordered residues and lower stability, and critically generate MHC-I peptides 5-fold more efficiently per translation event. Translating 5' "untranslated" regions hinders downstream translation of genes involved in transcription, translation, and antiviral responses. Novel protein isoforms show strong enrichment for signaling pathways deregulated in cancer. Only a small fraction of cryptic proteins detected in the proteome contribute to the MHC-I immunopeptidome, demonstrating the high preferential access of cryptic defective ribosomal products to the class I pathway.
Assuntos
Proteoma/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Fases de Leitura Aberta/genética , Isoformas de Proteínas/metabolismo , Proteoma/análise , Ribossomos/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/genética , Espectrometria de Massas em TandemRESUMO
The dominant paradigm holds that spontaneous and therapeutically induced anti-tumor responses are mediated mainly by CD8 T cells and directed against tumor-specific antigens (TSAs). The presence of specific TSAs on cancer cells can only be proven by mass spectrometry analyses. Bioinformatic predictions and reverse immunology studies cannot provide this type of conclusive evidence. Most TSAs are coded by unmutated non-canonical transcripts that arise from cancer-specific epigenetic and splicing aberrations. When searching for TSAs, it is therefore important to perform mass spectrometry analyses that interrogate not only the canonical reading frame of annotated exome but all reading frames of the entire translatome. The majority of aberrantly expressed TSAs (aeTSAs) derive from unstable short-lived proteins that are good substrates for direct major histocompatibility complex (MHC) I presentation but poor substrates for cross-presentation. This is an important caveat, because cancer cells are poor antigen-presenting cells, and the immune system, therefore, depends on cross-presentation by dendritic cells (DCs) to detect the presence of TSAs. We, therefore, postulate that, in the untreated host, most aeTSAs are undetected by the immune system. We present evidence suggesting that vaccines inducing direct aeTSA presentation by DCs may represent an attractive strategy for cancer treatment.