It has been reported that a strong interaction between the TcR and major histocompatibility complex (MHC) class II molecules triggers T_FH cell differentiation, which indicates that a strong TcR signal is essential for T_FH cell differentiation (22). In addition, among surface co-stimulatory molecules being expressed by T_FH cells, ICOS is induced when CD4 T cells become activated by recognizing antigen through TcRs, which then interact with ICOS-L that is expressed on B cells (7,11,23,24). Its binding to the ligand ICOS-L triggers activation signals in a similar way to other members of CD28 family co-stimulatory receptors (25,26). ICOS plays a significant role in increasing T cell proliferation and the production of cytokines, including IL-21 and IL-4 (11,27,28).

ICOS-mediated PI3K activation is crucial for T_FH cell differentiation, as a point mutation on the cytoplasmic tail of ICOS, where PI3K binds to and activates, led to a severely impaired T_FH cell differentiation of CD4 T cells (28). In contrast, overexpression of ICOS is sufficient to maintain T_FH cells in CD28-deficient mice (7). Among PI3K subunit p110γ appears to convey ICOS-mediated T_FH cell differentiation signaling pathway, as p110γ deficiency resulted in a defective T_FH cell differentiation, further strongly indicating that ICOS and PI3K are important for either differentiation or survival of T_FH cells. These results imply that ICOS-mediated PI3K signaling is crucial for the differentiation of T_FH cells (25). Moreover, Heping et al. reported that ICOS signaling is critical for motility of T_FH cells into the B cell follicle in a Bcl-6 independent manner (29).

PI3K signaling pathways following TcR and co-stimulation regulate the phosphorylation of Foxo1 to relocate it from the nucleus to the cytoplasm (30,31). A recent study revealed that Foxo1 negatively regulates T cell activation and contributes to T cell tolerance (32). Foxo1-deficient CD4 T cells contribute to the development of autoimmune phenotypes including increased autoantibody production with reduced Foxp3⁺ regulatory T cell development and function, and augmented generation of T_FH cells and GC formation. In addition, the presence of Foxo-binding elements has been identified in the promoter region of Bcl-6 (33), which suggests that Foxo1 might act as a transcriptional repressor of Bcl-6 and, if so, Foxo1 might negatively regulate T_FH differentiation. Thus, ICOS and PI3K signaling is crucial for T_FH differentiation and Foxo1 could be a regulator of GC reaction.

IL-6 and IL-21 are well-known pro-inflammatory cytokines with important roles in Bcl-6 expression and T_FH cell differentiation (4). IL-21 induces B cell proliferation and class switch recombination and IL-21R is required for antibody response and GC formation (34). The IL-6-mediated STAT3 activation is also important for IL-21 expression in human and mouse naïve CD4 T cells upon TcR stimulation (35,36). STAT3 is phosphorylated by JAK upon IL-6 stimulation, and activated STAT3 was shown to bind to Bcl-6 in T cells (33). IL-6 is an important factor for Bcl-6 induction in CD4 T cells during dendritic cell priming stage of CD4 T cell activation (37). However, other signaling pathways could compensate for IL-6 dependent T_FH differentiation pathway, as T_FH cells are normally found at the peak of the immune response to infection and immunization (38,39). IL-6 signaling is required for IL-21 expression via c-Maf (40-42). Once being produced, IL-21 further increases its own production through a positive feedback mechanism (43).

Augmented IL-21 was reported to induce the expression of the master regulator for T_FH cell differentiation, Bcl-6 (44), while controversies exist whether IL-21 is a critical factor for Bcl-6 induction in CD4 T cells (37-39). At a downstream level, Choi et al. showed that IL-6-mediated STAT1 signaling can also prime T_FH cells by compensating for STAT3 and inducing Bcl-6 expression. Another recent study demonstrated that IL-12-mediated STAT4 signaling can induce expression of both Bcl-6 and T-bet, and T-bet inhibits the function of Bcl-6 (45). The balance between T-bet and Bcl-6 expression might be regulated by IL-2 concentration (33). Furthermore, IFN-γ was accounted to lead to abnormal T_FH cell differentiation in the sanroque mouse model (46). Given that IFN-γ induced Bcl-6 via pSTAT1 which binds to an IRE in an exon of Bcl-6 (47), IFN-γ could function as a positive regulator by directly inducing Bcl-6 expression in CD4 T cells. This supported by recent study by Lee et al., which demonstrated that T cell specific deletion of IFN-γR resulted in decreased T_FH cell differentiation in sanroque mice (46). Further studies are needed to clarify how this complex cytokine network regulates Bcl-6 expression and T_FH cell differentiation.

The zinc-finger-containing transcriptional repressor Bcl-6 was originally described as a key molecule in GC formation and B cell response (48,49). Bcl-6-deficient mice cannot develop somatic hyper-mutation in B cell, result impaired GC formation (50,51). In addition, B cells from these mice do not undergo affinity maturation, somatic hyper-mutation, and class switch recombination of immunoglobulin (49). Recently, Bcl-6 was identified as a crucial factor for T_FH cell differentiation (3). Bcl-6-deficient mice show impaired T_FH cell differentiation (2) and non-T_FH CD4 T cells do not express increased levels of Bcl-6 (2,52). Bcl-6 directly inhibits a number of transcription factors, including T-bet and RORγt, which are key modulators of differentiation of Th1 and Th17 cells, respectively (3). Bcl-6 also inhibits expression of CCR7 and PSGL-1, which negatively regulate the migration of T cells into B cell follicles (39,53). Moreover, Bcl-6 regulates the expression of various T_FH cell-related molecules, including ICOS, PD-1, PTLA, CD200, and SAP (23). Turner et al. identified the mouse form of Blimp-1, which is induced by cytokine-mediated B cell differentiation (54). Recent studies reported that the transcription factor Blimp-1 has an antagonistic role of Bcl-6 (1,52,55) and inhibits T_FH cell differentiation (1). Blimp-1 is highly expressed in non-T_FH effector T cells such as Th1, Th2, and Th17 cells (1,52), whereas Bcl-6 is highly expressed only in T_FH cells. Moreover, constitutive expression of Blimp-1 inhibited T_FH cell formation (1) and Blimp-1 is important for terminal differentiation of both CD4⁺ and CD8⁺ T cells, which is characterized by high levels of effector molecule secretion and low proliferative potential (52). IL-2 mediated STAT5 signaling in activated CD4⁺ T cells induces expression of Blimp-1, which suppresses Bcl-6 and T_FH cell differentiation (56). High level of IL-2, especially in effector Th1 cells, induces T-bet, which also inhibits Bcl-6 expression and T_FH cell differentiation (33). Th1 cells might have the flexibility to regulate the expression of T-bet and Bcl-6 depending on environmental conditions (33). IL-6- and IL-21-mediated STAT3 signaling can also induce Blimp-1 or Bcl-6 (57) through the participation of additional transcription factors (5). To summarize, effector T cell fate seems to rely on the expression of Bcl-6 or Blimp-1 and they are reciprocally inhibit each other via complex signaling pathway, eventually act as a decision maker between T_FH cell and other effector T cell differentiation.

Foxp3-expressing regulatory T (T_reg) cells contribute to the maintenance of immune tolerance by suppressing the dysregulated immune response to self-antigens (58). Scurfy mice without Foxp3⁺ T cells demonstrate severe systemic autoimmune phenotype. In addition, CD4 T cells isolated from scurfy mouse are hyper-responsive to TcR stimulation (59,60).

It has been recently reported that the mice with CXCR5-deficient T_reg cells have more GC with augmented immunoglobulin production owing to the limited capability of these cells to migrate into B cell follicular region. This suggests that CXCR5 expression of T_reg cells is crucial for regulation of the GC reaction (61). In addition, T_reg cells expressing Bcl-6 and CXCR5, which originate from CXCR5-natural T_reg cell precursors, are found in GC (21). In the absence of CXCR5⁺Bcl-6⁺ T_reg cells, the GC reaction was not controlled efficiently leading to enhanced immunoglobulin production and increased B cell population in GCs. This result implies that T_reg cells expressing CXCR5 have important roles in regulation of the GC reaction. T_reg cells in GC are called follicular regulatory T (T_FR) cells, which share characteristics of both T_FH and T_reg cells since Bcl-6, CD28 and SAP also affect development of T_FR cells. 5~25% of T_FH cells expressing CXCR5 and PD-1 are also Foxp3⁺ T_FR cells and are located in the B cell follicle region (62). Recent study demonstrated that lack of the PD-1-PD-L1 pathway induced increase of T_FR cells and its suppressive ability, suggesting the regulatory role of PD-1 in the differentiation of T_FR cells (63). Co-transfer experiments with thymus-derived Foxp3⁺ CD4 T cells and Foxp3^- CD4 T cells into recipient demonstrated that Foxp3⁺ CD4 T cells become T_FR cells in mice immunized with antigen suggesting T_FR cells are derived from T_reg cell precursors. A recent study demonstrated Ag-specific antibody production was increased in the mice with Bcl-6-deficient T_reg cells (21). Furthermore, increased levels of high affinity antibody, plasma cells, and memory B cells are found in the mice demonstrating that T_FR cells expressing Bcl-6 control the GC reaction including plasma cell production and affinity maturation. By contrast, Blimp-1 down-regulates the number of T_FR cells, suggesting that Bcl-6 and Blimp-1 also reciprocally regulate differentiation of T_FH and T_FR cells to control the GC reaction (1). T_FR cells therefore seem to play a crucial role in the maintenance of immune tolerance, preventing autoimmune response by inhibiting the GC reaction and antibody production.

miRNAs are functional single stranded RNAs (ssRNAs), which are encoded endogenously, and are involved in immune cell development and differentiation (64,65). Recent study reported that the miR-17-92 cluster was regulated by Bcl-6 in CD4 T cells (3). T cells overexpressing Bcl-6 demonstrated diminished expression of the miR-17-92 cluster, as do T_FH cells, which, suppresses the expression of CXCR5. However, several studies have shown that the miRNA-17-92 cluster induces T_FH cell differentiation (16,17). T cell specific miR-17-92 transgenic mice demonstrate spontaneous Bcl-6 expression, T_FH cell differentiation, and GC formation (16). In contrast, miR-17-92-deficient mice show impaired T_FH cell differentiation during acute and chronic virus infection. The miR-17-92 cluster induces T_FH cell differentiation through suppression of PTEN and PHLPP2 expression, which regulate the ICOS-PI3K pathway. The miR-17-92 cluster also directly inhibits expression of RORα, which is involved in gene expression of non-T_FH effector T cell differentiation (17). In addition, miR-10a, which is specifically expressed in T_reg cells by TGF-β and retinoic acid, directly suppresses Bcl-6 expression (18). Some induced-T_reg (iT_reg) cells migrate to GC in Peyer's patch and have T_FH-like phenotypes. miR-10a expression is down-regulated in these iT_reg cells and overexpression of miR-10a significantly inhibits the conversion of iT_reg into T_FH-like cells. More studies on the role of T_FR cells and miRNA in T_FH differentiation are needed to improve our understanding on dynamic regulation of germinal center reaction.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex phenotype that includes systemic inflammation, fever, fatigue, and chills (66). Diagnosis of SLE is very difficult because its phenotype overlaps with other diseases. Recent studies have suggested that the pathogenesis of SLE is profoundly related to T_FH cells (44,67,68). Spontaneous GC formation and autoantibody production have been reported in many mouse models of SLE (44,67), suggesting that T_FH cells might be associated with pathogenesis of SLE. Indeed, recent studies demonstrated that T_FH cell differentiation is spontaneously induced in these mouse models (44,67,68). Also, dysregulated T_FH cell activity contributes to the pathogenesis of SLE through aberrant GC formation and massive production of autoantibodies, such as anti-dsDNA and ANA. T_FH cells induce these phenomena via cytokines and co-stimulatory molecules which stimulate B cells (69,70). Autoimmune phenotypes were alleviated when T_FH cell differentiation was inhibited in sanroque mice, which have increased GC formation and T_FH cell differentiation (70). Linterman et al. crossed sanroque mice with IL-21- or SAP-deficient mice, or mice heterozygous for Bcl-6 to examine the role of Bcl-6 in development of the lupus-like phenotype (71). They found that the deficiencies of Bcl-6 or SAP ameliorate the lupus-like phenotype in sanroque mice IL-21 independently. However, lupus-like autoimmune phenotypes were reduced in another study when IL-21 signaling is not present in BXSB-Yaa mice, another mouse model of human SLE (72), recapitulating the complexity of pathogenesis of SLE in human. Remarkably, IL-21 expression was up-regulated in SLE patients than in healthy controls (73), and elevated production of T_FH relating factors such as CXCL13, BAFF were reported in human SLE patients (74). These results suggest that abnormal T_FH cell differentiation strongly related to SLE pathogenesis.

Rheumatoid arthritis (RA) is an autoimmune disorder, which is recently studied that it is associated with dysregulated T_FH cell differentiation. Deborah et al. found that blockade of IL-21 signaling by IL-21R-Fc fusion protein treatment reduces disease severity in mouse and rat RA models (75). Furthermore, IL-21 blockade in animal models results in decreased IL-6 expression. A recent study by Victoratos et al. found that T_FH cells have a critical role in the maintenance of follicular dendritic cell (FDC)-mediated GC formation and autoantibody production in KRN/B mice that spontaneously develop RA (76,77). In addition, Jang et al. reported that IL-21 receptor-deficient KBx/N mice have less severe RA with reduced T_FH cell population in draining lymph node (43). An IL-21R-Fc fusion protein that inhibits IL-21 signaling can delay disease onset and progression. Platt et al. also found increased T_FH cells and antibody production in an OVA-induced RA mouse model (78). In this study, they showed that abatacept, a fusion protein composed of the Fc region of IgG and the extracellular domain of CTLA-4 has a role in regulation of T_FH cell differentiation in OVA-induced RA mouse models. In human RA patients, up-regulated IL-21 level was reported with increased T_FH-like cells that enhanced IL-21R expression (79). This increased T_FH-like cells population correlated with enhanced 28-joint count disease activity score and anti-CCP antibody, which indicate disease severity.

Synthetically, T_FH cells seem to be involved in the pathogenesis of human autoimmune diseases such as SLE, RA, etc., therefore, regulation of T_FH cell differentiation could be an important strategy for the suppression of autoimmune diseases.