Abstract
ObjectivesCD8+ T cells targeting 21-hydroxylase (21OH) are presumed to play a central role in the destruction of adrenocortical cells in autoimmune Addison’s disease (AAD). Earlier reports have suggested two immunodominant CD8+ T cell epitopes within 21OH: LLNATIAEV (21OH342-350), restricted by HLA-A2, and EPLARLEL (21OH431-438), restricted by HLA-B8. We aimed to characterize polyclonal CD8+ T cell responses to the proposed epitopes in a larger patient cohort with AAD.MethodsRecombinant fluorescent HLA-peptide multimer reagents were used to quantify antigen-specific CD8+ T cells by flow cytometry. Interferon-gamma (IFNγ) Elispot and biochemical assays were used to functionally investigate the 21OH-specific T cells, and to map the exactly defined epitopes of 21OH.ResultsWe found a significantly higher frequency of HLA-A2 restricted LLNATIAEV-specific cells in patients with AAD than in controls. These cells could also be expanded in vitro in an antigen specific manner and displayed a robust antigen-specific IFNγ production. In contrast, only negligible frequencies of EPLARLEL-specific T cells were detected in both patients and controls with limited IFNγ response. However, significant IFNγ production was observed in response to a longer peptide encompassing EPLARLEL, 21OH430-447, suggesting alternative dominant epitopes. Accordingly, we discovered that the slightly offset ARLELFVVL (21OH434-442) peptide is a novel dominant epitope restricted by HLA-C7 and not by HLA-B8 as initially postulated.ConclusionWe have identified two dominant 21OH epitopes targeted by CD8+ T cells in AAD, restricted by HLA-A2 and HLA-C7, respectively. To our knowledge, this is the first HLA-C7 restricted epitope described for an autoimmune disease.
Highlights
Patients with autoimmune Addison’s disease (AAD) suffer from adrenocortical insufficiency; an inability to produce adequate amounts of steroid hormones due to immune-mediated destruction of hormone-producing cells in the adrenal cortex. This potentially lethal disease can occur in isolation or as a component of autoimmune polyendocrine syndrome types 1 (APS-1) or 2 (APS-2), the former being a monogenic disorder caused by mutations in the AIRE gene [1]
Initial staining of peripheral blood mononuclear cells (PBMCs) from AAD patients with highly sensitive MHC dextramers revealed frequencies of LLNATIAEV- and EPLARLEL-specific T cells ranging from 0.002% to 0.183% of total CD8+ T cells (Figure 1A)
The highest individual frequency observed was for LLNATIAEVspecific cells (P5, 0.183% of CD8+ T cells)
Summary
Patients with autoimmune Addison’s disease (AAD) suffer from adrenocortical insufficiency; an inability to produce adequate amounts of steroid hormones due to immune-mediated destruction of hormone-producing cells in the adrenal cortex. This potentially lethal disease can occur in isolation or as a component of autoimmune polyendocrine syndrome types 1 (APS-1) or 2 (APS-2), the former being a monogenic disorder caused by mutations in the AIRE gene [1]. Among HLA class I genes, the B*0801 allele occurs more frequently in patients than controls [8] and increases the susceptibility to AAD in combination with DR3 [9]. A recently performed genome-wide association study (GWAS) has revelated AAD risk loci in several T cell related genes, including AIRE, CTLA4 and PTPN22, supporting the notion of T cell-driven pathology [10]
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