Discussion
Tfh and Tfreg cells are the latest members of CD4+ T cells that constitute a compact immune regulatory system and disturbance of this system can cause autoimmune disease [26, 29-31]. Despite ongoing research, little is known about the role of Tfh and Tfreg cells in MS pathogenesis.
In the present study, we showed an increased frequency of circulating CXCR5+PD-1+ Tfh cells in MS patients compared to HC. Along with higher frequency of cTfh cells, IL-21 secretion was also elevated significantly in MS patients. Subsequent analysis of IL-21 production by follicular and non-follicular Th cells revealed that cTfh cells were engaged with highest production of IL-21, where the frequency was remarkably increased in MS patients. The role of Tfh cells in autoimmune disease was initially described in murine model [39]. However, recent reports with human data in RA, SLE, MG, and autoimmune thyroid disease have revealed that frequency of Tfh cells and their cytokine IL-21 were elevated where they associated with disease activity as well as severity through the amplification of autoreactive B cells and pathogenic autoantibody production [28-30, 40]. Our data are consistent with those results and more importantly for MS, we convincingly connect cTfh cells to high levels of IL-21 production, suggesting that these cells may play a vital role in the immunopathogenesis of MS. Our results also supported by previous study in MS, where the frequency of memory Tfh and plasma as well as CSF IL-21 levels were higher in MS patients [33, 41, 42]. However, we have to admit that we lacked treatment-naïve MS patients who provide us clearer picture about cTfh and IL-21 in the disease context. Interestingly, despite our patients were on first-line injectable disease modifying therapy, the frequency of cTfh cells and the production of IL-21 remained elevated, while other subsets of Th cells showed no significant difference between HC and MS. These results suggest first-line injectable therapies may have minimal effects on cTfh and IL-21regarding their frequency and secretion in MS. Recently, Nicolas et al. reported that elevated cTfh cells can be reverted in patients with neuromyelitis optica after treatment with rituximab [43], suggesting to explore new therapeutic solution for MS.
Proportional bias among different subtypes of Tfh cells have been reported to be associated with several autoimmune diseases, such as SLE, MG, RA and IgG4-related disease [29-32]. cTfh17 cells have been reported to be enriched in primary progressive MS compared to HC and RRMS [41]. The exact role of cTfh17 cells in MS is not clear. Morita et al. have reported that cTfh17 cells proficiently help naïve and memory B cells to produce Igs (IgG, IgA, and IgM), a process that needs to be elucidated in MS [25]. Cunill et al. have recently described an increased percentage of cTfh17.1 cells in untreated RRMS compared to HC, but no significant difference was observed in the cTfh17 frequency [45]. In our study, both cTfh17 and cTfh17.1 cells were highly elevated in MS patients compared to HC. However, increased frequency of cTfh17.1 cells, but not of cTfh17 cells were positively associated with significantly higher production of IL-21 in MS patients. Additionally, among the four subtypes, only cTfh17.1 cells were involved in producing substantial amount of various cytokines including IFN-γ, IL-17 and IL-21, where the level of IL-21 secretion was greatest in MS patients. These observations collectively suggest cTfh17.1 cells may be one of the key players in disease pathogenesis. However, an extensive study with transcriptional profiling and evaluation of a broader range of cytokines may provide more insights on the role of cTfh17.1 cells in MS pathogenesis.
IL-17 producing Th17 cells were reported to be increased in untreated MS patients compared to HC [43, 45, 46]. In this study, there was no remarkable difference in the frequency of non-follicular Th17 cells and IL-17 secretion between HC and MS patients. This discrepancy might be attributed to enrolled patients who were continuously undergoing disease-modifying treatments. Indeed, recent reports have shown that IL-17 producing CD4+ T cells were reduced in MS patients after treatment with β-interferon or dimethyl fumarate [47, 48].
Alternatively, it is known that the pathogenesis of MS is linked to the failure of regulatory activities by Treg cells [15, 49, 50]. In this study, one of our main focuses was to investigate whether the proportion of CXCR5+PD-1+FoxP3+CD25+cTfreg cells is altered in MS patients. The data revealed that the frequency of cTfreg cells was significantly lower in MS patients compared to HC. Additionally, reduced CXCR5+PD-1+ phenotypes among cTreg cells in MS patients compared to HC implies the unique behavior of cTfreg cells in MS. Unlike GC-Tfreg, cTfreg does not express Bcl-6 and ICOS although they share the same functional property to control excessive humoral-mediated immunity [21]. Recently, an in vitro co-culture assay with responder T cells and resident Tfreg and cTfreg cells has shown that both cTfreg and resident Tfreg cells provide equal suppressive activity over the responder cells [34]. The frequency of cTfreg cells was found to be reduced in different autoimmune diseases [26, 29, 34]. These findings are consistent with our result and therefore suggest that impaired cTfreg cells may participate in uncontrolled immunoregulation in MS.
The mechanism underlying the suppressive activity of Tfreg cells over other effector T cells is still elusive. It is not clear whether Tfreg cells directly inhibit Tfh cells by blocking their transcriptional activation or they use the IL-10 signaling pathway to set up their inhibitory milieu. Our data has shown that the frequency of IL-10 producing cTfreg cells was significantly higher than their non-follicular cTreg counterpart in both HC and MS patients, suggesting that cTfreg cells were the primary producer of IL-10 among different subtypes of cTreg cells. Interestingly, we observed a decreased frequency of cTfreg cells associated with a parallel reduction of IL-10 in MS patients compared to HC. Taken together, these data demonstrate that the lower frequency of cTfreg is likely linked to reduced IL-10 secretion in MS patients. The exact role of IL-10 in MS remains elusive. Recently, depleted IL-10 secretion was reported to increase the severity of MS [51, 52]. One possible hypothesis might be that, IL-10 exerts beneficial effect in MS by regulating other cytokines, resulting in less tissue damage and minor lesion [53]. Failure of cytokine regulation by defective IL-10 has been reported in progressive MS [54]. Our findings together with previous reports suggest that lower level of IL-10 secreted by cTfreg cells may be involved in MS pathogenesis and might serve as a novel therapeutic target to treat MS.