Localisation of MHC Class II/HC gp-39 complexes in synovia of rheumatoid arthritis patients using complex-specific monoclonal antibodies

Objective

Recently HC gp-39 was identified as a candidate autoantigen in RA. The aim of this study is to generate and validate monoclonal antibodies (mAbs) to HLA DRαβ1*0401/HC gp-39^263–275 complexes to further investigate the relevance of this antigen in RA.

Methods

Spleen cells from mice immunised with HLA DRαβ1*0401/HC gp-39^263–275 complexes were selected and submitted to a B-cell culture and minielectrofusion procedures. The resulting set of mAbs were characterised for their specificity by ELISA and FACS. Functionally, they were tested for inhibition of HC gp-39^263–275-specific DRαβ1*0401-restricted T-cell hybridomas. Finally, one of the mAbs was used for staining of specific complexes in RA synovium.

Results

We have generated a set of five mAbs to a combination epitope of the specific MHC/peptide complexes. FACS studies revealed that these mAbs recognize specific complexes on homozygous DRαβ1*0401-positive BLCL pulsed with HC gp-39^263–275. The best mAb, 12A, was further characterized using a set of irrelevant DRαβ1*0401-binding peptides, and truncated/elongated versions of HC gp-39^263–275 itself. The minimal epitope recognized in combination with DRαβ1*0401 was HC gp-39^263–273. Peptides not encompassing HC gp-39^263–273 were not recognized. Three out of five mAbs were able to inhibit (up to 90%) the response of HC gp-39^263–275-specific DRαβ1*0401-restricted T-cell hybridomas to peptide-pulsed antigen-presenting cells or purified complexes. Moreover, mAb 12A localised and identified dendritic cells that present DRαβ1*0401/HC gp-39^263–275 complexes in synovial tissue of DRαβ1*0401-positive RA patients.

Conclusions

These data indicate local presentation of the HC gp-39^263–275 epitope in the inflamed target tissue by professional antigen-presenting cells and support a role of HC gp-39 in the local autoimmune response that leads to chronic inflammation and joint destruction.