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1.
Cell Rep ; 36(1): 109312, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34233181

RESUMO

Efforts to overcome resistance to immune checkpoint blockade therapy have focused on vaccination strategies using neoepitopes, although they cannot be applied on a large scale due to the "private" nature of cancer mutations. Here, we show that infection of tumor cells with Salmonella induces the opening of membrane hemichannels and the extracellular release of proteasome-generated peptides by the exacerbation of endoplasmic reticulum (ER) stress. Peptides released by cancer cells foster an antitumor response in vivo, both in mice bearing B16F10 melanomas and in dogs suffering from osteosarcoma. Mass spectrometry analysis on the supernatant of human melanoma cells revealed 12 peptides capable of priming healthy-donor CD8+ T cells that recognize and kill human melanoma cells in vitro and when xenotransplanted in vivo. Hence, we identified a class of shared tumor antigens that are generated in ER-stressed cells, such as tumor cells, that do not induce tolerance and are not presented by healthy cells.

3.
Front Immunol ; 12: 656451, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936085

RESUMO

Increasing evidence suggests that post-translational peptide splicing can play a role in the immune response under pathological conditions. This seems to be particularly relevant in Type 1 Diabetes (T1D) since post-translationally spliced epitopes derived from T1D-associated antigens have been identified among those peptides bound to Human Leucocyte Antigen (HLA) class I and II complexes. Their immunogenicity has been confirmed through CD4+ and CD8+ T cell-mediated responses in T1D patients. Spliced peptides theoretically have a large sequence variability. This might increase the frequency of viral-human zwitter peptides, i.e. peptides that share a complete sequence homology irrespective of whether they originate from human or viral antigens, thereby impinging upon the discrimination between self and non-self antigens by T cells. This might increase the risk of autoimmune responses triggered by viral infections. Since enteroviruses and other viral infections have historically been associated with T1D, we investigated whether cis-spliced peptides derived from selected viruses might be able to trigger CD8+ T cell-mediated autoimmunity. We computed in silico viral-human non-spliced and cis-spliced zwitter epitope candidates, and prioritized peptide candidates based on: (i) their binding affinity to HLA class I complexes, (ii) human pancreatic ß cell and medullary thymic epithelial cell (mTEC) antigens' mRNA expression, (iii) antigen association with T1D, and (iv) potential hotspot regions in those antigens. Neglecting potential T cell receptor (TCR) degeneracy, no viral-human zwitter non-spliced peptide was found to be an optimal candidate to trigger a virus-induced CD8+ T cell response against human pancreatic ß cells. Conversely, we identified some zwitter peptide candidates, which may be produced by proteasome-catalyzed peptide splicing, and might increase the likelihood of pancreatic ß cells recognition by virus-specific CD8+ T cell clones, therefore promoting ß cell destruction in the context of viral infections.


Assuntos
Antígenos Virais/genética , Antígenos Virais/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Células Secretoras de Insulina/imunologia , Splicing de RNA , Antígenos Virais/química , Autoimunidade , Diabetes Mellitus Tipo 1/metabolismo , Suscetibilidade a Doenças , Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Células Secretoras de Insulina/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
4.
Front Immunol ; 12: 614276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717099

RESUMO

The human immune system relies on the capability of CD8+ T cells to patrol body cells, spot infected cells and eliminate them. This cytotoxic response is supposed to be limited to infected cells to avoid killing of healthy cells. To enable this, CD8+ T cells have T Cell Receptors (TCRs) which should discriminate between self and non-self through the recognition of antigenic peptides bound to Human Leukocyte Antigen class I (HLA-I) complexes-i.e., HLA-I immunopeptidomes-of patrolled cells. The majority of these antigenic peptides are produced by proteasomes through either peptide hydrolysis or peptide splicing. Proteasome-generated cis-spliced peptides derive from a given antigen, are immunogenic and frequently presented by HLA-I complexes. Theoretically, they also have a very large sequence variability, which might impinge upon our model of self/non-self discrimination and central and peripheral CD8+ T cell tolerance. Indeed, a large variety of cis-spliced epitopes might enlarge the pool of viral-human zwitter epitopes, i.e., peptides that may be generated with the exact same sequence from both self (human) and non-self (viral) antigens. Antigenic viral-human zwitter peptides may be recognized by CD8+ thymocytes and T cells, induce clonal deletion or other tolerance processes, thereby restraining CD8+ T cell response against viruses. To test this hypothesis, we computed in silico the theoretical frequency of zwitter non-spliced and cis-spliced epitope candidates derived from human proteome (self) and from the proteomes of a large pool of viruses (non-self). We considered their binding affinity to the representative HLA-A*02:01 complex, self-antigen expression in Medullary Thymic Epithelial cells (mTECs) and the relative frequency of non-spliced and cis-spliced peptides in HLA-I immunopeptidomes. Based on the present knowledge of proteasome-catalyzed peptide splicing and neglecting CD8+ TCR degeneracy, our study suggests that, despite their frequency, the portion of the cis-spliced peptides we investigated could only marginally impinge upon the variety of functional CD8+ cytotoxic T cells (CTLs) involved in anti-viral response.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Tolerância Imunológica , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína , Sequência de Aminoácidos , Apresentação do Antígeno/imunologia , Deleção Clonal/imunologia , Epitopos de Linfócito T/imunologia , HIV/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Modelos Moleculares , Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma
5.
Sci Data ; 7(1): 146, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415162

RESUMO

Proteasomes are the main producers of antigenic peptides presented to CD8+ T cells. They can cut proteins and release their fragments or recombine non-contiguous fragments thereby generating novel sequences, i.e. spliced peptides. Understanding which are the driving forces and the sequence preferences of both reactions can streamline target discovery in immunotherapies against cancer, infection and autoimmunity. Here, we present a large database of spliced and non-spliced peptides generated by proteasomes in vitro, which is available as simple CSV file and as a MySQL database. To generate the database, we performed in vitro digestions of 55 unique synthetic polypeptide substrates with different proteasome isoforms and experimental conditions. We measured the samples using three mass spectrometers, filtered and validated putative peptides, identified 22,333 peptide product sequences (15,028 spliced and 7,305 non-spliced product sequences). Our database and datasets have been deposited to the Mendeley (doi:10.17632/nr7cs764rc.1) and PRIDE (PXD016782) repositories. We anticipate that this unique database can be a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing, with various future translational applications.


Assuntos
Bases de Dados de Proteínas , Complexo de Endopeptidases do Proteassoma/fisiologia , Isoformas de Proteínas/química , Antígenos/química , Linfócitos T CD8-Positivos , Humanos , Peptídeos/química
6.
Eur J Immunol ; 50(2): 270-283, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31729751

RESUMO

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.


Assuntos
Aminopeptidases/imunologia , Apresentação do Antígeno/imunologia , Retículo Endoplasmático/imunologia , Epitopos de Linfócito T/imunologia , Melanoma/imunologia , Melanoma/terapia , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Neoplasias , Linhagem Celular Tumoral , Células HeLa , Humanos , Fatores Imunológicos/imunologia , Imunoterapia/métodos , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Linfócitos T/imunologia
7.
Front Immunol ; 10: 2572, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803176

RESUMO

Targeting CD8+ T cells to recurrent tumor-specific mutations can profoundly contribute to cancer treatment. Some of these mutations are potential tumor antigens although they can be displayed by non-spliced epitopes only in a few patients, because of the low affinity of the mutated non-spliced peptides for the predominant HLA class I alleles. Here, we describe a pipeline that uses the large sequence variety of proteasome-generated spliced peptides and identifies spliced epitope candidates, which carry the mutations and bind the predominant HLA-I alleles with high affinity. They could be used in adoptive T cell therapy and other anti-cancer immunotherapies for large cohorts of cancer patients. As a proof of principle, the application of this pipeline led to the identification of a KRAS G12V mutation-carrying spliced epitope candidate, which is produced by proteasomes, transported by TAPs and efficiently presented by the most prevalent HLA class I molecules, HLA-A*02:01 complexes.


Assuntos
Processamento Alternativo , Biologia Computacional , Mapeamento de Epitopos , Epitopos/genética , Antígenos HLA-A/genética , Neoplasias/genética , Neoplasias/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Sítios de Ligação , Biologia Computacional/métodos , Epitopos/química , Epitopos/imunologia , Regulação Neoplásica da Expressão Gênica , Antígenos HLA-A/química , Antígenos HLA-A/imunologia , Humanos , Modelos Moleculares , Conformação Molecular , Neoplasias/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Relação Estrutura-Atividade
8.
J Biol Chem ; 294(19): 7740-7754, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30914481

RESUMO

An efficient immunosurveillance of CD8+ T cells in the periphery depends on positive/negative selection of thymocytes and thus on the dynamics of antigen degradation and epitope production by thymoproteasome and immunoproteasome in the thymus. Although studies in mouse systems have shown how thymoproteasome activity differs from that of immunoproteasome and strongly impacts the T cell repertoire, the proteolytic dynamics and the regulation of human thymoproteasome are unknown. By combining biochemical and computational modeling approaches, we show here that human 20S thymoproteasome and immunoproteasome differ not only in the proteolytic activity of the catalytic sites but also in the peptide transport. These differences impinge upon the quantity of peptide products rather than where the substrates are cleaved. The comparison of the two human 20S proteasome isoforms depicts different processing of antigens that are associated to tumors and autoimmune diseases.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/enzimologia , Simulação por Computador , Complexo de Endopeptidases do Proteassoma/química , Células A549 , Animais , Linfócitos T CD8-Positivos/imunologia , Catálise , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Células THP-1
9.
Cells ; 8(3)2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897778

RESUMO

The function of proteasomes in extracellular space is still largely unknown. The extracellular proteasome-osteopontin circuit has recently been hypothesized to be part of the inflammatory machinery regulating relapse/remission phase alternation in multiple sclerosis. However, it is still unclear what dynamics there are between the different elements of the circuit, what the role of proteasome isoforms is, and whether these inflammatory circuit dynamics are associated with the clinical severity of multiple sclerosis. To shed light on these aspects of this novel inflammatory circuit, we integrated in vitro proteasome isoform data, cell chemotaxis cell culture data, and clinical data of multiple sclerosis cohorts in a coherent computational inference framework. Thereby, we modeled extracellular osteopontin-proteasome circuit dynamics during relapse/remission alternation in multiple sclerosis. Applying this computational framework to a longitudinal study on single multiple sclerosis patients suggests a complex interaction between extracellular proteasome isoforms and osteopontin with potential clinical implications.


Assuntos
Espaço Extracelular/metabolismo , Esclerose Múltipla/metabolismo , Osteopontina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sistema Nervoso Central/patologia , Quimiotaxia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Leucócitos/patologia , Modelos Biológicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Osteopontina/sangue , Isoformas de Proteínas/metabolismo , Recidiva , Indução de Remissão , Trombina/farmacologia
10.
Cancer Immunol Res ; 7(1): 62-76, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30425108

RESUMO

Anticancer immunotherapies demand optimal epitope targets, which could include proteasome-generated spliced peptides if tumor cells were to present them. Here, we show that spliced peptides are widely presented by MHC class I molecules of colon and breast carcinoma cell lines. The peptides derive from hot spots within antigens and enlarge the antigen coverage. Spliced peptides also represent a large number of antigens that would otherwise be neglected by patrolling T cells. These antigens tend to be long, hydrophobic, and basic. Thus, spliced peptides can be a key to identifying targets in an enlarged pool of antigens associated with cancer.


Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Neoplasias do Colo/imunologia , Peptídeos/imunologia , Sequência de Bases , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Genes MHC Classe I , Humanos , Peptídeos/genética , Processamento de Proteína
11.
Curr Opin Immunol ; 52: 81-86, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29723668

RESUMO

The sequence of a large number of MHC-presented epitopes is not present as such in the original antigen because it has been re-shuffled by the proteasome or other proteases. Why do proteases throw a spanner in the works of our model of antigen tagging and immune recognition? We describe in this review what we know about the immunological relevance of post-translationally spliced epitopes and why proteases seem to have a second (dark) personality, which is keen to create new peptide bonds.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos/imunologia , Epitopos/imunologia , Peptídeo Hidrolases/metabolismo , Animais , Antígenos/química , Antígenos/genética , Epitopos/química , Epitopos/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Relação Estrutura-Atividade
12.
Bioinformatics ; 34(7): 1249-1250, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29228182

RESUMO

Motivation: Different experiments provide differing levels of information about a biological system. This makes it difficult, a priori, to select one of them beyond mere speculation and/or belief, especially when resources are limited. With the increasing diversity of experimental approaches and general advances in quantitative systems biology, methods that inform us about the information content that a given experiment carries about the question we want to answer, become crucial. Results: PEITH(Θ) is a general purpose, Python framework for experimental design in systems biology. PEITH(Θ) uses Bayesian inference and information theory in order to derive which experiments are most informative in order to estimate all model parameters and/or perform model predictions. Availability and implementation: https://github.com/MichaelPHStumpf/Peitho. Contact: m.stumpf@imperial.ac.uk or juliane.liepe@mpibpc.mpg.de.


Assuntos
Teoria da Informação , Software , Biologia de Sistemas/métodos , Teorema de Bayes
13.
Front Immunol ; 8: 1441, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163514

RESUMO

Efficient and safe induction of CD8+ T cell responses is a desired characteristic of vaccines against intracellular pathogens. To achieve this, a new generation of safe vaccines is being developed accommodating single, dominant antigens of pathogens of interest. In particular, the selection of such antigens is challenging, since due to HLA polymorphism the ligand specificities and immunodominance hierarchies of pathogen-specific CD8+ T cell responses differ throughout the human population. A recently discovered mechanism of proteasome-mediated CD8+ T cell epitope generation, i.e., by proteasome-catalyzed peptide splicing (PCPS), expands the pool of peptides and antigens, presented by MHC class I HLA molecules. On the cell surface, one-third of the presented self-peptides are generated by PCPS, which coincides with one-fourth in terms of abundance. Spliced epitopes are targeted by CD8+ T cell responses during infection and, like non-spliced epitopes, can be identified within antigen sequences using a novel in silico strategy. The existence of spliced epitopes, by enlarging the pool of peptides available for presentation by different HLA variants, opens new opportunities for immunotherapies and vaccine design.

14.
Trends Immunol ; 38(12): 904-915, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28830734

RESUMO

CD8+ T cell specificity depends on the recognition of MHC class I-epitope complexes at the cell surface. These epitopes are mainly produced via degradation of proteins by the proteasome, generating fragments of the original sequence. However, it is now clear that proteasomes can produce a significant portion of epitopes by reshuffling the antigen sequence, thus expanding the potential antigenic repertoire. MHC class I-restricted spliced epitopes have been described in tumors and infections, suggesting an unpredicted relevance of these peculiar peptides. We review current knowledge about proteasome-catalyzed peptide splicing (PCPS), the emerging rules governing this process, and the potential implications for our understanding and therapeutic use of CD8+ T cells, as well as mechanisms generating other non-canonical antigenic epitopes targeted by the T cell response.


Assuntos
Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/metabolismo , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apresentação do Antígeno , Antígenos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ativação Linfocitária , Peptídeos/imunologia , Proteólise
15.
Stem Cells ; 35(11): 2292-2304, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28833970

RESUMO

The hematopoietic stem cell (HSC) niche provides essential microenvironmental cues for the production and maintenance of HSCs within the bone marrow. During inflammation, hematopoietic dynamics are perturbed, but it is not known whether changes to the HSC-niche interaction occur as a result. We visualize HSCs directly in vivo, enabling detailed analysis of the 3D niche dynamics and migration patterns in murine bone marrow following Trichinella spiralis infection. Spatial statistical analysis of these HSC trajectories reveals two distinct modes of HSC behavior: (a) a pattern of revisiting previously explored space and (b) a pattern of exploring new space. Whereas HSCs from control donors predominantly follow pattern (a), those from infected mice adopt both strategies. Using detailed computational analyses of cell migration tracks and life-history theory, we show that the increased motility of HSCs following infection can, perhaps counterintuitively, enable mice to cope better in deteriorating HSC-niche microenvironments following infection. Stem Cells 2017;35:2292-2304.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Infecções/genética , Animais , Movimento Celular , Células-Tronco Hematopoéticas/citologia , Camundongos , Modelos Teóricos , Fenótipo
16.
Cell Rep ; 20(5): 1242-1253, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28768206

RESUMO

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Epitopos de Linfócito T/genética , Listeriose/genética , Listeriose/patologia , Camundongos , Complexo de Endopeptidases do Proteassoma/genética
17.
Sci Rep ; 7: 43718, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28276434

RESUMO

Osteopontin is a pleiotropic cytokine that is involved in several diseases including multiple sclerosis. Secreted osteopontin is cleaved by few known proteases, modulating its pro-inflammatory activities. Here we show by in vitro experiments that secreted osteopontin can be processed by extracellular proteasomes, thereby producing fragments with novel chemotactic activity. Furthermore, osteopontin reduces the release of proteasomes in the extracellular space. The latter phenomenon seems to occur in vivo in multiple sclerosis, where it reflects the remission/relapse alternation. The extracellular proteasome-mediated inflammatory pathway may represent a general mechanism to control inflammation in inflammatory diseases.


Assuntos
Esclerose Múltipla/metabolismo , Osteopontina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Quimiotaxia/imunologia , Células Endoteliais/metabolismo , Espaço Extracelular/metabolismo , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Modelos Moleculares , Esclerose Múltipla/imunologia , Osteopontina/química , Osteopontina/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Conformação Proteica , Relação Estrutura-Atividade
18.
Science ; 354(6310): 354-358, 2016 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-27846572

RESUMO

The proteasome generates the epitopes presented on human leukocyte antigen (HLA) class I molecules that elicit CD8+ T cell responses. Reports of proteasome-generated spliced epitopes exist, but they have been regarded as rare events. Here, however, we show that the proteasome-generated spliced peptide pool accounts for one-third of the entire HLA class I immunopeptidome in terms of diversity and one-fourth in terms of abundance. This pool also represents a unique set of antigens, possessing particular and distinguishing features. We validated this observation using a range of complementary experimental and bioinformatics approaches, as well as multiple cell types. The widespread appearance and abundance of proteasome-catalyzed peptide splicing events has implications for immunobiology and autoimmunity theories and may provide a previously untapped source of epitopes for use in vaccines and cancer immunotherapy.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Humanos , Ligantes , Peptídeos/imunologia , Peptídeos/metabolismo , Processamento de Proteína
19.
Cell Syst ; 3(1): 102-7, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27453447

RESUMO

Spatial structures often constrain the 3D movement of cells or particles in vivo, yet this information is obscured when microscopy data are analyzed using standard approaches. Here, we present methods, called unwrapping and Riemannian manifold learning, for mapping particle-tracking data along unseen and irregularly curved surfaces onto appropriate 2D representations. This is conceptually similar to the problem of reconstructing accurate geography from conventional Mercator maps, but our methods do not require prior knowledge of the environments' physical structure. Unwrapping and Riemannian manifold learning accurately recover the underlying 2D geometry from 3D imaging data without the need for fiducial marks. They outperform standard x-y projections, and unlike standard dimensionality reduction techniques, they also successfully detect both bias and persistence in cell migration modes. We demonstrate these features on simulated data and zebrafish and Drosophila in vivo immune cell trajectory datasets. Software packages that implement unwrapping and Riemannian manifold learning are provided.


Assuntos
Imageamento Tridimensional , Algoritmos , Imageamento por Ressonância Magnética , Microscopia , Imagens de Fantasmas , Reprodutibilidade dos Testes
20.
Curr Biol ; 26(15): 1975-1989, 2016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27426513

RESUMO

In the acute inflammatory phase following tissue damage, cells of the innate immune system are rapidly recruited to sites of injury by pro-inflammatory mediators released at the wound site. Although advances in live imaging allow us to directly visualize this process in vivo, the precise identity and properties of the primary immune damage attractants remain unclear, as it is currently impossible to directly observe and accurately measure these signals in tissues. Here, we demonstrate that detailed information about the attractant signals can be extracted directly from the in vivo behavior of the responding immune cells. By applying inference-based computational approaches to analyze the in vivo dynamics of the Drosophila inflammatory response, we gain new detailed insight into the spatiotemporal properties of the attractant gradient. In particular, we show that the wound attractant is released by wound margin cells, rather than by the wounded tissue per se, and that it diffuses away from this source at rates far slower than those of previously implicated signals such as H2O2 and ATP, ruling out these fast mediators as the primary chemoattractant. We then predict, and experimentally test, how competing attractant signals might interact in space and time to regulate multi-step cell navigation in the complex environment of a healing wound, revealing a period of receptor desensitization after initial exposure to the damage attractant. Extending our analysis to model much larger wounds, we uncover a dynamic behavioral change in the responding immune cells in vivo that is prognostic of whether a wound will subsequently heal or not. VIDEO ABSTRACT.


Assuntos
Fatores Quimiotáticos/metabolismo , Drosophila/fisiologia , Imunidade Inata , Animais , Análise de Sistemas
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