RESUMEN
OBJECTIVE: Rheumatoid arthritis (RA) immunopathogenesis revolves around the presentation of poorly characterised self-peptides by human leucocyte antigen (HLA)-class II molecules on the surface of antigen-presenting cells to autoreactive CD4 +T cells. Here, we analysed the HLA-DR-associated peptidome of synovial tissue (ST) and of dendritic cells (DCs) pulsed with synovial fluid (SF) or ST, to identify potential T-cell epitopes for RA. METHODS: HLA-DR/peptide complexes were isolated from RA ST samples (n=3) and monocyte-derived DCs, generated from healthy donors carrying RA-associated shared epitope positive HLA-DR molecules and pulsed with RA SF (n=7) or ST (n=2). Peptide sequencing was performed by high-resolution mass spectrometry. The immunostimulatory capacity of selected peptides was evaluated on peripheral blood mononuclear cells from patients with RA (n=29) and healthy subjects (n=12) by flow cytometry. RESULTS: We identified between 103 and 888 HLA-DR-naturally presented peptides per sample. We selected 37 native and six citrullinated (cit)-peptides for stimulation assays. Six of these peptides increased the expression of CD40L on CD4 +T cells patients with RA, and specifically triggered IFN-γ expression on RA CD4 +T cells compared with healthy subjects. Finally, the frequency of IFN-γ-producing CD4 +T cells specific for a myeloperoxidase-derived peptide showed a positive correlation with disease activity. CONCLUSIONS: We significantly expanded the peptide repertoire presented by HLA-DR molecules in a physiologically relevant context, identifying six new epitopes recognised by CD4 +T cells from patients with RA. This information is important for a better understanding of the disease immunopathology, as well as for designing tolerising antigen-specific immunotherapies.
Asunto(s)
Artritis Reumatoide , Epítopos de Linfocito T , Antígenos HLA-DR , Humanos , Leucocitos Mononucleares , PéptidosRESUMEN
We studied the association of human leukocyte antigen (HLA)-DRB1 and HLA-DQB1 alleles and HLA haplotypes with juvenile rheumatoid arthritis (JRA) in 65 patients and 65 controls from Colombia. The JRA subsets were distinguished on the basis of criteria established by the American College of Rheumatology. Two alleles were associated with protection, HLA-DRB1*1501 (p = 0.002) and HLA-DRB1*1402 (p = 0.01). HLA-DRB1*1602 (p = 0.0000002) was associated with susceptibility for systemic JRA and HLA-DRB1*1104 (p = 0.0002) for pauciarticular JRA. Amino acid sequences at residues 70-74 of DRB1 chain shared by HLA-DRB1 alleles (shared epitomes) were also informative. The polyarticular JRA subset revealed association with (70)QRRAA(74), which includes HLA-DRB1*04, 01, and (70)DRRAA(74), which includes DRB1*1601, 1602, 1101, and 1104. Two new findings of interest were the association of the haplotypes DRB1*1104, DQB1*0301(p = 0.0002) with pauciarticular JRA and DRB1*1602, DQB1*0301 (p = 0.0000002) association with systemic JRA. The DRB1 alleles of these two haplotypes share the epitope (70)DRRAA(74)and were associated with both the pauciarticular and the systemic subset of JRA. Our results suggest that studies of disease susceptibility in populations of admixed genetic background should take into account the contribution of different ethnic groups or nationalities in the recruitment of controls and patients studied in order to rule out genetic stratification.
Asunto(s)
Alelos , Artritis Juvenil/genética , Antígenos HLA-DR/genética , Adolescente , Artritis Juvenil/diagnóstico , Artritis Juvenil/etiología , Niño , Preescolar , Colombia/etnología , Epítopos/genética , Etnicidad/genética , Femenino , Marcadores Genéticos , Antígenos HLA-DQ/genética , Cadenas beta de HLA-DQ , Cadenas HLA-DRB1 , Haplotipos/genética , Humanos , Masculino , Análisis de Secuencia de ProteínaRESUMEN
Oligotypes of the human leukocyte antigen HLA Class II, DRB1 alleles were characterized at the molecular level in a group of Colombian children suffering juvenile rheumatoid arthritis (JRA). The distribution of these alleles was examined in a group of Colombian mestizo children (genetic admixture of Amerindians, Europeans and Africans) suffering from clinically distinct JRA subsets in order to detect HLA allele frequency differences in patients with different JRA subsets. A group of 65 patients with JRA and 65 controls were characterized for the subtypes of the HLA-DRB1 alleles using polymerase chain reaction with sequence-specific oligonucleotide probes (PCR-SSOP). The oligotyping protocol recommended by the 12th International Histocompatibility Workshop held in St. Malo, Paris, in 1996, was used. Subtype HLA-DRB1*1104 was the allele most strongly associated with susceptibility to JRA (Fisher's p = 0.013, odds ratio (OR) = 16.79, etiologic fraction (EF) = 0.93). HLA-DRB1*1602 was also associated with susceptibility to a lesser degree (Fisher's p = 0.016, OR = 8.98, EF = 0.88). HLA-DRB1 alleles participating in JRA protection were HLA-DRB1*1501 (preventive fraction (PF) = 0.466, p = 0.005) and HLA DRB1*1402 (PF = 0.49, p = 0.009). The relationship between some HLA-DRB1 alleles and clinical features was also compared. The presence of rheumatic factor was associated with the alleles HLA-DRB1*0407 (p = 0.05, OR = 11.2, EF = 0.45) and HLA-DRB1*1302 (p = 0.02, OR = 22.8, EF = 0.63). There was also an association between HLA-DRB1*0701 (p = 0.001, OR = 58, EF = 0.73) with expressing ANA +. We found that in the oligoarticular subset, the allele HLA-DRB1*1104 (p = 0.0034, OR = 41.53, EF = 0.97) was the one expressed most commonly. In the poliarticular group, the alleles most frequently expressed were HLA-DRB1*0404 (Fisher's p = 0.012, OR = 8.75, EF = 0.88). In patients with systemic JRA, the HLA-DRB1*1602 allele (p = 0.005, OR = 21.33, EF = 0.95) was most frequent. These results suggested that the MHC genes of mestizo children influence not only the clinical expression of the disease, but also the susceptibility to its development.
Asunto(s)
Alelos , Artritis Juvenil/genética , Antígenos HLA-DR/genética , Polimorfismo Genético , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Colombia , Femenino , Cadenas HLA-DRB1 , Humanos , Indígenas Sudamericanos , MasculinoRESUMEN
Se buscó tipificar molecularmente los alelos de los antígenos de leucocitos humanos HLADRB1 en un grupo de niños mestizos colombianos (población resultante de la mezcla genética de amerindios, europeos y africanos) con artritis reumatoidea juvenil (ARJ), así como analizar su frecuencia de expresión y compararla con sujetos clínicamente normales e investigar la asociación entre la frecuencia de los alelos con los diferentes subgrupos clínicos de ARJ. El estudio involucró 65 pacientes con ARJ y 65 controles sanos. La tipificación de los alelos HLADRB1 se realizó por medio de la metodología de la reacción en cadena de la polimerasa con sondas de oligonucleótidos de secuencias específicas (PCR-SSOP), utilizando el protocolo recomendado por el XII International Histocompatibilty Workshop, realizado en St. Malo, París, en 1996. Los alelos HLADRB1* 1104 (prueba exacta de Fisher, PEF)0,013, OR16,79, frecuencia etiológica (FE)0,93) y DRB1*1602 (PEF0,016, OR8,98, FE0,88) se evidenciaron como marcadores de susceptibilidad. HLA-DRB1*1501 (FP0,466; p0,005) y HLA DRB1*1402 (FP0,49; p0,009) se comportaron como alelos asociados con protección. Al comparar las asociaciones entre alelos y los diferentes subgrupos clínicos se encontró asociación entre ARJ oligoarticular con HLA-DRB1* 1104 ( p0,0034, OR41,53 , FE0,97), la ARJ poliarticular se asoció con el alelo HLADRB1* 0404 ( p0,012, OR8,75, FE0,88 ) y en el grupo sistémico, el alelo más expresado fue el HLA-DRB1*1602 ( p0,005, OR21,33, FE0,95). La presencia de factor reumatoide estuvo asociado con los alelos HLA-DRB1*0407 ( p0,05, OR11,2, FE0,45) y HLA-DRB1*1302 ( p0,02, OR22,8, FE0,63). En el grupo de pacientes con ANA+, sólo hubo significancia estadística para el alelo HLA-DRB1* 0701 ( p0,001, OR58, FE0,73). Nuestros resultados sugieren que los genes del MHC en este subgrupo étnico inciden no sólo en la susceptibilidad a desarrollar ARJ sino también en la expresión clínica de la enfermedad.