Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis

Abstract Follicular helper T (TFH) cell provides germinal centre (GC) B cell with critical signals for autoantibody production in the immunopathogenesis and progression of autoimmune hepatitis (AIH). However, the immunoregulatory functions of follicular regulatory T (TFR) cell in AIH are still unclear. The numbers of circulating TFR/TFH cells were measured in AIH patients. Moreover, we established experimental autoimmune hepatitis (EAH) model to examine the function of TFR cells on B‐cell differentiation and autoantibody production in vivo and vitro. AIH patients had significantly increased numbers of TFH cells and decreased numbers of TFR cells as well as imbalanced TFR/TFH‐type cytokines (IL‐10, TGF‐β1 and IL‐21) compared with healthy controls (HCs). In addition, TFR cell numbers negatively correlated with TFH cell numbers. Also, serum hypergammaglobulinaemia (IgG and IgM) concentration negatively correlated the levels of serum IL‐21, but positively correlated with the levels of serum IL‐10 in AIH patients. Furthermore, in comparison with control group, significantly higher frequencies of spleen TFR cells but lower frequencies of spleen TFH cells were detected in the EAH group. Further analysis found that TFR cells simultaneously express the phenotypic characteristics of Treg and TFH cells, but exercise as negative regulators of autoantibody production in vitro culture. Our findings demonstrated that dysregulated between TFR and TFH cells might cause excessive production of autoantibodies and destruction of the immune homeostasis, leading to the immunopathological process in AIH.


| INTRODUC TI ON
Autoimmune hepatitis (AIH) is a severe progressive chronic autoimmune disease. AIH is characterized by high levels of hypergammaglobulinaemia and hepatic inflammatory infiltrates, leading to potential cirrhosis. 1 While environmental parameters are implicated, 2,3 immunological dysfunction plays a crucial role in the pathogenesis of AIH. [4][5][6][7] However, the exact mechanisms of autoimmune responses remain unclear.
It is generally believed that abnormal selection of high-affinity autoantibody-producing plasma cells in germinal centre (GC) plays a central role of AIH. 8 We previously characterized hyperactive B-cell immunity in AIH patients and found that original B cells can differentiate into CD27 + memory and CD138 + plasma cells that produce autoantibodies, such as anti-nuclear antibodies. 4 This differentiation process of B cells requires the help of TFH cells, 4,[9][10][11] which is one of the important regulators of humoural immune responses.

TFH cells express high levels of inducible T-cell co-stimulator (ICOS),
programmed death 1(PD-1) and B-cell lymphoma 6, a master transcriptional activator of TFH differentiation. [9][10][11] In the GC, TFH cell provides signals for B-cell survival and differentiation via the coreceptor ICOS and CD40L expression. 12,13 Besides direct B-TFH contact, TFH cell also promotes the generation of Abs with high affinity through soluble mediators like interleukin-4 (IL-4) and IL-21. [9][10][11] Uncontrolled accumulation of TFH cell might activate autoreactive B cells to produce excessive autoantibodies that cause autoimmune responses. 14,15 Our previous study also confirmed that TFH cell has been detected in the peripheral circulation, and the excessive activation of TFH cells accelerated immunopathological process of AIH. 4 In addition, circulating activated TFH cells are associated with hypergammaglobulinaemia. 4 Hence, the exact regulation of TFH cell-mediated antibody production is essential for preventing AIH.
More notably, a newly identified T-cell subset, termed follicular regulatory T(TFR) cell, propose themselves as ideal candidates for preventing emergence of autoreactive B cells and regulating the normal GC response. 16,17 TFR cell combining phenotypic characteristics with conventional Foxp3 + Tregs and TFH cell yet are not identical to both. [16][17][18] Similar to TFH cell, TFR cell migrates to the GC by expressing CXCR5, which is a chemokine receptor for CXCL13. Furthermore, both subsets commonly express ICOS, PD-1 and Bcl6. 16,17 Unlike TFH cell, which normally differentiate from naive CD4 + T cell precursors, TFR cell originated from thymic Foxp3 + Treg precursors. 17 Therefore, in comparison with TFH cell, TFR cell has unique characteristics by the expressions of Treg-related molecules. 18 Research confirmed that TFR cell simultaneously expresses the Treg master regulator Foxp3 and other Treg-related molecules such as CD25 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). [16][17][18] Several studies have showed that abnormal TFR cells activity might cause the destruction of immune tolerance and thereby lead to the immunopathogenesis of autoimmune diseases. 19,20 Additionally, like TFH cell, TFR cells are also present in the peripheral circulation, the levels of which are directly correlated with a variety of human immunological disorders, such as primary biliary cholangitis (PBC) and multiple sclerosis(MS). 21,22 However, to the best of our knowledge, there are no reports of circulating TFR cells in patients with newly diagnosed AIH. Furthermore, the potential mechanisms underlying the function of TFR cells remain to be elucidated.
In the current study, our data indicated that imbalance of TFR/ TFH ratio involves in the pathogenesis of AIH and provides new insights into how TFR cell regulates humoural immune responses and potential therapeutic targets for the development of novel therapies for AIH.

| Participants
We recruited 32 AIH patients at the inpatient clinic of the

| Clinical index assay
The concentrations of serum alanine transaminase (ALT) and aspartate aminotransferase (AST), anti-nuclear and smooth muscle antibody (ANA/SMA), IgG, IgM and IgA were determined according to the manufacturers' instruction (Beckman Coulter).

| Establishment of experimental autoimmune hepatitis (EAH) model
Liver antigens were always prepared freshly as described previously from C57BL/6 female mice after perfusion of livers with phosphate-buffered saline (PBS). Livers were homogenized on ice, and nuclei and remaining intact cells were centrifuged at 150 g for 10 minutes. Subsequently, the supernatants were centrifuged for 1 hour at 100 000 g, and the remaining supernatants used for immunization (called S100). Induction of experimental autoimmune hepatitis (EAH) was achieved by intraperitoneal injection of the mice with freshly prepared S-100 antigen at a dose of 0.5-2 mg/mL in 0.5 mL PBS that had been emulsified in an equal volume of complete Freund's adjuvant (CFA) on day 0. A booster dose was given on day 7 as well. 5,6,20 Disease severity was assessed histologically on day 28 when the peak of disease activity was observed. Disease severity was graded on a scale of 0-3 by a researcher who was blinded to the sample identity, as follows: grade 0, none; grade 1, mild-scattered foci of lobular-infiltrating lymphocytes; grade 2, moderate-numerous foci of lobular-infiltrating lymphocytes; grade 3, severe-extensive pan-lobular-infiltrating lymphocytes. 5

| Enzyme-linked Immunosorbent assay for IL-10, TGF-β1 and IL-21
The concentrations of serum/supernatant IL-10, TGF-β1 and IL-21 in individual subjects were determined by ELISA using commercially available IL-10, TGF-β1 and IL-21 ELISA kits (R&D system) according to the manufacturer's instructions.

| Cell culture in vitro
We pre-purified CD4 + CXCR5 + GITR -TFH and CD4 + CXCR5 + GITR + TFR cell subsets from spleen cells of wild-type mice and B cells from

| Statistical analysis
The difference between two independent groups was analysed by Fisher's exact test or the Kruskal-Wallis nonparametric test using the SPSS 18.0 software(SPSS, Inc). The relationship between variables was evaluated using the Spearman rank correlation test. A two-sided P value <.05 was considered statistically significant.

| Patient characteristics
The clinical and sociodemographic characteristics of recruited subjects were described in Table 1. In comparison to HCs, patients had significantly higher concentrations of serum liver enzymes (ALT/AST/γ-GT and ALP), and higher the levels of serum immunoglobulin (IgG, IgM and IgA). Furthermore, the majority of AIH patients were seropositive for anti-ANAs and anti-SMA antibodies. Consistently, all AIH patients displayed active disease and hypergammaglobulinaemia.

| Decreased numbers of circulating TFR cells and increased numbers of circulating TFH cells in AIH patients
Follicular helper T cell originates from peripheral Foxp3-T cells, in contrast to TFR cell, which originate from thymic-derived Foxp3 + T cell. 18 According to the expression patterns of FoxP3, peripheral blood CD4 + CXCR5 + T cells were divided into circulating TFR and TFH cell subsets. The gating strategy for flow cytometric analysis of TFR (CD4 + CXCR5 + FoxP3 + ) and TFH (CD4 + CXCR5 + FoxP3 -) cells was shown in Figure 1A. In contrast to HCs, TFR cells expression and TFR/TFH ratio declined, but TFH cells expression increased in AIH patients ( Figure 1B). Hence, imbalanced between TFR and TFH cells may be associated with the pathogenesis of AIH.

| Circulating TFR express markers of both TFH and Treg differentiation in AIH patients
In the GC, TFR cell shares phenotypic, ontological and functional characteristics with conventional Tregs and TFH cell. [16][17][18] This led us to question whether circulating follicular cells are phenotypically similar to those derived from tonsils using both follicular (ICOS and PD-1) and regulatory (CTLA-4 and CD25) markers ( Figure 1C, 1). We found that circulating TFH cells express higher levels of PD-1 and ICOS, and similar levels of CTLA-4 and CD25 in AIH patients compared with HCs. As a comparison, circulating TFR cells express higher levels of PD-1 and ICOS, and lower levels of CTLA-4 and CD25 in AIH patients.
In addition, we also detect a lower numbers of IL-10 + TFR cells and a greater numbers of IL-21 + TFH cells in AIH patients compared to that in the HC ( Figure 1C, 1). These data suggest that TFR cell that simultaneously expresses the phenotypic characteristics of TFH and Tregs played a negative regulatory role in the pathogenesis of AIH.

| TFR/TFH-Related Cytokines in AIH Patients
Follicular regulatory T cell secretes IL-10 and TGF-β1, which are functionally important for the regulatory/suppressive activities of TFR cell, 19,20 whereas TFH cell produces IL-21, which is critical for GC formation as well as TFH cell generation. [9][10][11] We further tested the levels of these serum cytokines and found a lower concentrations of serum IL-10 and a higher concentrations of serum IL-21 in the patients compared with those in HCs. However, the serum levels of TGF-β1 showed no significant difference between HCs and AIH patients ( Figure 2). These data clearly indicated that elevated levels of serum IL-21 and reduced levels of serum IL-10 may be associated with the development of AIH.

| Circulating TFR cells are inversely associated with TFH cells in AIH patients
To further analyse the relationship among the numbers of TFR/TFH cells and the levels of serum TFR/TFH-related cytokines, we next performed a set of correlation analyses and found that the numbers of circulating TFR cells in AIH patients are positively associated with the levels of serum IL-10 ( Figure 3D) and TGF-β1 ( Figure 3E), but negatively correlated with the numbers of circulating TFH cells ( Figure 3A) as well as the levels of serum IL-21 ( Figure 3C). In addition, the numbers of circulating TFH cells are associated with the concentrations of serum IL-21 in those patients ( Figure 3B). These data suggest that TFR cell negatively affects the formation of TFH cell and the production of IL-21 during the development of AIH.

| Correlations of TFR/TFH-Related cytokines with clinical parameters in AIH patients
Next, we further determined the associations among the levels of serum IL-21/IL-10 and clinical parameters of AIH patients. We

| Statistical decrease in TFR cells and increase in TFH cells in EAH mice
To assess the effects of follicular cells in vivo, our previous studies had successfully established the autoimmune hepatitis model of EAH mice ( Figure 5A, 5). 5,6 Considering that GITR by TFR cells has enabled sorting strategies to highly purify TFH and TFR cells, 16 we further analysed the percentages of CD4 + CXCR5 + GITR + TFR and CD4 + CXCR5 + GITR -TFH cells in EAH mice ( Figure 5C). We observed that CD4 + CXCR5 + GITR + TFR cells in the experimental group significantly decreased from 14th to 28th days of post-EAH induction. On the other hand, CD4 + CXCR5 + GITR -TFH cells in the experimental group gradually increased from 7th to 28th days of post-EAH induction compared with control group ( Figure 5D). These results indicated that the imbalance of TFR-to-TFH ratios might favour the pathogenesis of EAH.

| TFR regulates B-cell subset differentiation and immunoglobulin production in vitro
To identify potential mechanisms by which TFH cell and B cell are regulated by TFR cell 24 , we co-cultured spleen B cells from EAH mice with TFH cells from wild-type mice for 7 days in the absence and/ or presence of TFR cells from wild-type mice, anti-CTLA antibodies ( Figure 6A). Next, we characterized the expression of CD138 and CD27 on CD19 + B cells from cultures by flow cytometry analysis ( Figure 6B). As shown in the Figure 1C-E, we observed higher percentage of memory B and plasma cells and higher levels of IgG when B cell was co-cultured with TFH cells. However, after TFR cells were added to the wells together with TFH cells, the percentages of memory B and plasma cells as well as the production of IgG were significantly dramatically decreased. On the other hand, when anti-CTLA antibodies were added, the percentages of these cells and the production of IgG were slightly increased ( Figure 6B-E). Together, these data suggest that TFR cell can suppress B-cell proliferation and antibody production in vitro.

| D ISCUSS I ON
Although TFR cells are known to be important regulators of autoimmune responses in human, 19   Taken together, the presented data described the subset phenotypes of circulating TFR and TFH cells and their relationships with clinical parameters in AIH patients. In addition, our study also showed that dysregulated between TFR and TFH cells might contribute to the immunopathological process in AIH. In vitro culture experiments demonstrated that TFR cells could inhibit the secretion of autoantibody by indirectly inhibiting the activation of TFH cells, or directly regulating the differentiation of B cells via the coreceptor CTLA-4. We recognized the limitations of this study including a relatively small sample size and the lack of vivo functional study of TFR cells. Therefore, further studies of these findings in a larger populations are warranted.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no financial or commercial conflicts.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available upon request by contact with the corresponding author.