Transcriptional regulation of Tfh dynamics and the formation of immunological synapses

Inside germinal centers (GCs), antigen-specific B cells rely on precise interactions with immune cells and strategic localization between the dark and light zones to clonally expand, undergo affinity maturation, and differentiate into long-lived plasma cells or memory B cells. Follicular helper T (Tfh) cells, the key gatekeepers of GC-dependent humoral immunity, exhibit remarkable dynamic positioning within secondary lymphoid tissues and rely on intercellular interactions with antigen-presenting cells (APCs) during their differentiation and execution of B-cell-facilitating functions within GCs. In this review, we briefly cover the transcriptional regulation of Tfh cell differentiation and function and explore the molecular mechanisms governing Tfh cell motility, their interactions with B cells within GCs, and the impact of their dynamic behavior on humoral responses.


INTRODUCTION
In the intricate landscape of secondary lymphoid tissues, a remarkable choreography unfolds, orchestrated by components of the innate and adaptive immune system.This immunological program involves sophisticated interactions between innate and adaptive immune cells in an antigen (Ag)-specific manner, culminating in the generation of effector cells 1 .At the core of this intricate program lies the germinal center (GC), an immunological engine compelling Ag-specific B cells clonally expand and evolve their Ag receptors through AID (activationinduced cytidine deaminase)-mediated somatic hypermutation.This process leads to a gradual increase in the Ag-binding strength of B-cell receptors (BCRs).Within GCs, long-lived plasma cells or memory B cells emerge 2 .The complex behavior of GC B cells is meticulously regulated by the immunological functions of CD4 T cells 1,2 .
Histological findings of CD4 T cells in the GC areas of human tonsils introduced the practical concept that CD4 T cells, which aid B cells, are colocalized in follicles with B cells 3 .While this may seem obvious now, limited information was available about the specific positioning of B helper CD4 T cells, despite their existence being known for decades.Transcriptomic analyses of follicle-resident CD4 T cells in humans and mice opened Pandora's box of a new lineage of effector CD4 T cells.These B-cell helper effector CD4 T cells, known as follicular helper CD4 T (Tfh) cells, exhibited a unique gene expression profile in comparison to those of other effector CD4 T cells, and B-cell lymphoma-6 (Bcl6) was identified as a major lineage-determining transcription factor 1,4 .
In secondary lymphoid organs, naive Ag-specific CD4 T cells develop into effectors in the T-cell zone during interaction with Ag-presenting dendritic cells (DCs).Early bifurcation between Tfh and non-Tfh cells occurs during this stage 5 , and these effector CD4 T cells exhibit differential migratory and intercellular interaction behaviors.Tfh cells stay and migrate to the B:T border, where they interact with cognate B cells to further polarize into germinal center Tfh (GC-Tfh) cells, while non-Tfh cells migrate to peripheral effector sites to fulfill immunological functions.GC-Tfh cells induce GC formation in B-cell follicles.Via recurring interactions with B cells, GC-Tfh cells play important roles in the selection of clones that express affinity-matured BCRs, the provision of signals required for B cells to undergo GC reactions, and B cell differentiation into memory B cells or longlived plasma cells 1 .
Tfh differentiation necessitates a judicious control given the critical roles in inducing GC responses, one of the most powerful responses of the immune system.The pivotal process requires Tfh cells to distinguish cognate B cells from myriad B-cell clones with irrelevant antigen specificities in lymphoid tissues [6][7][8] .To guarantee this important checkpoint, a delicate balance of signaling pathways and transcription factor activity is required to control the expression of chemokine receptors and molecules regulating CD4 T-cell dynamics, such as positioning and migration, as well as the expression of costimulatory molecules involved in the interaction of CD4 T cells with B cells.
In this review, we explore the complex dynamics of Tfh cells, beginning with an exploration of the transcriptional regulation that underpins their differentiation and function.We then review the current status of our understanding of the molecular regulation of Tfh cell positioning and the intricacies of Tfh cell interactions with B cells.We will then discuss the consequences of dysregulated Tfh cell dynamics and B:T synapse formation in various physiological contexts, such as vaccination and autoimmune diseases.
TFH CELL DIFFERENTIATION Ag-specific CD4 T cells develop into pre-Tfh cells during interactions with antigen-presenting cells (APCs) in the T-cell zone, similar to other effector CD4 T cells (Th1, Th2, or Th17) 1 .Pre-Tfh cells migrate toward the boundary between the T-cell zone and B-cell follicles (B:T border), where Tfh cells interact with B cells that present cognate peptide Ags.Cognate interaction is an important checkpoint for Tfh cells to further polarize into GC-Tfh cells.This process leads to further enhancement of Tfh identity, resulting in proper localization within the B-cell follicle for interaction with GC B cells 6 .

Positive regulators of Tfh differentiation
The Tfh differentiation pathway necessitates stable and sequential interactions with APCs, such as non-B-cell APCs in the T-cell zone and B cells at the B:T border and inside GCs.This notion implies that signaling pathways downstream of T-cell receptors (TCRs) and costimulatory molecules could create a conducive environment for Tfh differentiation.TCRs with greater affinity for peptide Ags enable prolonged interactions with DCs in the T-cell zone, enhancing Tfh differentiation 9,10 .Inducible T-cell costimulator (ICOS), which interacts with ICOS ligand (ICOS-L) expressed on DCs and B cells, acts as a positive regulator of Tfh differentiation 5 .ICOS-mediated activation signals not only induce Bcl6 and c-Maf, transcription factors that positively regulate Tfh differentiation (discussed below) but also inhibit Foxo1, Blimp1, and T-bet, transcription factors that antagonize Tfh differentiation (discussed in the following section), partly via PI3K-mediated activation of Akt and TBK1 [11][12][13] .
The identification of Bcl6 as a fate-determining transcription factor for Tfh differentiation led to the acknowledgment of Tfh cells as a distinct lineage of effector CD4 T cells.Bcl6 centrally locates in Tfh cell development by shaping their distinct gene expression profiles and functional attributes by upregulating genes related to Tfh identity, follicular migration, and B-cell interactions 4,[14][15][16][17][18] .In addition to Bcl6, numerous transcription factors, including Ascl2, TCF1, c-Maf, Batf, and IRF4 4,8 , also play critical roles in Tfh differentiation.Ascl2, a member of the basic helix-loop-helix (bHLH) transcription factor family, contributes to Tfh cell differentiation by collaborating with Bcl6 to enhance CXCR5 expression 19 .TCF1 cooperates with LEF1 to drive early Tfh differentiation by activating pro-Tfh genes such as Bcl6, Icos, and Il6st while inhibiting anti-Tfh genes such as Il2ra and Prdm1 [20][21][22][23] .c-Maf, a member of the Maf family of transcription factors, and IRF4 were also found to positively regulate Tfh differentiation via IL-21 cytokine production and its signaling pathway in CD4 T cells [24][25][26][27][28][29] .Batf acts during the early stage of Tfh differentiation by actively inducing Bcl6 and Maf in CD4 T cells 27 .
Tfh differentiation is also regulated by cytokines.IL-6 induces c-Maf, which in turn activates IL-21 production in activated CD4 T cells 30 .By sharing downstream STAT1 and STAT3 transcription factors, IL-6 and IL-21 synergistically induce Tfh differentiation via transcriptional activation of target genes, including Bcl6 31,32 .Recently, IL-6 was revealed to inhibit the upregulation of the beta subunit of the IL-2 receptor complex (IL-2Rβ) by impeding STAT5 binding to the Il2rb locus, rendering Tfh and GC-Tfh cells, which are strong IL-2 producers, less responsive to autocrine IL-2-mediated inhibition of Tfh differentiation 33 .In humans, Tfh differentiation can be induced by IL-12 via STAT4 activation during in vitro stimulation 8,34 .
While IL-2 was originally identified as a T-cell growth factor 42 , recent studies revealed its important role in inhibiting Tfh differentiation 9,43,44 .Robust IL-2 signaling due to enhanced expression of IL-2Rα, the high-affinity IL-2 receptor, or high IL-2 bioavailability leads to strong phosphorylation of STAT5, which represses Bcl6 via Blimp1 induction 16,44 .Anti-IL-2 monoclonal antibodies enhanced Tfh differentiation of murine CD4 T cells following exogenous stimulation and maximal Tfh differentiation during in vitro stimulation of human CD4 T cells, highlighting the physiological relevance of IL-2-dependent inhibition of Tfh differentiation 44,45 .By sharing the common gamma chain (γc) with IL-2, IL-7 also negatively regulates Tfh differentiation.It functions in a slightly different manner from IL-2.STAT5 activated by IL-7 competes with STAT3 and inhibits STAT3-mediated Bcl6 induction rather than activating Blimp1 expression 46 .

Interplay of transcription factors in Tfh differentiation
The dynamic balance between positive and negative regulators ensures the stage-specific control of Tfh differentiation, the maintenance of Tfh integrity, and the proper localization of Tfh cells.A recent study revealed that Bcl6 is central to this regulatory network.As a nexus transcriptional repressor, Bcl6 orchestrates the Tfh differentiation program by (1) directly repressing transcription factors such as T-bet, GATA-3, RORγt, STAT5, and Blimp1, which are crucial inducers of alternative helper T-cell lineages, and (2) inducing molecules essential for Tfh differentiation (i.e., CXCR5, SAP, PD-1, ICOS, and CD40L) through a repression-of-repressor mechanism 4,14 .Bcl6 inhibits Blimp1, Id2, and Runx3 from repressing CXCR5 expression, whereas Klf2-mediated suppression of PD-1 and S1PR1 is released via direct repression of Klf2 by Bcl6 4 .

Epigenetic regulation of Tfh differentiation
Epigenetic studies have ushered in a new era in our efforts to understand how CD4 T cells control the development of effector CD4 T cells.In the context of the Tfh differentiation program, epigenetic regulators have been shown to collaborate with transcription factors or act independently to control genes involved in the Tfh differentiation pathway.For instance, Teneleven translocation 2 (Tet2), one of the three Tet methylcytosine dioxygenases, negatively regulates Tfh differentiation 47 .Tet2 deletion in CD4 T cells hence leads to enhanced differentiation of CD4 T cells into GC-Tfh cells.Mechanistically, Tet2 coordinates with Foxo1 to mediate demethylation at the Runx2 and Runx3 loci, inducing the expression of these genes 47 .Other chromatin regulators also impact Tfh differentiation.Ezh2 histone methyltransferase is recruited by TCF1 to activate Bcl6 transcription and facilitate Tfh differentiation 48 .The histone methyltransferase mixed lineage leukemia 1 (Mll1) also functions as a positive regulator of Tfh differentiation.Deletion of Mll1 in CD4 T cells led to significant reductions in Bcl6, LEF-1 and TCF-1 expression and consequently resulted in impaired Tfh differentiation of Agspecific CD4 T cells during acute viral infections or protein immunizations 49 .Given the spatiotemporal nature inherent to Tfh differentiation and function, which are orchestrated by a series of transcription factors, it is imperative to elucidate how epigenetic modifications of Tfh-inducing or -antagonizing transcription factors may modulate the target genes governing Tfh differentiation.

DYNAMICS OF TFH CELLS
To elicit T cell-dependent (TD) humoral immunity from Ag-specific B cells, Tfh cells need to meet at least two conditions: colocalization with B cells and GC B cells and provision of immunological nutrients.Inside GCs, GC B cells repeatedly migrate between the dark and light zones, where Ag-specific B cells undergo somatic hypermutation during proliferation and are clonally selected by GC-Tfh cells via BCR affinity-dependent competition 2,50 .GC-Tfh cells employ chemokine receptors and B-cell-interacting molecules to properly localize in the light zone and to ensure that GC B cells that have successfully acquired affinity maturation for Ags are selected 2 .
Cellular dynamics of GCs GC-Tfh cells, GC B cells, and follicular dendritic cells (FDCs) actively interact inside GCs 51 .After undergoing cell division and somatic hypermutation in the dark zone, GC B cells migrate back to the light zone.In the light zone, GC B cells with diverse BCR sequences compete for Ags captured by FDCs, and cells with higher Ag affinity BCRs can take up and process Ags, presenting peptide Ags on MHC II molecules.GC B cells expressing cognate peptide Ags interact with GC-Tfh cells, receive signals to migrate back to the dark zone and undergo another round of the cell division-somatic hypermutation-positive selection cycle 7,51,52 .
The dynamic migration of GC B cells is controlled by chemokine receptors.Sphingosine-1-phosphate receptor 2 (S1PR2), coupled with the heterotrimeric G protein Gα13, is important for maintaining GC B-cell localization inside GCs.A deficiency in the Gα13 gene led to rapid dissemination of GC B cells into circulation 53 .The cyclic movement of GC B cells between the dark and light zones is controlled by the modulation of CXCR4 (dark zone homing) and CXCR5 (light zone homing) chemokine receptors 54 .Changes in the surface expression of these chemokine receptors occur within several hours, as 10% of GC B cells that migrate from the dark zone to the light zone return to the dark zone within 6 hours 52,54 .

Tfh dynamics
Tfh and GC-Tfh cells continuously migrate between GCs in lymphoid tissues 55 .Tfh and GC-Tfh cells were recently revealed to migrate into neighboring GCs, unlike GC B cells, which are mostly confined to a single GC in the context of acute protein immunization 55 .Moreover, recently developed Tfh and GC-Tfh cells can participate in ongoing GC reactions, extending the duration of GC responses 55 .This migratory behavior is strictly governed by the regulation of various chemokine receptors and adhesion molecules, including CCR7, CXCR5, PSGL-1, and EBI2, as well as surface receptors such as S1PR2 and PD-1 8,[56][57][58][59] .
Tfh cells migrate toward CXCL13-rich B-cell follicles by inducing the surface expression of CXCR5 and downregulating the expression of CCR7 and PSGL-1, the membrane proteins required for T-cell homing and retention in the T-cell zone 56,60 .While EBI2, a G protein-coupled receptor activated by 7α,25dihydroxycholesterol, is required for Tfh cell positioning at the B:T border, it is downregulated when Tfh cells migrate deep into the follicles to induce GCs 61 .The dramatic change in EBI2 expression is controlled partly by Bcl6.Bcl6 also induces CXCR5 expression by inhibiting Blimp1 and Id2, which suppress CXCR5 expression 14,15,62 .Bcl6 simultaneously represses the expression of CCR7, PSGL-1, and EBI2 14,58 , leading Tfh cells to migrate from the T-cell zone to the B:T border and subsequently into B-cell follicles.In addition to Bcl6, Ascl2, Tox2, and STAT1 have been identified as important regulators of these chemokine receptors.Ascl2 acts as a transcriptional activator of the Cxcr4 and Cxcr5 genes and as a repressor of the Ccr7 and Selplg genes, respectively 19 .Tox2 directly enhances Cxcr5 gene expression by augmenting chromatin accessibility 63 , while STAT1 upregulates CXCR5 expression via p300 recruitment 64 .Conversely, Bach2 serves as a transcriptional repressor by binding directly to a regulatory region of the murine Cxcr5 gene promoter 38 .
Within B-cell follicles, GC-Tfh cells upregulate S1PR2 and PD-1 to adequately support cognate GC B cells by maintaining their localization.This process is partially mediated by S1PR2.The retention of S1PR2-deficient GC-Tfh cells within GCs is reduced without disrupting Tfh differentiation 57 .Furthermore, PD-1 plays an additional role in regulating Tfh positioning by limiting the upregulation of CXCR3.Considering that CXCR3 ligands (CXCL9 and CXCL10) are abundantly expressed in the interfollicular areas outside the follicles 65 , the differential surface expression of PD-1 between Tfh and GC-Tfh cells could determine whether the corresponding cells effectively accumulate inside the GC territory or migrate from the GCs 66 .Although how S1pr2 gene expression is regulated in Tfh cells remains to be elucidated, both Tox2 and STAT1 have been revealed to induce Pdcd1 gene expression 63,64 and therefore might be involved in regulating Tfh and GC-Tfh localization and positioning inside GCs.
Inside GCs, strong CXCR5 expression enables GC-Tfh cells to localize in the light zone, where they place selection pressure on GC-B cells 2 .Interestingly, a fraction of human and mouse GC-Tfh cells were also revealed to express CXCR4 54,67 , suggesting that CXCR4 and CXCR5 potentially cooperate in GC-Tfh positioning, similar to the cyclic migration of GC B cells between the dark zone and the light zone.Indeed, GC-Tfh cells expressing both CXCR4 and CXCR5 preferentially localize to the dark zone 68 .Moreover, experiments using CD4 T-cell-specific Cxcr4-or Cxcr5-knockout mice revealed that CXCR4-deficient CD4 T cells preferentially localize to the light zone, while CXCR5 deficiency leads to the preferential positioning of GC-Tfh cells to the dark zone 68 .Thus, it is crucial to identify the transcriptional regulation circuits controlling the surface expression of CXCR4 and CXCR5 on GC-Tfh cells to further elucidate the mechanisms of GC-Tfh cells positioning in TD humoral immune responses 68,69 .

SYNAPSE FORMATION BETWEEN TFH CELLS AND B CELLS
Intravital imaging studies revealed that cells dynamically move across the compartmentalized areas of secondary lymphoid tissues.Inside GCs, Ag-specific GC B cells migrate between the two zones, repeatedly undergoing cell division-somatic hypermutation-positive selection cycles.At the same time, numerous nonspecific B cells migrate in and out of GCs.Thus, the pivotal step occurs when GC B cells, which have successfully evolved their BCR affinity, need to be adequately recognized by GC-Tfh cells in the light zone.
Identification of the correct binding partners is determined via a cell-to-cell interaction-based scanning process of GC-Tfh cells.These cells form immune synapses with B cells (hereafter referred to as B:T synapses) through the engagement of the TCR/MHCpeptide Ag complex, costimulatory molecules such as CD40 ligand (CD40L)/CD40 and ICOS/ICOS-L, and adhesion molecules such as LFA-1/ICAM-1 and SLAMs 70 .In fact, the B:T synapse is crucial for the full maturation of Tfh cells into GC-Tfh cells at the B:T border 71,72 .Thus, the precise control of molecules participating in B:T synapse formation is essential to induce GC formation in B cells in an Ag-specific manner and to guarantee the qualitative evolution of their Ag-binding strength.
Following TCR and CD28 stimulation, the early growth response (Egr) family of transcription factors, including NFAT and possibly Egr-1, binds to the 3' region of the TATA-proximal NFAT site of the Cd40lg gene, inducing its transcription 73 .In contrast, PU.1, an ETS family transcription factor, negatively regulates Cd40lg expression by directly binding to the promoter region of the gene.PU.1 deletion in CD4 T cells thus led to a significant increase in Tfh differentiation, enhancing GC formation and TD humoral immunity 74 .TCR-and CD28-mediated signals also trigger AP-1 activation.The AP-1 transcription factor binds to an AP-1 responsive site within the Icos promoter, leading to the upregulation of ICOS expression in activated CD4 T cells 75 .The LEF-1 and TCF-1 transcription factors also contribute to the positive regulation of Icos expression, ultimately enhancing Tfh differentiation 20 .TCF-1-mediated Icos activation can also be induced by Bcl6 via transcriptional repression of Runx2, Runx3, and Klf2, which antagonize ICOS expression in CD4 T cells during Tfh differentiation 14 .
The expression of CD11A, the α subunit of LFA-1, can be regulated by epigenetic modifications.DNA hypomethylation and hyperacetylation of histone H3 at the CD11A promoter can be induced by reduced expression of regulatory factor X-box 1 (RFX1), which functions via interaction with DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) and consequently results in augmented expression of CD11A on CD4 T cells 76 .Additionally, CD11A expression can be enhanced by reduced methylation in the promoter region of the CD11A gene by the histone demethylase Jumonji domain-containing protein D3 (JMJD3) 77 .
SAP (SLAM-associated protein), a small signaling adapter protein that functions downstream of SLAM family receptors, facilitates the ability of CD4 T cells to form immune synapses with cognate B cells 78 .SAP-deficient CD4 T cells fail to form stable synapses with cognate B cells and undergo GC-Tfh differentiation; thus, they are not able to support GC formation or induce long-lived plasma cells (LLPCs) or memory B cells [78][79][80][81] .Given the critical roles of SAP in Tfh and GC-Tfh cell differentiation and TD humoral immunity, it is very important to understand the transcriptional regulation of the SH2D1A (Sh2d1a) gene, which encodes SAP.Ets-1 and Ets-2 bind to the Ets consensus site within the SH2D1A promoter region.Dominant-negative mutants containing only the DNA binding domain led to a significant decrease in the basal promoter of SH2D1A, indicating the promotive roles of Ets-1 and Ets-2 in SAP expression 82 .BCL6 is a positive regulator of SAP expression in human CD4 T cells.Ectopic BCL6 expression in tonsillar non-Tfh (CXCR5 -) and Tfh (CXCR5 int ) cells results in substantial upregulation of SAP expression 25 .
Recently, we revealed a novel mechanism illustrating how CD4 T cells negatively regulate SAP expression to modulate B:T synapse-dependent TD humoral immunity.Mef2d, a member of the myocyte enhancer factor-2 transcription factor family known for its negative regulatory function in neuronal synapse development 83 , plays a similar inhibitory role within the immune system.By binding to a putative Mef2 binding sequence within the murine Sh2d1a gene, Mef2d directly suppresses SAP expression in CD4 T cells, inhibiting SAP-dependent B:T synapse formation and TD humoral immune responses 84 .

TFH DYNAMICS AND B:T SYNAPSE FORMATION IN VACCINES AND DISEASES
B cell help signals are provided by costimulatory molecules and cytokines.CD40L, expressed on Tfh and GC-Tfh cells, triggers the NF-κB pathway downstream of CD40 in the interacting B cells, leading to B-cell proliferation inside GCs 6,85,86 .The interaction between CD40L and CD40 also contributes to a temporal increase in SLAM and CD40 expression on a subset of GC B cells in the light zone, which contributes to further activation of CD40-mediated signals and prolonged interaction with GC-Tfh cells, supporting the division of GC B cells in the dark zone while inhibiting plasma cell differentiation 87 .ICOS-L is upregulated on GC B cells by CD40-mediated activation, which increases the surface area available for engagement with ICOS hi GC-Tfh cells, reinforcing Tfh integrity 71 .
Inside GCs, GC-Tfh cells are an important cellular source of cytokines 88,89 , which are required for proliferation, isotype switching, and antibody-secreting plasma cell differentiation of GC B cells.IL-21 enhances Bcl6 expression in GC B cells and supports GC maintenance, leading to maximal affinity maturation in Ag-specific GC B cells 8,88,90 .This process is supported by IL-4, which exerts antiapoptotic effects on GC B cells that are highly susceptible to apoptosis due partly to genomic instability or nonfunctional BCR expression after somatic hypermutation 3 .These cytokines are also important for the generation of plasma cells 2,89 .IL-21 induces early plasma cell differentiation in IRF4 hi Bcl6 lo GC B cells via STAT3-mediated Blimp1 induction 6,87,91 .With CD40-mediated signals, IL-21 also promotes the positive selection of GC B cells in the light zone 92 .IL-21 and IL-4, together with IFN-γ and IL-17, play important roles in enabling GC B cells to switch their heavy chain constant regions 3,93,94 .
The B-cell help functions of Tfh cells rely on the proper localization of both Tfh and GC-Tfh cells, along with stable cell-tocell interactions with (GC) B cells.The precise spatiotemporal regulation of the positioning of CD4 T cells and interaction with (GC) B cells is crucial for orchestrating TD humoral immunity.Errors in these regulatory circuits are expected to contribute to poor GC formation or antibody production against exogenous Ags or to elicit uncontrolled GC formation and antibody responses to harmless substances or self-antigens.
EBI2-dependent chemotaxis toward the outer T-cell zone and to the B:T border is important for CD4 T cells to interact with ICOS-L hi CD25 + DCs, the potent inducers of Tfh differentiation during the priming stage, as well as to make contact with cognate B cells 61 .Ebi2 deficiency thus severely impairs Tfh differentiation and decreases GC formation and plasma cell differentiation after acute bacterial infection or immunization with SRBCs 61 .After GCs form, GC-Tfh cells downregulate EBI2 expression for GC retention rather than migrating toward the T-cell zone 58 .At the same time, GC-Tfh cells induce the expression of S1PR2, which binds to sphingosine 1-phosphate (S1P), the ligand chemoattracting B cells toward the inner follicle.Although the lack of the S1pr2 gene encoding S1PR2 is insufficient to impede GC-Tfh retention inside GCs, S1PR2 appears to play redundant roles with CXCR5 in controlling the positioning of GC-Tfh cells, enabling interactions with GC B cells in GCs 57 .
Positive selection of GC B cells occurs in the light zone of GCs 2,6 .Thus, the mislocalization of GC-Tfh cells outside the light zone could have profound impacts on the quantity or quality of Ag-specific B-cell responses.This process is negatively controlled partly by the CXCR4 chemokine receptor 68 .CXCR4 expression in GC-Tfh cells was significantly enhanced in aged mice, which led to the preferential positioning of GC-Tfh cells in the CXCL12-rich dark zone.The mislocalization of GC-Tfh cells in the dark zone led to reduced cellular outputs of GCs in aged mice after immunization, which was restored by adoptive transfer of young CD4 T cells 68 .
As explained above, positive selection guarantees that Agspecific B cells clonally expand and undergo affinity maturation.Tfh and GC-Tfh cells scan B cells and GC B cells whether they present appropriate peptide Ags and form stable interactions.Molecules contributing to the stable interaction between Tfh cells and B cells at the B:T border and between GC-Tfh cells and GC B cells in the light zone thus function as limiting factors for B cells to form GC responses.Genetic ablation of ICOS and CD40L 95,96 or blocking antibodies interfering with the CD40-CD40L or ICOS-ICOS-L interaction 97,98 results in poor GC formation and impaired production of isotype-switched affinity-matured Abs.In contrast, enhanced ICOS expression in CD4 T cells or CD40LG duplication is strongly associated with the unchecked induction of autoantibodies in autoimmune disorders such as systemic lupus erythematosus and rheumatoid arthritis [99][100][101] .Moreover, failures in the epigenetic regulation of CD11A expression (discussed above) are associated with enhanced Tfh differentiation in SLE patients 76 , fueling T and B-cell hyperactivity and exacerbating SLE pathogenesis 77 .
Among the molecules functioning in the B:T synapse, SAP is very important.CD4 T cells require SAP expression to interact with B cells but not with non-B-cell APCs 78 .Inside GCs, the presence of SAP determines whether GC-Tfh cells make sufficient contact with B cells to provide positive selection signals in the light zone.Loss-of-function mutations in the SH2D1A gene contribute to the development of type 1 X-linked lymphoproliferative (XLP) syndrome 102 , leading patients to suffer from clinical manifestations, such as hypogammaglobulinemia and recurrent bacterial or viral infections.Thus, transcriptional regulation of SAP expression in CD4 T cells might control TD humoral immunity.Intriguingly, we recently found that CD4 T-cell-specific deletion of Mef2d led to a significant increase in Ag-specific GC formation and antibody production after immunization, while a profound reduction in MEF2D mRNA expression in CD4 T cells was strongly correlated with various autoimmune parameters in patients with SLE 84 .
A comprehensive understanding of the transcriptional regulation of Tfh cells, particularly its impact on Tfh dynamics (Fig. 1) and synapse formation with B cells (Fig. 2), would provide insights for the development of innovative therapeutic strategies for the treatment of infectious diseases and autoimmune disorders.

Fig. 1
Fig. 1 Tfh dynamics and synapse formation with B cells and GC B cells.Stage 0: Naive CD4 T cells are activated in the T-cell zone of lymphoid tissues after recognition of antigens presented by DCs.These cells express high levels of receptors for chemokines and chemoattractant molecules present in the T-cell zone.Stage 1: Pre-Tfh cells, which are formed during interaction with DCs, upregulate CXCR5 while downregulating CCR7 and PSGL-1 and migrate to the B:T border.Stage 2: At the B:T border, pre-Tfh cells interact with cognate B cells.This interaction is supported by ICOS, CD40L, and SAP, which drive full maturation of Tfh cells into GC-Tfh cells.Stage 3: To ensure further migration into GC areas, these cells increase CXCR5 and PD-1 expression while decreasing the expression of chemoattractant receptors, including EBI2 and PSGL-1.Stage 4: Inside GCs, GC-Tfh cells upregulate CXCR5, S1PR2, and PD-1 to maintain their localization in the light zone of the GC and upregulate synapse-related molecules to stably interact with GC B cells that present cognate peptide Ags.

Fig. 2
Fig. 2 Transcriptional regulation of immune synapse formation molecules by Tfh cells.The transcriptional regulatory network controls the expression of molecules involved in immune synapse formation between Tfh and B cells and between GC-Tfh cells and GC B cells.Activators (Egr-1, TCF-1, RFX1, and BCL6): upregulate the expression of the Cd40lg, Icos, Cd11a, and Sh2d1a genes, respectively.Repressors (PU.1, Runx3, and Mef2d): downregulate the expression of Cd40lg, Icos, and Sh2d1a.The positive and negative regulators of these genes highlight that the B:T immune synapse requires an intricate genetic program to properly elicit powerful TD humoral immune responses.