Background
IgG4-related disease (IgG4-RD) is a new disease entity characterized by elevated serum IgG4 and increased infiltration of IgG4
+ plasma cells in lesions, such as in the salivary and lacrimal glands, pancreas, bile duct, lung, kidney, aorta, retroperitoneum, and lymph nodes [
1‐
5]. IgG4-related dacryoadenitis and sialoadenitis, so-called Mikulicz disease, was originally considered a subtype of primary Sjögren syndrome (pSS) based on the similarity of organ involvement. However, Mikulicz disease was recently recognized as a subtype of IgG4-RD due to high serum IgG4 concentrations and the infiltration of IgG4
+ plasma cells in glandular tissues [
6]. Elevated serum IgG4 and IgG4
+plasma cell infiltration in lesions have also been reported in multicentric Castleman’s disease (MCD), a rare and poorly understood lymphoproliferative disorder [
7‐
12]. As they satisfy the diagnostic criteria of IgG4-RD, the differentiation between IgG4-RD and MCD on the basis of serum IgG4 level or the histologic finding alone is recognized to be difficult [
7‐
12].
Interestingly, recent reports have shown that the number of circulating plasmablasts positively correlates with disease activity in IgG4-RD [
13,
14]. The expansion of plasmablasts is accompanied by enhanced somatic mutation and the emergence of distinct plasmablast clones related to relapse of IgG4-RD, suggesting the linkage of disease pathogenesis to
de novo recruitment of naïve B cells into T-cell-dependent responses [
15]. Collaboration of follicular helper T (Tfh) cells and B cells at the germinal center plays a major role in antibody production, immunoglobulin-isotype switching, affinity maturation, and plasmablast and plasma-cell genesis [
16,
17]. Indeed, in IgG4-RD, germinal centers are often observed within affected organs [
18] and are presumably the source of plasmablasts. In general, bona fide Tfh cells have initially been identified in secondary lymphoid organs but their counterparts and subsets (Tfh1, Tfh2, or Tfh17 cells) have only been recognized in peripheral blood [
19]. We previously reported that the number of circulating Tfh2 cells is increased in IgG4-RD in correlation with elevated serum IgG4 and the number of plasmablasts, suggesting the important role of Tfh2 cells in IgG4-RD pathogenesis [
20,
21]. However, the question of whether Tfh2 cells actually induce B cells to differentiate into plasmablasts and to produce IgG4 in patients with IgG4-RD remains unanswered. Functional analysis by in vitro assay is thus desired.
Simpson et al. initially described the expansion of circulating Tfh cells in patients with systemic lupus erythematosus that is the prototype of human autoimmune disease [
22]. Recently, circulating Tfh cells have been reported to be a valuable biomarker for the monitoring of dysregulated antibody responses and disease activity in autoimmune diseases [
22‐
25]. Defining therapeutic targets for IgG4-RD requires a clear understanding of the pathogenic pathways and corresponding biomarkers of disease activity. Recent reports have shown that detection of the CCR7
lowPD-1
high subset, the “activated Tfh cells” in circulation, is a useful tool in monitoring the activation status of Tfh cells in autoimmunity, human immunodeficiency virus infection, and vaccination [
22‐
26]. Indeed, a high percentage of activated Tfh cells was observed in Tfh-biased autoimmune sanroque mice and patients with systemic lupus erythematosus with high autoantibody titers and severe disease activity [
26]. These observations suggest that circulating activated Tfh cells may link to disease activity in Tfh-biased diseases. To date, however, this question is uncertain in patients with IgG4-RD.
Thus, herein we sought to investigate the functional role of Tfh cell subsets in helping B cells, and assessed the expansion of activated Tfh cell subsets for correlation with disease activity, in the blood of patients with active, untreated IgG4-RD, and comparing this to patients with pSS or MCD and to healthy controls (HCs).
Discussion
This study demonstrated that Tfh2 cells, but not Tfh1 or Tfh17 cells, induced naïve B cells to differentiate into plasmablasts and to produce IgG4 in patients with active, untreated IgG4-RD. Of note, IgG4 production by Tfh2 cells from patients with active, untreated IgG4-RD was enhanced compared to those from HCs. Further, activated Tfh2 cells were linked to disease activity in patients with IgG4-RD in the baseline active, untreated phase. Moreover, activated Tfh2 cells decreased with GC treatment and with improvement in disease activity. Interestingly, the number of activated Tfh1 cells was increased in IgG4-RD, and correlated with disease activity, but not with serum IgG4. These results suggest that both activated Tfh1 and Tfh2 cells are the underlying pathological abnormalities playing different roles in IgG4-RD.
Given that the immunoglobulin genes of expanded plasmablasts exhibit somatic hypermutation in patients with IgG4-RD, the expansion of plasmablasts appears to be generated by a T-cell-dependent immune response [
15]. Previous studies have assumed that T helper 2 cells and regulatory T cells contribute to IgG4 class-switching in IgG4-RD pathogenesis [
35‐
40], but empirical evidence to show whether these cells actually induce the differentiation of naïve B cells into plasmablasts and the production of IgG4 in patients with IgG4-RD is still lacking. In contrast, Tfh cells are increasingly recognized as CD4
+ T cells that play an essential role in T-cell-dependent immune response [
16,
17], and consist of three subsets (Tfh1, Tfh2, and Tfh17) [
19]. Here, our finding that Tfh2 cells, but not Tfh1 and Tfh17 cells, induce the differentiation of naïve B cells into plasmablasts and the production of IgG4 in patients with active, untreated IgG4-RD suggests that Tfh2 cells are pathogenic CD4
+ T cells that play a pivotal role in the T-cell-dependent immune response in patients with IgG4-RD. To our knowledge, this is the first study to describe the functional role of Tfh2 cells in helping B cells in a vitro assay in patients with IgG4-RD.
We observed that activated Tfh2 cells (CCR7
lowPD-1
high) were increased in patients with IgG4-RD compared to patients with pSS or MCD, and compared to HCs. Importantly, the expression of CCR7 on T cells is well-known to be generally downregulated when they are activated [
26]. Therefore, the low CCR7 expression on Tfh2 cells indicates their recent activation [
26]. Meanwhile, the expression of PD-1 on Tfh cells is essential for B cell selection and survival in the germinal centers and for further maturation of B cells into plasmablasts and plasma cells to produce antibodies [
41]. Collectively, our finding that activated Tfh2 cells are expanded in patients with active, untreated IgG4-RD suggests that Tfh2 cells are recently activated and functionally important for B cell maturation in IgG4-RD. Our feasible model of the pathogenesis of IgG4-RD is that chronic stimulation by an unknown antigen induces the polarization and activation of Tfh2 cells that are the disease-causing CD4
+ T cells, which may result in the development of germinal centers at affected sites and the generation of IgG4-secreting plasmablasts and plasma cells. One of the factors that could contribute to the increased activated Tfh2 cells in IgG4-RD may be interferon (IFN)-α. In fact, IFN-α is greatly increased in serum and in the affected tissues in patients with IgG4-related pancreatitis [
42]. As a matter of fact, the transcription factor IFN regulatory factor 9 activated by IFN-α is known to bind to the promoter region of the
PD-1 gene and induce
PD-1 mRNA transcription [
43,
44].
The establishment of appropriate biomarkers that are linked to disease activity in IgG4-RD is important. The occasional finding of IgG4-seronegative IgG4-RD despite active disease indicates that elevated levels of serum IgG4 are not sufficiently sensitive or specific for disease activity [
45,
46]. Meanwhile, the plasmablast count has been recently reported to coincide with both the active phase and disease relapse, and has been suggested to be a useful biomarker of disease activity in IgG4-RD [
13‐
15]. In the present study, we have empirically shown that the number of activated Tfh2 cells correlates with disease activity in the baseline active phase in patients with IgG4-RD. Furthermore, we have demonstrated that the number of activated Tfh2 cells parallels disease activity during GC treatment. These results suggest the potential of activated Tfh2 cells as a biomarker in IgG4-RD. We await corroboration of our results in an analysis of a larger cohort.
Although serum sIL-2R is reported to reflect disease activity in IgG4-RD [
31‐
33], the pathologic significance of the serum level is unclear. Here, we demonstrated that serum sIL-2R is positively correlated with the number of activated Tfh1 cells or activated Tfh2 cells, but not with the number of total Tfh2 cells or total Tfh1 cells, serum CRP, or LDH in patients with IgG4-RD. This suggests that sIL-2R is released from activated Tfh1 and activated Tfh2 cells in IgG4-RD and thus reflects disease activity.
Previous studies report that circulating activated Tfh cells are generated from germinal centers in the spleen or lymph nodes [
26]. Hyperplastic germinal centers have been rarely observed in patients with pSS or MCD [
47,
48] but have been shown to develop in affected organs in patients with IgG4-RD [
18]. Here, we found strong correlation between the number of circulating activated Tfh2 cells and the number of affected organs in patients with IgG4-RD, suggesting that these cells are generated from germinal centers in affected organs in patients with IgG4-RD. Further elucidation of Tfh2 cells in affected tissues would be required for the future.
Because IgG4-RD and MCD are both systemic diseases that present with hypergammaglobulinemia and elevated serum IgG4 [
7‐
12], differentiation between these two diseases is sometimes difficult. The hallmark of the pathogenesis of MCD is considered the hyper interleukin (IL)-6 syndrome, which induces B cell differentiation into plasmablasts and plasma cells, resulting in polyclonal hypergammaglobulinemia [
49]. On the other hand, IL-4, IL-5, IL-10, IL-13, IL-21, transforming growth factor (TGF)-β and B cell activating factor (BAFF) are considered the key cytokines in the pathogenesis of IgG4-RD [
1‐
3]. Additionally, in the present study, we showed that the number of total Tfh2 cells was significantly higher in IgG4-RD than in MCD. Furthermore, the number of activated Tfh2 cells was also increased in IgG4-RD compared to MCD. Our findings suggest that IgG4-RD is pathogenetically distinct from MCD from the viewpoint of Tfh2 cell immunology in addition to cytokine profiles. To our knowledge, this is the first study that investigated the differences in Tfh2 cells in IgG4-RD and MCD.
Previous reports have shown that IFN-γ is expressed in local affected lesions and circulating IFN-γ-producing CD4
+cells are increased in IgG4-RD [
37,
50,
51]. Interestingly, in our present study the number of Tfh1 cells and their activated phenotype was increased in patients with IgG4-RD compared to patients with pSS or MCD, or HCs. Although the precise function of Tfh1 cells remains to be elucidated, they are known to express CXCR5 and the transcription factor T-bet, and to produce IFN-γ [
19]. Thus, they migrate to the germinal centers that express CXCL13, the ligand for CXCR5, and produce IFN-γ in the local affected lesions of IgG4-RD. On that note, in our present study, the number of activated Tfh1 cells correlated with disease activity, but not with serum IgG4, which was reproduced in our co-culture experiment (Fig.
4). Taken together, both Tfh1 and Tfh2 cells are involved in the pathogenesis of IgG4-RD, while Tfh2 cells are rather important in the process of IgG4 production. Importantly, we have observed patients with active IgG4-RD with normal serum IgG4, suggesting that not only Tfh2 cells but also Tfh1 cells play a role in the pathogenesis of IgG4-RD.
In our previous study [
20] we observed positive correlation between serum IL-4, the number of Tfh2 cells, and serum IgG4 in IgG4-RD. Therefore, we measured IL-4 in the in vitro experiments. IL-4 was detectable, but unexpectedly, there was no difference between patients with IgG4-RD and HCs. This may be ascribed to the time point of IL-4 measurements; IL-4 was measured at day 7 but not at a shorter time interval from baseline. Thus, our observation may be the result of IL-4 being partly decreased due to the consumption through the IL-4 receptor of naïve B cells to promote the differentiation of naïve B cells into plasmablasts and the production of IgG4.
Our study demonstrated that Tfh2 cells from patients with active, untreated IgG4-RD induced enhanced production of IgG4 compared to those from HCs. The conceivable mechanisms of this phenomenon are: (1) an increased number of activated Tfh2 cells; (2) involvement of altered co-stimulatory molecules in the process of collaboration between Tfh2 cells and B cells [
52]; (3) enhanced cytokine production, such as IL-4, IL-10, IL-13 and IL-21 from Tfh2 cells known for IgG4 class-switching [
53]; and (4) an intrinsic alteration of naïve B cells in IgG4-RD patients. Although alteration in naïve B cells is a possibility for the underlying abnormality in IgG4-RD, treatment with an anti-CD20 antibody targeting B cells is known to result in relapse in some patients [
54]. Therefore, these possible defects of Tfh2 cells in IgG4-RD suggest that targeting activated Tfh2 cells could lead to the development of novel pharmacological strategies aimed at disrupting the T-B-dependent immune response.
Acknowledgements
We thank the patients and healthy individuals who participated in this study. We specially thank Mr. Noriyasu Seki, Mr. Humitsugu Yamane, Mrs. Yoshiko Yogiashi, and Ms. Yuki Otomo for their technical assistance.
Competing interests
MA, HY, KS, HK, RM, and AY declare that they have no competing interests. TT has received consultancies, speaking fees, and honoraria from Astellas Pharma, Bristol–Myers K.K., Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Eisai Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahikasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Takeda Pharmaceutical Co., Ltd, Nipponkayaku Co., Ltd, Astra Zeneca K.K., Eli Lilly Japan K.K., and Novartis Pharma K.K. KY has received consultancies, speaking fees, and honoraria from Pfizer Japan Inc., Chugai Pharmaceutical Co, Ltd., Mitsubishi Tanabe Pharma Co., Takeda Pharmaceutical Co., Ltd, GlaxoSmithkline, Nipponkayaku Co., Ltd, Eli Lilly Japan K.K., Janssen Pharmaceutical K.K., Eisai Co., Ltd., Astellas Pharma, and Acterlion Pharmaceuticals. YK has received consultancies, speaking fees, and honoraria from Astellas Pharma, Chugai Pharmaceutical Co, Ltd., Bristol–Myers K.K., Eisai Co., Ltd., Kissei Co., Ltd., Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Taisho Toyama Pharma Co., and UCB. YK, KK, and TM are employees of Takeda Pharmaceutical Co., Ltd.