鉴定反应性关节炎和未分化脊柱关节病患者中沙门氏菌外膜蛋白的免疫原性 HLA-B*27:05 结合肽。

Identification of immunogenic HLA-B*27:05 binding peptides of salmonella outer membrane protein in patients with reactive arthritis and undifferentiated spondyloarthropathy.

机构信息

Department of Clinical Immunology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.

出版信息

J Rheumatol. 2013 Feb;40(2):173-85. doi: 10.3899/jrheum.110849. Epub 2013 Jan 15.

DOI:10.3899/jrheum.110849

PMID:23322470

Abstract

OBJECTIVE

Salmonella outer membrane proteins (OMP) are major immunogenic targets to synovial fluid lymphocytes of patients with reactive arthritis (ReA)/undifferentiated spondyloarthropathy (uSpA). Because these patients have genetic predisposition to HLA-B27 and its subtype HLA-B27:05, we sought to identify immunogenic HLA-B*27:05-binding salmonella OMP peptides in patients with ReA/uSpA.

METHODS

A total of 125 HLA-B*27:05-binding salmonella OMP peptides identified using ProPred-I software were synthesized and grouped in 23 pools. The peptide pools, along with crude enteric bacterial lysates and salmonella OMP, were cultured with synovial fluid (SF) or peripheral blood mononuclear cells (PBMC) from 23 patients with ReA/uSpA, 10 with rheumatoid arthritis (RA), and 10 healthy individuals in 96-well culture plates. Proliferation was measured by tritiated thymidine uptake and interferon-γ (IFN-γ) levels in culture supernatant. Individual peptides from pools having significant responses were retested with cryopreserved cells. Immunogenic peptides thus identified were further tested in 5 additional new patients with ReA/uSpA by flow cytometry. A Basic Local Alignment Search Tool program was used to search for similar peptides from a protein bank of arthritogenic bacteria and human protein.

RESULTS

Nineteen of 23 SFMC from ReA/uSpA showed a significant proliferative response to salmonella OMP, with minimal response of PBMC (1/10) from ReA/uSpA, SFMC from RA (1/10), or PBMC from controls (1/10). Nine salmonella OMP peptides, QRAEMLPTL, SRSGLNIAL, LRFLYAKSL, RLEGTWVKL, ARCIAPYAL, KLFLTTAAL, YRNSDFFGL, QRPAVRVKL, and YRVGPGDVL, were identified. Response to QRAEMLPTL was seen in 6/7 HLA-B*27:05-positive patients. All immunogenic peptides had sequence similarity with peptides from arthritogenic bacterial proteins, while 5 had similarity with peptides from human proteins.

CONCLUSION

Nine novel immunogenic OMP peptides binding to HLA-B*27:05 were identified that showed sequence similarity with other arthritogenic bacteria.

摘要

目的

沙门氏菌外膜蛋白（OMP）是反应性关节炎（ReA）/未分化脊柱关节病（uSpA）患者滑液淋巴细胞的主要免疫原性靶标。由于这些患者具有 HLA-B27 及其亚型 HLA-B27:05 的遗传易感性，我们试图在 ReA/uSpA 患者中鉴定免疫原性 HLA-B*27:05 结合的沙门氏菌 OMP 肽。

方法

使用 ProPred-I 软件共鉴定出 125 种与 HLA-B*27:05 结合的沙门氏菌 OMP 肽，并将其分为 23 个池。将这些肽池与粗肠细菌裂解物和沙门氏菌 OMP 一起与 23 例 ReA/uSpA、10 例类风湿关节炎（RA）和 10 例健康个体的滑液（SF）或外周血单核细胞（PBMC）在 96 孔培养板中孵育。通过放射性氚摄取和培养上清液中的干扰素-γ（IFN-γ）水平测量增殖。对具有显著反应的池中的个别肽进行冷冻保存细胞的重新测试。通过流式细胞术进一步在 5 名新的 ReA/uSpA 患者中测试鉴定出的免疫肽。使用 Basic Local Alignment Search Tool 程序从关节炎细菌和人类蛋白质的蛋白质库中搜索相似的肽。

结果

23 例 ReA/uSpA 的 SFMC 中有 19 例对沙门氏菌 OMP 表现出显著的增殖反应，而 ReA/uSpA 的 PBMC（1/10）、RA 的 SFMC（1/10）或对照的 PBMC（1/10）反应最小。鉴定出 9 种沙门氏菌 OMP 肽，分别为 QRAEMLPTL、SRSGLNIAL、LRFLYAKSL、RLEGTWVKL、ARCIAPYAL、KLFLTTAAL、YRNSDFFGL、QRPAVRVKL 和 YRVGPGDVL。在 6/7 HLA-B*27:05 阳性患者中观察到对 QRAEMLPTL 的反应。所有免疫肽都与关节炎细菌蛋白的肽具有序列相似性，而 5 个肽与人类蛋白的肽具有相似性。