IgG4-related disease (IgG4-RD) is a newly recognized condition characterized by mass-forming lesions involving various tissues (1). The disease more frequently affects males over 50 years of age (2–4). The unifying finding of the disease is the pathological lesion characterized by dense lymphoplasmacytic infiltrates mainly consisting of CD4⁺ T cells and numerous IgG4⁺ plasma cells associated with fibrosis (5). Tertiary germinal center formation is also frequently observed in diseased tissues.

Mikulicz’s disease, otherwise considered as a disease manifestation of primary Sjögren’s syndrome (pSS) (6) is also part of the spectrum of IgG4-RD. It has now been clearly demonstrated that despite common features shared by both diseases, clinical, biological, pathological, and immunohistological findings differ between pSS and IgG4-RD (7). Yet, in daily practice, pSS remains a frequent differential diagnosis of IgG4-RD.

Despite ongoing genetic and mechanistic studies, the pathogenesis of IgG4-RD remains poorly understood. Rather than a vector of tissue damage, IgG4 is considered to be a marker of the disease. Yet, elevated levels of serum IgG4 have also been reported in other conditions, and approximately 30% of patients with biopsy-proven IgG4-RD have normal serum levels of IgG4 (8). Hence, the identification of novel biomarkers is a timely topic in the field of IgG4-RD.

Besides studies regarding B cells and immunoglobulin biology, careful attention is currently being paid on the role of T cells in the disease. Conflicting results have been reported regarding the polarization of T helper (T_H) cells in IgG4-RD, especially in patients with pancreatic involvement and Mikulicz’s disease (9, 10). Because regulatory T cells (Treg) are a source of IL-10 and TGF-β (i.e., key cytokines involved, respectively, in the differentiation of IgG4-producing B cells and in the genesis of fibrosis), the roles of these cells have also been investigated in a few preliminary studies regarding IgG4-related hepatic and pancreatic involvements (11–13) More recently, T follicular helper cells (T_FH), an important cell subset involved in the development of germinal centers as well as in antibody production, have also been studied in a small series of IgG4-RD patients (14) suggesting changes of this compartment in the disease. Yet, these results need to be confirmed in a new and larger cohort, including patients with other than salivary and lachrymal-restricted involvements, and with a more precise phenotypic definition of T_FH subsets, especially concerning PD1 molecule expression.

In this study, we investigated circulating lymphocytes in untreated patients with IgG4-RD, with a focus on T_H1/T_H2/T_H17 balance and PD1⁺ T_FH cells, and compared them to pSS patients.

IgG4-related disease is characterized by tissue infiltration by numerous IgG4⁺ plasmocytes, increased numbers of circulating plasmablasts and elevated titers of both serum IgG4, IgE, as well as other IgG subclasses (2). B cell activation appears to be T cell-dependent because activated B cells harbor enhanced somatic mutation, and relapses after rituximab (a B-cell-depleting agent) are characterized by the emergence of new plasmablast clones (17). T CD4⁺ cells are numerous in disease infiltrates and are thought to trigger B cell polyclonal expansion and fibrosis. Prior studies regarding blood and/or tissue analysis of IgG4-RD patients suggest changes of T helper cells, Treg, and more recently oligoclonal CD4⁺ effector/memory cytotoxic T lymphocytes (9–11, 18, 19).

Hence, we conducted a systematic analysis of lymphocyte subsets in untreated patients with IgG4-RD. Major T cell subsets, including memory and naïve T cells, NK, and B cells were normal in patients with IgG4-RD compared to HC. Concordant with previous reports, plasmablast cell counts were higher in patients with IgG4-RD as compared to HC (17). Albeit not reaching significance, plasmablast cells were also higher in patients with IgG4-RD as compared to pSS patients. Next, in line with a previous study, there was a clear trend toward an increase of circulating CD4⁺Foxp3⁺ Tregs in IgG4-RD patients, suggesting that in such patients Treg expansion is not restricted to diseased tissues (18). Treg were also shown to be increased in blood, without reaching significance, in an independent study (19). In IgG4-RD, Treg expansion might contribute to the genesis of fibrosis through TGF-β release and to the increase of IL-10 release by PBMCs. From that perspective, IgG4-RD is different than most autoimmune diseases where Treg are commonly decreased (20).

Over-production of T_H2-related cytokines (IL-4, IL-13, and IL-5) in IgG4-RD tissues has previously been reported (11) and correlates with IgE and IgG4 class switch, blood eosinophilia, and eosinophilic infiltrates that are common features of IgG4-RD (4, 11). Hence, and despite previous studies that provided conflicting results regarding the T_H1/T_H2 balance in PBMCs from IgG4-RD patients, it has been suggested that T_H2 cells might be key players in the disease’s pathophysiology (9, 10). Yet, the central role of such cells is debated because IL-4 production upon re-stimulation has been shown to be restricted to CD4⁺GATA3⁺ T cells in patients with known atopy, suggesting that the role of T_H2 responses in IgG4-RD might be confounded by concomitant allergic disease (21). The present analysis of T_H1, T_H2, and T_H17 cells in IgG4-RD showed an increase of both T_H2 and T_H17 cells. Interestingly, the increase of T_H2 cells in the present study was not restricted to a unique organ involvement. Moreover, only 4 out of 28 patients (16%) with IgG4-RD also complied with the definition of the European Academy of Allergy and Clinical Immunology for atopy (22). T_H2 and T_FH2 cell number, IgE levels, and eosinophil numbers were not significantly different between atopic and non-atopic patients (data not shown). Hence, according to our data, T_H2 cell expansion in IgG4-RD patients cannot be restricted only to patients with concomitant atopy. Next, the analysis of cytokine release by PBMCs upon stimulation were in line with these findings and showed that patients with IgG4-RD produced higher amounts of IL-4 but also of IL-10 and IL-17 as compared with HC and patients with pSS. Both T_H2 and Treg are thought to contribute to IL-4 and IL-10 release, but we did not expect to report on both high IL-17 levels and elevated T_H17 cells. Analysis of the co-expression of intracellular IL-17 and IL-4 after stimulation in CD4⁺T cells showed that double positive cells were extremely rare, and therefore that the source of both cytokines were differentiated T_H2 or T_H17 cells (data not shown).

Extranodal germinal centers are often found in IgG4-RD tissues, and IgG4⁺ plasmablasts harbor somatic hypermutation (17), a process occurring during B cell differentiation in germinal centers. Because T_FH are essential for germinal center formation and Ig class switch in humans and mice, they could be implicated in the pathogenesis of IgG4-RD (23). The circulating T_FH compartment in human is heterogeneous based on PD1, CCR6, and CXCR3 expression (23, 24). Here, we show that the CD4⁺CXCR5⁺PD1⁺ T_FH cells are specifically expanded in IgG4-RD. Interestingly, since only one patient presented with an increase of T_FH after treatment with rituximab, expansion is reversible in patients with IgG4-RD that are efficiently treated. Expanded PD1⁺ T_FH have been previously reported in human autoimmune diseases such as systemic lupus erythematosus, pSS, and juvenile dermatomyositis (25, 26). In the latter, both T_FH2 and T_FH17 are increased and correlate with plasmablast numbers and disease activity (26). In our analysis, CXCR5⁺PD1⁺ T_FH cells were not expanded in untreated pSS patients but were expanded in IgG4-RD. T_FH expansion was mostly related to the T_FH2 CCR6⁻CXCR3⁻ subset and to a lesser extent to the T_FH17 CCR6⁺CXCR3⁻ subset. T_FH2 and T_FH17 cells have been shown to efficiently induce in vitro naïve B cells to proliferate and differentiate into plasmablasts and produce all IgG subclasses, in contrast to T_FH 1 (25). T_FH2 specifically produces IL-4, IL-5, and IL-13, which are important cytokines for the class switching to IgE and IgG4. The expansion of T_FH 2 is consistent with pathological and biological abnormalities reported in IgG4-RD patients. Our study showed that T_FH2 cell numbers correlated positively with serum IgG4 (r = 0.64; p = 0.0004), IL-4 (r = 0.55; p = 0.01), and IL-10 (r = 0.49; p = 0.03) (Table 3). Moreover, an increase of the CD4⁺CXCR5⁺CD45RA⁻ T_FH and T_FH 2 cells in IgG4-RD has been reported in another series of 15 patients (14). However, PD1 expression was not analyzed. The specific expansion of CXCR5⁺PD1⁺ T_FH observed in our study could be related to some unique functional properties inherent to IgG4-RD’s pathogenesis. Indeed, PD1⁺ T_FH require less activation than PD1⁻ T_FH to differentiate into functional helpers and, by opposition to PD1⁻ T_FH, PD1⁺ T_FH express low levels of CCR7 (24). The PD1⁺CCR7^low T_FH population is required for T cells to migrate into B cell follicles (27). Thus the specific expansion of PD1⁺ T_FH in IgG4-RD could be an important trigger to B cell activation, class switch, and plasmablast generation. Interestingly, it has been shown in rheumatoid arthritis that PD1⁺ T_FH is maintained by plasmablasts by an IL-6-dependent positive feedback loop that should be investigated in IgG4-RD (28).

The findings reported in our study consist of correlations and causation of these T cells changes in the pathophysiology of IgG4-RD have to be confirmed by further functional studies. It has been recently shown in Japanese patients with predominant salivary and lachrymal glands involvement that CD4⁺CD45RA⁻CXCR5⁺CCR6⁻CXCR3⁻ T_FH2 cells were more efficient in inducing differentiation into plasmablasts and led to higher IgG4 production by autologous naïve B cells in active, untreated IgG4-RD patients than in HC (29), suggesting a functional role of these cells in the disease. The same authors found in a previous study a correlation between the increased number of circulating T_FH2 cells and the number of plasmablasts (14), which was not found in our study. Conversely it is also plausible that these T cells changes are secondary to yet other unknown factor(s) (e.g., a source of TGF-β) that drives T cell differentiation and IgG4 production. In this line, mast cells have recently been shown to express IL-4, IL-10, and TGF-β (30), as well as IL-13 (31) in IgG4-RD tissues, and these innate cells could contribute to the T_H2/T regulatory cytokines orientation reported in the disease.

Major cytokines involved in the early T_FH differentiation process from CD4⁺ T cells in human, including IL-12, IL-23, and TGF-β, are also supported by other STAT3-activating cytokines including IL-6, IL-21, and IL1-β (23). In human autoimmune diseases, both T_H17 and T_FH co-emerge and share a developmental mechanism induced by TGF-β. It has been proposed that abundant expression of TGF-β in inflammatory sites in human autoimmune diseases (28), where tertiary lymphoid organs are often formed, contribute to the generation of T_FH and T_H17 cells (24). Hence, the expansion of these cells could be the consequence of an initial inflammatory process. In tissues, T_H17-related molecules have been reported in salivary glands of patients with IgG4-RD, albeit at low levels (32). The site where the differentiation and expansion of T_FH occurs in IgG4-RD is unknown, and no link has been established with IL-1β and TGF-β producing clonal expanded CD4⁺SLAMF7⁺ CTLs (19).

Interestingly, our results showed that PD1⁺ T_FH cells were significantly decreased in patients with IgG4-RD who were treated with either steroids alone, steroids plus azathioprine, or steroids plus rituximab, and that the decrease in PD1⁺ T_FH cells was always associated with clinical improvement of the disease. Significant variation of T_FH cells after treatment with steroids has also been recently reported in Japanese patients with modification of T_FH1 cells, which we did not observe in our larger study (29). However, organ involvement was significantly different from our study. Interestingly, the PD1⁺ T_FH subset also comprises T_FH regulatory cells co-expressing Foxp3 and an imbalance of T_FH/T_FH Foxp3 regulatory could also be implicated in the disease’s pathophysiology and should be further explored (33).

We show that patients with active, untreated IgG4-RD presenting with variable organ involvements present specific changes of T cells in peripheral blood. Circulating PD1⁺ T_FH are expanded together with T_H2 and T_H17, associated with elevated IL-4, IL-10, and IL-17 release. Both PD1⁺ Tfh2 and Tfh17 subsets have been shown to be prone to migrate into B cell follicles and/or inflammatory sites and could therefore contribute to B cell activation and IgG4 class switch in IgG4-RD tissues. Yet, the precise role of these T cell changes together with the role of clonal expanded cytotoxic T cells, their interactions with B cells and other immune cells, and the induction of fibrosis remains to be further assessed in IgG4-RD. Indeed, in a near future, it is likely that specific treatments targeting T cells will be assessed in IgG4-RD.

AG, ME, FV, and NS: conception and design of the work; acquisition, analysis, and interpretation of data for the work; drafting the work and revising it critically for important intellectual content. CP, CF, J-RH, and NC-C: acquisition, analysis, and interpretation of data for the work; revising the work critically for important intellectual content. MG, AR, MS, BT, NM, SA, FM, JG, MH, AF, SP, EB, and BB: acquisition of data for the work; revising the work critically for important intellectual content. All authors meet following criteria: final approval of the version to be published and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.