The main function of Tfh cell is to help B lymphocytes to proliferate, differentiate and promote antibody production, and regulate humoral immunity [5, 6]. When CD4+ naïve-T cells are activated, they increase CXCR5expression, while decrease CCR7, which allows them to migrate into the B-cell follicles in response to CXCR5 ligand, CXCL13. This enables Tfh to localize in close proximity to and interact with B cell, and promotes GC formation and class switched high affinity antibody production [6, 9]. However, Tfh deregulations can drive abnormal germinal center, B-lymphocyte differentiation and survival, autoantibody generation, and thus could be related to the development of autoimmune diseases [12, 47]. Due to the difficulty in the sampling from human lymphoid tissue [6, 8], and since PB Tfh cells in terms of phenotypic and B lymphocyte helper functions are similar to the GC Tfh cells [16], analysis of cTfh cells in patients has become an important clinically significant alternative strategy [48]
RA is a systemic autoimmune disease characterized by the production of a large number of autoantibodies, such as ACPAs, RF and others [3], and this autoantibody production may be related to Tfh cell abnormality. In this line, while several studies have evaluated the frequency of Tfh in the PB of patients with RA compared to HCs, conflicting conclusions have been reached in some cases. The purpose of this work was to systematically evaluate the proportion of Tfh in PB of RA patients compared to HCs to clarify the proportion of cTfh in RA patients.
Meta-analysis of all included studies neglecting the definition markers, treatment status, disease activity status and serological status of the RA patients revealed a significantly higher Tfh cells proportion in the PB of patients with RA in comparison with HCs. However, significantly high degree of heterogeneity was found between studies. Thus, sub-group analyses were subsequently performed based on different factors including surface markers used to define Tfh cells, treatment status, disease activity status, and serological status of the patients as well as the geographical location of the included studies from which the participants originated. Our sub-analyses revealed that subgrouping the studies based on these criteria were associated with reduction in the strength of heterogeneity in most of analyses which reached to moderate, low or even no heterogeneity (Tables 2, 3, 4, 6 and 8).
In the sub-group analyses, at first we performed the meta-analysis based on only definition markers used to identify Tfh cells. According to the results, the proportion of Tfh cells with the all definition markers was significantly higher in the PB of RA patients compared to HCs (Table 2). From different definitions, “CD4+CXCR5+PD-1high” and then “CD4+CXCR5+PD-1+” cells showed the largest SMD, and while “CD4+CXCR5+” were significantly increased in RA, it showed the lowest SMD, suggesting the importance of considering the expression level of PD-1 in RA patients.
PD-1 can promote GC B cells survival and high-affinity long-lived plasma cell formation through its interaction with programmed death-ligand 1 (PD-L1) and PD-L2 on the surface of GC B cells [49, 50]. Studies have shown that the expression of PD-1 is elevated on Tfh cells in patients with autoimmune diseases including RA [40, 51], and PD-1high Tfh cells have a stronger ability to activate B lymphocytes [51]. Consistently, a positive correlation has been found between the expression of PD-1 on Tfh cells, and disease activity of RA [40]. However, the negative regulatory function of this molecule on T cells, that is essential for maintaining peripheral immune-tolerance, should not be ignored [49, 52]. Blocking the interaction of PD-1 with its ligands leads to increase differentiation and number of Tfh cells [50], suggesting the negative regulatory function of PD-1 signals in the differentiation of Tfh cells. Thus, the expression level of PD-1 could mirror the activation status of the Tfh cells, and the increasing in the expression of PD-1 on Tfh cells in RA patients might represent a spontaneous compensatory regulation mechanisms for controlling its excessive activation. But this regulatory feedback fails to inhibit Tfh cells, which is due to increase in soluble form of PD-1 (sPD-1) in the RA patients [33, 53]. Consistently, there is also a significant positive correlation between serum level of the sPD-1 and the frequency of cTfh cells, titer of auto-antibodies and DAS in RA patients [33, 53]. Indeed, the inhibitory function of the increased membrane-bound PD-1 on Tfh cells is blocked in the presence of its soluble form, while its humoral assistance to antibody producing cells is intact or even hyper activated. The results of our meta-analyses revealed significantly higher proportion of Tfh cells in all the comparisons which have included PD-1high for Tfh cells (RA versus HC, u-RA versus HC, S+RA versus S−RA and S+RA versus HC). Furthermore, based on subgroup analyses, the definitions which included PD-1 (“CD4+CXCR5+PD-1+” and especially “CD4+CXCR5+PD-1high”) had the largest SMDs in comparison with the other definitions suggesting more associations between the proportion of these phenotypes and RA. Thus, in RA patients, the higher expression of PD-1 on the Tfh cells could be associated with the higher proportion of these cells.
ICOS is another surface molecule that has a pivotal role in the development of Tfh cells and also in the production of IL-21 as signature cytokine of Tfh [54]. In addition, ICOS signaling is essential for maintenance of anatomical localization of Tfh cells in B cell follicles through preserving the expression of homing receptor pattern on Tfh cells. Blocking of ICOS has also been shown to lead to reverse differentiation of Tfh through CCR7 up-regulation and CXCR5 down-regulation, hence relocation of Tfh cells to the T cell zone and subsequently collapsing of the GC response [55, 56]. ICOS also promotes survival and functional maturation of GC B cells [57]. Furthermore, Tfh cells expressing the highest expression level of ICOS have the most capacity of inducing IgG production [58]. Our study demonstrated that Tfh cells expressing ICOS (CD4+CXCR5+ICOS+ and CD4+CXCR5+ICOShigh) (Table 2) were higher in RA versus HCs. In addition, “CD4+CXCR5+ICOShigh” proportion had higher association with RA than “CD4+CXCR5+ICOS+” proportion as was evident from SMDs (Table 2) which, could be justified by the above-mentioned roles defined for ICOS molecule. Considering the importance of ICOS in antibody production, unfortunately there were not at least 2 studies with CD4+CXCR5+ICOS+/high definitions for comparing Tfh cells proportion based on serostatus subgroup analyses.
We also analyzed the studies that only included untreated patients to exclude the effect of immuno-suppressive drugs. The results further confirmed the elevated proportions of Tfh cells in untreated patients, regardless of their definitions. Furthermore, subgroups analyses demonstrated higher proportions of “CD4+CXCR5+”, “CD4+CXCR5+PD-1+”, “CD4+CXCR5+PD-1high”, “CD4 + CXCR5 + ICOShigh”, “CD4+CXCR5+PD-1+ICOS+” and “CD4+CXCR5+CCR7lowPD-1high” cells in u-RA compared with HCs. From these comparisons the highest association was found for “CD4+CXCR5+PD-1high”, and “CD4+CXCR5+” had the lowest SMD, again suggesting the importance PD-1 molecules in defining the Tfh cells.
The proportion of Tfh cell was also evaluated based on the stage of the disease course. According to 2015 ACR guideline, RA patients with disease duration less than 6 months are considered “early RA”, and those with disease duration ≥ 6 months are considered as “established RA” [59]. Studies have shown that the immunological aberrations during the first few months after the disease onset differ from those during later phases [60], which may explain why response rate to treatment in early RA patients is different from those of established RA (early treatment of the RA patients is beneficial and can improve disease outcome) [61, 62]. Because of the importance of the early diagnosis of RA and also to clarify the status of Tfh cell in the early phase of the disease, we also compared Tfh cells proportion in e-RA patients with that of HCs. All e-RA patients included in the selected articles were treatment naïve. The pooled results of the all Tfh definitions exhibited that cTfh cells proportion in untreated e-RA patients was significantly greater than that of the HC group. Subgroup analysis of Tfh cells with definitions of “CD4+CXCR5+”, CD4 + CXCR5 + PD-1+ also showed higher Tfh cells proportion in untreated e-RA compared to HCs, suggesting pathogenetic role of Tfh in initial stage of the RA development.
In the next step, Tfh cells proportion in patients with a-RA was compared to r-RA patients and HCs. The pooled results regardless of definition markers revealed that the proportion of Tfh cells in a-RA patients was significantly greater than those of both r-RA patients and HCs. Altogether these results suggest pathogenic association of the Tfh frequency with RA. We then evaluated the Tfh cell proportion based on Tfh definitions. The results revealed that there was no significant difference in the proportion of “CD4+CXCR5+”, and “CD4+CXCR5+ICOS+” cells when a-RA was compared to the both r-RA and HCs, though higher SMD was observed for comparison of a-RA versus HCs than the SMD derived from comparing a-RA with r-RA. It should be kept in mind that since a maximum of two studies have been used for sub-meta analyses comparing a-RA with r-RA and HCs, the power of the test is low in these comparisons and meta-analysis on higher number of studies in the future could be more valuable.
To evaluate the link between the frequency of Tfh cells and presence of auto-antibodies in RA, we next performed meta-analysis with respect to the serological status of the patients. S+RA is defined as positive for RF or ACPAs. Seropositivity is associated with more severe disease, joint destruction, and even higher mortality [3]. The pooled meta-analysis results, regardless of the Tfh definition demonstrated that S+RA patients had a significantly higher proportion of Tfh cells in their PB compared to the either S− RA patients or HCs. Also, markers-based subgroup analysis showed that the proportion of Tfh cells was significantly elevated in S+RA in comparison with the both S−RA patients and HCs when Tfh cells were defined as CD4+CXCR5+PD-1high. This can further emphasize the previously mentioned importance of PD-1 expression level in the frequency of Tfh cells and autoantibody production in RA patients. Due to insufficient study number, we unfortunately couldn’t perform subgroup analysis on the other Tfh definitions especially ICOS-expressing Tfh cells to compare S+RA and S−RA patients. Altogether, these results demonstrate that the production of auto-antibodies in RA patients is associated with the proportion of CD4+CXCR5+PD-1high Tfh cells, which provides evidence for the connection between the seropositivity and the proportion of these cTfh cells.
Looking at different definitions used in all comparisons demonstrates that the largest SMDs for proportion of Tfh cells in comparison groups belong to the Tfh cells expressing PD-1 (PD-1+ and especially PD-1high), while the lowest association was related to the “CD4+CXCR5+” cells.
The present work has some limitations. First, as disease duration was inconsistent across the studies we couldn’t consider duration of the disease in the analysis. Second, the disease activity score of RA patients was not uniform through the studies. Third, due to heterogeneity of drugs used for treatment of patient, we couldn’t evaluate the effect of treatment on the proportion of Tfh cells in RA. Fourth, insufficient number of studies with particular Tfh definitions prevented their evaluation in some of the comparisons.