Background/Purpose: HLA class II is the major susceptibility region for systemic lupus erythematosus (SLE) and other autoimmune disorders such as rheumatoid arthritis, multiple sclerosis and type 1 diabetes. The current paradigm is that autoimmune response is the result of breaking tolerance. However, it’s difficult to explain the presence of autoreactive Abs and T cells in normal people. Previously we showed HLA-DR3 (DR3) transgenic mice respond best to immunization with SmD and Ro60. To clarify the mechanisms for auto-Ab generation to SLE-related Ags, we undertook a detailed analysis of the anti-SmD T cell responses in DR3⁺AE⁰ mice.

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. Two hundred blood samples of healthy donors from Virginia Blood Services were analyzed for their HLA-DR types and auto-Abs titers.  

Results: 47 stable T-T hybridomas reactive to SmD were generated by 7 fusions. They reacted with seven SmD antigenic regions. By alanine substitutions, they reacted with varied core epitopes. In some, the flanking sequences contributed to the core epitopes. The TCR of 17 hybridomas were sequenced. They used unique combinations of TCRα and TCRβ. By bioinformatics, we identified more than 10,000 potential bacterial peptide mimics of SmD T cell epitopes. Some of them are from commensal bacteria residing in the mucosal surfaces and skin. The ranking of affinities for SmD T epitopes to DR3 were in the mid-ranges among those of all potential bacterial mimics. 12 bacterial mimics from one of the SmD T epitopes with high, medium, and low affinities for DR3 were selected. Only those with modest binding affinities were able to stimulate relevant hybridomas and to induce auto-Ab in a similar fashion as the corresponding SmD peptide. The 200 blood donors were typed for HLA-DR2, DR3 and DR4. DR3⁺ individuals had higher anti-SmD Abs than those who were negative for DR3 and DR2.  

Conclusion: SmD has multiple T cell epitopes and the immune response to SmD is similar of those to conventional Ags with diverse TCRs. The T cells with cross-reactivity to auto-epitopes and to bacterial mimics are those with moderate binding affinities to the relevant DR molecules. They are likely to be positively selective for the purpose of host defense against pathogens. This thesis is supported by the finding of auto-Abs with high titers to SLE-related auto-Ags. These results provide us insight into the origin of SLE-related auto-Abs. On a basic level, they support the thesis that auto-reactive T cells are an inherent part of the normal immune repertoire.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hla-dr3-restricted-response-to-lupus-related-auto-antigen-smd-autoreactive-t-cells-are-inherent-in-normal-immune-repertoires/