The role of CCR9-expressing T cell subsets in the immunopathology of primary Sjögren’s syndrome

Summary

The main aim of this thesis was to gain a better understanding of the role of CCR9-expressing T cells in pSS. So far in pSS patients CCR9+ CD4 T cells had been studied, but other CCR9-expressing cell subsets that could play a role in pSS immunopathology had not been considered. Using novel techniques the molecular, phenotypic, and functional features of several CCR9+ T cell subsets were studied, as described in chapters 2-6. Additionally, in chapter 7 a new minimal invasive method using sebum metabolomics was investigated to evaluate its potential to monitor immunopathology and to discover novel biomarkers in pSS. The main findings reported in this thesis are summarized below.

In chapter 2 the transcriptome of CCR9+ Tfh-like cells was studied. CCR9+, CXCR5+, and CXCR5-CCR9- CD4 T cells from pSS patients and healthy controls (HC) were sorted using flow cytometry, and RNA sequencing was performed. Computational analysis was used to identify differentially expressed genes (DEGs), coherent gene expression networks (modules), and differentially regulated pathways. From the three subsets CCR9+ CD4 T cells showed most DEGs between pSS patients and HC. For further analysis one module was selected that showed high eigengene expression in CCR9+ CD4 T cells, and pathways enriched for genes involved in Th1 function (cytokine and chemokine production), and migration and adhesion. Target genes were validated in additional cohorts by qPCR or on protein level by flow cytometry. However, although the findings in CCR9+ CD4 T cells as compared to CXCR5+, and CXCR5-CCR9- CD4 T cells were validated, the differences between HC and pSS patients were not.
One gene of interest was CCL5. CCR9+ CD4 T cells were shown to express higher levels of the chemokine CCL5 than CXCR5+ and CXCR5-CCR9- CD4 T cells, with the highest expression of CCL5 found in effector CCR9+ CD4 T cells. Antigenic triggering and stimulation with IL-7 of the Th cell subsets co-cultured with monocytes strongly induced CCL5 secretion in CCR9+ CD4 T cell co-cultures. Additionally, effector CCR9+ CD4 T cells rapidly released CCL5 and secreted the highest CCL5 levels upon stimulation. Given the increased numbers of CCR9+ CD4 T 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.

CD8 T cells previously were shown to secrete more CCL5 than CD4 T cells. Furthermore, using epigenetic cell counting we found that CD8 T cell presence in labial salivary gland tissue was associated with the LFS and presence of Tfh cells, implicating that CD8 T cells can play a role in lymphocytic infiltration/organization and ectopic follicular responses. The finding that CCL5 is highly expressed by CCR9+ effector CD4 T cells, led to the hypothesis that CCL5 expression by CCR9+ CD8 T cells might also be elevated in pSS patients and contribute more to total circulating CCL5 levels. In chapter 3 we were the first to show that CCR9-expressing CD8 T cells are more abundant in pSS patients as compared to HC. Antigen-experienced CD8 T cells, and especially CCR9+ effector CD8 T cells, expressed the highest CCL5 levels, and released the highest levels of CCL5 upon stimulation. CCR9+ CD8 T cells rapidly released CCL5, and released significantly more CCL5 than CCR9+ CD4 T cells. pSS patients expressed less CCL5 in their effector and memory CD8 T cells compared to HC, and subsequently released lower levels of CCL5 upon stimulation, which could point to increased release in vivo. Together these findings suggest a role for CCL5-releasing (CCR9+) CD8 T cells in pSS immunopathology.

Tph cells and CCR9+ Tfh-like cells are two CXCR5- Tfh-like cell subsets with B cell activating potential and higher frequencies in the circulation of pSS patients compared to controls. In chapter 4 the question was answered if in fact these Tfh-like cell subsets are the same or separate subsets. It was demonstrated that the two populations of cells are largely distinct and only have limited overlap. Only a small percentage of CXCR5-PD-1hi memory cells expressed CCR9. Using flow cytometry the expression of activation markers PD-1 and ICOS was compared between both CXCR5- cell subsets and CXCR5+ cells, taking the differentiation status of the cells into account. PD-1/ICOS expression was higher in memory cells expressing CXCR5 or CCR9. The highest expression of PD-1/ICOS was found in CXCR5/CCR9 co-expressing T cells, which were enriched in the circulation of pSS patients. Since the ligands for CCR9 and CXCR5 (CCL25 and CXCL13, respectively) are upregulated in pSS salivary gland tissue, cells expressing either or both chemokine receptors will migrate to the inflammatory site, and may contribute to the ongoing immune responses.

Previously we have demonstrated that CCR9-expressing CD4 T cells have increased expression of IL-7R and subsequently strongly respond to IL-7. In recent years it has become clear that besides adaptive immune cell subsets such as CD4 T cells, also innate and innate-like cell subsets can play a role in IL-7-related immunopathology, including B cell hyperactivity and local inflammation as are seen in patients with pSS. In chapter 5 the potential of IL-7 to activate IL-7R-expressing innate(-like) cells is summarized.
As pSS is characterized by inflammation in mucosa-associated tissues, in chapter 6 innate-like mucosal-associated invariant T (MAIT) cells were studied for their expression of CCR9, CXCR5, IL-18Rα and IL-7R in pSS. Both CD161+ and IL-18Rα+ MAIT cell numbers were decreased in pSS patients as compared to HC. pSS patients showed a significantly increased CD4/CD8 MAIT cell ratio, due to elevated CD4 MAIT cell frequencies compared to controls. The number of CCR9+ and CXCR5+ MAIT cells was significantly higher in pSS patients. Furthermore, expression of IL-7R, IFN-γ, and IL-21 was higher in MAIT cells as compared to non-MAIT cells. IL-7R expression on CD8 CD161+ MAIT cells correlated to lymphocytic focus scores, percentage of IgA-expressing plasma cells in salivary gland tissue, and serum IgG levels. Production of IL-21 by MAIT cells was significantly inhibited by LEF/HCQ exposure. Given the enriched CCR9/CXCR5 expression of MAIT cells, this suggests that overexpressed CCL25 and CXCL13 in the inflamed salivary glands of pSS patients could facilitate increased migration of these cells. Given the pivotal role of IL-7 and IL-21 in inflammation in pSS this also indicates a potential role for MAIT cells in driving pSS immunopathology.

Although many new technologies require limited amounts of biological material to study disease, most still do require invasive procedures to obtain the material. Therefore, the development of new minimal invasive technologies might be of interest to patients, physicians and researchers. In chapter 7 a new minimal invasive method of high throughput metabolomics technology of sebum, the product of exocrine sebaceous glands, was investigated. Sebum was easily collected from the forehead of individuals using sebutapes, and the extracted sebum was analyzed. Using untargeted mass spectrometry several thousands of metabolite ions were found in all samples. Modest differences were observed between the sebum metabolome of pSS patients as compared to controls. However, correlation analyses of metabolic changes and disease activity markers identified a few metabolite ions with a good correlation with disease activity. Sebum metabolome analysis of SSc patients showed a more outspoken difference compared to controls, with several identified metabolites involved in neurotransmission. When analyzing differentially expressed metabolite ions of pSS and SSc patients (relative to controls) strong correlations between the metabolites were observed. Altogether, this pilot study demonstrated that sebum metabolomics might be a novel strategy to identify biomarkers in pSS and other (systemic) autoimmune diseases.

Full text