RESUMO
CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Antígeno HLA-DR1 , Epitopos de Linfócito T , Peptídeos , Linfócitos T CD4-Positivos , Linfócitos T CD8-PositivosRESUMO
To exemplify autoimmune-associated genetic influence on the colonization of bacteria frequently used in probiotics, microbial composition of stool from 1326 one-year-old infants was analyzed in a prospective general-population cohort, All Babies In Southeast Sweden (ABIS). We show that an individual's HLA haplotype composition has a significant impact on which common Bifidobacterium strains thrive in colonizing the gut. The effect HLA has on the gut microbiome can be more clearly observed when considered in terms of allelic dosage. HLA DR1-DQ5 showed the most significant and most prominent effect on increased Bifidobacterium relative abundance. Therefore, HLA DR1-DQ5 is proposed to act as a protective haplotype in many individuals. Protection-associated HLA haplotypes are more likely to influence the promotion of specific bifidobacteria. In addition, strain-level differences are correlated with colonization proficiency in the gut depending on HLA haplotype makeup. These results demonstrate that HLA genetics should be considered when designing effective probiotics, particularly for those at high genetic risk for autoimmune diseases.
Assuntos
Antígeno HLA-DR1 , Humanos , Lactente , Estudos Prospectivos , SuéciaRESUMO
La enfermedad antimembrana basal glomerular (EMBG) es un trastorno autoinmune caracterizado por la presencia de anticuerpos antimembrana basal glomerular (AMBG), hemorragia pulmonar, glomerulonefritis necrotizante y depósito lineal de inmunoglobulinas en inmunofluorescencia directa. La predisposición genética, entre otros factores, posee un papel importante en el desarrollo de la enfermedad. Estudios previos han demostrado que el antígeno leucocitario humano (HLA), HLA-DR15 y HLA- DR4, se asocian con mayor riesgo de presentarla, mientras que el HLA-DR1 y HLA-DR7 han demostrado ser factor de protección frente a su desarrollo.Describimos el primer caso de dos hermanos no gemelos con EMBG con tipaje HLA idéntico, con factor de riesgo HLA-DR4 y factor de protección HLA-DR7. Planteamos la importancia de analizar el tipaje de histocompatibilidad en hermanos de pacientes con EMBG, para determinar el grado de susceptibilidad genética y plantear en ellos un seguimiento estrecho, con el objetivo de lograr un diagnóstico y tratamiento precoces en caso de presentar la enfermedad. (AU)
Anti-glomerular basement membrane disease (AGBM) is an autoinmune disorder characterized by the presence of anti-glomerular basement membrane (anti-GBM) antibodies, alveolar hemorrhage, necrotizing glomerulonephritis, and linear deposition of immunoglobulins through direct inmunofluorescence. Genetic predisposition, among other factors, plays an important role in the development of the disease. Previous studies have shown that HLA-DR15 and HLA-DR4 increase the risk of presenting it, while HLA-DR1 and HLA-DR7 protect against its development.We describe the first case of two non-twin siblings with AGBM and identical HLA, with HLA-DR4 as risk factor and HLA-DR7 as protection factor. We propose the importance of analyzing HLA in siblings of patients with AGBM, to determine the degree of genetic susceptibility and to carry out a close follow-up on them, with the aim of achieving an early diagnosis and treatment in case of presenting the disease. (AU)
Assuntos
Humanos , Masculino , Feminino , Adulto Jovem , Doença Antimembrana Basal Glomerular , Predisposição Genética para Doença , Antígeno HLA-DR1 , Antígeno HLA-DR4 , Antígeno HLA-DR7RESUMO
BACKGROUND: Multiple sclerosis (MS) can be induced upon successful presentation of myelin antigens by MHC I/II. Antigenic similarity between the myelin and viral proteins may worsen the immunological responses. METHODOLOGY: Antigenic regions within myelin proteins; PLP1, MBP, MOG, and MAG were analyzed using SVMTrip and EMBOSS. Homology search identified sequence similarity between the predicted host epitopes and viral proteins. NetMHCpan predicted MHC I/II binding followed by peptide-protein docking through the HPEPDOCK server. Thereafter we analyzed conformational flexibility and stability of 15 protein-peptide complexes based on high docking scores. The binding free energy was calculated using conventional (MD) and Gaussian accelerated molecular dynamics simulation. RESULTS: PLP1, MBP, MAG and MOG contained numerous antigenic epitopes. MBP and MOG epitopes had sequence similarity to HHV-6 BALF5; EBNA1 and CMV glycoprotein M (gM), and EBV LMP2B, gp350/220; HHV-8 ORFs respectively. Many herpes virus proteins like tegument, envelope glycoproteins, and ORFs of EBV, CMV, HHV-6, and HHV-8 demonstrated sequence similarity with MAG and PLP1. Some antigenic peptides were also linear B-cell epitopes and influenced cytokine production by T-cell. MHC I allele HLA-B*57:01 bound to PLP1 peptide and HLA-A*68:02 bound to a MAG peptide strongly. MHC II alleles HLA-DRB1*04:05 and HLA-DR1*01:01 associated with MAG- and MOG-derived peptides, respectively, demonstrating high HPEPDOCK scores. MD simulations established stable binding of certain peptides with the MHC namely HLA-B*51:01-MBP(DYKSAHKGFKGVDAQGTLSKIFKL), HLA-B*57:01-PLP1(PDKFVGITYALTVVWLLVFACSAVPVYIYF), HLA-DR1*01:01-MOG(VEDPFYWVSPGVLVLLAVLPVLLLQITVGLVFLCLQYR) and HLA-DRB1*04:05-MAG(TWVQVSLLHFVPTREA). CONCLUSIONS: Cross-reactivity between self-antigens and pathogen derived immunodominant epitopes may induce MS. Our study supported the role of specific MHC alleles as a contributing MS risk factor.
Assuntos
Infecções por Citomegalovirus , Esclerose Múltipla , Epitopos de Linfócito B , Antígeno HLA-DR1 , Cadeias HLA-DRB1 , Histocompatibilidade , Humanos , Glicoproteína Mielina-Oligodendrócito , Peptídeos , Proteínas ViraisRESUMO
BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.
Assuntos
Vacinas Anticâncer , Glioma , Animais , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Glutaratos , Antígeno HLA-A2/genética , Antígeno HLA-DR1/genética , Humanos , Inibidores de Checkpoint Imunológico , Isocitrato Desidrogenase/genética , Camundongos , Camundongos Transgênicos , Microambiente Tumoral , Regulação para Cima , Vacinas de Subunidades AntigênicasRESUMO
Objectives: The principal purpose of this meta-analysis was to assess the association between HLA-DRB1 (HLA-DR1, HLA-DR13, and HLA-DR16) polymorphisms and SLE susceptibility. Methods: We searched published case-control studies on the association between HLA-DRB1 polymorphisms and SLE susceptibility from PubMed and Web of Science databases. The pooled ORs with 95% CIs were utilized to estimate the strength of association of HLA-DR1, HLA-DR13, and HLA-DR16 polymorphisms and SLE susceptibility by fixed effect models. We also performed sensitivity analysis, trial sequential analysis, Begg's test, and Egg's test in this meta-analysis. Results: A total of 18 studies were included in this meta-analysis. Overall analysis showed that HLA-DR1 and HLA-DR13 polymorphisms were associated with a decreased risk of SLE (OR = 0.76, 95% CI: 0.65-0.90, P < 0.01; OR = 0.58, 95% CI: 0.50-0.68, P < 0.01), and HLA-DR16 polymorphism was associated with an increased risk of SLE (OR = 1.70, 95% CI: 1.24-2.33, P < 0.01). In subgroup analysis of ethnicity, the results were as follows: HLA-DR1 polymorphism in Caucasians (OR = 0.76, 95% CI: 0.58-0.98,P = 0.04) and North Americans (OR = 0.64, 95% CI: 0.42-0.96,P = 0.03); HLA-DR13 polymorphism in Caucasians (OR = 0.62, 95% CI: 0.47-0.82,P < 0.01) and East Asians (OR = 0.44, 95% CI: 0.34-0.57,P < 0.01); and HLA-DR16 polymorphism in East Asians (OR = 2.62, 95% CI: 1.71-4.03,P < 0.01). Conclusions: This meta-analysis showed that HLA-DR1 and HLA-DR13 are protective factors for SLE, and HLA-DR16 is a risk factor. Due to the limitations of this meta-analysis, the association between HLA-DRB1 polymorphisms and SLE susceptibility needs to be further researched before definitive conclusions are proved.
Assuntos
Antígeno HLA-DR1 , Lúpus Eritematoso Sistêmico , Predisposição Genética para Doença , Subtipos Sorológicos de HLA-DR , Cadeias HLA-DRB1/genética , Humanos , Lúpus Eritematoso Sistêmico/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Ag-specific immunotherapy is a long-term goal for the treatment of autoimmune diseases; however developing a means of therapeutically targeting autoimmune T cells in an Ag-specific manner has been difficult. Through the engineering of an HLA-DR1 chimeric Ag receptor (CAR), we have produced CD8+ CAR T cells that target CD4+ T cells in an Ag-specific manner and tested their ability to inhibit the development of autoimmune arthritis in a mouse model. The DR1 CAR molecule was engineered to contain CD3ζ activation and CD28 signaling domains and a covalently linked autoantigenic peptide from type II collagen (CII; DR1-CII) to provide specificity for targeting the autoimmune T cells. Stimulation of the DR1-CII CAR T cells by an anti-DR Ab induced cytokine production, indicating that the DR1-CAR functions as a chimeric molecule. In vitro CTL assays using cloned CD4+ T cells as target cells demonstrated that the DR1-CII CAR T cells efficiently recognize and kill CD4+ T cells that are specific for the CII autoantigen. The CTL function was highly specific, as no killing was observed using DR1-restricted CD4+ T cells that recognize other Ags. When B6.DR1 mice, in which autoimmune arthritis had been induced, were treated with the DR1-CII CAR T cells, the CII-specific autoimmune CD4+ T cell response was significantly decreased, autoantibody production was suppressed, and the incidence and severity of the autoimmune arthritis was diminished. These data demonstrate that HLA-DR CAR T cells have the potential to provide a highly specific therapeutic approach for the treatment of autoimmune disease.
Assuntos
Artrite Experimental/terapia , Artrite Reumatoide/terapia , Doenças Autoimunes/terapia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/genética , Animais , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Engenharia Genética , Antígeno HLA-DR1/genética , Antígeno HLA-DR1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos Quiméricos/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.
Assuntos
Leishmania major/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose/imunologia , Metaloendopeptidases/imunologia , Peptídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Proteção Cruzada , Antígeno HLA-A2/imunologia , Antígeno HLA-DR1/imunologia , Imunidade Celular , Imunidade Humoral , Leishmania infantum/imunologia , Leishmaniose/prevenção & controle , Vacinas contra Leishmaniose/administração & dosagem , Vacinas contra Leishmaniose/química , Mesocricetus , Metaloendopeptidases/química , Óleo Mineral/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/químicaRESUMO
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos/imunologia , Região Variável de Imunoglobulina/genética , Vírus da Influenza A/imunologia , Adulto , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Aves/virologia , Regiões Determinantes de Complementaridade/química , Sequência Conservada , Epitopos/química , Feminino , Células Germinativas/metabolismo , Antígeno HLA-DR1/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Suínos/virologia , Doadores de Tecidos , Proteínas Virais/imunologia , Adulto Jovem , Zoonoses/imunologia , Zoonoses/virologiaRESUMO
BACKGROUND: The negative role of HLA class II donor-specific antibody on graft outcome is well recognized. However, the potentially negative cardiovascular effects of preformed HLA class II antibodies and donor HLA in kidney transplant recipients (KTRs) remain unestablished. METHODS: We conducted a single-center, retrospective cohort study including 1115 KTRs (2003-2016) with up to 4449 person-years of follow-up after transplantation and a median follow-up time of 5.1 years (interquartile range, 2.7-7.6). We evaluated the unadjusted and multivariable-adjusted association between pretransplant HLA class I and II antibodies, as well as HLA-DR1 donor/recipient genotype and the primary (major adverse cardiac and cerebrovascular event [MACCE] or all-cause mortality) and secondary (MACCE or cardiovascular mortality) outcome. RESULTS: In a multivariate Cox proportional hazard model, HLA class II antibodies before transplantation were associated with increased adjusted hazard ratio (aHR) for MACCE or all-cause mortality (aHR, 1.71 [1.13-2.60]; P = 0.012) even after adjustment for time-varying covariate graft loss (aHR, 1.68 [1.08-2.62]; P = 0.022) and biopsy-proven acute rejection (aHR, 1.71 [1.13-2.60]; P = 0.012). HLA class II antibodies were also associated with increased aHR for the secondary outcome, MACCE, or cardiovascular mortality (aHR, 1.92 [1.12-3.30]; P = 0.018). We investigated the effect of donor and recipient HLA-DR1 on these outcome parameters and demonstrated that KTRs with HLA-DR1 positive donors had an increased aHR for MACCE with all-cause (aHR, 1.45 [1.09-1.94]; P = 0.012) and cardiovascular mortality (aHR, 1.49 [1.00-2.22]; P = 0.05). CONCLUSIONS: Prior sensitization against HLA class II antigens is associated with unfavorable long-term cardiovascular outcome in KTRs independent of graft loss or rejection. Recipients of an HLA-DR1 donor also have an impaired cardiovascular outcome.
Assuntos
Doenças Cardiovasculares/imunologia , Antígenos HLA/imunologia , Histocompatibilidade , Isoanticorpos/sangue , Transplante de Rim/efeitos adversos , Adulto , Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/mortalidade , Causas de Morte , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/mortalidade , Sobrevivência de Enxerto , Antígenos HLA/classificação , Antígeno HLA-DR1/imunologia , Humanos , Isoanticorpos/classificação , Transplante de Rim/mortalidade , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR-pHLA-II interactions.
Assuntos
Epitopos/imunologia , Antígeno HLA-DR1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Cristalografia por Raios X , Epitopos/química , Epitopos/metabolismo , Antígeno HLA-DR1/química , Antígeno HLA-DR1/imunologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Introduction: Early detection of sepsis is a critical step to improve patient's survival and cellular markers effective diagnosis tools. The aim of this work was to evaluate HLA-DR expression on peripheral T-lymphocytes (CD3+), a marker associate to T-cell activation, as an early sepsis detection tool. Patients and methods: A cross-sectional study was conducted in twenty-six patients with confirmed sepsis by blood culture, eighteen healthy individuals and four patients with systemic inflammatory response syndrome. The analysis of the HLA-DR expression was carried by flow cytometry. Results: The patients with confirmed sepsis had significantly higher percentage of CD3+/HLA-DR+ lymphocytes compared with both, patients with SIRS (20.37±9.42 vs. 8.7±2.9; p<0.005) and healthy individuals (20.37±9.42 vs. 6.58±3.89; p<0.005). Moreover, the average amount of HLA-DR expressed was higher when caused by gram-positive than by gram-negative bacterias (216.61±131.35 vs. 135.05±31.82; p=0.041). A ROC curve analysis showed the utility of HLA-DR expression on T-cells to identify patients with sepsis. Discussion: Our results suggest that surface expression of HLA-DR on T-lymphocytes could be an early marker for the presence of sepsis in non-surgical septic patients
Introducción: La detección temprana de la sepsis es un paso crítico para mejorar la supervivencia del paciente. Nuestro objetivo fue evaluar la expresión de HLA-DR en linfocitos T periféricos (CD3+), marcador asociado a la activación de células T, como herramienta de detección temprana de la sepsis. Pacientes y métodos: Se realizó un estudio en 26 pacientes con sepsis confirmada, 18 sanos y 4 con síndrome de respuesta inflamatoria sistémica(SIRS). La expresión de HLA-DR se midió por citometría de flujo. Resultados: Los pacientes con sepsis tenían un porcentaje significativamente mayor de linfocitos CD3+/HLA-DR+ en comparación con los otros grupos, pacientes con SIRS (20,37±9,42 vs. 8,7±2,9; p<0,005) y sanos (20,37±9,42 vs. 6,58±3,89; p<0,005). La cantidad media de HLA-DR fue mayor cuando la sepsis estaba causada por bacterias gram-positivas que por gram-negativas (216,61±131,35 vs. 135,05±31,82; p=0,041). El análisis mediante curva ROC mostró la utilidad de la expresión de HLA-DR en células T para identificar pacientes con sepsis. Discusión: Nuestros resultados sugieren que la expresión de HLA-DR en linfocitos T podría ser un marcador temprano de sepsis en pacientes sépticos no quirúrgicos
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Sepse/sangue , Antígeno HLA-DR1/sangue , Antígeno HLA-DR1/metabolismo , Linfócitos T/metabolismo , Biomarcadores/sangue , Estudos Transversais , Estudos de Casos e ControlesRESUMO
The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HCaliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) >> P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.
Assuntos
Antígeno HLA-DR1/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Teoria Quântica , Algoritmos , Sítios de Ligação , Antígeno HLA-DR1/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Ligação de Hidrogênio , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Relação Quantitativa Estrutura-AtividadeRESUMO
Visceral leishmaniasis is a tropical and neglected disease with an estimated 200 000-400 000 cases and 60 000 deaths worldwide each year. Currently, no clinically valid vaccine is available for this disease. In this study, we formulated DNA and protein vaccines encoding HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2 against visceral leishmaniasis. We predicted the secondary and tertiary structures, surface properties, subcellular localization, potential binding sites and HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2. The best candidate CpG ODN (2395, M362, D-SL03 or 685) was screened out as a DNA vaccine adjuvant. We also prepared Kmp-11 and Kmp-11/CaNA2 DNA and protein vaccines, respectively, for comparison. BALB/c mice were immunized with a DNA prime-protein boost immunization strategy and challenged with a newly isolated Leishmania strain from an individual with visceral leishmaniasis. The IgG antibody titers showed that our vaccine had strong immunogenicity with a long duration, especially cellular immunity. The spleen parasite burden of each group demonstrated that the CaNA2 vaccine had a certain immune protective effect on visceral leishmaniasis in BALB/c mice, and the amastigote reduction rate reached 76%. Preliminary safety tests confirmed the safety of the vaccine. Our work demonstrates that the HLA-A2, HLA-A24 and HLA-DR1 restricted epitope CaNA2 DNA prime-protein boost vaccine may be a safe and effective epitope vaccine candidate against visceral leishmaniasis.
Assuntos
Antígenos de Protozoários/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Vacinas Protozoárias/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Células Cultivadas , Epitopos/genética , Epitopos/metabolismo , Feminino , Antígeno HLA-A2/metabolismo , Antígeno HLA-A24/metabolismo , Antígeno HLA-DR1/metabolismo , Humanos , Imunidade Humoral , Imunização Secundária , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Ligação Proteica , Células RAW 264.7 , Vacinas de DNARESUMO
Major Histocompatibility Complex class II (MHC-II) molecules bind peptides and present them to receptors on CD4+ T cells as part of the immune system's surveillance of pathogens and malignancy. In the absence of peptide, MHC-II equilibrates between peptide-receptive and peptide-averse conformations. The conversion between these forms has been postulated to be important in regulating cellular antigen presentation but has been difficult to study. In order to generate the MHC-II molecule HLA-DR1 in the peptide-receptive form, we designed and tested a series of photocleavable peptides that included the UV-sensitive 3-amino-3-(2-nitrophenyl)-propionate amino acid analog. They were intended to bind tightly to the HLA-DR1 MHC molecule, but to generate low-affinity fragments after UV exposure that would be released to yield HLA-DR1 in the peptide-receptive conformation. We were able to identify photocleavable peptides that bound tightly to HLA-DR1 and generated the peptide-receptive conformation after UV exposure. However, slow release of photocleaved peptide fragments from the binding site limited the rate of binding of an incoming labeled peptide and complicated kinetic measurements of the individual steps of the overall peptide binding reaction. Modification of the N-terminal region of the photocleavable peptide to reduce MHC-II pocket or H-bonding interactions allowed for generation of the peptide receptive form immediately after UV exposure with peptide fragments neither retained within the site nor interfering with binding of an incoming peptide. However this was achieved only at the expense of a substantial reduction in overall peptide binding affinity, and these peptides had such weak interaction with HLA-DR1 that they were easily exchanged by incoming peptide without UV exposure. These results show that photocleavable peptides can be used to generate peptide-receptive HLA-DR1 and to facilitate peptide exchange in generation of specific peptide-MHC-II complexes, but that usage of these peptides for kinetic studies can be constrained by slow fragment release.
Assuntos
Antígeno HLA-DR1/química , Antígeno HLA-DR1/metabolismo , Fragmentos de Peptídeos/metabolismo , Sítios de Ligação , Humanos , Cinética , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/efeitos da radiação , Processos Fotoquímicos , Ligação Proteica , Conformação Proteica , Raios UltravioletaRESUMO
OBJECTIVES: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. RESULTS: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR12xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. CONCLUSIONS: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.
Assuntos
Técnicas de Cultura de Células/métodos , Antígeno HLA-DR1/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Reatores Biológicos , Biotecnologia/métodos , Linhagem Celular , Proliferação de Células , Drosophila , Antígeno HLA-DR1/genética , Proteínas Recombinantes/genéticaRESUMO
Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239-1253-loaded HLA-DR tetramers.
Assuntos
Proteína ADAMTS13/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-DR1/imunologia , Epitopos Imunodominantes/imunologia , Fragmentos de Peptídeos/imunologia , Proteína ADAMTS13/química , Alelos , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Epitopos de Linfócito T/química , Antígeno HLA-DR1/química , Antígeno HLA-DR1/metabolismo , Humanos , Imunização , Epitopos Imunodominantes/química , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Púrpura Trombocitopênica Trombótica/genética , Púrpura Trombocitopênica Trombótica/imunologia , Púrpura Trombocitopênica Trombótica/metabolismoRESUMO
Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cell self-epitope derived from the α3 chain of type IV collagen (α3135-145). While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive α3135-145-specific T cells expand in patients with Goodpasture disease and, in α3135-145-immunized HLA-DR15 transgenic mice, α3135-145-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the α3135-145 epitope in different binding registers. HLA-DR15-α3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135-145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Treg cells) expressing tolerogenic cytokines. HLA-DR1-induced Treg cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors display altered α3135-145-specific T-cell antigen receptor usage, HLA-DR15-α3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-α3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded α3135-145-specific CD4+ T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Treg cells that leads to protection or causation of autoimmunity.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Autoimunidade/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doença Antimembrana Basal Glomerular/patologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Colágeno Tipo IV/química , Colágeno Tipo IV/imunologia , Citocinas/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Subtipos Sorológicos de HLA-DR/imunologia , Antígeno HLA-DR1/imunologia , Humanos , Epitopos Imunodominantes , Masculino , Camundongos , Camundongos Transgênicos , Modelos MolecularesRESUMO
The immune system focuses on and responds to very few representative immunodominant epitopes from pathogenic insults. However, due to the complexity of the antigen processing, understanding the parameters that lead to immunodominance has proved difficult. In an attempt to uncover the determinants of immunodominance among several dominant epitopes, we utilized a cell free antigen processing system and allowed the system to identify the hierarchies among potential determinants. We then tested the results in vivo; in mice and in human. We report here, that immunodominance of known sequences in a given protein can change if two or more proteins are being processed and presented simultaneously. Surprisingly, we find that new spacer/tag sequences commonly added to proteins for purification purposes can distort the capture of the physiological immunodominant epitopes. We warn against adding tags and spacers to candidate vaccines, or recommend cleaving it off before using for vaccination.
Assuntos
Apresentação de Antígeno , Epitopos Imunodominantes , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígeno HLA-DR1/genética , Antígeno HLA-DR1/imunologia , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , VacinaçãoRESUMO
BACKGROUND: Luminex panel reactive antibody (PRA) screening assays using microbeads are widely used for organ transplantation. Anti-HLA serum reactivity is calculated by correcting for non-specific binding to the negative control (NC) beads. High mean fluorescence intensity (MFI) value of NC beads are observed in some patients and can result in false negative results in the PRA screening assay. We analyzed the clinical characteristics and HLA types of those patients with high MFI values of NC beads. METHODS: Sixty-six patients with high MFI values of NC beads (> 300) in the PRA LABScreen Mixed assay (One Lambda) tested were included as the high NC group. Age and gender matched controls with low MFI values of NC beads (< 100) (n = 132), tested with PRA, were selected as the low NC group and 207 healthy Koreans were used as normal controls. Association of clinical characteristics and HLA types with the high NC group were analyzed using Chi-square test or Fischer's exact test, as appropriate. RESULTS: The proportion of patients with underlying liver disease was higher in the high NC group compared to the low NC group (18.1% vs. 1.5%, p < 0.001, OR = 14.2). The seropositivity of anti-nuclear antibody and rheumatoid factor, the frequency of use of intravenous immunoglobulin G, anti-thymocyte globulin, and rituximab showed no difference between two groups. The phenotype frequency (PF) of HLA-B46 was higher in the high NC group than in the low NC group (8.0% vs. 3.2%, p = 0.036, OR = 2.8). The PF of HLA-B7 was lower in the high NC group than in the healthy controls (0.0% vs. 6.5%, p = 0.008, OR = 0.1). The PF of HLA-DR1 was lower in the high NC group than in the low NC group (0.8% vs. 6.6%, p = 0.015, OR = 0.1) or healthy controls (0.8% vs. 7.4%, p = 0.003, Pc = 0.042, OR = 0.1). CONCLUSIONS: Increased non-specific binding to NC beads was associated with underlying liver disease and HLAB46. HLA-B7 and HLA-DR1 were related to a lower chance of non-specific binding to NC beads. The mechanism of those associations, such as differences in non-specific antibody response according to HLA phenotype or underlying disease, needs to be elucidated in further studies.