Mapping of class II HLA restriction of SepSecS-specific CD4 T-cell epitopes in autoimmune hepatitis

Autoreactive CD4 T cells, which recognize liver self-antigens such as SepSecS, are potentially main drivers of the chronic inflammatory response during autoimmune hepatitis (AIH). Previous studies have revealed SepSecS epitopes often associated with HLA-DRB1∗03 or HLA-DRB1∗04 restriction, two alleles enriched in AIH population. However, human leukocyte antigen (HLA) restriction of numerous SepSecS epitopes remains incomplete, and whether they could be presented by non-HLA-DR molecules remains unclear. Here, we investigated epitope recognition and HLA restriction of SepSecS-specific CD4 T cells from AIH patients.

We cloned 17 T-cell receptor αβ (TCRαβ) from the most expanded SepSecS-specific CD4 T cells of five AIH patients from our previous studies into a murine hybridoma T-cell line. The epitope reactivity of each TCR cell line was identified via IL-2 secretion following culture with 20-mer overlapping peptides of the SepSecS protein and immortalized B-cell lines. HLA restriction was defined using HLA blocking antibodies. We analyzed 484 SepSecS-specific TCRs from nine AIH patients using GLIPH2 algorithm. CD154 activation-induced marker assay was used to evaluate the SepSecS epitope reactivity in AIH patients (n = 11).

Autoreactive TCRs recognized six SepSecS amino acid regions in vitro, with four distinct class II HLA restrictions, including non-HLA-DR restrictions. The GLIPH2 algorithm clustered SepSecS-specific TCRs into six groups. Notably, 6% of the total SepSecS-specific TCR clonotypes shared common CDR3β motifs linked to the recognition of HLA-DPA1∗02:01/HLA-DPB1∗01:01-restricted SepSecS_152-179 epitope. Ex vivo peptide stimulation assay revealed that SepSecS_152-179 epitope represents 15% to 67% of total SepSecS CD4 T-cell reactivity in HLA-DPA1∗02:01∼HLA-DPB1∗01:01 AIH patients (n = 5).

We highlighted that SepSecS epitopes could be presented by HLA-DR and non-HLA-DR molecules to autoreactive CD4 TCRs with potentially conserved CDR3 motifs for common epitope recognition.

In AIH, SepSecS is a key autoantigen targeted by the adaptive immune system, but only few T-cell epitopes are defined and associated with a precise HLA restriction. By reconstructing TCRs from the most expanded SepSecS-specific CD4 T cells of AIH patients, we identified new T-cell epitopes and their specific class II HLA restriction, including peptides presented by non-HLA-DR molecules. From a clinical perspective, these findings could be used to develop tools to track the frequency of autoreactive T cells in patients. This work could also pave the way for self-antigen-specific immunotherapies, such as peptide-based desensitization or the development of HLA-multimer technologies to selectively deplete or reprogram pathogenic T cells without inducing systemic immunosuppression.