4.1.3. Th17 Cells

The role of Th17 cells, has been studied extensively in the past decade in the pathogenesis of SS [130]. Both IL-6 and transforming growth factor (TGF)-β are required to induce naive murine CD4<sup>+</sup> T cells to develop into Th17 cells, which are characterized by the expression of retinoic acid receptor-related orphan receptor γ (RORγ)t. In humans, the differentiation of Th17 cells occurs by activation of T cell receptor (TCR) signaling in the presence of TGF-β and IL-6 or IL-21 stimulation [131]. Other critical cytokines that play a role in the progression of the disease include IL-22 and IL-23. IL-22 is derived primarily from natural killer cells, but it is also produced by Th17 cells, and it has been identified in the mSG tissue of pSS patients [74]. IL-23, while not required for differentiation of Th17 cells, is a cytokine that is necessary for their survival and maintenance [132]. Th17 cells produce IL-17A (referred to here as IL-17) and five other IL-17 members which have also been described that are termed as IL-17B, C, D, E (or IL-25), and F with conserved residues in the c-terminal region that form homodimers [133]. Local IL-17 protein production and mRNA levels, together with IL-6 and IL-23 mRNA, have been shown to increase with the progression of lesion severity in mSGs of pSS patients [134]. Th17 cells are the primary producers of IL-17A and IL17F and other cytokines such as TGFβ, IL-6, and IL-12, which have been detected in the plasma and saliva of pSS patients [134]. The nuclear receptor RORγt plays an indispensable role in the differentiation of Th17 cells as increased presence and activation indicates an increase in autoimmunity [135]. PBMCs from SS patients have the capacity to secrete IL-17 and IL-12 which skew naïve CD4<sup>+</sup> T cells to Th1 and Th17 cells respectively, thereby facilitating initiation of the auto-immune cascade [136]. IL-21 expression in SGs has also been associated with hypergammaglobulinemia and patients with primary SS [137]. Th17 cells display the CD4<sup>+</sup> CD161<sup>+</sup> phenotype in circulation and have been found to be increased at advanced stages of the disease [138]. There are other subsets of marker specific T cells that contribute to disease progression. CD4−CD8− double negative T cells are a subset that is capable of producing IL-17 and has been correlated with more severe glandular infiltration and is present during the formation of GCs [139]. Another direct set of Th17 cells that secrete IL-17 consistently in the periductal infiltrates of all mSGs,

has been identified with the level of expression directly correlating with the severity of glandular inflammation and as a result destruction of healthy gland tissue [134].

Other Th17 cytokines that include IL-17 and IL-23 expression in SGs cause an increase in Tbet expression in the pre-disease phase in the C57BL/6.NOD-*Aec1Aec2* model [140]. The systemic effect of IL-17 on sexual dimorphism has been elucidated by genetically ablating IL-17 in C57BL/6.NOD-*Aec1Aec2* mice. It has been observed that the elimination of IL-17 reduces sialadenitis more drastically in females than in males [141]. The TCR repertoires of Th1 and Th17 cells in SG infiltrates have been found to be restricted, with an increase in the number of pathogenic effector T cells in the glands with a sex-based selection bias of TCR repertoires [142]. Furthermore, it has been observed that transferring Th17 cells in IL-17 deficient mice, restores the SS disease phenotype, highlighting the key role of Th17 cells in the inflammatory cascade and subsequent disease progression [143].

The function of RORγt overexpression in naive CD4<sup>+</sup> T cells has been elucidated in RAG deficient mice showing the development of pSS phenotype upon transfer of RORγt-overexpressing CD4+T cells that induce sialadenitis. The findings in IL-17-deficient mice therefore, suggest that IL-17 is essential for the development of sialadenitis [144]. Gene therapy studied in the C57BL/6.NOD-*Aec1Aec2* mice has explored the role of cytokines like IL-27. Induction of IL-27, a natural inhibitory cytokine of Th17 expression, was found to down-regulate or reverse SS in C57BL/6.NOD-*Aec1Aec2* mice via a recombinant adeno-associated virus (rAAV) 2-IL27 vector injection. Th1 activation and inhibitory activity of Th17 cells was observed [145].

M3R-reactive CD3<sup>+</sup> T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics SS [146]. M3R is the primary receptor subtype that promotes fluid secretion in salivary acinar cells. Both interferon IFN-γ and IL-17 are required for induction of SS in MIS, indicating that M3R-reactive Th1 and Th17 cells contribute to the pathogenesis of autoimmune sialadenitis. Thus, MIS is used to analyze the effectiveness of RORγt antagonists [147]. As mentioned, anti-M3R autoantibodies have been proposed to contribute to secretory dysfunction in SS. Iizuka et al. showed that transferring the M3R deficient splenocytes to RAG deficient mice lead to Th1 and Th17 infiltration in SGs and pSS like symptoms. Lymphocytic infiltration and destruction of epithelial cells in the SGs indicated that M3R reactive CD3<sup>+</sup> T cells played a pathogenic role in the development of autoimmune sialadenitis [146].

In the lacrimal glands, the lymphocytic infiltration and the presence of IL-17 can also be observed. IL-17 conjunctival mRNA and protein expression in tears is observed to be higher in pSS as compared to non-SS patients exhibiting dry eye disease [148], whereas percentages of peripheral IL-17-producing CD4<sup>+</sup> T cells are shown to be similar between pSS patients and controls. The importance of Th17 was further supported in animal models of SS. IL-2Rα (CD25) knockout mice develop autoimmunity and lymphoproliferative disorders and produce significantly higher levels of IL-6, TGF-β1, IL-23R, IL-17, IL-17F, IL-21, IL-10, and IFN-γ mRNA in the cornea and conjunctiva. This promotes autoimmune lacrimal-keratoconjunctivitis with symptoms closely resembling SS. Th-17 cells are shown to produce IL-17 that overlap with the peak severity of corneal epithelial disease [149]. A clinical trial using anti-IL-17 failed to improve dry eye in SS patients, which makes the role of Th17 cells in disease progression within LGs ambiguous [150].

## 4.1.4. T Regulatory Cells (Tregs)

Tregs possess suppressive activity towards autoreactive lymphocytes via either cell-cell contact or the release of soluble mediators including IL-10 and TGF-β. The commitment of a naïve T lymphocyte towards a Treg phenotype is dependent on a specific cytokine microenvironment and of the expression of the forkhead box protein P3 (FoxP3) transcription factor [151]. Understanding the role of Treg cells in SS pathogenesis has been complicated by studies reporting mixed and controversial results. The inconsistencies in results can be explained at least in part by the different strategies employed to assess Treg cells in the course of disease progression. Studies follow two approaches of either enumerating the proportion of circulating Treg cells according to the high surface expression of

CD25high cells or combining surface expression with the co-expression of FoxP3, the most specific marker of Treg cells. An increase of circulating FoxP3<sup>+</sup> cells in pSS biopsies correlates with worse clinical disease has been observed as shown by Sarigul et al., similar to FoxP3<sup>+</sup> cells circulating in patients with RA [143,152,153]. Several studies report a reduction of peripheral blood Treg cells [78,154–157] and highlight an association between the reduction of these cells and exacerbated clinical symptoms. Szodoray et al. have proven that Treg cell reduction resulted in prevention of extra-glandular manifestations [78]. Contrary to these results, other groups report increased circulating Treg cells in pSS patients that show clinical symptoms, with no glandular manifestation and no serological features [152,158], and in a few cases CD4+CD25high cell percentages are similar in the peripheral blood of pSS patients and controls [153,159,160].

Disease activity does not influence the number of circulating Treg cells and the disease presents as either being a mild stable polyclonal hypergammaglobulinemia, as was the case for one group (inactive) or a more severe polyclonal hypergammaglobulinemia (active) [160], as was the case for another group. Other murine studies on Tregs in SS include the treatment of TSP1-KO mice with TSP1-derived peptide to prove attenuation of the clinical symptoms of SS-associated dry eye in TSP-1 deficient mice. This demonstrates that an increase in Treg cells, which reduce Th17 cells, can attenuate disease symptoms. TGF-β plays a pivotal role in differentiation for immunosuppressive FoxP3+Tregs, where an increase is evident in biopsy specimens with mild and moderate inflammation which is disproportionate to escalating pro-inflammatory Th17 populations in advanced disease [134].

There is an increase in the frequency of CD4+Foxp3<sup>+</sup> Tregs observed with age in the cervical lymph node (CLN), spleen, and LG of NOD.B10.H2b mice. These CD4+CD25<sup>+</sup> cells lose suppressive ability, while maintaining expression of Foxp3 and producing IL-17 and IFN-γ. Furthermore, an increase of Foxp3+IL-17<sup>+</sup> or Foxp3+IFN-γ<sup>+</sup> cells was observed in the LG and LG-draining CLN of these mice [161]. The role of Tregs is uncertain because of a balance in between Tregs and Th17 cells [162]. Further soluble mediators, such as TGF-β, the level of which is increased in SGs of SS patients compared to controls, is required for both Treg and Th17 cell development [163,164].

#### 4.1.5. T Follicular Helper Cells (Tfh)

T follicular helper (Tfh) cells are specialized providers of T cell help to B cells, and are essential for GC formation, affinity maturation, and the development of high affinity antibodies and memory B cells. Tfh cell differentiation is a multi-factorial process involving B cell lymphoma 6 (Bcl6) and other transcription factors that are usually upregulated in autoimmunity [165]. B cell depletion therapy by Rituximab has been used in patients with pSS, where it decreased the elevated levels of circulating Tfh cells and improved the symptoms of patients, illustrating the crucial role of the crosstalk between B cells and Tfh cells in pSS [166]. Tfh cell differentiation is driven by the transcription factor Bcl-6 and activates Tfh cells to express high levels of Inducible T-cell costimulator (ICOS) and Programmed cell death protein 1 (PD-1) [167]. Tfh cells are important in the formation of GCs and primarily show presence of CD84 as a cell surface marker. CD84 <sup>+</sup> PD-1+Bcl6<sup>+</sup> Tfh cells have been identified in organized structures with high focus scores and are in close proximity with Bcl6<sup>+</sup> B cells, suggesting an association with increased disease severity in SS [168]. Tfh cells facilitate T cell–dependent B cell responses, mainly by secretion of IL-21, a primary driver of B cell activation and differentiation towards plasma cells. Increased frequencies of Tfh cells have been associated with several autoimmune diseases [169,170]. Cohorts of Tfh cells have been defined where the frequencies of circulating Tfh (cTfh) cells, defined as CD4+CD45RA−CXCR5+PD-1+cells, and are increased in pSS patients. Tfh cells within glandular tissue cannot be easily identified due to overlapping CXCR5 expression with B cells. Detection by immunohistochemistry and quantification of these cells by flow cytometry is difficult because biopsies are processed into cell suspensions using enzymatic digestion, and in the process CXCR5 expression is lost [171]. The function of CXCR5 positive Tfh cells is thus directly related to the secretion of IL-21 mediating B cell maturation, proliferation, and GC formation.
