For naïve CD4⁺ T cell to differentiate into effector T cell, it requires antigenic peptide presentation through MCH-II and CD28 costimulation by antigen-presenting cells such as dendritic cells, macrophage, or B cells along with lineage driving cytokines. Signaling from CD28 by binding with its ligand CD80 (B7-1) or CD86 (B7-2) is critical for antibody class switching and GC formation (53, 54). Studies suggest that B7-1 and B7-2 have overlapping function as evidence from unaltered antibody response in mice lacks either of the molecules (53, 55). However, during vaccinia virus infection, expression of B7-2, but not B7-1, is critical for the generation of Tfh cells and thus plasma cell generation and virus specific antibodies (56). CD28 signaling positively regulates early stage of Tfh differentiation (expression of ICOS and BCL-6), and its signaling is also important at late stage of Tfh differentiation as exemplified in blocking B7 ligand during ongoing infection impaired Tfh response (39, 57, 58). Sustained signaling from CD28 is critical for Tfh survival as evidence from apoptosis of Tfh cells during influenza viral or Citrobacter rodentium infection into a mice strain that after initial priming selectively lose CD28 expression on CD4 T cells (58). Although CD28 signaling plays critical role in Tfh differentiation and maintenance, its downstream PI3k activation is dispensable for its generation and during infection with Salmonella in Roquin mutant mice, the generation Tfh does not depend on CD28 signaling (59). Thus, requirement of CD28 downstream signaling for Tfh generation may be infection specific, and still there is scope to determine unknown CD28 downstream signaling molecules.

The presence of CXC chemokine receptor 5 (CXCR5), a hallmark Tfh cell surface marker, differentiates these cells from other T cell lineages (60, 61). Migration of T cells to the B cell zone is mediated by CXCR5, which is attracted toward CXCL13 ligand of B cell follicle (62), thereby promoting T:B cell contact. Although T cells are found in B cell follicle of immunized mice (selectively null for CXCR5 T cells), these mice were still found to have low GC number and decreased humoral immune response (63). Thus, CXCR5 is important for T cell positioning to follicular GC. However, it is also important that these cells should express low CCR7 expression as its signaling promotes retention of T cells to the T cell area of GC (64, 65). Signaling through activin A plays critical role in downregulation of CCR7 and upregulation of CXCR5 expression (26). Moreover, Tfh cells express high NFAT2 transcription factor that is involved in CXCR5 expression (66). Reduced CXCR5 expression was observed in NFAT2, NFAT1/2-deficient mice-infected LCMV Armstrong strain, indicating that nuclear factor of activated T cell (NFAT) plays critical role in Tfh differentiation by regulating CXCR5 expression (67).

Upon antigen challenge and CD28 costimulation, ICOS is expressed on all effector T cells, and its signaling through ICOS-ICOSL is critical for Tfh generation and GC formation (18, 68). In patients with ICOS deficiency, the GC is severely damaged, and reduction in CXCR5⁺ CD45RO⁺ memory cells validates the importance of the ICOS in Tfh differentiation (69). During an acute viral infection, T cells that intermediately express IL-2 receptor α induces the expression of BCL-6 and CXCR5 through ICOS signaling leading to Tfh differentiation by 72 h of antigen challenge, without the help of B cell, whereas T cells expressing higher IL-2 receptor α induces Blimp-1 expression driving the cells toward other effector T cells subsets (70). After 72 h, the differentiated Tfh cells die due to the absence of ICOSL signal from B cells. Thus, sequential ICOS signaling is important for Tfh generation as well as maintenance.

Although PI3k is activated by signaling through T cell receptor (TCR), CD28, the ICOS-driven PI3k activation, is critical for Tfh cell as suggested by impaired Tfh generation in mice selectively defective in ICOS downstream signaling of the PI3k activation (71). Moreover, conditional deletion of catalytic subunit P110delta of PI3k also validate that the ICOS downstream PI3k pathway is important for Tfh generation (72). Other than known PI3K binding motif Tyr-x-x-Met (YxxM, where ‘x’ indicates any amino acid) of ICOS, Pedros et al. using SILAC (stable isotope labeling) approach showed that ICOS cytoplasmic tail has other two conserved motifs, one Proximal SSSVHDPNGE (IProx) and second AVNTAKK motif. Retroviral transduction of CD4⁺ T cells (TCR transgenic for LCMV peptide gp 61–80) with ICOS mutant in PI3k-binding site of IProx motif and adoptive transfer into ICOS^−/− mice and then LCMV infection fail to generate Tfh cells illustrating that IProx plays a critical positive role in Tfh differentiation (73). Importantly, IProx motif has homology with the conserved sequences with TRAF2 and TRAF3. Furthermore, IProx motif is involved in recruitment of serine threonine kinase TBK-1, and depletion of TBK-1 is associated with impaired Tfh generation. Strong TCR and ICOS signaling is needed for activation of TBK-1 pathway. Moreover, ICOS signaling also regulates IL-21 production through c-Maf, which in turn regulates Tfh differentiation (74). Recently, ICOS signaling has been associated with Tfh phenotype maintenance by FOXO1-mediated downregulation of transcription factor Kruppel-like factor 2 (KLF2) (39). KLF2 negatively regulates Tfh differentiation by directly binding to Cxcr5, Cccr7, Psgl-1, and S1pr1 receptors, which leads to suppression of CXCR5 expression. In addition to Klf2, FOXO1 is also repressed by ICOS-induced mTORc2, thereby promoting Tfh differentiation (75). Although FOXO1 degradation is necessary for early-stage Tfh differentiation, however during late stage, it is required for transcription of several genes that help in Tfh differentiation (76).

The adaptor molecule SAP is very critical for the generation of antigen-specific long-lived plasma and memory B cells (77) by stabilizing T:B cell interaction (78). Two-photon imaging (79) showed that SAP deficiency selectively impairs T:B cell interaction rendering impaired T cell migration and retention in GC. SLAM/SAP signaling not only regulates stable T:B cell interaction but also exhibits a regulatory role in the expression of ICOS and CD40L (80). Thus, SLAM/SAP signaling positively regulates Tfh differentiation by stabilizing T and B cell interaction as well as the expression of ICOS and CD40L.

Signaling through costimulatory molecule OX40 (CD138) and its ligand OX40L (CD252) is critical for T cell survival, proliferation, and differentiation (81–83). Classically, OX40 expression on T cells is considered to be transient with the exception during chronic LCMV clone 13 and Plasmodium infection wherein it is constantly expressed (84, 85). Enhancing signaling through OX40 is critical to control rodent malaria parasite infection by Tfh generation and GC response. Increasing OX40 signaling with OX40 agonistic antibody along with blocking PD-1 signaling leads to loss of Tfh generation by Blimp-1 expression and excessive IFN-γ production and thus impaired malaria parasite clearance (85). Therefore, it seems that the crosstalk between costimulatory and co-inhibitory receptor signaling is important for Tfh generation. In contrast, enhancing OX40 signaling during LCMV Cl 13 infection and initial priming of Listeria monocytogenes leads to inhibition of Tfh differentiation by increasing transcriptional repressor Blimp-1 (86, 87). Whereas following L. monocytogenes infection, enhancing OX40 signaling does not alter the Tfh number stating that the time of OX40 stimulation is an important factor for regulating Tfh differentiation. Moreover, infection with L. monocytogenes in mice deficient in OX40 and CD30 showed normal Tfh generation (86). However, OX40-deficient mice with LCMV Cl 13 infection showed impaired generation of Tfh cell, GC, and plasma cells (84). Thus, it seems that infection-specific tight regulation of OX40 signal is required for Tfh cell generation. Further, despite low memory CD4 T cell and low circulating memory B cells, intact antibody response to vaccination was observed in patient with OX40 deficiency (88). Aberrantly increased Tfh response leading to SLE is contributed by signaling through OX40 ligand (OX40L)–OX40 axis by augmentation of TCR signal via PI3K–Akt pathway (89). Thus, robust regulation of OX40/OX40L signaling is associated with positive and negative regulation of Tfh differentiation.

During chronic LCMV infection and in collagen-induced arthritis (CIA) mice, the expression of glucocorticoid-induced TNF receptor family-related protein (GITR) was elevated on Tfh cells than non-Tfh cells (90, 91). Impaired Tfh generation and loss in control of chronic LCMV infection was observed in GITR^−/− mice. Moreover, administration of GITR-Fc protein greatly reduced CIA and joint damage by inhibiting Tfh generation and thereby autoantibody production (90). Mechanistically, GITR cell intrinsically expands Tfh number, thereby promoting antibody production to control chronic LCMV Cl13 infection (91). Thus, GITR signaling positively regulates Tfh generation.

Programed death 1, a surface molecule expressed on exhausted T cells, is also expressed by Tfh cells. PD-1/PD-L1 and PDL-2 signaling is associated with the dephosphorylation of molecules downstream of TCR signaling. Mechanistically, PD-1 engagement inhibits ICOS signaling thereby regulating Tfh generation (92). However, IL-2 through STAT5 rescues inhibition of ICOS signaling by PD-1, and additionally, IL-2 itself through STAT5 negatively regulates Tfh differentiation. Therefore, it seems that many positive and negative regulatory pathways of Tfh differentiation are interlinked. The Tfr cells also express PD-1, regulate Tfh cells, and hence humoral immune response (93).

The cytotoxic T lymphocyte antigen 4 (CTLA-4), a regulatory T cell marker involved in regulation of effector T cells, was recently shown to be expressed by Tfh (42) and Tfr (42, 94) cells and negatively regulates Tfh differentiation. Downregulation or loss of CTLA-4 in Tfh cells and Treg cells resulted in augmented B cell response, while loss of CTLA-4 in Tfr was not able to augment B cell response. Mechanistically, Tfr and Treg control Tfh and humoral immunity by downregulating costimulatory molecule B7-1 and B7-2 through CTLA-4 (Figure 3) (42, 94). In contrast to this, earlier studies have shown that CTLA-4 activates ubiquitin ligase Itch resulting in inhibition of T cell activity (95), whereas recent studies have shown Itch has a positive regulator of Tfh differentiation (96). However, it is possible that CTLA-4 function may be controversial and milieu dependent or it may have new Tfh regulatory signaling pathways which are yet to be identified.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role as a positive regulator in Tfh differentiation (97). Mice with T cell-specific STAT3 deficiency showed reduced B cell response (98). Moreover, studies in human patients with STAT3 deficiency were reported with reduced circulating Tfh cells, defective IL-21 expression, and B helper activity supporting that STAT3 is a critical positive regulator of Tfh differentiation (40). Mechanistically, IL-6 enhances Tfh generation through expression of STAT3, which in turn regulates IL-21 production. In CD8 cells, IL-6-induced NFAT2/STAT3 was found to interact with Pdcd1 gene promoter thereby regulating PD-1 (36). Thus, it is possible that IL-6-induced NFAT2/STAT3 might also be involved in the expression of PD-1 of Tfh cells.

Follicular helper T cells cannot be generated in the absence of the transcriptional repressor BCL-6, implicating its importance in Tfh differentiation and persistence (99, 100). Ectopic BCL-6 expression leads to induction of CXCR5, ICOS, and PD-1 on CD4⁺ T cells (37, 101). BCL-6 promotes Tfh differentiation by inhibiting other T cell subset differentiation in a DNA binding-dependent manner. BCL-6 also regulates Th1 and Th17 cells by binding to T-bet and RORγt promoter thereby regulating IFN-γ and IL-17 production (101). Furthermore, BCL-6 directly binds with the AP-1 and blocks or subverts downstream TCR signaling of AP-1 and helps in Tfh generation (21). Yu et al. showed that the miRNAs, including miR-17–92 involved in CXCR5 repression, are repressed by BCL-6, driving the cells toward Tfh differentiation (101). BCL-6 has also been implicated in downregulation of P-selectin glycoprotein ligand 1 (PSGL1) simultaneously upregulating CXCR5 and PD-1 (102). Mechanistically, out of bric-a-brac, tramtrack, broad-complex (BTB) domain, a middle domain (also known as RDII), and a zinc finger domain of BCL-6, the BTB domain through interacting with BCOR (103), and middle domain RDII by associating with metastasis-associated protein 3 (MTA3) play critical role in Tfh differentiation (104, 105). Further acetylation of RDII domain at K379 prevents binding of MTA3, thereby inhibiting Tfh differentiation.

Cytokine IL-6 (44), IL-27 (106), and ICOS (74) induced c-Maf, which regulates IL-21 production, which in turn regulates Tfh generation. BCL-6 controls expression of most of the genes related to Tfh differentiation but does not have any role in IL-21 expression. Over-expression of c-Maf induces IL-21 production without signaling from IL-6/IL-4/STAT6 signaling (44). Mechanistically, c-Maf regulates IL-21 production through MARE site binding to the activated IL-21p and the CNS-2 enhancer (44). c-Maf induction is also involved in the expression of CXCR5 (107).

Aschaete scute homolog 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, is specifically upregulated in Tfh cells. It positively regulates Tfh generation as blockade of Ascl2 signaling with Id3 leads to impaired Tfh generation (108). Ascl2 is involved in upregulation of the CXCR5 and downregulation of CCR7, thus helping in migration of these cells to the GC (108).

Interferon regulatory factor 4 (IRF4) controls expression of BCL-6, as Irf4^−/− CD4⁺ T cells failed to express BCL-6, thus regulating the Tfh generation (109).

The transcription factor NFAT consists of five members, namely, NFAT1 (NFATp or NFATc2), NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3), and NFAT5. Signaling through Ca²⁺ regulates NFAT1, NFAT2, NFAT3, and NFAT4. The Ca²⁺ signaling-regulated NFAT1, NFAT2, and NFAT4 is critical for T cell activation, differentiation, and maintenance of tolerance (110–112). Recently, it has been shown that Tfh cells express high NFAT2 transcription factor that is involved in CXCR5 expression. Reduced expression of Tfh markers CXCR5, ICOS, and PD-1 was observed in NFAT2, NFAT1, 2-deficient mice infected with LCMV Armstrong strain, which illustrates important positive role for NFAT1 and NFAT2 in Tfh differentiation (67). In contrast, unexpectedly, augmented GC reaction was observed upon immunization in mice deficient in NFAT2. However, in these NFAT2-deficient mice, only Tfr population was hampered, indicating that NFAT2 intrinsically downregulates CXCR5 in Tfr than Tfh (66).

During T cell activation, continuous STAT5 signaling inhibits Tfh generation by regulating expression of CXCR5, c-Maf (113), and BCL-6 (113, 114). Therefore, in vivo inhibition of STAT5 signaling leads to enhanced Tfh cell and GC formation. In the absence of Blimp-1, STAT 5 failed to inhibit Tfh cell formation, whereas over-expression of Blimp-1 in STAT5-deficient T cells resulted in Tfh differentiation. Thus, IL-2/STAT5 axis negatively regulates Tfh differentiation by regulating Blimp-1 (113).

B lymphocyte maturation protein 1, a transcriptional repressor, plays a critical role in the regulation of terminal B (115) as well as T cell differentiation, maturation, survival, and homeostasis (116, 117). It negatively regulates Tfh differentiation by opposing the action of BCL-6 (100) by binding to multiple loci of Bcl-6 genes (118). Blimp-1 also inhibits PD-1 expression by directly repressing NFAT2 (a positive regulator of PD-1), or by binding to PD-1 locus changing the chromatin structure, thereby resulting in the dissociation of NFAT2 from its site (78). Thus, Blimp-1 negatively regulates Tfh differentiation by downregulating BCL-6 and PD-1.

The master transcription factor T-bet and BCL-6 for Th1 and Tfh cells, respectively, have been shown to be co-expressed during early and late stages of differentiation and regulate each other to drive the cells either toward Th1 or Tfh type (119). In the early stages of Th1 cell differentiation, IL-12 priming through STAT4 induces IL-21 and BCL-6, but during late phase of differentiation, STAT4-induced T-bet represses BCL-6 leading naive CD4⁺ T cell to differentiate into Th1. Recent finding has shown that during late event of Tfh cell differentiation, low expression of KLF-2 at early stages leads to BCL-6 expression and high expression of KLF-2 during later phase results in downregulation of BCL-6 by upregulating BCL-6 antagonistic Blimp-1 and high expression of T-bet (40). Thus, KLF2 may regulate T-bet and thereby negatively regulate Tfh differentiation, but still our knowledge is unclear for molecules that are involved during late event of Th1 differentiation that negatively regulates Tfh differentiating molecules.

E protein and its natural repressors, the inhibitor of DNA-binding protein (Id1–Id4), play a critical role in Tfh differentiation. The generation of Tfh cells was increased upon depletion of Id2 via RNA-mediated interference. Moreover, over-expression of Id3 inhibits Tfh generation during LCMV infection or following immunization with protein (120). Ectopic expression of E protein E47 enhanced CXCR5 expression, while Id2 inhibits CXCR5 expression. Chromatin immunoprecipitation (ChIP) followed by deep sequencing has revealed that Bcl-6 binds with Id2 locus repressing Id2 expression thereby positively regulating Tfh differentiation (120).

Kruppel-like factor 2, member of zinc finger transcription factor, is involved in T cell homeostasis, trafficking, and survival, and KLF2-deficient mice showed reduction in peripheral T cells (126–128). Lee et al. showed that the KLF2 deficiency or low levels of KLF2 have been shown to be associated with enhanced Tfh cell differentiation. Moreover, over-expression of KLF2 leads to blocking or reduction of Tfh cell differentiation and thereby resulting in reduced GC formation (40). Mechanistically, KLF2 negatively regulates Tfh differentiation by directly binding to Cxcr5, Ccr7, Psgl-1, and S1pr1 and thus negatively regulating homing receptor (39, 40). The other mechanism by which KLF2 regulates Tfh differentiation is through the induction of Blimp-1, T-bet, and GATA3 expression (40).

The transcription factors LEF-1 and TCF-1 act as a positive regulator of Tfh differentiation and expressed before the expression of Tfh marker CXCR5. During early stage of viral infection, virus-specific Tfh cells reciprocally express TCF-1 and Blimp-1 (129). The TCF-1 deficiency led to poor differentiation of Tfh cells and their maintenance. Mechanistically, TCF-1 regulates Tfh differentiation by directly binding to the Bcl6 promoter and prdm 5′ regulatory region enhancing BCL-6 and repression of Blimp-1 protein (130). Moreover, LEF-1, by enhancing IL-6 receptor and ICOS expression, promotes Tfh differentiation (131).