Background/Purpose: Sjögren’s syndrome (SS) is an autoimmune disease which is characterized by lymphocytic infiltration including CD4⁺ IL-17 producing helper T (Th17) cells to the lacrimal and salivary glands. We previously detected anti-M3 muscarinic acetylcholine receptor (M3R) antibodies and M3R reactive CD4⁺ IFNγ producing helper T (Th1) cells in patients with SS. Moreover, we clarified that M3R reactive Th1 and Th17 cells had pathogenic roles in the development of auto-immune sialadenitis in SS mice model. The purpose of this study was to identify circulating M3R reactive Th17 cells, those T cell epitopes, and the relationship between these cells and clinical features in patients with primary SS (pSS).

Methods:

1. Peripheral blood mononuclear cells (PBMCs) were isolated from 10 pSS patients and age-gender matched 10 healthy controls (HCs). According to their HLA-DRB1 typing, top 10 ranked 20 mer peptides from the full length of M3R, which were highly predicted to bind to each HLA molecules by using the immune epitope database website, were selected for each case. PBMCs were stimulated with these selected M3R peptides mixture for 40 hours, and M3R reactive IL-17 producing cells were detected by IL-17 enzyme-linked immunospot assay (ELISpot).
2. Clinical features were compared between M3R reactive IL-17 producing cells positive and negative pSS patients.
3. PBMCs from 5 pSS patients positive for M3R reactive IL-17 producing cells, were stimulated with each selected 12-20 mer M3R peptide separately, to identify the dominant M3R peptides responsible for IL-17 secretion.
4. To confirm that detected IL-17 producing cells were Th17 cells, peripheral CD4⁺ T cells from 3 pSS patients positive for M3R reactive IL-17 producing cells, were co-cultured with dendritic cells (DCs) generated from peripheral CD14⁺ monocytes in each case, and stimulated with the dominant M3R peptides identified in Methods 3.

Results:

1. 5 of 10 (50%) pSS patients, while none of 10 (0%) HCs, showed significantly increased IL-17 positive spots against selected M3R peptides mixture stimulation compared with non-stimulation in ELISpot. M3R reactive IL-17 producing cells were detected significantly more frequently in pSS than in HCs (p=0.03).
2. 5 pSS patients positive for M3R reactive IL-17 producing cells had significantly higher ESSDAI score than 5 negative pSS patients (8.4±4.8 vs 2.0±0.0, p=0.031).
3. In all 5 pSS patients positive for M3R reactive IL-17 producing cells described in Results 1, IL-17 was produced against M3R AA76-95 stimulation, showing that the sequence might be the dominant M3R peptide responsible for IL-17 secretion.
4. Co-cultured CD4⁺ T cells with DCs under stimulation with the dominant M3R peptide identified in Results 3, showed significantly increased IL-17 positive spots than non-stimulation, clarifying that M3R reactive Th17 cells were detected.

Conclusion: We detected circulating M3R reactive Th17 cells in pSS patients using ELISpot for the first time. Moreover, T cell epitope of these cells was shown to be M3R AA76-95 in all M3R reactive Th17 cells positive pSS patients. Interestingly, M3R reactive Th17 cells might associate with higher ESSDAI score.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/detection-and-clinical-significance-of-circulating-m3-muscarinic-acetylcholine-receptor-reactive-th17-cells-in-patients-with-primary-sjogrens-syndrome/