Transcriptome Analysis of CCR9+ T Helper Cells From Primary Sjögren’s Syndrome Patients Identifies CCL5 as a Novel Effector Molecule

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.

CCL5 Release by CCR9+ CD8 T Cells: A Potential Contributor to Immunopathology of Primary Sjögren’s Syndrome

“…Although CCR9+ effector CD8 T cells expressed the highest CCL5 levels, also in all other CD27/CD45RO-defined CD8 T cell subsets a significantly higher CCL5 expression was found in CCR9+ cells as compared to CCR9- CD8 T cells, which we previously also demonstrated for CD4 T cells ( 17 ). Since CCR9 is a chemokine receptor that initiates migration of T cells towards CCL25 under both physiological and inflammatory conditions and CCL5 is a chemokine that recruits several types of leukocytes to inflamed sites, the high expression of CCL5 in CCR9-expressing cells indicates that facilitating recruitment of other inflammatory cells is a main feature of these cells in immunopathology.…”

“…Earlier work in our group has shown that CCR9+CXCR5- “Tfh-like” CD4 T cells form a distinct group of cells, which show a potent pro-inflammatory phenotype and have an increased frequency in peripheral blood and salivary gland tissue of pSS patients ( 15 ). Chemokine CCL5 was identified as a potential target, and was highest expressed by effector CCR9+ CD4 T cells ( 17 ). CD8 T cells, however, are described as a more potent producer of CCL5 ( 36 , 37 ): a finding we confirmed in this study for both HC and pSS patients.…”

“…One study has found elevated serum CCL5 in pSS patients with an elevated ESR ( 40 ). In addition, several studies have shown increased expression of CCR5 by pDCs, cDC2s, and CCR9+ CD4 T cells ( 17 , 20 ). Both CCL5 and CCR5 were shown to be increased in salivary glands of pSS patients and thus increased release of CCL5 by CD8 T cells could play a key role in regulation of chemotaxis and subsequent inflammation ( 21 , 22 ).…”

“…In our previous work studying CCR9+ CD4 T cells in pSS we identified genes that are specifically upregulated in CCR9+ CD4 T cells and were involved in effector T cell pathways compared to CXCR5+ and CCR9-CXCR5- CD4 T cells ( 17 ). One of the identified genes is CCL5 .…”

“…T cells, monocytes and NK cells. It is associated with Th1 activity, just like its receptor CCR5 ( 17 – 19 ). The main producers of type I IFNs, pDCs, have an elevated expression of CCR5 in pSS patients compared to healthy controls (HC) ( 20 ).…”