Background/Purpose:  Complex auto-Abs targeting nuclear proteins is a hallmark of systemic lupus erythematosus (SLE). It is documented that this complexity is the result of “B cell epitope spreading” with unique patterns. The generation of SLE-related auto-Abs is T cell dependent and preferentially restricted to HLA-DR3 (DR3) and DR2. The current hypotheses for the generation of these auto-Abs such as the “B cell epitope mimicry hypothesis” and the “particle hypothesis” in that a limited auto-reactive T cell population is able to provide T cell help to B cells of multiple specificities, are inadequate to account for the above-stated observations. The results of our analysis of DR3 restricted T cell response to SmD and Ro60 provides an alternative to the current paradigms.

Methods: T-T hybridomas were generated by fusing lymph node cells from DR3⁺AE⁰ mice immunized with SmD in IFA with BW5147TCR^-/-. IL-2 in the supernatants of T-T hybridomas stimulated by SmD with syngeneic splenic cells as APC was determined by ELISA. SmD T cell epitope regions were determined with 15mers overlapping by 3 amino acids covering the span of SmD with the established T-T hybridomas. The core epitopes were determined by alanine substitutions. TCR utilized by T-T hybridomas were sequenced. For response to SmD peptides and its mimics DR3⁺AE⁰ mice were immunized with 100µg peptide in CFA on day 1 and 50 µg of the immunogen in IFA on days 14 and 28. Sera were collected at 42, 56, 70, 90 and 120 days and assayed for Abs to SLE-related auto-Ags.

Results: Multiple T-T hybridomas reactive to SmD were generated by 7 fusions. Many hybridomas were found to be reactive with more than one T cell epitope within SmD. Some of them reacted with SmB and the A protein (ARNP) of the snRNA particle. The core epitopes of 7 SmD peptides were ascertained by alanine substitution and they have no sequence homology. Several of 15mers of these core epitopes induced B cell epitope spreading to SmB, ARNP and certain proteins in the Ro60/La system. Some bacterial peptides induced B cell epitope spreading in a manner similar to that of the SmD peptide. Interestingly, cross reactive B cell epitopes were demonstrated among the SmD peptide and its bacterial mimics. Similar findings were obtained regarding the T cell responses to Ro60 and its 15mers.

Conclusion: The results showed that SLE-related auto-Ags have multiple cross-reactive intramolecular T cell epitopes. This is a feature shared by other auto-Ags such as GAD65 in type 1 DM and myelin basic protein in EAE, a model for multiple sclerosis. Although not the subject of this abstract, there are cross-reactive inter and intra molecular B cell epitopes among the SLE-related Ags. These structural features are likely the reasons why they are the targeted in autoimmunity. The presence of multiple cross reactive intermolecular T cell epitopes provides the basis for DR3 restricted B cell epitope spreading in SLE. The cross-reactivities at both T and B cell levels of the bacterial molecular mimics and their auto-epitopes provide a scenario for the presence of SLE-related auto-Abs in normal DR3 individuals.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hla-dr3-restricted-t-cell-response-to-smd-and-ro60-reveals-multiple-cross-reactive-intra-and-inter-molecular-t-cell-epitopes-unique-antigenic-structures-of-lupus-related-auto-antigens-and-the-basis/