Epigenetically quantified immune cells in salivary glands of Sjögren's syndrome patients: a novel tool that detects robust correlations of T follicular helper cells with immunopathology.

OBJECTIVE: To investigate whether epigenetic cell counting represents a novel method to quantify immune cells in salivary glands of patients with different forms of Sjögren's and sicca syndrome and to capture immunopathology and potentially aid in diagnosis. METHODS: DNA from frozen salivary gland tissue sections of sicca patients was used for bisulphite conversion of demethylated DNA cytosine residues, followed by cell-specific quantitative PCR to calculate cell percentages in relation to total tissue cell numbers as quantified by housekeeping gene demethylation. The percentages of epigenetically quantified cells were correlated to RNA expression of matched salivary gland tissue and histological and clinical parameters. RESULTS: The percentages of epigenetically quantified CD3, CD4, CD8, T follicular helper (Tfh) cells, FoxP3+ regulatory T cells and B cells were significantly increased in the salivary glands of patients with SS. Unsupervised clustering using these percentages identified patient subsets with an increased lymphocytic focus score and local B cell hyperactivity and classifies patients different from conventional classification criteria. In particular, Tfh cells were shown to strongly correlate with the expression of CXCL13, lymphocytic focus scores, local B cell hyperactivity and anti-SSA positivity. CONCLUSION: Epigenetic cell counting is a promising novel tool to objectively and easily quantify immune cells in the labial salivary gland of sicca patients, with a relatively small amount of tissue needed. In view of the potential of this technique to include a huge number of (cell-specific) biomarkers, this opens up new standardized ways of salivary gland analysis with high relevance for patient classification, understanding of immunopathology and monitoring of drug responses in clinical trials.

Salivary gland secretome: a novel tool towards molecular stratification of patients with primary Sjögren's syndrome and non-autoimmune sicca.

Objective: To explore the potential of salivary gland biopsy supernatants (the secretome) as a novel tool to aid in stratification of patients with sicca syndrome and to study local immunopathology in Sjögren's syndrome. Methods: Labial salivary gland biopsies were incubated in saline for 1 hour. In these tissue supernatants from a discovery cohort (n=16) of patients with primary Sjögren's syndrome (pSS) and non-Sjögren's sicca (nSS), 101 inflammatory mediators were measured by Luminex. Results were validated in a replication cohort (n=57) encompassing patients with pSS, incomplete SS and nSS. Results: The levels of 23 cytokines were significantly increased in patients with pSS versus nSS in the discovery cohort. These 23 and 3 additional cytokines were measured in a second cohort. Elevated concentrations of 11 cytokines were validated and the majority correlated with clinical parameters. Classification tree analysis indicated that the concentrations of CXCL13, IL-21, sIL-2R and sIL-7Rα could be used to classify 95.8% of patients with pSS correctly. Conclusion: Labial salivary gland secretomes can be used to reliably assess mediators involved in immunopathology of patients with pSS, potentially contributing to patient classification. As such, this method represents a novel tool to identify therapeutic targets and markers for diagnosis, prognosis and treatment response.