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1.
Nat Commun ; 12(1): 2061, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824318

RESUMO

Stress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains.


Assuntos
Evasão da Resposta Imune , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Virais/química , Proteínas Virais/metabolismo , Linhagem Celular , Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/imunologia , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Proteólise , Solubilidade
2.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802650

RESUMO

As an essential modulator of IgG disposition, the neonatal Fc receptor (FcRn) governs the pharmacokinetics and functions many therapeutic modalities. In this review, we thoroughly reexamine the hitherto elucidated biological and thermodynamic properties of FcRn to provide context for our assessment of more recent advances, which covers antigen-binding fragment (Fab) determinants of FcRn affinity, transgenic preclinical models, and FcRn targeting as an immune-complex (IC)-clearing strategy. We further comment on therapeutic antibodies authorized for treating SARS-CoV-2 (bamlanivimab, casirivimab, and imdevimab) and evaluate their potential to saturate FcRn-mediated recycling. Finally, we discuss modeling and simulation studies that probe the quantitative relationship between in vivo IgG persistence and in vitro FcRn binding, emphasizing the importance of endosomal transit parameters.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/química , Receptores Fc/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Receptores Fc/imunologia , Distribuição Tecidual/imunologia
3.
Nat Commun ; 12(1): 2308, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863906

RESUMO

Conventional T cells are selected by peptide-MHC expressed by cortical epithelial cells in the thymus, and not by cortical thymocytes themselves that do not express MHC I or MHC II. Instead, cortical thymocytes express non-peptide presenting MHC molecules like CD1d and MR1, and promote the selection of PLZF+ iNKT and MAIT cells, respectively. Here, we report an inducible class-I transactivator mouse that enables the expression of peptide presenting MHC I molecules in different cell types. We show that MHC I expression in DP thymocytes leads to expansion of peptide specific PLZF+ innate-like (PIL) T cells. Akin to iNKT cells, PIL T cells differentiate into three functional effector subsets in the thymus, and are dependent on SAP signaling. We demonstrate that PIL and NKT cells compete for a narrow niche, suggesting that the absence of peptide-MHC on DP thymocytes facilitates selection of non-peptide specific lymphocytes.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunidade Inata , Timócitos/imunologia , Timo/imunologia , Animais , Diferenciação Celular/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Células T Invariáveis Associadas à Mucosa/imunologia , Células T Matadoras Naturais/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Timócitos/metabolismo , Timo/citologia
4.
Methods Mol Biol ; 2224: 123-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606211

RESUMO

Proteinuria is a widely used marker of renal disease and is strongly associated with renal and cardiovascular outcomes. The molecular mechanisms underlying filtration of serum proteins through the glomerular filtration barrier (GFB) remain to be determined. Since the GFB is a complex structure, studies of albumin or IgG trafficking in cultured cells in vitro may not fully recapitulate these processes in vivo. In other epithelial cells including renal proximal tubular cells, the neonatal Fc receptor (FcRn) is required to divert albumin and IgG from the degradative pathway which allows these proteins to be recycled or transcytosed. To examine the role of podocyte FcRn in albumin and IgG trafficking in vivo, we detail the creation of a podocyte-specific FcRn knockout mouse and describe methods for examining intraglomerular detection of albumin and IgG in these mice.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Podócitos/metabolismo , Receptores Fc/metabolismo , Albuminas/metabolismo , Animais , Células Epiteliais/metabolismo , Feminino , Imunoglobulina G/metabolismo , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico/fisiologia , Proteinúria/metabolismo , Transcitose/fisiologia
5.
Hum Immunol ; 82(3): 177-185, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33597096

RESUMO

Hepatitis C virus usually produces chronic infection and liver damage. Considering that: i) the human leukocyte antigen-E (HLA-E) molecule may modulate the immune response, and ii) little is known about the role of HLA-E gene variability on chronic hepatitis C, we studied the impact of HLA-E polymorphisms on the magnitude of HLA-E liver expression and severity of hepatitis C. HLA-E variability was evaluated in terms of: i) single nucleotide polymorphism (SNP) alleles and genotypes along the gene (beginning of the promoter region, coding region and 3'UTR), and ii) ensemble of SNPs that defines the coding region alleles, considered individually or as genotypes. The comparisons of the HLA-E variation sites between patients and controls revealed no significant results. The HLA-E + 424 T > C (rs1059510), +756 G > A (rs1264457) and + 3777 G > A (rs1059655) variation sites and the HLA-E*01:01:01:01 and HLA-E*01:03:02:01 alleles, considered at single or double doses, were associated with the magnitude of HLA-E liver expression in Kupfer cell, steatosis, inflammatory activity and liver fibrosis. Although these associations were lost after corrections for multiple comparisons, these variable sites may propitiate biological clues for the understanding of the mechanisms associated with hepatitis C severity.


Assuntos
Genótipo , Hepacivirus/fisiologia , Hepatite C Crônica/genética , Antígenos de Histocompatibilidade Classe I/genética , Fígado/metabolismo , Adulto , Idoso , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto Jovem
6.
BMC Bioinformatics ; 22(1): 7, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407098

RESUMO

BACKGROUND: Accurate prediction of binding between class I human leukocyte antigen (HLA) and neoepitope is critical for target identification within personalized T-cell based immunotherapy. Many recent prediction tools developed upon the deep learning algorithms and mass spectrometry data have indeed showed improvement on the average predicting power for class I HLA-peptide interaction. However, their prediction performances show great variability over individual HLA alleles and peptides with different lengths, which is particularly the case for HLA-C alleles due to the limited amount of experimental data. To meet the increasing demand for attaining the most accurate HLA-peptide binding prediction for individual patient in the real-world clinical studies, more advanced deep learning framework with higher prediction accuracy for HLA-C alleles and longer peptides is highly desirable. RESULTS: We present a pan-allele HLA-peptide binding prediction framework-MATHLA which integrates bi-directional long short-term memory network and multiple head attention mechanism. This model achieves better prediction accuracy in both fivefold cross-validation test and independent test dataset. In addition, this model is superior over existing tools regarding to the prediction accuracy for longer ligand ranging from 11 to 15 amino acids. Moreover, our model also shows a significant improvement for HLA-C-peptide-binding prediction. By investigating multiple-head attention weight scores, we depicted possible interaction patterns between three HLA I supergroups and their cognate peptides. CONCLUSION: Our method demonstrates the necessity of further development of deep learning algorithm in improving and interpreting HLA-peptide binding prediction in parallel to increasing the amount of high-quality HLA ligandome data.


Assuntos
Biologia Computacional/métodos , Antígenos de Histocompatibilidade Classe I , Redes Neurais de Computação , Peptídeos , Ligação Proteica , Algoritmos , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Modelos Estatísticos , Peptídeos/química , Peptídeos/metabolismo
7.
Nat Immunol ; 22(2): 140-153, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33349708

RESUMO

Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8+ T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity.


Assuntos
Apresentação do Antígeno , Apresentação Cruzada , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Fagossomos/metabolismo , Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Linfócitos T/metabolismo , Animais , Morte Celular , Técnicas de Cocultura , Células Dendríticas/imunologia , Células HEK293 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Lectinas Tipo C/genética , Ligantes , Camundongos , NADPH Oxidases/metabolismo , Fagossomos/genética , Fagossomos/imunologia , Fosforilação , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/genética , Receptores Mitogênicos/genética , Transdução de Sinais , Quinase Syk/metabolismo , Linfócitos T/imunologia
8.
Biochem Biophys Res Commun ; 536: 32-37, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33360096

RESUMO

The neonatal Fc receptor (FcRn) interacts with IgG and albumin at acidic pH within endosomes, thus protecting these plasma proteins from degradation. Recently, we proposed fibrinogen as a new binding partner of FcRn. This work was aimed at providing a direct demonstration of FcRn-fibrinogen binding at acidic pH by Fluorescence Correlation Spectroscopy. The increase in diffusion time between free and fibrinogen-bound FITC-labelled FcRn was assumed as the binding indicator. We observed that, at acidic pH (pH = 5.3), FcRn diffusion time shifted from ≈730 µs (FITC-labelled FcRn alone) to >1200 µs (FITC-labelled FcRn added with fibrinogen). A similar trend was exhibited by albumin, a known FcRn interactor, while no significant variations in diffusion time were observed upon incubation with catalase as negative control. Our results demonstrate a binding interaction between fibrinogen, one of the most abundant plasma proteins, and FcRn, a receptor involved in the regulation of the levels of IgG and albumin. This interaction is likely responsible for fibrinogen protection from intracellular degradation and recycling in plasma. Fibrinogen is crucial not only in haemostasis but also in acute inflammatory response and in some pathological conditions. The interaction with FcRn can influence not only the levels of fibrinogen in plasma and other tissues, but also the levels of other FcRn binding partners, among which are some plasma proteins of clinical relevance.


Assuntos
Fibrinogênio/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/metabolismo , Espectrometria de Fluorescência , Catalase/metabolismo , Difusão , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Ligação Proteica , Albumina Sérica Humana/metabolismo
9.
Mol Immunol ; 130: 148-153, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33358568

RESUMO

MR1 is an MHC class I-like molecule with unique structural and biological features that make it an important member among the molecules involved in antigen presentation to T cells. Distinctive features include ubiquitous expression of the MR1 gene and its monomorphism. Another relevant property is that the MR1 protein appears at very low levels on the plasma membrane and its surface expression is regulated by antigen binding. Finally, the nature of presented antigens differs from those that bind other presenting molecules and includes small metabolites of microbial and self-origin, small drugs and tumor-associated antigens. This opinion paper describes in detail some of those features and discusses recent literature in the field.


Assuntos
Apresentação do Antígeno , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/metabolismo , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ligantes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T/imunologia
10.
Mol Immunol ; 130: 64-68, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33360378

RESUMO

The monomorphic MHC-class I-like molecule, MR1, presents small metabolites to T cells. MR1 is the restriction element for microbe-reactive mucosal-associated invariant T (MAIT) cells. MAIT cells have limited TCR usage, including a semi-invariant TCR alpha chain and express high levels of CD161 and CD26. In addition to microbial lumazine metabolites, recent studies have demonstrated that MR1 is able to capture a variety of diverse chemical entities including folate-derivatives, a number of drug-like and other synthetic small molecules, and as yet undefined compounds of self-origin. This capacity of MR1 to bind distinct ligands likely accounts for the recent identification of additional, non-canonical, subsets of MR1-restricted T (MR1T) cells. These subsets can be defined based on their ability to recognize diverse microbes as well as their reactivity to non-microbial cell-endogenous ligands, including tumor-associated antigens. Herein, we will discuss our current understanding of MR1T cell diversity in terms of TCR usage, ligand recognition and functional attributes.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariáveis Associadas à Mucosa/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade nas Mucosas/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Células T Invariáveis Associadas à Mucosa/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta
11.
PLoS One ; 15(12): e0230401, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33370294

RESUMO

Podocytes have been proposed to be antigen presenting cells (APCs). In traditional APCs, the neonatal Fc receptor (FcRn) is required for antigen presentation and global knockout of FcRn protects against glomerulonephritis. Since podocytes express FcRn, we sought to determine whether the absence of podocyte FcRn ameliorates immune-mediated disease. We examined MHCII and costimulatory markers expression in cultured wild type (WT) and FcRn knockout (KO) podocytes. Interferon gamma (IFNγ) induced MHCII expression in both WT and KO podocytes but did not change CD80 expression. Neither WT nor KO expressed CD86 or inducible costimulatory ligand (ICOSL) at baseline or with IFNγ. Using an antigen presentation assay, WT podocytes but not KO treated with immune complexes induced a modest increase in IL-2. Induction of the anti-glomerular basement membrane (anti-GBM) model resulted in a significant decrease in glomerular crescents in podocyte-specific FcRn knockout mouse (podFcRn KO) versus controls but the overall percentage of crescents was low. To examine the effects of the podocyte-specific FcRn knockout in a model with a longer autologous phase, we used the nephrotoxic serum nephritis (NTS) model. We found that the podFcRn KO mice had significantly reduced crescent formation and glomerulosclerosis compared to control mice. This study demonstrates that lack of podocyte FcRn is protective in immune mediated kidney disease that is dependent on an autologous phase. This study also highlights the difference between the anti-GBM model and NTS model of disease.


Assuntos
Glomerulonefrite/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Podócitos/metabolismo , Receptores Fc/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Citometria de Fluxo , Membrana Basal Glomerular/metabolismo , Glomerulonefrite/genética , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Camundongos Knockout , Receptores Fc/genética
12.
Scand J Immunol ; 92(5): e12978, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32969499

RESUMO

MHC class I molecules on the cellular surface display peptides that either derive from endogenous proteins (self or viral), or from endocytosis of molecules, dying cells or pathogens. The conventional antigen-processing pathway for MHC class I presentation depends on proteasome-mediated degradation of the protein followed by transporter associated with antigen-processing (TAP)-mediated transport of the generated peptides into the endoplasmic reticulum (ER). Here, peptides are loaded onto MHC I molecules before transportation to the cell surface. However, several alternative mechanisms have emerged. These include TAP-independent mechanisms, the vacuolar pathway and involvement of autophagy. Autophagy is a cell intrinsic recycling system. It also functions as a defence mechanism that removes pathogens and damaged endocytic compartments from the cytosol. Therefore, it appears likely that autophagy would intersect with the MHC class I presentation pathway to alarm CD8+ T cells of an ongoing intracellular infection. However, the importance of autophagy as a source of antigen for presentation on MHC I molecules remains to be defined. Here, original research papers which suggest involvement of autophagy in MHC I antigen presentation are reviewed. The antigens are from herpesvirus, cytomegalovirus and chlamydia. The studies point towards autophagy as important in MHC class I presentation of endogenous proteins during conditions of immune evasion. Because autophagy is a regulated process which is induced upon activation of, for example, pattern recognition receptors (PRRs), it will be crucial to use relevant stimulatory conditions together with primary cells when aiming to confirm the importance of autophagy in MHC class I antigen presentation in future studies.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos/imunologia , Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Animais , Antígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/imunologia
13.
Proc Natl Acad Sci U S A ; 117(34): 20597-20606, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788370

RESUMO

The major histocompatibility complex class-I (MHC-I) peptide-loading complex (PLC) is a cornerstone of the human adaptive immune system, being responsible for processing antigens that allow killer T cells to distinguish between healthy and compromised cells. Based on a recent low-resolution cryo-electron microscopy (cryo-EM) structure of this large membrane-bound protein complex, we report an atomistic model of the PLC and study its conformational dynamics on the multimicrosecond time scale using all-atom molecular dynamics (MD) simulations in an explicit lipid bilayer and water environment (1.6 million atoms in total). The PLC has a layered structure, with two editing modules forming a flexible protein belt surrounding a stable, catalytically active core. Tapasin plays a central role in the PLC, stabilizing the MHC-I binding groove in a conformation reminiscent of antigen-loaded MHC-I. The MHC-I-linked glycan steers a tapasin loop involved in peptide editing toward the binding groove. Tapasin conformational dynamics are also affected by calreticulin through a conformational selection mechanism that facilitates MHC-I recruitment into the complex.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Calreticulina/metabolismo , Microscopia Crioeletrônica , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Polissacarídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo
14.
Crit Rev Immunol ; 40(2): 173-184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32749095

RESUMO

Mucosa-associated invariant T cells (MAIT cells) are unconventional, innate-like T lymphocytes with remarkable effector and immunoregulatory functions. They are abundant in the human peripheral blood and also enriched in mucosal layers and in the lungs, SARS-CoV-2's main ports of entry. Once activated, MAIT cells produce inflammatory cytokines and cytolytic effector molecules quickly and copiously. MAIT cells are best known for their antibacterial and antifungal properties. However, they are also activated during viral infections, typically in a cytokine-dependent manner, which may promote antiviral immunity. On the other hand, it is plausible to assume active roles for MAIT cells in infection-provoked cytokine storms and tissue damage. SARS-CoV-2 infection may be asymptomatic, mild, severe, or even fatal, depending on sex, age, the presence of preexisting morbidities, and the individual's immunological competence, or lack thereof, among other factors. Based on the available literature, I propose that MAIT cells regulate the host response to SARS-CoV-2 and constitute attractive targets in the prevention or clinical management of coronavirus disease 19 (COVID-19) and some of its complications. Unlike mainstream T cells, MAIT cells are restricted by a monomorphic antigen-presenting molecule called MHC-related protein 1 (MR1). Therefore, MR1 ligands should modify MAIT cell functions relatively uniformly in genetically diverse subjects and may be tested as immunotherapeutic agents or vaccine adjuvants in future studies.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariáveis Associadas à Mucosa/imunologia , Pneumonia Viral/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/patologia , Citocinas/imunologia , Humanos , Células T Invariáveis Associadas à Mucosa/metabolismo , Pandemias , Pneumonia Viral/patologia
15.
Cells ; 9(9)2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32859121

RESUMO

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells' exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Doadores de Sangue , Brônquios/citologia , Degranulação Celular/genética , Técnicas de Cocultura , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Células Epiteliais/metabolismo , Humanos , Interferon gama/metabolismo , Células K562 , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , RNA Viral/genética , Vírus da SARS/química , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Transfecção
16.
BMC Bioinformatics ; 21(1): 279, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32615972

RESUMO

BACKGROUND: Immunotherapy is a promising route towards personalized cancer treatment. A key algorithmic challenge in this process is to decide if a given peptide (neoepitope) binds with the major histocompatibility complex (MHC). This is an active area of research and there are many MHC binding prediction algorithms that can predict the MHC binding affinity for a given peptide to a high degree of accuracy. However, most of the state-of-the-art approaches make use of complicated training and model selection procedures, are restricted to peptides of a certain length and/or rely on heuristics. RESULTS: We put forward USMPep, a simple recurrent neural network that reaches state-of-the-art approaches on MHC class I binding prediction with a single, generic architecture and even a single set of hyperparameters both on IEDB benchmark datasets and on the very recent HPV dataset. Moreover, the algorithm is competitive for a single model trained from scratch, while ensembling multiple regressors and language model pretraining can still slightly improve the performance. The direct application of the approach to MHC class II binding prediction shows a solid performance despite of limited training data. CONCLUSIONS: We demonstrate that competitive performance in MHC binding affinity prediction can be reached with a standard architecture and training procedure without relying on any heuristics.


Assuntos
Algoritmos , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Modelos Genéticos , Alelos , Área Sob a Curva , Sequência de Bases , Bases de Dados Genéticas , Humanos , Peptídeos/metabolismo , Ligação Proteica , Curva ROC
17.
Proc Natl Acad Sci U S A ; 117(32): 19399-19407, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719124

RESUMO

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/genética
18.
Nat Commun ; 11(1): 2760, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488085

RESUMO

Peptides bound to class I major histocompatibility complexes (MHC) play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer. Surface MHC levels can be modulated by anticancer agents, altering immunity. However, understanding the peptide repertoire's response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking normalization controls. We describe an experimental platform that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging to quantify peptide repertoire alterations using low sample input. HipMHCs improve quantitative accuracy of peptide repertoire changes by normalizing for variation across analyses and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens, which can inform immunotherapy design. Applying this platform in melanoma cell lines to profile the immunopeptidome response to CDK4/6 inhibition and interferon-γ - known modulators of antigen presentation - uncovers treatment-specific alterations, connecting the intracellular response to extracellular immune presentation.


Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Apresentação do Antígeno , Antígenos , Linhagem Celular , Humanos , Imunoterapia , Interferon gama/farmacologia , Espectrometria de Massas , Peptídeos/imunologia , Proteômica
19.
J Vis Exp ; (159)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32510500

RESUMO

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.


Assuntos
Citometria de Fluxo/métodos , Hipocampo/citologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Feminino , Hipocampo/embriologia , Camundongos Endogâmicos C57BL , Neurônios/citologia
20.
Immunogenetics ; 72(5): 295-304, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32577798

RESUMO

Tumor-specific neoantigens are mutated self-peptides presented by tumor cell major histocompatibility complex (MHC) molecules and are necessary to elicit host's anti-cancer cytotoxic T cell responses. It could be specifically recognized by neoantigen-specific T cell receptors (TCRs). However, current wet-lab assays for identifying peptide MHC binding are too expensive and time-consuming to meet the clinical needs. In this study, we developed an in silico method with a deep convolutional neural network (CNN) model, iConMHC, to predict peptide MHC binding affinity. Unlike other in silico methods that only learn from properties of amino acid in neoantigen peptides alone and/or MHCs alone, iConMHC learns from physical and chemical interaction properties between pairwise amino acids from the two molecules. These properties, such as contact potentials and distances in folded proteins, directly affect neoantigen-MHC binding affinity. In addition, IConMHC is a pan-allele model that is capable of making predictions for all the MHC alleles. Even for those rare MHC alleles without training data, iConMHC can make predictions with reasonable accuracy. We benchmarked iConMHC with other commonly used MHC-I binding predictors and found our model performs better than most of the pan-allele models.


Assuntos
Aprendizado Profundo , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Alelos , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Simulação por Computador , Bases de Dados de Proteínas , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Redes Neurais de Computação , Peptídeos/química , Ligação Proteica , Reprodutibilidade dos Testes
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