CD4+ T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren’s syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4+CD45RA− T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies.

Objective Determine the presence and assess the extent of fatty infiltration of the minor salivary glands (SG) of primary SS patients (pSS) as compared to those with non SS sicca (nSS). Methods Minor SG biopsy samples from 134 subjects with pSS (n= 72) or nSS (n = 62) were imaged. Total area and fatty replacement area for each glandular cross-section (n=4–6 cross-sections per subject) were measured using Image J (National Institutes of Health, Bethesda, MD, USA). The observer was blinded to subject classification status. The average area of fatty infiltration calculated per subject was evaluated by logistic regression and general linearized models (GLM) to assess relationships between fatty infiltration and clinical exam results, extent of fibrosis and age. Results The average area of fatty infiltration for subjects with pSS (median% (range) 4.97 (0.05–30.2)) was not significantly different from that of those with nSS (3.75 (0.087–41.9). Infiltration severity varied widely, and subjects with fatty replacement greater than 6% were equivalently distributed between pSS and nSS participants (χ2 p=0.50). Age accounted for all apparent relationships between fatty infiltration and fibrosis or reduced saliva flow. The all-inclusive GLM for prediction of pSS versus non-SS classification including fibrosis, age, fatty replacement and focus score was not significantly different from any desaturated model. In no iteration of the model did fatty replacement exert a significant effect on the capacity to predict pSS classification. Conclusions Fatty infiltration is an age-associated phenomenon and not a selective feature of Sjögren’s syndrome. Sicca patients who do not fulfill pSS criteria have similar rates of fatty infiltration of the minor SG.

To assess the types of salivary gland (SG) T cells contributing to Sjögren’s syndrome (SS), we evaluated SG T cell subtypes for association with disease features and compared the SG CD4+ memory T cell transcriptomes of subjects with either primary SS (pSS) or non-SS sicca (nSS). SG biopsies were evaluated for proportions and absolute numbers of CD4+ and CD8+ T cells. SG memory CD4+ T cells were evaluated for gene expression by microarray. Differentially-expressed genes were identified, and gene set enrichment and pathways analyses were performed. CD4+CD45RA− T cells were increased in pSS compared to nSS subjects (33.2% vs. 22.2%, p < 0.0001), while CD8+CD45RA− T cells were decreased (38.5% vs. 46.0%, p = 0.0014). SG fibrosis positively correlated with numbers of memory T cells. Proportions of SG CD4+CD45RA− T cells correlated with focus score (r = 0.43, p < 0.0001), corneal damage (r = 0.43, p < 0.0001), and serum Ro antibodies (r = 0.40, p < 0.0001). Differentially-expressed genes in CD4+CD45RA− cells indicated a T follicular helper (Tfh) profile, increased homing and increased cellular interactions. Predicted upstream drivers of the Tfh signature included TCR, TNF, TGF-β1, IL-4, and IL-21. In conclusion, the proportions and numbers of SG memory CD4+ T cells associate with key SS features, consistent with a central role in disease pathogenesis.

The ELISPOT is a powerful functional assay used to detect biological activity and immunological secretions from immune cells. In this chapter, we specifically discuss T cell ELISPOT methods for the detection of secreted cytokines. A detailed protocol is given enabling the detection of interferon gamma-secreting CD8(+) T cells and/or peripheral blood mononuclear cells following their isolation and polyclonal activation. Included is a brief discussion on choosing the activation method for your T cell ELISPOT assay, as well as additional instructions for the adaptation of this protocol for the study of memory and antigen-specific T cell responses.

Objective To assess the types of salivary gland (SG) T cells contributing to Sjögren’s syndrome (SS), we evaluated SG T cell subtypes for association with disease features and compared the SG CD4+ memory T cell transcriptomes of primary SS (pSS) and sicca subjects not meeting SS criteria (nSS). Methods SG biopsies of pSS and nSS subjects were evaluated for proportions (n=51 pSS, n=69 nSS) and absolute numbers (n=35 pSS, n=57 nSS) of CD4+ and CD8+ T cells. SG memory CD4+ T cells from focus score positive pSS (n=17) and focus score negative nSS subjects (n=15) were evaluated for gene expression by microarray. Differentially-expressed genes were identified and gene set enrichment and pathways analyses were performed. Results CD4+CD45RA− T cell frequencies were increased in pSS compared to nSS subjects (33.2 vs. 22.2%, p&lt;0.0001), while CD8+CD45RA− T cells were decreased (38.5 vs. 46.0%, p=0.0014). SG fibrosis positively correlated with numbers of memory T cells, regardless of phenotype. Proportions of SG CD4+CD45RA− T cells correlated with focus score (r=0.43, p&lt;0.0001), corneal damage (r=0.43, p&lt;0.0001), and serum Ro antibodies (r=0.40, p&lt;0.0001). Differentially expressed genes in CD4+CD45RA− cells of pSS versus nSS subjects indicated a T follicular helper (Tfh) profile, increased homing and increased cellular interactions. Predicted upstream drivers of the Tfh signature included TCR, TNF, TGF-β1, IL-4 and IL-21. Conclusions The proportions and numbers of SG memory CD4+ T cells associate with key SS features, consistent with a central role in disease pathogenesis. SG memory CD4+ T cells express a germinal center Tfh profile.

Sjögren’s syndrome is a systemic rheumatic disorder characterized by dry eyes, dry mouth and T cell infiltration of exocrine gland tissue. To determine the extent of salivary gland (SG) CD4+ memory T cell clonal expansion and whether these expansions are as frequent in peripheral blood (PB), a multiplex PCR method was used to amplify both the α and β T cell receptor (TCR) sequences from memory CD4+ T cells sorted from SG lip biopsy tissue and PB of 10 primary Sjögren’s syndrome patients. Over 3,000 TCR sequences were obtained from 50-115 (median 91) individual SG and 75-121 (median 104) individual PB T cells per patient. The percentage of cells that were part of clonal expansions was significantly higher in SG (median 11%, range 0-28%) compared to PB (median 0.9%, range 0-7%, p=0.003). Sequence analysis revealed: 1) highly homologous CDR3s among different expanded SG T cell clones within single patients, suggesting antigen-driven expansion and 2) unique cases of convergent recombination among unrelated patients, where different V segments/additions/deletions were utilized to make identical CDR3 amino acid sequences. The percentages of clonally expanded SG T cells correlated significantly with degree of SG fibrosis and reduced saliva production but not with systemic features of disease. SG clonal expansions detected in this study likely identify T cells involved in recognition of common antigen(s) and glandular dysfunction.