Abstract
IntroductionCCR9+ Tfh-like pathogenic T helper (Th) cells are elevated in patients with primary Sjögren’s syndrome (pSS) and indicated to play a role in pSS immunopathology. Here we delineate the CCR9+ Th cell-specific transcriptome to study the molecular dysregulation of these cells in pSS patients.MethodsCCR9+, CXCR5+ and CCR9-CXCR5- Th cells from blood of 7 healthy controls (HC) and 7 pSS patients were FACS sorted and RNA sequencing was performed. Computational analysis was used to identify differentially expressed genes (DEGs), coherent gene expression networks and differentially regulated pathways. Target genes were replicated in additional cohorts.Results5131 genes were differentially expressed between CCR9+ and CXCR5+ Th cells; 6493 and 4783 between CCR9+ and CCR9-CXCR5- and between CXCR5+ and CCR9-CXCR5-, respectively. In the CCR9+ Th cell subset 2777 DEGs were identified between HC and pSS patients, 1416 and 1077 in the CXCR5+ and CCR9-CXCR5- subsets, respectively. One gene network was selected based on eigengene expression differences between the Th cell subsets and pathways enriched for genes involved in migration and adhesion, cytokine and chemokine production. Selected DEGs of interest (HOPX, SOX4, ITGAE, ITGA1, NCR3, ABCB1, C3AR1, NT5E, CCR5 and CCL5) from this module were validated and found upregulated in blood CCR9+ Th cells, but were similarly expressed in HC and pSS patients. Increased frequencies of CCR9+ Th cells were shown to express higher levels of CCL5 than CXCR5+ and CCR9-CXCR5- Th cells, with the highest expression confined to effector CCR9+ Th cells. Antigenic triggering and stimulation with IL-7 of the Th cell subsets co-cultured with monocytes strongly induced CCL5 secretion in CCR9+ Th cell cocultures. Additionally, effector CCR9+ Th cells rapidly released CCL5 and secreted the highest CCL5 levels upon stimulation.ConclusionTranscriptomic analysis of circulating CCR9+ Th cells reveals CCR9-specific pathways involved in effector T cell function equally expressed in pSS patients and HC. Given the increased numbers of CCR9+ Th cells in the blood and inflamed glands of pSS patients and presence of inflammatory stimuli to activate these cells this suggests that CCR9-specific functions, such as cell recruitment upon CCL5 secretion, could significantly contribute to immunopathology in pSS.
Highlights
chemokine receptor 9 (CCR9)+ T follicular helper (Tfh)-like pathogenic T helper (Th) cells are elevated in patients with primary Sjögren’s syndrome and indicated to play a role in Primary Sjögren’s syndrome (pSS) immunopathology
The isolated Th subsets were robustly distinguished based on their transcriptomic profile as shown in the multidimensional scaling (MDS) plot in Figure 1C. 5131 genes were differentially expressed between CCR9+ and CXCR5+ Th cells; 6493 genes were differentially expressed between CCR9+ and DN and 4783 genes were differentially expressed between CXCR5+ and DN (Figure 1D)
Transcriptomic profiles differed between healthy controls (HC) and pSS, with the largest number of Differentially expressed genes (DEGs) in the CCR9+ subset, followed by the CXCR5+ subset and the DN subset (2777, 1416 and 1077, respectively Figure 1E)
Summary
CCR9+ Tfh-like pathogenic T helper (Th) cells are elevated in patients with primary Sjögren’s syndrome (pSS) and indicated to play a role in pSS immunopathology. A hallmark feature of pSS is B cell hyperactivity, reflected by autoantibody production, elevated serum IgG levels and increased risk of lymphoma development (in 5-10% of patients) [1,2,3] Both the presence of germinal center-like structures (GCs) and a high number of lymphocytic infiltrates in the salivary glands are associated with lymphoma development [5]. CCR9+Th cells migrate to mucosal sites in response to their ligand, the C-C Motif Chemokine Ligand 25 (CCL25) and are important for mucosal immune homeostasis [15, 16] These cells are indicated to have a function in mucosal inflammation, contributing to inflammatory bowel disease (IBD) [17, 18]. In Crohn’s disease patients, inconsistent results were demonstrated, possibly due to poor pharmacokinetic properties of the small molecule inhibitors used for therapy [21,22,23]
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