T cell epitope mimicry between Sjögren’s syndrome Antigen A (SSA)/Ro60 and oral, gut, skin and vaginal bacteria (1)
Cross-reactivity between Ro60 and EBV protein EBNA-1 has been proposed as a mechanism for the initiation of anti-Ro60 antibody response in lupus patients. Similarly, B cell cross-reactivity between Coxsackie virus 2B protein and Ro60 has been suggested to play a role in the initiation and perpetuation of anti-Ro60 autoantibody response in Sjögren’s syndrome.
A mouse strain that lacks murine MHC class II and expresses the HLA DR3 (DRB1*0301 and DRA1*0101 transgenes) was used. Mice were immunized in one foot pad and at the base of the tail with 100 μg of recombinant human Ro60 emulsified in Freund’s incomplete adjuvant. On day 10 after immunization, the draining lymph node cells were harvested and stimulated for 3 days with recombinant Ro60. Cells were fused with BW5147 TCR−/− fusion partner and T cell hybrids were selected using standard HAT medium. A panel of T cell hybrids was generated from HLA-DR3 transgenic mice immunized with Ro60 and epitope mapping was performed using 52 synthetic peptides. Four hybridomas (Hyb-9, Hyb-24, Hyb-34, and Hyb-26) showed that the T cell epitopes localized to 3 regions on Ro60.
Fine epitope mapping was done by using overlapping synthetic peptides covering each epitope region to stimulate the respective T cell hybridoma.The hyb-24.1 and hyb-34.14 recognize the same epitope region on Ro60, their core epitopes are different. For hyb-24.1 it is Ro60228-237 (YLEAVEKVKR), whereas for hyb-34.14 it is Ro60229-238 (LEAVEKVKRT).
In the 13 peptide mimics that activated Ro60 reactive T cells, 3 are found in oral flora (Prevotella disiens , Capnocytophaga sputigena and Capnocytophaga ochracea); 4 are in the gut flora (Bacterioides finegoldii, Bacteroides intestinalis, Bacteroides fragilis and Alistipes finegoldii) and 2 are found on the skin (Corynebacteriium amycolatum and Acinetobacter johnsonii). While Prevotella disiens is also found in the vagina and is associated with vaginosis, Pseudomonas mendocina is an opportunistic pathogen linked with endocarditis. Two mimics (Flavobacteria bacterium and Listeria grayi) are found in marine flora and in contaminated food respectively. Purified recombinant vWFA protein was generated and its ability to activate hyb-9.5 investigated. However, C. ochracea can activate hyb-9.5 were not successful. This failure might be due to the presence of a suppressor factor produced by C. ochracea that has been shown to suppress mitogen stimulated lymphocyte proliferation.
Cross-reactive B cells recognizing Epstein bar virus nuclear antigen 1 (EBNA1) peptide 58-72 and Ro60 peptide 169-180 have been suggested to initiate the anti-Ro60 antibody responses in lupus patients. Anti-Ro60169-180 affinity purified antibodies reacted with EBNA-1 and recognized the EBNA-1 peptide 58-72. Moreover, immunization of rabbits with the EBNA-1 peptide 58-72, led to the generation of anti-Ro60 antibodies. Anti-Ro60216-232 antibodies from Sjögren’s syndrome patients were cross-reactive with Coxsackie virus 2B protein peptide 31-47.
B cells cross-reactive with Coxsackie virus 2B protein and Ro60 might be involved in the initiation of anti-Ro60 antibody response.
The vWFA domain was originally described in the blood coagulation protein von Willebrand factor and is present in a large number of prokaryotic and eukaryotic proteins. C. ochracea, is a gram negative, facultative anaerobic bacterium that was originally isolated from dental plaque obtained from both healthy and diseased gingival sites.