Background/Purpose: Selective modulation of autoimmune responses through antigen-specific therapies represents a promising direction for improving treatment specificity and safety. Genetic associations with HLA class II alleles, combined with evidence of tissue-infiltrating T cells and T cell–mediated cytokine signatures, highlight the central role of CD4⁺ T cells in driving autoimmune pathology. However, the targets of these pathogenic T cells remain poorly defined, limiting development of antigen-specific therapies. TScan’s proprietary TargetScan platform offers an unbiased, high-throughput method to identify natural targets of disease-relevant T cell receptors and support development of antigen-specific tolerizing modalities.

Methods: To identify HLA class II T cell targets for CD4⁺ T cells, the TargetScan platform was adapted to present genetically encoded peptide libraries on ectopically expressed HLA class II molecules. We initially applied this approach to scleroderma. CD4⁺ T cells were isolated from affected skin biopsies of patients and analyzed using 10x Genomics single-cell RNA sequencing. A total of 764 TCRs were identified, with candidates selected for screening based on pathogenic gene expression profiles and CDR3 sequence clustering shared by TCRs found in more than one patient. 61 candidate TCRs were screened for antigen recognition in the context of disease-associated Class II HLA alleles, and identified targets were assessed for disease relevance using follow-up validation assays. Similarly, we used the TargetScan platform to evaluate TCRs from inflamed colon samples derived from patients with ulcerative colitis.

Results: Screening of the candidate TCRs using the Class II TargetScan platform identified eight novel targets presented by multiple HLA Class II alleles associated with scleroderma. These reactive TCRs belonged to shared CD3⁺ clusters found in multiple patients and were characterized by high expression of activation and tissue-homing markers, as well as genes associated with Th2 and Th17 profiles. The identified targets exhibited expression patterns consistent with disease relevance and were validated through orthogonal assays, including functional T cell activation assays. In ulcerative colitis, two novel targets were identified with expression patterns consistent with disease pathology.

Conclusion: TScan’s Class II TargetScan platform enables unbiased identification of novel autoantigens in CD4⁺ T cell–driven autoimmune diseases where antigenic drivers have remained poorly defined. These discoveries may inform the development of antigen-specific therapeutic strategies, including tolerogenic approaches aimed at restoring immune homeostasis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-novel-hla-class-ii-restricted-autoantigens-in-scleroderma-and-ulcerative-colitis-using-targetscan/