Follicular helper T cells in the pathogenesis of IgG4-related disease

Follicular helper T cells in the pathogenesis of IgG4-related disease Abstract IgG4-related disease (IgG4-RD) is a recently recognized disease entity characterized by high serum IgG4 concentrations and infiltration of IgG4+ plasma cells with hyperplastic ectopic germinal centres at affected sites. Although the underlying immune mechanism of this disease remains unclear, T cells are abundantly present at affected sites and key players in IgG4-RD pathogenesis. The role of T cell subsets has been investigated thoroughly in this disease. Recent advances in this field have clarified the importance of T follicular helper cells. In this review, we describe the role of T follicular helper cells in the disease process of IgG4-RD, in particular, for IgG4 class-switching, plasmablast and plasma cell differentiation, and germinal centre formation. IgG4-related disease, pathogenesis, T lymphocyte, T follicular helper cell, germinal centre, plasmablast, somatic hypermutation, interleukin-4, interleukin-21 Rheumatology key messages T follicular helper 2 cells lead to enhanced IgG4 production in IgG4-related disease. T follicular helper 2 cells induce plasmablast differentiation and germinal centre formations in IgG4-related disease. Activated T follicular helper 2 cells are a potential biomarker for IgG4-related disease activity. Introduction IgG4-related disease (IgG4-RD) affects various organ systems and is characterized by elevated serum IgG4 levels, tissue infiltration by IgG4+ plasma cells and storiform fibrosis [1–4]. The underlying pathogenesis of IgG4-RD remains largely unknown. A better understanding of this disease is necessary for identifying therapeutic targets. Since large numbers of T cells are present at affected sites, T cells are involved in the pathogenesis of this disease. Evidence implicating T cells in IgG4-RD has been increasing, and the role of T cell subsets have been clarified. Ectopic germinal centres appear at affected sites and local B cells produce IgG4. T follicular helper (Tfh) cells help B cell maturation and Ig isotype-switching at germinal centres. Of note, recent investigations have revealed the importance of Tfh cells in the pathophysiology of IgG4-RD. In this review we discuss the pathogenic role of Tfh cells as well as the other T cell subsets in IgG4-RD. The differences between T helper cells and Tfh cells It would be helpful to understand the major differences between T helper (Th) and Tfh cells because Tfh cells are specialized providers of B cell help and germinal centre development [5, 6]. B cell lymphoma 6 (Bcl-6) has been demonstrated as the master regulator of Tfh cell differentiation because the deletion of Bcl-6 results in the absence of Tfh cells, but normal development of the other Th cell subsets [7–9]. Accordingly, Bcl-6 is highly expressed in Tfh cells and is also known to be an antagonistic transcription factor for B lymphocyte–induced maturation protein 1 (Blimp-1), the transcription factor highly expressed in Th cell subsets other than Tfh cells [7–9]. The chemokine receptor CXC chemokine receptor 5 (CXCR5) is a central marker of Tfh cells, which is required for Tfh cells to migrate into germinal centres via interactions with its ligand CXC chemokine ligand 13 (CXCL13) [6]. CXCL13 is also expressed at high levels by Tfh cells (but not by other Th cells) in humans [6]. The transcription factor achaete-scute homologue 2 (Ascl2) is selectively upregulated in Tfh cells in vivo [10]. Ascl2 upregulates CXCR5 (but not Bcl-6) in T cells to induce T cell migration into the germinal centres [10], whereas Ascl2 acts as a suppresser of Th1, Th2 and Th17 cell differentiation. At the T–B border, B cells provide another signal for precursor Tfh cells to upregulate Bcl-6 expression for the completion of Tfh cell polarization and germinal centre formation [10]. Signalling lymphocytic activation molecule (SLAM)-associated protein (SAP) is critical for Tfh cell and germinal centre development [6]. SAP plays a role in Tfh–B interaction by binding to the intracellular domains of SLAM family surface receptors [6]. Programmed death (PD)-1 is generally expressed on activated T cells, including Tfh cells. PD-1 is an inhibitory receptor essential for T cell tolerance, but PD-1 expressed by Tfh cells plays a role in B cell selection via direct inductive signals to B cells [6]. Inducible T-cell co-stimulator (ICOS) has roles in Tfh cell differentiation, migration, cytokine production and germinal centre formation [11, 12]. B cells also express ICOS ligand, which is required for germinal centre formation. IL-21 is the most important Tfh cell cytokine for driving plasmablast and plasma cell differentiation in germinal centres [13]. IL-4 from Tfh cells (but not from Th2 cells) plays a role in class-switch recombination [14, 15]. Moreover, the deletion of Th2-related genes does not result in a loss of germinal centres [16, 17], whereas the deletion of both IL-4 and the IL-21 receptor resulted in a loss of germinal centres [13], suggesting that both IL-4 and IL-21 from Tfh cells are required for germinal centre development and maintenance. Germinal centres are the primary location for antigen-specific B cell proliferation, mutation and differentiation and Ig class-switching [6]. In summary, distinguishing features of Tfh cells from the other Th cells include the expression of Bcl-6, CXCR5, Ascl2, SAP, PD-1, ICOS, IL-21 and IL-4, in concert with the absence of Blimp-1 (Table 1). Table 1 The differences between T helper cells and T follicular helper cells Characteristics  Th cells  Tfh cells  Functions          Antigen-specific B cell proliferation, affinity mutation and somatic hypermutation  −  +      Class switch recombination  −  +      Plasmablast and plasma cell differentiation  −  +      Germinal centre development  −  +  Molecule expressions          Bcl-6  −  +      Blimp-1  +  −      Ascl2  −  +      CXCR5  −  +      CXCL13  −  +      SAP  Normal  High      PD-1  Variable  +      ICOS  Variable  +  Cytokines          IL-21  Variable  +      IL-4  + (Th2)  +  Characteristics  Th cells  Tfh cells  Functions          Antigen-specific B cell proliferation, affinity mutation and somatic hypermutation  −  +      Class switch recombination  −  +      Plasmablast and plasma cell differentiation  −  +      Germinal centre development  −  +  Molecule expressions          Bcl-6  −  +      Blimp-1  +  −      Ascl2  −  +      CXCR5  −  +      CXCL13  −  +      SAP  Normal  High      PD-1  Variable  +      ICOS  Variable  +  Cytokines          IL-21  Variable  +      IL-4  + (Th2)  +  Bcl-6: B cell lymphoma-6; Blimp-1: B lymphocyte-induced maturation protein 1; Ascl2: achaete-scute homologue 2; CXCR5: CXC chemokine receptor 5; CXCL13: CXC chemokine ligand 13; SAP: signalling lymphocytic activation molecule–associated protein; PD-1: programmed cell death-1; ICOS: inducible T cell co-stimulator; +: positive; −: negative. Th1 cells Th1 cells play a role in cell-mediated immune responses, including production of IFN-γ (which leads to the activation of macrophages and cytotoxic T cells). Th1-type immune responses have been reported to be involved in the pathogenesis of IgG4-RD in the last decade. Circulating Th1-like cells (as indicated by IFN-γ+ CD4+ T cells) have been reported as increased in IgG4-RD [18]. Moreover, an increased level of serum IFN-γ has been reported [19–21]. IFN-γ+ CD4+ cells and IFN-γ mRNA expression were also significantly increased at affected sites [19, 22]. Of note, even in the cases that showed considerable skewing towards Th2-type immune responses, a substantial number of Th1-type immune responses were identified, suggesting that Th1-type immune responses play a role in IgG4-RD [23, 24]. However, it remained unclear which type of CD4+ T cells that secrete IFN-γ [Th1 cells, cytotoxic CD4+ T cells or T follicular helper 1 (Tfh1) cells] are involved in this disease. Recent studies have revealed that cytotoxic CD4+ T cells or Tfh1 cells are likely candidates for IFN-γ–producing CD4+ T cells in IgG4-RD [25, 26]. Th2 cells IgG4-RD patients frequently have an atopic history, serum IgE elevation and eosinophilia [27, 28]. Atopic features are induced by Th2 cytokines, such as IL-4, IL-5 and IL-13. Therefore, Th2 cells have been considered to play a role in the pathogenesis of IgG4-RD. Several studies that examined peripheral blood have found that circulating Th2 cells, characterized by GATA-3 transcription factor mRNA, CRTH2 expression or IL-4 and IL-5 expression on CD4+ T cells, are increased in IgG4-RD [29–32]. From the viewpoint of serum cytokine levels, a remarkable increase in serum IL-4 has been demonstrated in IgG4-RD [21, 33]. Bile samples from patients with IgG4-related cholangitis showed significantly increased IL-4, IL-5 and GATA-3 mRNA [34]. The cytokine profiles in bronchoalveolar fluids from IgG4-related respiratory disease showed significantly higher IL-5 and IL-13 [24]. The expression of Th2 cytokines such as IL-4, IL-5 and IL-13 at affected sites was also increased [23, 35–37]. However, controversial results have been reported. One report demonstrated increased circulating GATA-3+ Th2 cells only in patients with IgG4-RD who had an atopic history [38], and another one demonstrated that GATA-3+ Th2 cells were sparse at sites of IgG4-RD, although IL-4 mRNA expression was significantly increased [25]. These results suggest that the increased IL-4 at affected sites is produced by non-Th2 cells [25]. Further, atopic asthma patients showed significantly lower levels of serum IgG4 compared with IgG4-RD patients, while elevated serum IgE levels and increased circulating Th2 cells were comparable between the two diseases, also suggesting that non-Th2 cells play a role in IgG4 class-switching [29, 30]. In line with these reports, we have observed that Th2 cells from patients with IgG4-RD did not induce the in vitro differentiation of naïve B cells into plasmablasts, or IgG4 production (Akiyama M, et al., unpublished data). Importantly, more recent studies have revealed that Tfh2 cells are a likely candidate as IL-4–producing cells in IgG4-RD [26, 39]. Treg cells Treg cells play an important role in the regulation of self-tolerance and the maintenance of tissue homeostasis [40]. Accumulating evidence of increased Treg cells at affected sites and also in peripheral blood has been reported in IgG4-RD [35, 37, 41–45], although a more recent report has shown only a marginal increase in circulating Treg cells [46]. On the other hand, there has been no report of decreased Treg cells in IgG4-RD, in contrast to the situation in other autoimmune diseases, such as type 1 diabetes, multiple sclerosis, lupus and RA [47]. Involvement in IgG4 class-switching Although several studies suggest that IL-10 secreted by Treg cells plays a role in IgG4 class-switching [36, 37, 41, 42], IL-10 is also known to be secreted by other immune cells such as Th cells, Tfh cells, B cells, mast cells, eosinophils, monocytes and macrophages [48]. Another important consideration is that eosinophilic granulomatosis with polyangiitis showed a decrease in circulating Treg cells [49–51] despite its remarkable elevation of serum IgG4 and IgG4/IgG ratio [52, 53]. No study has reported a functional analysis of Treg cells from patients with IgG4-RD, and direct evidence of IgG4 class-switching by Treg cells is required in the future. Involvement in fibrosis Another important point about the presence of Treg cells in IgG4-RD is their possible involvement in the pathogenesis of fibrosis through TGF-β secretion. Although the induction of extracellular matrix components by TGF-β is crucial during wound healing and tissue repair, overproduction of TGF-β leads to fibrosis [54]. Since fibrosis at affected sites is an important characteristic of IgG4-RD, TGF-β may play an important role. Indeed, several studies have shown increased TGF-β expression, particularly at fibrosclerosing lesions of IgG4-RD [34, 36, 42, 55]. Considering the capability of a broad range of cells to produce TGF-β [56], Treg cells may be one of the immune cell types contributing to fibrosis in IgG4-RD. CD8+ T cells Tissue destruction caused by the cytolytic mediator perforin, the serine protease granzyme or Fas lytic pathways from CD8+ T cells is more important in SS than IgG4-related dacryoadenitis and sialadenitis, so-called Mikulicz disease. However, the number of IFN-γ+CD8+ T cells in peripheral blood was reported to be increased in IgG4-RD [18] with a similar extent of CD8+ T cell infiltration at affected sites between IgG4-RD and SS [57]. Importantly, the frequency of expression of perforin and granzyme B by CD8+ T cells was lower in IgG4-RD, whereas the frequency of expression of the immunoinhibitory receptor PD-1 by the same cells was higher in IgG4-RD compared with in SS [57], suggesting that CD8+ T cells are exhausted in IgG4-RD. In fact, expression of the apoptosis mediator Fas (CD95)–Fas ligand in lymphocytes was higher at affected sites in patients with SS compared with in patients with Mikulicz disease [58, 59]. Moreover, circulating lymphocytes in Mikulicz disease showed a defect in cytotoxic capacity [59]. As a result, although both diseases involve lymphocytic infiltration, only the acinar cells in SS undergo apoptosis [58]. Thus, acinar cell apoptosis by CD8+ T cells is involved in the pathogenesis of SS but not of Mikulicz disease. Cytotoxic CD4+ T cells Cytotoxic CD4+ T cells are classically known as the CD28low subpopulation among CD4+ T cells, and they secret Th1-like and proinflammatory cytokines [60]. The expansion of this T cell subset at affected sites and in peripheral blood has been initially described in patients with granulomatosis with polyangiitis, and their numbers correlate with the total number of affected organs, suggesting that cytotoxic CD4+ T cells are important in the pathogenesis of this disease [60–63]. Clonally expanded cytotoxic CD4+ T cells that secrete IFN-γ and perforin have also been reported in peripheral blood and at affected sites in RA [64, 65]. Oligoclonal expansion of circulating CD4+SLAMF7+ T cells in patients with IgG4-RD, and their infiltration at affected sites, has recently been described [25, 46]. These cells also express low levels of CD28 and secrete granzyme B, TGF-β, IL-1β and IFN-γ, suggesting that this T cell subset consists of so-called cytotoxic CD4+ T cells [46]. TGF-β, IL-1β and IFN-γ are believed to be important mediators of fibrosis. Thus, these cytotoxic CD4+ T cells may play a role in fibrosis in IgG4-RD. Importantly, the number of circulating CD4+SLAMF7+ T cells is also increased in systemic sclerosis patients, suggesting that the increase in this T cell subset is not specific for IgG4-RD [46]. Tfh cells Molecules associated with Tfh cell functions Activation-induced cytidine deaminase (AID) is necessary for somatic hypermutation and class-switch recombination of antibodies [66–69], but the specific target of the heavy-chain constant-region gene recombination requires additional cytokines and CD40 signalling [70]. CD40 ligand is the most important protein expressed by Tfh cells, and it provides signals to B cells (through CD40) essential to their differentiation, Ig class-switching and germinal centre formation [71, 72]. IL-4 was long considered to be exclusively produced by Th2 cells, and thus Th2 cells were believed to be the main providers of ‘help’ to B cells. However, it is now well accepted that Tfh cells are the specialized providers of ‘help’ to B cells. It has been shown that Tfh cells secrete IL-21 and are the primary producers of IL-4 in secondary lymphoid organs [73–75]. Thus, the combination of AID, CD40L, IL-4 and IL-21 is important for controlling B cell differentiation, somatic hypermutation and Ig class-switching, and germinal centre development [66]. Tfh-cell associated events at affected sites of IgG4-RD The germinal centre is composed of two regions: the dark zone is the site of rapid germinal centre B cell proliferation, whereas the light zone is where Tfh cells reside and interact with germinal centre B cells [76]. Zaidan et al. [77] reported the localization of Tfh cells in relation to B cells in a typical pathology of IgG4-RD. The topography of the IgG4-RD patient’s T cells was quite unusual as compared with controls, with mostly Tfh cells (62% of total T cells) infiltrating the light zones; these findings were not seen in the disease controls [77]. The Tfh cells were co-localized with CD20+ B cells within germinal centres. Maehara et al. [78] also reported that the infiltration of IL-4–secreting Tfh cells was predominantly outside the ectopic germinal centres in IgG4-RD, while IL-21–secreting Tfh cells were also observed inside the ectopic germinal centres, suggesting that Tfh cells are present both inside and outside the ectopic germinal centres of the affected sites, but the cytokines produced by Tfh cells differ according to their localization. These findings are important because Tfh cells in the germinal centres progressively differentiate from IL-21–secreting Tfh cells to IL-4–secreting Tfh cells through stages of localization in vivo [76, 79]. IL-21–secreting Tfh cells predominantly localized in the dark zone of the germinal centres, while IL-4–secreting Tfh cells localized in the light zone [76, 79]. In functional analysis, IL-4–secreting Tfh cells promoted class-switch recombination of antibodies, whereas IL-21–secreting Tfh cells were essential for promoting somatic hypermutation in B cells [76, 79]. Thus, enhanced Tfh cell activity (via distinct cytokine profiles with different localization) participates in B cell selection and proliferation, germinal centre formation and IgG4 class-switching. IgG4-RD patients have a significantly higher number and size of ectopic germinal centres and higher expression of Bcl-6 mRNA at affected sites [78, 80, 81]. CXCL13 and CXCR5 are also upregulated at affected sites, indicating that CXCL13–CXCR5 interactions play a role in migration of Tfh cells into affected sites [78, 82, 83]. Indeed, follicular dendritic cells that produce CXCL13 were present within ectopic germinal centres at sites of IgG4-RD [84]. As expected from the prominent infiltration of IgG4+ plasma cells, AID is expressed at sites of IgG4-RD, suggesting that IgG4 class-switching is indeed induced at local affected sites [85–87]. Enhanced expression of both CD40 and CD40 ligand has also been observed in IgG4-RD lesions, suggesting an enhanced Tfh–B interaction pathway as a mechanism for IgG4 induction [32]. Tfh-related cytokines such as IL-4 and IL-21 were the most upregulated two cytokines in a microarray analysis of affected tissues [88]. Furthermore, both IL-4 and IL-21 mRNA positively correlated with the IgG4/IgG mRNA ratio, suggesting that these cytokines are involved in IgG4 class-switching [37, 78]. Collectively, these findings strongly suggest that Tfh cells at affected sites play a pivotal role in the pathogenesis of IgG4-RD. Circulating Tfh cells It is always an issue whether circulating cells reflect those events at affected sites. As a matter of fact, bona fide Tfh cells are found in secondary lymphoid organs, and more recent studies have demonstrated that circulating Tfh cells share phenotypic and functional features with bona fide Tfh cells [89]. Since assessing the role of Tfh cells in affected sites is challenging due to the difficulty of access, many researchers have examined the circulating Tfh cells to evaluate Tfh–cell responses. The value of circulating Tfh cells as a biomarker for the monitoring of dysregulated antibody responses and disease activity in autoimmune diseases has been confirmed [89–91]. Circulating Tfh cells contain three major subsets according to their differential expression of the chemokine receptors CXCR3 and CCR6: Tfh1, Tfh2 and Tfh17 cells [92]. These three subsets are functionally distinct with respect to their transcriptional and cytokine properties and their abilities to support a humoral response. PD-1 is a member of the CD28 family, negative regulators of T cell activation. Importantly, PD-1 is highly expressed by activated Tfh cells, and is involved in B cell selection and survival in germinal centres [93]. CCR7 controls T cell migration towards lymphoid tissues through interaction with its ligands CCL19 and CCL21. The expression of CCR7 on T cells is downregulated when they are activated to migrate into sites of inflammation [94]. Recent work has demonstrated that circulating CCR7lowPD-1high Tfh cells, i.e. activated Tfh cells, are a useful biomarker for monitoring the activation status of Tfh cells in autoimmunity, virus infection and vaccination [94]. Since circulating activated Tfh cells show low expression of CD45RA, this population is an effector–memory phenotype that comes from local affected sites. Circulating Tfh cells and their capacity for promoting IgG4 production in IgG4-RD More recently, several studies have demonstrated the expansion of circulating plasmablasts, correlating with IgG4-RD disease activity [39, 95–98]. Of note, these increased circulating plasmablasts coincided with somatic hypermutation, and the appearance of distinct plasmablast clones were associated with relapse of IgG4-RD, suggesting that plasmablasts of IgG4-RD are derived from germinal centres assisted by Tfh cells [98]. The highly abundant IgG4+ B cell receptor clones in the blood and tissues of IgG4-RD patients also suggest that Tfh cell–dependent responses play a role in the plasmablast expansion in IgG4-RD [99, 100]. Elucidating therapeutic targets for IgG4-RD requires a clear understanding of the pathogenic pathways and corresponding biomarkers of disease activity. As a result, substantial interest has been shown in the evaluation of circulating Tfh cells and their relationship with disease activity in IgG4-RD. We have recently shown that IgG4-RD patients have alterations in the composition of circulating Tfh cell subsets. The number of circulating Tfh2 cells was significantly and specifically increased in IgG4-RD compared with in primary SS, multicentric Castleman’s disease, allergic rhinitis and healthy controls [26, 39]. This increased number of circulating Tfh2 cells positively correlated with serum IgG4 level, IgG4/IgG ratio and the number of circulating plasmablasts [26, 39]. Interestingly, the number of circulating Tfh2 cells also positively correlated with the proportion of IgG4+ plasma cells at affected sites [101]. On the other hand, we found no correlation between the number of Tfh1 or Tfh17 cells and serum IgG4 level or the number of circulating plasmablasts. In line with these observations, Tfh2 cells, but not Tfh1 or Tfh17 cells, from IgG4-RD patients efficiently induced the differentiation of naïve B cells into plasmablasts in vitro [26]. Moreover, IgG4 class-switching was exclusively induced by Tfh2 cells [26]. Remarkably, while IgG production induced by Tfh2 cells was comparable between IgG4-RD and healthy controls, IgG4 production was significantly higher with Tfh2 cells from IgG4-RD than with those from healthy controls [26]. These results suggest that Tfh2 cells are the culprit cell type for inducing plasmablast expansions and IgG4 class-switching in IgG4-RD. This is the only immune cell type for inducing plasmablast differentiation and IgG4 production found in IgG4-RD. Activated circulating Tfh cells and disease activity in IgG4-RD To evaluate the mechanism of enhancement of IgG4 production by Tfh2 cells from IgG4-RD, we also conducted an analysis of the activation status of circulating Tfh2 cells. Importantly, circulating activated Tfh2 cells were significantly increased in IgG4-RD, positively correlating with disease activity [26]. Moreover, the number of activated Tfh2 cells was positively correlated with the number of affected organs and serum IgG4 level [26], indicating that these cells come from ectopic germinal centres at affected sites. Following treatment with glucocorticoids, the number of activated Tfh2 cells decreases, concomitant with clinical resolution [26]. Of note, we experienced a case whose number of activated Tfh2 cells was again elevated at disease relapse, suggesting the potential of activated Tfh2 cells as a biomarker for disease activity [26]. We found a significant positive correlation between the number of Tfh2 cells and serum levels of IL-4, and that the two patients with the highest levels of IL-21 had the highest numbers of plasmablasts, serum IgG4 levels and Tfh2 cell numbers in active, untreated IgG4-RD [26, 39]. Thus, activated Tfh2 cells more efficiently secrete IL-4 and IL-21 to induce enhanced IgG4 production through plasmablast differentiation [26, 39]. IL-10 is also known as a potential cytokine for IgG4 class-switching, but there was no correlation between serum IgG4 and IL-10 levels [39]. The other pathological role of Tfh2 cells in IgG4-RD IL-21 from Tfh cells is essential for germinal centre formation [6, 13]. Indeed, IL-21 mRNA expression was increased, positively correlating with the number of hyperplastic ectopic germinal centre formations in IgG4-RD [78, 80]. IL-21–secreting lymphocytes also expressed IL-4, Bcl-6 and CXCR5 [78], suggesting that IL-21–secreting lymphocytes are Tfh2 cells. Thus, excessive IL-21 production from Tfh2 cells may induce hyperplastic germinal centre formations in IgG4-RD. The possible mechanism of increased Tfh2 cells in IgG4-RD IgG4-RD responds well to glucocorticoid treatment, but tapering or cessation of treatment often leads to relapse [102]. In this regard, while activated Tfh2 cell counts, plasmablast counts and levels of serum IgG4 and IL-4 decrease after glucocorticoid treatment in parallel with disease improvement, total Tfh2 cell counts remain unchanged [26, 39], suggesting that increased total Tfh2 cells are the underlying pathogenic mechanism and contribute to incomplete cure of IgG4-RD. Tfh cell differentiation is induced by chronic antigen stimulation in secondary lymphoid organs and by a combination of cytokines [103]. On this point, since circulating Tfh2 cells in IgG4-RD have a memory phenotype, as evidenced by their decreased expression of CD45RA [26, 39], chronic stimulation by unknown antigens (such as allergens) is one factor promoting Tfh2 cell differentiation. Interestingly, chronic exposure to occupational antigens may play a role in the initiation and/or maintenance of IgG4-RD [104]. The increased IgG4 response to food and animal antigens also deserves attention [105]. IgG4 itself is generally thought to be non-inflammatory because it does not crosslink antigens and fix complement by the process called Fab-arm exchange [106]. Furthermore, from the viewpoint of allergy (which is frequently observed in IgG4-RD), the emergence of IgG4 is associated with a decrease in allergic symptoms through an allergen-blocking effect [106]. However, according to recent evidence showing pathogenic effects of IgG4 itself from IgG4-RD [107], Tfh2 cells that induce IgG4 production from plasmablasts are the potential therapeutic target in this disease. Indeed, the efficacy of abatacept in a patient with IgG4-RD may be explained by inhibition of Tfh cells with respect to B cell interaction and subsequent disruption of the formation of ectopic germinal centres [108, 109]. Although it is still unclear whether IgG4-RD is an autoimmune disease or an allergic disorder, the detection of IgG4-recognizing autoantigens or exogenous antigens may contribute to a more thorough elucidation of the pathophysiology of IgG4-RD. Circulating Tfh1 cells in IgG4-RD We have made another important observation: that Tfh1 cells were also increased and activated in IgG4-RD [26]. Activated Tfh1 cells positively correlated with disease activity, but not with serum IgG4 levels, suggesting that they play a role in the pathogenesis of IgG4-RD, but are not involved in IgG4 production. Although the precise function of Tfh1 cells remains unclear, they express CXCR5 and the transcription factor T-bet, and produce IFN-γ [92]. Thus, they migrate to the ectopic germinal centres that express CXCL13 (the ligand for CXCR5) and produce IFN-γ at affected sites, resulting in tissue fibrosis. Circulating Tfh17 cells in IgG4-RD The number of circulating Tfh17 cells and their activated phenotype was not increased in IgG4-RD [26, 39]. Furthermore, the number of these Tfh17 cells did not correlate with the level of serum IgG4 or the number of circulating plasmablasts [26]. Moreover, IL-17 (which is the hallmark of Tfh17 cells) is rarely expressed at affected sites [78]. Collectively, Tfh17 cells may not be involved in the pathogenesis of IgG4-RD. Conclusions Recently accumulated findings have clarified that T cells are involved in the pathogenesis of IgG4-RD (Table 2). Tfh2 cells are important in germinal centre formation, as well as in plasmablast and plasma cell differentiation and IgG4 class-switching (Fig. 1). Future insights into T cell immunology and pathophysiology should lead to novel treatments for this disease. Table 2 Overview of T cell subsets in IgG4-related disease T cell phenotype  Principle findings in IgG4-RD  Reference  Tfh cells  Increased expression of BCL6 (the master regulator of Tfh cells) at affected sites  [78, 80]    Increased expression of Tfh-related chemokines (CXCL13) and chemokine receptors (CXCR5) at affected sites  [78, 82, 83]    Increased expression of IL-21 at affected sites  [78, 80, 88]    Increased expression of CD40 ligand at affected sites  [32]    Increased formation of germinal centres at affected sites  [77, 78, 80, 81]    Increased circulating Tfh cells and their activated phenotype  [26, 39]    Association with IgG4 production at affected sites  [78]  Tfh2 cells  Increased circulating Tfh2 cells and their activated phenotype  [26, 39, 101]    Capacity to help naïve B cells to differentiate into plasmablasts and IgG4 production in vitro  [26]    Association with disease activity and the number of affected organs  [26]    Association with IL-4 and IL-21 production  [26, 39]  Tfh1 cells  Increased circulating Tfh1 cells and their activated phenotype  [26]    Association with disease activity and the number of affected organs  [26]  Cytotoxic CD4+ T cells  Oligoclonally increased circulating cytotoxic CD4+ T cells and their infiltration at affected sites  [25, 46]    Capacity to produce IFN-γ, IL-1β, TGF-β and granzyme  [25, 46]  Treg cells  Increased expression of FOXP3 (the master regulator of Treg), IL-10 and TGF-β at affected sites  [35, 37, 41–43]    Increased circulating Treg cells  [44, 45]    Association of TGF-β with fibrosis at affected sites  [34, 36, 42, 55]  CD8+ T cells  Exhausted cytotoxic capacity  [57−59]    Increased circulating CD8+ T cells  [18]  T cell phenotype  Principle findings in IgG4-RD  Reference  Tfh cells  Increased expression of BCL6 (the master regulator of Tfh cells) at affected sites  [78, 80]    Increased expression of Tfh-related chemokines (CXCL13) and chemokine receptors (CXCR5) at affected sites  [78, 82, 83]    Increased expression of IL-21 at affected sites  [78, 80, 88]    Increased expression of CD40 ligand at affected sites  [32]    Increased formation of germinal centres at affected sites  [77, 78, 80, 81]    Increased circulating Tfh cells and their activated phenotype  [26, 39]    Association with IgG4 production at affected sites  [78]  Tfh2 cells  Increased circulating Tfh2 cells and their activated phenotype  [26, 39, 101]    Capacity to help naïve B cells to differentiate into plasmablasts and IgG4 production in vitro  [26]    Association with disease activity and the number of affected organs  [26]    Association with IL-4 and IL-21 production  [26, 39]  Tfh1 cells  Increased circulating Tfh1 cells and their activated phenotype  [26]    Association with disease activity and the number of affected organs  [26]  Cytotoxic CD4+ T cells  Oligoclonally increased circulating cytotoxic CD4+ T cells and their infiltration at affected sites  [25, 46]    Capacity to produce IFN-γ, IL-1β, TGF-β and granzyme  [25, 46]  Treg cells  Increased expression of FOXP3 (the master regulator of Treg), IL-10 and TGF-β at affected sites  [35, 37, 41–43]    Increased circulating Treg cells  [44, 45]    Association of TGF-β with fibrosis at affected sites  [34, 36, 42, 55]  CD8+ T cells  Exhausted cytotoxic capacity  [57−59]    Increased circulating CD8+ T cells  [18]  Tfh cells: T follicular helper cells; Tfh2 cells: T follicular helper 2 cells; Tfh1 cells: T follicular helper 1 cells. Fig. 1 View largeDownload slide Schematic model of involvements of T cells in the pathogenesis of IgG4-related disease Chronic stimulation by unknown antigens activates APCs, and they induce the differentiation of Tfh2 cells. FDCs (or Tfh2 cell accumulation themselves) secrete CXCL13, which induces Tfh2 cells and B cell accumulation through interaction with CXCR5, resulting in the formation of ectopic germinal centres in affected tissues. Tfh2 cells induce the differentiation of naïve B cells into plasmablasts and plasma cells, and IgG4 class-switching through cell-to-cell contact with the elaboration of IL-4 and IL-21. IgG4 itself and the cytokines from Tfh1 cells, cytotoxic CD4+ T cells and Treg cells may contribute to tissue damage and fibrosis. Tfh2: T follicular helper 2 cells; Tfh1: T follicular helper 1 cells; APC: antigen-presenting cell; FDC: follicular dendritic cell; SHM: somatic hypermutation; CXCL13: CXC chemokine ligand 13; Bcl-6: B cell lymphoma-6; PD-1: programmed death-1; AID: activation-induced cytidine deaminase; CD40L: CD40 ligand. Fig. 1 View largeDownload slide Schematic model of involvements of T cells in the pathogenesis of IgG4-related disease Chronic stimulation by unknown antigens activates APCs, and they induce the differentiation of Tfh2 cells. FDCs (or Tfh2 cell accumulation themselves) secrete CXCL13, which induces Tfh2 cells and B cell accumulation through interaction with CXCR5, resulting in the formation of ectopic germinal centres in affected tissues. Tfh2 cells induce the differentiation of naïve B cells into plasmablasts and plasma cells, and IgG4 class-switching through cell-to-cell contact with the elaboration of IL-4 and IL-21. IgG4 itself and the cytokines from Tfh1 cells, cytotoxic CD4+ T cells and Treg cells may contribute to tissue damage and fibrosis. Tfh2: T follicular helper 2 cells; Tfh1: T follicular helper 1 cells; APC: antigen-presenting cell; FDC: follicular dendritic cell; SHM: somatic hypermutation; CXCL13: CXC chemokine ligand 13; Bcl-6: B cell lymphoma-6; PD-1: programmed death-1; AID: activation-induced cytidine deaminase; CD40L: CD40 ligand. Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this manuscript. Disclosure statement: Y.T. has received consultant fees from Pfizer, Chugai Pharma, Mitsubishi-Tanabe Pharma and AbbVie, received honoraria from Pfizer, Chugai Pharma, Mitsubishi-Tanabe Pharma, Bristol-Myers Squibb, Takeda Industrial Pharma, GlaxoSmithkline, Nippon Shinyaku, Eli Lilly, Janssen Pharma, Eisai Pharma, Astellas Pharma and Actelion Pharmaceuticals and received research support from Chugai Pharma and Mitsubishi-Tanabe Pharma. T.T has received consulting fees, speaking fees and/or honoraria from Pfizer Japan, Mitsubishi Tanabe Pharma, Eisai, Astellas Pharma and UCB (<$10 000 each) and from Chugai Pharmaceutical, Bristol-Myers K.K., Daiichi Sankyo, AbbVie, Janssen Pharmaceutical K.K., Pfizer Japan, Asahi Kasei Pharma, Takeda Pharmaceutical, AstraZeneca K.K., Eli Lilly Japan K.K. and Novartis Pharma K.K. (>$10 000 each). All other authors have declared no conflicts of interest. References 1 Kamisawa T, Zen Y, Pillai S, Stone JH. IgG4-related disease. 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Follicular helper T cells in the pathogenesis of IgG4-related disease

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Oxford University Press
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© The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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1462-0324
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1462-0332
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10.1093/rheumatology/kex171
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Abstract

Abstract IgG4-related disease (IgG4-RD) is a recently recognized disease entity characterized by high serum IgG4 concentrations and infiltration of IgG4+ plasma cells with hyperplastic ectopic germinal centres at affected sites. Although the underlying immune mechanism of this disease remains unclear, T cells are abundantly present at affected sites and key players in IgG4-RD pathogenesis. The role of T cell subsets has been investigated thoroughly in this disease. Recent advances in this field have clarified the importance of T follicular helper cells. In this review, we describe the role of T follicular helper cells in the disease process of IgG4-RD, in particular, for IgG4 class-switching, plasmablast and plasma cell differentiation, and germinal centre formation. IgG4-related disease, pathogenesis, T lymphocyte, T follicular helper cell, germinal centre, plasmablast, somatic hypermutation, interleukin-4, interleukin-21 Rheumatology key messages T follicular helper 2 cells lead to enhanced IgG4 production in IgG4-related disease. T follicular helper 2 cells induce plasmablast differentiation and germinal centre formations in IgG4-related disease. Activated T follicular helper 2 cells are a potential biomarker for IgG4-related disease activity. Introduction IgG4-related disease (IgG4-RD) affects various organ systems and is characterized by elevated serum IgG4 levels, tissue infiltration by IgG4+ plasma cells and storiform fibrosis [1–4]. The underlying pathogenesis of IgG4-RD remains largely unknown. A better understanding of this disease is necessary for identifying therapeutic targets. Since large numbers of T cells are present at affected sites, T cells are involved in the pathogenesis of this disease. Evidence implicating T cells in IgG4-RD has been increasing, and the role of T cell subsets have been clarified. Ectopic germinal centres appear at affected sites and local B cells produce IgG4. T follicular helper (Tfh) cells help B cell maturation and Ig isotype-switching at germinal centres. Of note, recent investigations have revealed the importance of Tfh cells in the pathophysiology of IgG4-RD. In this review we discuss the pathogenic role of Tfh cells as well as the other T cell subsets in IgG4-RD. The differences between T helper cells and Tfh cells It would be helpful to understand the major differences between T helper (Th) and Tfh cells because Tfh cells are specialized providers of B cell help and germinal centre development [5, 6]. B cell lymphoma 6 (Bcl-6) has been demonstrated as the master regulator of Tfh cell differentiation because the deletion of Bcl-6 results in the absence of Tfh cells, but normal development of the other Th cell subsets [7–9]. Accordingly, Bcl-6 is highly expressed in Tfh cells and is also known to be an antagonistic transcription factor for B lymphocyte–induced maturation protein 1 (Blimp-1), the transcription factor highly expressed in Th cell subsets other than Tfh cells [7–9]. The chemokine receptor CXC chemokine receptor 5 (CXCR5) is a central marker of Tfh cells, which is required for Tfh cells to migrate into germinal centres via interactions with its ligand CXC chemokine ligand 13 (CXCL13) [6]. CXCL13 is also expressed at high levels by Tfh cells (but not by other Th cells) in humans [6]. The transcription factor achaete-scute homologue 2 (Ascl2) is selectively upregulated in Tfh cells in vivo [10]. Ascl2 upregulates CXCR5 (but not Bcl-6) in T cells to induce T cell migration into the germinal centres [10], whereas Ascl2 acts as a suppresser of Th1, Th2 and Th17 cell differentiation. At the T–B border, B cells provide another signal for precursor Tfh cells to upregulate Bcl-6 expression for the completion of Tfh cell polarization and germinal centre formation [10]. Signalling lymphocytic activation molecule (SLAM)-associated protein (SAP) is critical for Tfh cell and germinal centre development [6]. SAP plays a role in Tfh–B interaction by binding to the intracellular domains of SLAM family surface receptors [6]. Programmed death (PD)-1 is generally expressed on activated T cells, including Tfh cells. PD-1 is an inhibitory receptor essential for T cell tolerance, but PD-1 expressed by Tfh cells plays a role in B cell selection via direct inductive signals to B cells [6]. Inducible T-cell co-stimulator (ICOS) has roles in Tfh cell differentiation, migration, cytokine production and germinal centre formation [11, 12]. B cells also express ICOS ligand, which is required for germinal centre formation. IL-21 is the most important Tfh cell cytokine for driving plasmablast and plasma cell differentiation in germinal centres [13]. IL-4 from Tfh cells (but not from Th2 cells) plays a role in class-switch recombination [14, 15]. Moreover, the deletion of Th2-related genes does not result in a loss of germinal centres [16, 17], whereas the deletion of both IL-4 and the IL-21 receptor resulted in a loss of germinal centres [13], suggesting that both IL-4 and IL-21 from Tfh cells are required for germinal centre development and maintenance. Germinal centres are the primary location for antigen-specific B cell proliferation, mutation and differentiation and Ig class-switching [6]. In summary, distinguishing features of Tfh cells from the other Th cells include the expression of Bcl-6, CXCR5, Ascl2, SAP, PD-1, ICOS, IL-21 and IL-4, in concert with the absence of Blimp-1 (Table 1). Table 1 The differences between T helper cells and T follicular helper cells Characteristics  Th cells  Tfh cells  Functions          Antigen-specific B cell proliferation, affinity mutation and somatic hypermutation  −  +      Class switch recombination  −  +      Plasmablast and plasma cell differentiation  −  +      Germinal centre development  −  +  Molecule expressions          Bcl-6  −  +      Blimp-1  +  −      Ascl2  −  +      CXCR5  −  +      CXCL13  −  +      SAP  Normal  High      PD-1  Variable  +      ICOS  Variable  +  Cytokines          IL-21  Variable  +      IL-4  + (Th2)  +  Characteristics  Th cells  Tfh cells  Functions          Antigen-specific B cell proliferation, affinity mutation and somatic hypermutation  −  +      Class switch recombination  −  +      Plasmablast and plasma cell differentiation  −  +      Germinal centre development  −  +  Molecule expressions          Bcl-6  −  +      Blimp-1  +  −      Ascl2  −  +      CXCR5  −  +      CXCL13  −  +      SAP  Normal  High      PD-1  Variable  +      ICOS  Variable  +  Cytokines          IL-21  Variable  +      IL-4  + (Th2)  +  Bcl-6: B cell lymphoma-6; Blimp-1: B lymphocyte-induced maturation protein 1; Ascl2: achaete-scute homologue 2; CXCR5: CXC chemokine receptor 5; CXCL13: CXC chemokine ligand 13; SAP: signalling lymphocytic activation molecule–associated protein; PD-1: programmed cell death-1; ICOS: inducible T cell co-stimulator; +: positive; −: negative. Th1 cells Th1 cells play a role in cell-mediated immune responses, including production of IFN-γ (which leads to the activation of macrophages and cytotoxic T cells). Th1-type immune responses have been reported to be involved in the pathogenesis of IgG4-RD in the last decade. Circulating Th1-like cells (as indicated by IFN-γ+ CD4+ T cells) have been reported as increased in IgG4-RD [18]. Moreover, an increased level of serum IFN-γ has been reported [19–21]. IFN-γ+ CD4+ cells and IFN-γ mRNA expression were also significantly increased at affected sites [19, 22]. Of note, even in the cases that showed considerable skewing towards Th2-type immune responses, a substantial number of Th1-type immune responses were identified, suggesting that Th1-type immune responses play a role in IgG4-RD [23, 24]. However, it remained unclear which type of CD4+ T cells that secrete IFN-γ [Th1 cells, cytotoxic CD4+ T cells or T follicular helper 1 (Tfh1) cells] are involved in this disease. Recent studies have revealed that cytotoxic CD4+ T cells or Tfh1 cells are likely candidates for IFN-γ–producing CD4+ T cells in IgG4-RD [25, 26]. Th2 cells IgG4-RD patients frequently have an atopic history, serum IgE elevation and eosinophilia [27, 28]. Atopic features are induced by Th2 cytokines, such as IL-4, IL-5 and IL-13. Therefore, Th2 cells have been considered to play a role in the pathogenesis of IgG4-RD. Several studies that examined peripheral blood have found that circulating Th2 cells, characterized by GATA-3 transcription factor mRNA, CRTH2 expression or IL-4 and IL-5 expression on CD4+ T cells, are increased in IgG4-RD [29–32]. From the viewpoint of serum cytokine levels, a remarkable increase in serum IL-4 has been demonstrated in IgG4-RD [21, 33]. Bile samples from patients with IgG4-related cholangitis showed significantly increased IL-4, IL-5 and GATA-3 mRNA [34]. The cytokine profiles in bronchoalveolar fluids from IgG4-related respiratory disease showed significantly higher IL-5 and IL-13 [24]. The expression of Th2 cytokines such as IL-4, IL-5 and IL-13 at affected sites was also increased [23, 35–37]. However, controversial results have been reported. One report demonstrated increased circulating GATA-3+ Th2 cells only in patients with IgG4-RD who had an atopic history [38], and another one demonstrated that GATA-3+ Th2 cells were sparse at sites of IgG4-RD, although IL-4 mRNA expression was significantly increased [25]. These results suggest that the increased IL-4 at affected sites is produced by non-Th2 cells [25]. Further, atopic asthma patients showed significantly lower levels of serum IgG4 compared with IgG4-RD patients, while elevated serum IgE levels and increased circulating Th2 cells were comparable between the two diseases, also suggesting that non-Th2 cells play a role in IgG4 class-switching [29, 30]. In line with these reports, we have observed that Th2 cells from patients with IgG4-RD did not induce the in vitro differentiation of naïve B cells into plasmablasts, or IgG4 production (Akiyama M, et al., unpublished data). Importantly, more recent studies have revealed that Tfh2 cells are a likely candidate as IL-4–producing cells in IgG4-RD [26, 39]. Treg cells Treg cells play an important role in the regulation of self-tolerance and the maintenance of tissue homeostasis [40]. Accumulating evidence of increased Treg cells at affected sites and also in peripheral blood has been reported in IgG4-RD [35, 37, 41–45], although a more recent report has shown only a marginal increase in circulating Treg cells [46]. On the other hand, there has been no report of decreased Treg cells in IgG4-RD, in contrast to the situation in other autoimmune diseases, such as type 1 diabetes, multiple sclerosis, lupus and RA [47]. Involvement in IgG4 class-switching Although several studies suggest that IL-10 secreted by Treg cells plays a role in IgG4 class-switching [36, 37, 41, 42], IL-10 is also known to be secreted by other immune cells such as Th cells, Tfh cells, B cells, mast cells, eosinophils, monocytes and macrophages [48]. Another important consideration is that eosinophilic granulomatosis with polyangiitis showed a decrease in circulating Treg cells [49–51] despite its remarkable elevation of serum IgG4 and IgG4/IgG ratio [52, 53]. No study has reported a functional analysis of Treg cells from patients with IgG4-RD, and direct evidence of IgG4 class-switching by Treg cells is required in the future. Involvement in fibrosis Another important point about the presence of Treg cells in IgG4-RD is their possible involvement in the pathogenesis of fibrosis through TGF-β secretion. Although the induction of extracellular matrix components by TGF-β is crucial during wound healing and tissue repair, overproduction of TGF-β leads to fibrosis [54]. Since fibrosis at affected sites is an important characteristic of IgG4-RD, TGF-β may play an important role. Indeed, several studies have shown increased TGF-β expression, particularly at fibrosclerosing lesions of IgG4-RD [34, 36, 42, 55]. Considering the capability of a broad range of cells to produce TGF-β [56], Treg cells may be one of the immune cell types contributing to fibrosis in IgG4-RD. CD8+ T cells Tissue destruction caused by the cytolytic mediator perforin, the serine protease granzyme or Fas lytic pathways from CD8+ T cells is more important in SS than IgG4-related dacryoadenitis and sialadenitis, so-called Mikulicz disease. However, the number of IFN-γ+CD8+ T cells in peripheral blood was reported to be increased in IgG4-RD [18] with a similar extent of CD8+ T cell infiltration at affected sites between IgG4-RD and SS [57]. Importantly, the frequency of expression of perforin and granzyme B by CD8+ T cells was lower in IgG4-RD, whereas the frequency of expression of the immunoinhibitory receptor PD-1 by the same cells was higher in IgG4-RD compared with in SS [57], suggesting that CD8+ T cells are exhausted in IgG4-RD. In fact, expression of the apoptosis mediator Fas (CD95)–Fas ligand in lymphocytes was higher at affected sites in patients with SS compared with in patients with Mikulicz disease [58, 59]. Moreover, circulating lymphocytes in Mikulicz disease showed a defect in cytotoxic capacity [59]. As a result, although both diseases involve lymphocytic infiltration, only the acinar cells in SS undergo apoptosis [58]. Thus, acinar cell apoptosis by CD8+ T cells is involved in the pathogenesis of SS but not of Mikulicz disease. Cytotoxic CD4+ T cells Cytotoxic CD4+ T cells are classically known as the CD28low subpopulation among CD4+ T cells, and they secret Th1-like and proinflammatory cytokines [60]. The expansion of this T cell subset at affected sites and in peripheral blood has been initially described in patients with granulomatosis with polyangiitis, and their numbers correlate with the total number of affected organs, suggesting that cytotoxic CD4+ T cells are important in the pathogenesis of this disease [60–63]. Clonally expanded cytotoxic CD4+ T cells that secrete IFN-γ and perforin have also been reported in peripheral blood and at affected sites in RA [64, 65]. Oligoclonal expansion of circulating CD4+SLAMF7+ T cells in patients with IgG4-RD, and their infiltration at affected sites, has recently been described [25, 46]. These cells also express low levels of CD28 and secrete granzyme B, TGF-β, IL-1β and IFN-γ, suggesting that this T cell subset consists of so-called cytotoxic CD4+ T cells [46]. TGF-β, IL-1β and IFN-γ are believed to be important mediators of fibrosis. Thus, these cytotoxic CD4+ T cells may play a role in fibrosis in IgG4-RD. Importantly, the number of circulating CD4+SLAMF7+ T cells is also increased in systemic sclerosis patients, suggesting that the increase in this T cell subset is not specific for IgG4-RD [46]. Tfh cells Molecules associated with Tfh cell functions Activation-induced cytidine deaminase (AID) is necessary for somatic hypermutation and class-switch recombination of antibodies [66–69], but the specific target of the heavy-chain constant-region gene recombination requires additional cytokines and CD40 signalling [70]. CD40 ligand is the most important protein expressed by Tfh cells, and it provides signals to B cells (through CD40) essential to their differentiation, Ig class-switching and germinal centre formation [71, 72]. IL-4 was long considered to be exclusively produced by Th2 cells, and thus Th2 cells were believed to be the main providers of ‘help’ to B cells. However, it is now well accepted that Tfh cells are the specialized providers of ‘help’ to B cells. It has been shown that Tfh cells secrete IL-21 and are the primary producers of IL-4 in secondary lymphoid organs [73–75]. Thus, the combination of AID, CD40L, IL-4 and IL-21 is important for controlling B cell differentiation, somatic hypermutation and Ig class-switching, and germinal centre development [66]. Tfh-cell associated events at affected sites of IgG4-RD The germinal centre is composed of two regions: the dark zone is the site of rapid germinal centre B cell proliferation, whereas the light zone is where Tfh cells reside and interact with germinal centre B cells [76]. Zaidan et al. [77] reported the localization of Tfh cells in relation to B cells in a typical pathology of IgG4-RD. The topography of the IgG4-RD patient’s T cells was quite unusual as compared with controls, with mostly Tfh cells (62% of total T cells) infiltrating the light zones; these findings were not seen in the disease controls [77]. The Tfh cells were co-localized with CD20+ B cells within germinal centres. Maehara et al. [78] also reported that the infiltration of IL-4–secreting Tfh cells was predominantly outside the ectopic germinal centres in IgG4-RD, while IL-21–secreting Tfh cells were also observed inside the ectopic germinal centres, suggesting that Tfh cells are present both inside and outside the ectopic germinal centres of the affected sites, but the cytokines produced by Tfh cells differ according to their localization. These findings are important because Tfh cells in the germinal centres progressively differentiate from IL-21–secreting Tfh cells to IL-4–secreting Tfh cells through stages of localization in vivo [76, 79]. IL-21–secreting Tfh cells predominantly localized in the dark zone of the germinal centres, while IL-4–secreting Tfh cells localized in the light zone [76, 79]. In functional analysis, IL-4–secreting Tfh cells promoted class-switch recombination of antibodies, whereas IL-21–secreting Tfh cells were essential for promoting somatic hypermutation in B cells [76, 79]. Thus, enhanced Tfh cell activity (via distinct cytokine profiles with different localization) participates in B cell selection and proliferation, germinal centre formation and IgG4 class-switching. IgG4-RD patients have a significantly higher number and size of ectopic germinal centres and higher expression of Bcl-6 mRNA at affected sites [78, 80, 81]. CXCL13 and CXCR5 are also upregulated at affected sites, indicating that CXCL13–CXCR5 interactions play a role in migration of Tfh cells into affected sites [78, 82, 83]. Indeed, follicular dendritic cells that produce CXCL13 were present within ectopic germinal centres at sites of IgG4-RD [84]. As expected from the prominent infiltration of IgG4+ plasma cells, AID is expressed at sites of IgG4-RD, suggesting that IgG4 class-switching is indeed induced at local affected sites [85–87]. Enhanced expression of both CD40 and CD40 ligand has also been observed in IgG4-RD lesions, suggesting an enhanced Tfh–B interaction pathway as a mechanism for IgG4 induction [32]. Tfh-related cytokines such as IL-4 and IL-21 were the most upregulated two cytokines in a microarray analysis of affected tissues [88]. Furthermore, both IL-4 and IL-21 mRNA positively correlated with the IgG4/IgG mRNA ratio, suggesting that these cytokines are involved in IgG4 class-switching [37, 78]. Collectively, these findings strongly suggest that Tfh cells at affected sites play a pivotal role in the pathogenesis of IgG4-RD. Circulating Tfh cells It is always an issue whether circulating cells reflect those events at affected sites. As a matter of fact, bona fide Tfh cells are found in secondary lymphoid organs, and more recent studies have demonstrated that circulating Tfh cells share phenotypic and functional features with bona fide Tfh cells [89]. Since assessing the role of Tfh cells in affected sites is challenging due to the difficulty of access, many researchers have examined the circulating Tfh cells to evaluate Tfh–cell responses. The value of circulating Tfh cells as a biomarker for the monitoring of dysregulated antibody responses and disease activity in autoimmune diseases has been confirmed [89–91]. Circulating Tfh cells contain three major subsets according to their differential expression of the chemokine receptors CXCR3 and CCR6: Tfh1, Tfh2 and Tfh17 cells [92]. These three subsets are functionally distinct with respect to their transcriptional and cytokine properties and their abilities to support a humoral response. PD-1 is a member of the CD28 family, negative regulators of T cell activation. Importantly, PD-1 is highly expressed by activated Tfh cells, and is involved in B cell selection and survival in germinal centres [93]. CCR7 controls T cell migration towards lymphoid tissues through interaction with its ligands CCL19 and CCL21. The expression of CCR7 on T cells is downregulated when they are activated to migrate into sites of inflammation [94]. Recent work has demonstrated that circulating CCR7lowPD-1high Tfh cells, i.e. activated Tfh cells, are a useful biomarker for monitoring the activation status of Tfh cells in autoimmunity, virus infection and vaccination [94]. Since circulating activated Tfh cells show low expression of CD45RA, this population is an effector–memory phenotype that comes from local affected sites. Circulating Tfh cells and their capacity for promoting IgG4 production in IgG4-RD More recently, several studies have demonstrated the expansion of circulating plasmablasts, correlating with IgG4-RD disease activity [39, 95–98]. Of note, these increased circulating plasmablasts coincided with somatic hypermutation, and the appearance of distinct plasmablast clones were associated with relapse of IgG4-RD, suggesting that plasmablasts of IgG4-RD are derived from germinal centres assisted by Tfh cells [98]. The highly abundant IgG4+ B cell receptor clones in the blood and tissues of IgG4-RD patients also suggest that Tfh cell–dependent responses play a role in the plasmablast expansion in IgG4-RD [99, 100]. Elucidating therapeutic targets for IgG4-RD requires a clear understanding of the pathogenic pathways and corresponding biomarkers of disease activity. As a result, substantial interest has been shown in the evaluation of circulating Tfh cells and their relationship with disease activity in IgG4-RD. We have recently shown that IgG4-RD patients have alterations in the composition of circulating Tfh cell subsets. The number of circulating Tfh2 cells was significantly and specifically increased in IgG4-RD compared with in primary SS, multicentric Castleman’s disease, allergic rhinitis and healthy controls [26, 39]. This increased number of circulating Tfh2 cells positively correlated with serum IgG4 level, IgG4/IgG ratio and the number of circulating plasmablasts [26, 39]. Interestingly, the number of circulating Tfh2 cells also positively correlated with the proportion of IgG4+ plasma cells at affected sites [101]. On the other hand, we found no correlation between the number of Tfh1 or Tfh17 cells and serum IgG4 level or the number of circulating plasmablasts. In line with these observations, Tfh2 cells, but not Tfh1 or Tfh17 cells, from IgG4-RD patients efficiently induced the differentiation of naïve B cells into plasmablasts in vitro [26]. Moreover, IgG4 class-switching was exclusively induced by Tfh2 cells [26]. Remarkably, while IgG production induced by Tfh2 cells was comparable between IgG4-RD and healthy controls, IgG4 production was significantly higher with Tfh2 cells from IgG4-RD than with those from healthy controls [26]. These results suggest that Tfh2 cells are the culprit cell type for inducing plasmablast expansions and IgG4 class-switching in IgG4-RD. This is the only immune cell type for inducing plasmablast differentiation and IgG4 production found in IgG4-RD. Activated circulating Tfh cells and disease activity in IgG4-RD To evaluate the mechanism of enhancement of IgG4 production by Tfh2 cells from IgG4-RD, we also conducted an analysis of the activation status of circulating Tfh2 cells. Importantly, circulating activated Tfh2 cells were significantly increased in IgG4-RD, positively correlating with disease activity [26]. Moreover, the number of activated Tfh2 cells was positively correlated with the number of affected organs and serum IgG4 level [26], indicating that these cells come from ectopic germinal centres at affected sites. Following treatment with glucocorticoids, the number of activated Tfh2 cells decreases, concomitant with clinical resolution [26]. Of note, we experienced a case whose number of activated Tfh2 cells was again elevated at disease relapse, suggesting the potential of activated Tfh2 cells as a biomarker for disease activity [26]. We found a significant positive correlation between the number of Tfh2 cells and serum levels of IL-4, and that the two patients with the highest levels of IL-21 had the highest numbers of plasmablasts, serum IgG4 levels and Tfh2 cell numbers in active, untreated IgG4-RD [26, 39]. Thus, activated Tfh2 cells more efficiently secrete IL-4 and IL-21 to induce enhanced IgG4 production through plasmablast differentiation [26, 39]. IL-10 is also known as a potential cytokine for IgG4 class-switching, but there was no correlation between serum IgG4 and IL-10 levels [39]. The other pathological role of Tfh2 cells in IgG4-RD IL-21 from Tfh cells is essential for germinal centre formation [6, 13]. Indeed, IL-21 mRNA expression was increased, positively correlating with the number of hyperplastic ectopic germinal centre formations in IgG4-RD [78, 80]. IL-21–secreting lymphocytes also expressed IL-4, Bcl-6 and CXCR5 [78], suggesting that IL-21–secreting lymphocytes are Tfh2 cells. Thus, excessive IL-21 production from Tfh2 cells may induce hyperplastic germinal centre formations in IgG4-RD. The possible mechanism of increased Tfh2 cells in IgG4-RD IgG4-RD responds well to glucocorticoid treatment, but tapering or cessation of treatment often leads to relapse [102]. In this regard, while activated Tfh2 cell counts, plasmablast counts and levels of serum IgG4 and IL-4 decrease after glucocorticoid treatment in parallel with disease improvement, total Tfh2 cell counts remain unchanged [26, 39], suggesting that increased total Tfh2 cells are the underlying pathogenic mechanism and contribute to incomplete cure of IgG4-RD. Tfh cell differentiation is induced by chronic antigen stimulation in secondary lymphoid organs and by a combination of cytokines [103]. On this point, since circulating Tfh2 cells in IgG4-RD have a memory phenotype, as evidenced by their decreased expression of CD45RA [26, 39], chronic stimulation by unknown antigens (such as allergens) is one factor promoting Tfh2 cell differentiation. Interestingly, chronic exposure to occupational antigens may play a role in the initiation and/or maintenance of IgG4-RD [104]. The increased IgG4 response to food and animal antigens also deserves attention [105]. IgG4 itself is generally thought to be non-inflammatory because it does not crosslink antigens and fix complement by the process called Fab-arm exchange [106]. Furthermore, from the viewpoint of allergy (which is frequently observed in IgG4-RD), the emergence of IgG4 is associated with a decrease in allergic symptoms through an allergen-blocking effect [106]. However, according to recent evidence showing pathogenic effects of IgG4 itself from IgG4-RD [107], Tfh2 cells that induce IgG4 production from plasmablasts are the potential therapeutic target in this disease. Indeed, the efficacy of abatacept in a patient with IgG4-RD may be explained by inhibition of Tfh cells with respect to B cell interaction and subsequent disruption of the formation of ectopic germinal centres [108, 109]. Although it is still unclear whether IgG4-RD is an autoimmune disease or an allergic disorder, the detection of IgG4-recognizing autoantigens or exogenous antigens may contribute to a more thorough elucidation of the pathophysiology of IgG4-RD. Circulating Tfh1 cells in IgG4-RD We have made another important observation: that Tfh1 cells were also increased and activated in IgG4-RD [26]. Activated Tfh1 cells positively correlated with disease activity, but not with serum IgG4 levels, suggesting that they play a role in the pathogenesis of IgG4-RD, but are not involved in IgG4 production. Although the precise function of Tfh1 cells remains unclear, they express CXCR5 and the transcription factor T-bet, and produce IFN-γ [92]. Thus, they migrate to the ectopic germinal centres that express CXCL13 (the ligand for CXCR5) and produce IFN-γ at affected sites, resulting in tissue fibrosis. Circulating Tfh17 cells in IgG4-RD The number of circulating Tfh17 cells and their activated phenotype was not increased in IgG4-RD [26, 39]. Furthermore, the number of these Tfh17 cells did not correlate with the level of serum IgG4 or the number of circulating plasmablasts [26]. Moreover, IL-17 (which is the hallmark of Tfh17 cells) is rarely expressed at affected sites [78]. Collectively, Tfh17 cells may not be involved in the pathogenesis of IgG4-RD. Conclusions Recently accumulated findings have clarified that T cells are involved in the pathogenesis of IgG4-RD (Table 2). Tfh2 cells are important in germinal centre formation, as well as in plasmablast and plasma cell differentiation and IgG4 class-switching (Fig. 1). Future insights into T cell immunology and pathophysiology should lead to novel treatments for this disease. Table 2 Overview of T cell subsets in IgG4-related disease T cell phenotype  Principle findings in IgG4-RD  Reference  Tfh cells  Increased expression of BCL6 (the master regulator of Tfh cells) at affected sites  [78, 80]    Increased expression of Tfh-related chemokines (CXCL13) and chemokine receptors (CXCR5) at affected sites  [78, 82, 83]    Increased expression of IL-21 at affected sites  [78, 80, 88]    Increased expression of CD40 ligand at affected sites  [32]    Increased formation of germinal centres at affected sites  [77, 78, 80, 81]    Increased circulating Tfh cells and their activated phenotype  [26, 39]    Association with IgG4 production at affected sites  [78]  Tfh2 cells  Increased circulating Tfh2 cells and their activated phenotype  [26, 39, 101]    Capacity to help naïve B cells to differentiate into plasmablasts and IgG4 production in vitro  [26]    Association with disease activity and the number of affected organs  [26]    Association with IL-4 and IL-21 production  [26, 39]  Tfh1 cells  Increased circulating Tfh1 cells and their activated phenotype  [26]    Association with disease activity and the number of affected organs  [26]  Cytotoxic CD4+ T cells  Oligoclonally increased circulating cytotoxic CD4+ T cells and their infiltration at affected sites  [25, 46]    Capacity to produce IFN-γ, IL-1β, TGF-β and granzyme  [25, 46]  Treg cells  Increased expression of FOXP3 (the master regulator of Treg), IL-10 and TGF-β at affected sites  [35, 37, 41–43]    Increased circulating Treg cells  [44, 45]    Association of TGF-β with fibrosis at affected sites  [34, 36, 42, 55]  CD8+ T cells  Exhausted cytotoxic capacity  [57−59]    Increased circulating CD8+ T cells  [18]  T cell phenotype  Principle findings in IgG4-RD  Reference  Tfh cells  Increased expression of BCL6 (the master regulator of Tfh cells) at affected sites  [78, 80]    Increased expression of Tfh-related chemokines (CXCL13) and chemokine receptors (CXCR5) at affected sites  [78, 82, 83]    Increased expression of IL-21 at affected sites  [78, 80, 88]    Increased expression of CD40 ligand at affected sites  [32]    Increased formation of germinal centres at affected sites  [77, 78, 80, 81]    Increased circulating Tfh cells and their activated phenotype  [26, 39]    Association with IgG4 production at affected sites  [78]  Tfh2 cells  Increased circulating Tfh2 cells and their activated phenotype  [26, 39, 101]    Capacity to help naïve B cells to differentiate into plasmablasts and IgG4 production in vitro  [26]    Association with disease activity and the number of affected organs  [26]    Association with IL-4 and IL-21 production  [26, 39]  Tfh1 cells  Increased circulating Tfh1 cells and their activated phenotype  [26]    Association with disease activity and the number of affected organs  [26]  Cytotoxic CD4+ T cells  Oligoclonally increased circulating cytotoxic CD4+ T cells and their infiltration at affected sites  [25, 46]    Capacity to produce IFN-γ, IL-1β, TGF-β and granzyme  [25, 46]  Treg cells  Increased expression of FOXP3 (the master regulator of Treg), IL-10 and TGF-β at affected sites  [35, 37, 41–43]    Increased circulating Treg cells  [44, 45]    Association of TGF-β with fibrosis at affected sites  [34, 36, 42, 55]  CD8+ T cells  Exhausted cytotoxic capacity  [57−59]    Increased circulating CD8+ T cells  [18]  Tfh cells: T follicular helper cells; Tfh2 cells: T follicular helper 2 cells; Tfh1 cells: T follicular helper 1 cells. Fig. 1 View largeDownload slide Schematic model of involvements of T cells in the pathogenesis of IgG4-related disease Chronic stimulation by unknown antigens activates APCs, and they induce the differentiation of Tfh2 cells. FDCs (or Tfh2 cell accumulation themselves) secrete CXCL13, which induces Tfh2 cells and B cell accumulation through interaction with CXCR5, resulting in the formation of ectopic germinal centres in affected tissues. Tfh2 cells induce the differentiation of naïve B cells into plasmablasts and plasma cells, and IgG4 class-switching through cell-to-cell contact with the elaboration of IL-4 and IL-21. IgG4 itself and the cytokines from Tfh1 cells, cytotoxic CD4+ T cells and Treg cells may contribute to tissue damage and fibrosis. Tfh2: T follicular helper 2 cells; Tfh1: T follicular helper 1 cells; APC: antigen-presenting cell; FDC: follicular dendritic cell; SHM: somatic hypermutation; CXCL13: CXC chemokine ligand 13; Bcl-6: B cell lymphoma-6; PD-1: programmed death-1; AID: activation-induced cytidine deaminase; CD40L: CD40 ligand. Fig. 1 View largeDownload slide Schematic model of involvements of T cells in the pathogenesis of IgG4-related disease Chronic stimulation by unknown antigens activates APCs, and they induce the differentiation of Tfh2 cells. FDCs (or Tfh2 cell accumulation themselves) secrete CXCL13, which induces Tfh2 cells and B cell accumulation through interaction with CXCR5, resulting in the formation of ectopic germinal centres in affected tissues. Tfh2 cells induce the differentiation of naïve B cells into plasmablasts and plasma cells, and IgG4 class-switching through cell-to-cell contact with the elaboration of IL-4 and IL-21. IgG4 itself and the cytokines from Tfh1 cells, cytotoxic CD4+ T cells and Treg cells may contribute to tissue damage and fibrosis. Tfh2: T follicular helper 2 cells; Tfh1: T follicular helper 1 cells; APC: antigen-presenting cell; FDC: follicular dendritic cell; SHM: somatic hypermutation; CXCL13: CXC chemokine ligand 13; Bcl-6: B cell lymphoma-6; PD-1: programmed death-1; AID: activation-induced cytidine deaminase; CD40L: CD40 ligand. Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this manuscript. Disclosure statement: Y.T. has received consultant fees from Pfizer, Chugai Pharma, Mitsubishi-Tanabe Pharma and AbbVie, received honoraria from Pfizer, Chugai Pharma, Mitsubishi-Tanabe Pharma, Bristol-Myers Squibb, Takeda Industrial Pharma, GlaxoSmithkline, Nippon Shinyaku, Eli Lilly, Janssen Pharma, Eisai Pharma, Astellas Pharma and Actelion Pharmaceuticals and received research support from Chugai Pharma and Mitsubishi-Tanabe Pharma. T.T has received consulting fees, speaking fees and/or honoraria from Pfizer Japan, Mitsubishi Tanabe Pharma, Eisai, Astellas Pharma and UCB (<$10 000 each) and from Chugai Pharmaceutical, Bristol-Myers K.K., Daiichi Sankyo, AbbVie, Janssen Pharmaceutical K.K., Pfizer Japan, Asahi Kasei Pharma, Takeda Pharmaceutical, AstraZeneca K.K., Eli Lilly Japan K.K. and Novartis Pharma K.K. (>$10 000 each). All other authors have declared no conflicts of interest. References 1 Kamisawa T, Zen Y, Pillai S, Stone JH. IgG4-related disease. 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RheumatologyOxford University Press

Published: Feb 1, 2018

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