The differentiation courses of the Tfh cells: a new perspective on autoimmune disease pathogenesis and treatment

Abstract The follicular helper T cells are derived from CD4+T cells, promoting the formation of germinal centers and assisting B cells to produce antibodies. This review describes the differentiation process of Tfh cells from the perspectives of the initiation, maturation, migration, efficacy, and subset classification of Tfh cells, and correlates it with autoimmune disease, to provide information for researchers to fully understand Tfh cells and provide further research ideas to manage immune-related diseases.


Early stage of Tfh cells differentiation
The process of naïve CD4 + T cells differentiating into Tfh cells mainly occurs in the T cell region of lymphoid tissue, and is jointly regulated by immune cells such as dendritic cells (DCs), transcription factors such as Bcl-6, and cytokines such as achaete-scute homologue 2 (Ascl2).

DC acts on early differentiation of Tfh cells through signal pathways such as IL-6/STAT3 and IL-12/STAT4
DCs and B cells both produce interleukin-6 (IL-6).After the combination of IL-6 and IL-21, the signal transducer and activator of transcription 3 (STAT3) could be activated to start Tfh cells differentiation [3].
In the lungs of asthmatic mice, conventional type 2 dendritic cells (cDC2) is the main DC subset responsible for Tfh cells differentiation.cDC2 produced a large amount of IL-6 and IL-21, which can more strongly induce the differentiation of Bcl-6-producing Tfh cells.Block the expression of IL-6, then the differentiation of Tfh cells was inhibited [4].Compared with healthy people, Tfh cells expression level in rheumatoid arthritis (RA) patients was increased, and Tfh cells proportion was positively correlated with disease activity score in 28 joints (DAS28), phosphorylated STAT3 (pSTAT3) -Tyr705, and plasma IL-6 concentration.IL-6 induced STAT3' Tyr705 phosphorylation, resulted in the overactivation of STAT3, and then drove Tfh cells differentiation [5].This indicates the important role of the IL-6-pSTAT3-Tfh cells immunomodulatory axis in the pathological process of RA.
In inflammatory models, the induction of optimal Tfh cells differentiation and B-cell response requires the involvement of IL-6 [6].Type I interferon (IFN-I) can up-regulate and increase the expression of CD86 on the surface of DCs and B cells, mediating the production of IL-6 by DCs, thereby enhancing the differentiation of Tfh cells [7].Toll-like receptors (TLRs) specifically recognize pathogens and endogenous pro-inflammatory factors and trigger adaptive immune and inflammatory responses.Cytosine-phosphodiester-guanine (CpG)-B oligonucleotides belong to TLR ligands.The addition of CpG-B oligonucleotides to vaccine adjuvants can promote the production of IL-6 by DCs, which in turn enhances antigen-specific Tfh cells differentiation in vivo and promotes T cell-dependent B-cell responses [8].Cutaneous DCs expand CXCR5 + Tfh cells through IL-6 and IFN-α/β receptor non-dependent mechanisms, providing new ideas for vaccination [9].Transcription factor T-cell factor 1 (TCF-1) is important for early T-cell development.TCF-1 promoted IL-6 receptor expression and increased the responsiveness of naïve CD4 + T cells to Tfh cells signaling, and then promoted early Tfh cells differentiation by mediating the sustained expression of IL-6 receptor α (IL-6Rα) and IL-6/IL-6Rα downstream signal anti-IL-6Rβ: glycoprotein130 (gp130) [10].The IL-6/STAT3 pathway promotes Thymocyte selection-associated high mobility group box 2 (Tox2) expression, while Tox2 inversely enhances IL-6 signaling and promotes Tfh cells differentiation [11].The B lymphocyte-inducible maturation protein 1 (Blimp-1) is encoded by PR Domain Containing Protein 1 (PRDM1) [12].In female mice, Blimp-1 deficiency led to more IL-6 secretion by DCs, promoted Tfh cells differentiation and germinal center (GC) responses, and produced lupus-like autoantibodies [13].Total glucosides of paeony (TGP) improved symptoms and bone destruction in collagen-induced arthritis (CIA) model mice, and the expression of Tfh cells, IL-6 and IL-21, as well as STAT3 signaling pathways were also inhibited.It is speculated that TGP's anti-arthritic effect is due to its inhibition of spleen Tfh cells differentiation and GC formation through the IL-6-STAT3 signaling pathway, thereby suppressing autoimmune responses [14].Niclosamide is a potent STAT3 signaling inhibitor.In systemic lupus erythematosus (SLE) model mice, Niclosamide significantly reduced IL-6, p-STAT3, and TCF-1 levels, down-regulated Tfh cells and IL-21 expression, and ameliorated mesangial matrix increase, moderate perivascular mononuclear cell infiltration, glomerular basement membrane thickening, and C3 immune complex deposition and other renal pathological features [15].IL-6, p-STAT3, IFN-γ, IL-21 and Tfh cells levels were significantly elevated in SLE model mice.Artesunate is a semisynthetic derivative of artemisinin with anti-inflammatory and immunosuppressive effects.After artesunate intervention, IL-6, p-STAT3, IFN-γ, IL-21 and Tfh cells levels were significantly reduced and artesunate inhibited STAT3 signaling in a dose-dependent manner [16].The up-regulation of Tfh cells differentiation mediated by abnormal IL-6/STAT3 signaling pathway is involved in the pathological process of AID.Inhibition of the IL-6/STAT3 signaling pathway can down-regulate Tfh cells differentiation and thus improve the state of AID.
IL-12 is also involved in the early differentiation phase of Tfh cells.IL-12 secreted by DCs, which are activated by TLRs or CD40L, induced the differentiation of naïve CD4 + T cells into Tfh cells via STAT4 in a dose-dependent manner.After IL-12 inhibition, Tfh cells differentiation was also inhibited, IL-21 and IFN-γ expression was reduced, and the ability of B cells to produce antibodies was correspondingly diminished [17,18].
The Non-obese diabetic (NOD) mice displayed similar lymphocyte alterations to Sjögren's syndrome (SS) patients.Using IL-12 antibody to intervene in the NOD mice can reduce the serum IL-12 level and the number of Tfh cells.Mesenchymal stem cell transplantation has also been found a similar phenomenon in the treatment of SS patients.Targeted regulation of IL-12 may be a new idea for SS treatment [19].Hydroxychloroquine (HCQ) is a commonly used drug for the treatment of RA.HCQ downregulated the level of IL-12 in vitro experiment [20].HCQ can inhibit the differentiation of Tfh cells induced by IL-12, and improve the incidence rate and score of arthritis in model mice [21].The DCs level increased in RA patients, and HCQ further interfered with DC maturation by blocking TLR9 signal transduction [20].The above research results suggest that the mechanism of HCQ in the treatment of RA may include inhibiting DCs and IL-12 secretion, and down-regulating the expression of Tfh cells, thereby improving the symptoms of RA.
In summary, DCs can act on the early differentiation of Tfh cells and participate in the disease process through signaling pathways such as IL-6/STAT3 or IL-12/STAT4.Biological agents play an important role in the treatment of AID, and tocilizumab, a monoclonal antibody targeting IL-6, has been approved for the treatment of RA [22].However, there is a lack of clinical studies on the inhibitory effect of tocilizumab on Tfh cells, and the biologics targeting IL-12 have not been applied in the treatment of AID.Targeting the regulation of the early differentiation stage of Tfh cells in which DCs are involved has a promising future in the treatment of AID.

Bcl-6 is a core transcription factor for the early differentiation of Tfh cells
The expression of Bcl-6 in Tfh cells was significantly higher than that in other Th cell subsets.Bcl-6 deficient mice cannot form GC and Bcl-6 deficient T cells cannot develop into Tfh cells [2].Bcl-6 affects the level of Tfh cells in a gene dose-dependent manner.Blimp-1 and Bcl-6 antagonize each other and jointly regulate the differentiation of Th cell subsets [2].Bcl-6 is regulated by factors such as STAT signals and affects Tfh cells differentiation through related miRNAs and cytokines.
The STAT signaling family regulates Bcl-6 and is closely related to the early differentiation of Tfh cells.IL-12 promoted the differentiation of Tfh-Th1-like cells by binding STAT1 and STAT4 on Bcl-6 and T-Box protein 21 (TBX21) genes.Silence the STAT1 or STAT4 could reduce the expression of Bcl-6, CXCR5, CXCR3 and T-box expressed in T cells (T-bet), and inhibit IL-12 mediated Tfh-Th1-like cells differentiation [23].The regulation effect of the STAT3 signaling pathway on Bcl-6 is complex.STAT3 promoted Tfh cells differentiation by inducing Bcl-6 and inhibiting Blimp-1 expression [24].In mouse CD4 + T cells, leptin activated the STAT3 and mammalian target of rapamycin (mTOR) pathways, upregulated the Bcl-6 expression, and enhanced the Tfh cells differentiation and function [25].However, another study showed that STAT3 inhibited the activity of Bcl-6, and proposed that the inhibition or activation of Bcl-6 by STAT3 depends on the activation of other STAT signals and cytokines [26].In the presence of IFN-I, both STAT5 and STAT3 can bind to BCL-6 loci, and STAT5 has a strong binding force.STAT5 includes STAT5A and STAT5B.STAT5B inhibits Tfh cells through the Blimp-1/Bcl-6 axis.STAT5B deficiency can impair the generation of regulatory T (Treg) and follicular regulatory T (Tfr) cells, enhance the differentiation of naïve CD4 + T cells into memory cells, and thus lead to the expansion and persistence of the circulating Tfh (cTfh) cells population [27].Both phosphorylated STAT1 and STAT5 bound to the BCL-6 site.IL-2 induced high levels of STAT5 phosphorylation, and pSTAT5 binding to the BCL-6 gene locus can reduce pSTAT1 levels, thus inhibiting Tfh cells differentiation [28].STAT5 also upregulated the antagonistic transcription factor Blimp-1 of Bcl-6, promoted Th1 cell differentiation, and inhibit Tfh cells differentiation [24].Erythropoietin can also directly prevent Tfh cells differentiation and the production of GC B cells and autoantibodies in a STAT5-dependent manner, and the inhibiting effect of erythropoietin on Tfh cells may be related to the decreased expression of Bcl-6 [29].In conclusion, in terms of affecting the expression of Bcl-6, STAT1 and STAT4 promote the early differentiation of Tfh cells, STAT5 inhibits the early differentiation of Tfh cells, and STAT3 plays a complex regulatory role.
The extracellular signal-regulated kinase (ERK) pathway is involved in Tfh cells differentiation, and inhibition of ERK2 promoted Tfh cells development.Zinc Finger Protein 831 (Zfp831) is a functional molecule downstream of CD28 and ICOS-ERK axes, and its expression is inhibited by ERK.Zfp831 promoted Tfh cells differentiation by directly up-regulating the expression of transcription factors Bcl-6.It was revealed that Zfp831 is bound to the BCL-6 promoter and enhanced its expression, thus promoting Tfh cells differentiation [30].Overexpression of Tox2 led to a substantial increase in BCL-6 mRNA and protein expression, while Bcl-6 directly drove Tox2 expression during Tfh cells development.Clustering analysis revealed that IL-6 signaling and Tox2 together induced BCL-6 gene expression [11].IFN-γ promoted CD4 + T cell proliferation, enhanced Bcl-6 expression, and promote pre-Tfh cells differentiated to Tfh cells.While inhibition of IFN-γ expression, Tfh cells and autoantibody levels decreased.IFN-γR signal did not affect Bcl-6 expression in GC B cells [31].Guanine nucleotide-binding protein subunit α13 (Gα13) signaling in T cells also decreased Bcl-6 and CXCR5 expression, thereby affecting Tfh cells differentiation [32].In resting CD4 + T cells, protein kinase D2 (Prkd2) restricted the entry of Bcl-6 into the nucleus, thereby inhibiting Tfh cells differentiation.Following immunization, Bcl-6 was up-regulated in CD4 + T cells, overcoming the inhibitory effect of Prkd2 and entering the nucleus to drive Tfh cells differentiation [33].IκBNS belongs to the nuclear factor κB (NF-κB) repressor proteins.IκBNS promoted Bcl-6 expression by binding to the promoter region of BCL-6, thereby promoting Tfh cells differentiation [34].The methyltransferase nuclear receptor binding SET domain protein 2 (Nsd2) -mediated modification of histone H3 lysine 36 dimethylation (H3K36me2) is required for Bcl-6 expression and Tfh cells differentiation.Lack of Nsd2 in T cells resulted in decreased Bcl-6 expression, impaired Tfh cells production and GC response, while Nsd2 overexpression increased Bcl-6 expression, and promoted Tfh cells differentiation and GC response [35].
Regulation of Bcl-6 expression may be used in the treatment of autoimmune diseases.After the intervention of TLR7 agonist imiquimod, SLE model mice and Tfh cells significantly decreased BCL-6 mRNA expression and upregulated PRDM1 (encoding Blimp-1) and STAT5b mRNA expression, and the number of Tfh cells and GC B cells were significantly reduced, while the anti-double-stranded DNA (anti-dsDNA) antibody and antinuclear antibody titers in serum were also significantly decreased [36].Cyanidin is a natural pigment that can significantly down-regulate the activation of STAT3 and the expression of Bcl-6, increase the phosphorylation level of STAT5, and thus inhibit inflammation and joint destruction, as well as IL-21 and immunoglobulin G (IgG) production in adjuvant induced arthritic rats.In vitro, the expression of Rho associated coiled-coil forming protein kinase-2 (ROCK-2) in cells treated with cyanidin was also reduced, indicating that ROCK-2 is involved in controlling STATs phosphorylation and Bcl-6 expression to control T-cell differentiation [37].The artemisinin analogue SM934 can alleviate the severity of arthritis in CIA mice, reduce bone erosion, reduce the levels of Tfh cells and Th17 cells, and inhibit the production of pathogenic antibodies.In vitro, it has been found that the therapeutic effect of SM934 may be related to inhibiting the expression of Bcl-6, thereby reducing the differentiation level of Tfh cells [38].According to the study on ulcerative colitis model mice, curcumin inhibited the expression of Tfh cells related transcription factors Bcl-6 and p-STAT3, and significantly increased the protein levels of Blimp-1 and STAT3 in colon tissue, so as to achieve a therapeutic effect [39].
The expression of Bcl-6 is influenced by various signal transduction pathways and cytokines, specifically participate in the early differentiation of Tfh cells, and designating the Tfh cells lineage typing.Intervention in the above-mentioned signal transduction pathways and corresponding cytokines can regulate the expression of Bcl-6 and the differentiation of Tfh cells, which is of great significance for the treatment of immune related diseases.

Ascl2 is another key transcription factor for the early differentiation of Tfh cells
Ascl2 is highly expressed in Tfh cells, and its expression may precede that of Bcl-6.The high expression state of Ascl2 increased CXCR5 mRNA expression by approximately 60 times but did not affect the expression of BCL-6, ICOS, and IL-21.At the same time, it down-regulated C-C chemokine receptor 7 (CCR7) and IL-2 in T cells, accelerating T-cell migration to lymphatic follicles and differentiation of Tfh cells.Genome-wide analysis showed that Ascl2 directly regulated Tfh cells related genes and inhibited the expression of Th1 and Th17 characteristic genes.Knocking out the Ascl2 gene can lead to absolute damage to Tfh cells development [40].After genetic mutations in Ascl2, the production of CXCR5 in CD4 + T cells was significantly reduced, and Ascl2 could also up-regulate the expression of IκBNS levels in CD4 + T cells to affect Tfh cells differentiation [34].Ascl2 acts on the early stage of Tfh cells differentiation and may be a key transcription factor for Tfh cells early differentiation.In the SS mice model, it was found that the Ascl2 mRNA levels in model mice were significantly higher than those in control mice, which promoted the differentiation of Tfh cells in model mice and thus affected the autoimmune response of the SS model [41].
The naïve T cells differentiate toward Tfh cells in the T-cell region.DCs, Bcl-6, and Ascl2 play essential roles in this course.Various signaling pathways, cells, and cytokines are also involved in this process.Physiologically, the differentiation and maturation of Tfh cells are beneficial for the body to produce protective antibodies.Pathologically, excessive differentiation of Tfh cells can promote the expression of pathogenic antibodies in the body.The intervention of pathological early differentiation of Tfh cells by regulating signaling pathways, cell and cytokine levels is of great significance for disease treatment.

Tfh cells migration and maturation stage
The second stage of Tfh cells differentiation occurs mainly in the T-B cell border and follicles of lymphoid tissue.Pre-Tfh cells are influenced by ICOS and PD-1 to migrate to the T-B cell border and interact with B cells.Activated B cells contribute to GC formation, and pre-Tfh cells further differentiate into mature Tfh cells within the GC, entering the final stage of Tfh cells differentiation.Relative to pre-Tfh cells, mature Tfh cells have increased expression of Bcl-6, CXCR5 and ICOS, which are involved in auxiliary B cell differentiation into mature plasma cells and antibody production.Tfh cells also have a secretory function, secreting factors such as IL-4, IL-10, IL-21 and IFN-γ.

Bcl-6 promotes T-B cell interactions
In addition to influencing the early differentiation of Tfh cells, Bcl-6 also promotes T-B cell interactions.It was found that an intact BCL-6 allele was sufficient to maintain long-term contact between activated T cells and cognate B cells.Enhanced T-and B-cell contact time was associated with enhanced calcium signaling, which mediated CD40L externalization from pre-Tfh cells, while T-and B-cell interactions depended on the delivery of CD40L from pre-Tfh cells to cognate B cells.Although T cells with insufficient Bcl-6 expression could express CD40L, they could not effectively deliver CD40L during brief contacts with cognate B cells, which might be associated with impaired calcium signaling pathways in T cells when Bcl-6 expression was impaired, and insufficient Bcl-6 expression in T cells also led to impaired CD40L signaling pathways in B cells [42].

ICOS affects the differentiation and migration of Tfh cells and maintains their phenotype
ICOS externalizes CD40L in pre-Tfh cells by enhancing TCR dependent calcium signaling and binds to CD40 on the surface of B cells.At the same time, ICOS on the surface of Tfh cells also binds to ICOS ligand (ICOSL) on the surface of B cells [43].The combination of ICOS and ICOSL can enhance the activation of phosphatidylinositol 3-kinase (PI3K), promoting Tfh cells to migrate across the T-B boundary into follicles.Further research has found that ICOS relies on its transmembrane domain to promote binding to tyrosine kinases lymphocyte cellspecific protein tyrosine kinase (LCK), thereby enhancing calcium signaling and activating PI3K [44].ICOS promotes the formation of coordinated pseudopodia in T cells and promotes their persistent movement in a PI3K-dependent manner, thereby promoting T-cell recruitment from the T-B boundary to the follicles [45].During the interaction between T and B cells, T cells secrete helper cytokines such as IL-4 and IL-21.Existing studies have shown that ICOS activates PI3K signaling through its tyrosine-based signaling motif, while demonstrating that the ICOS-PI3K signaling pathway plays a dominant role in increasing the expression of IL-21, IL-4, and promoting Tfh cells development [46].Compared with Bcl-6, the upregulation of CXCR5 expression is more dependent on ICOS stimulation [47].CXCR5 signal can also activate PI3K.ICOS deficient T cells cannot normally express CXCR5 and do not affect the expression of Bcl-6 and Ascl2, so ICOS can directly promote T-cell migration to follicles through the CXCR5 signal pathway [48].Krüppel-like transcription factor 2 (Klf2) is an inhibitor of CXCR5, an activator of CCR7, and a transcription factor inhibiting Tfh cells differentiation.Research has shown that ICOS inhibits Klf2 through Forkhead box O1 (Foxo1) to maintain the Tfh cells phenotype.After blocking the ICOS signal of the fully developed Tfh cells, Tfh cells transformed into other Th subsets and migrates back to the T cell zone, resulting in the disappearance of subsequent GC reactions [49].B cells expressing ICOSL are also crucial for optimal collaboration between antigen-specific T cells and B cells, promoting the production of normal Tfh cells and GC [45].OX40 is coexpressed with ICOS on Tfh cells in and around GC.The interaction of OX40-OX40L and ICOS-ICOSL is critical to the later development of Tfh cells and the maintenance of GC B cells [50].In SS patients, Tfh cells are enriched in salivary glands with GC and express ICOS.Blocking the expression of ICOS in vitro can effectively reduce the expression of IL-21, TNF-α, IL-6 and IL-8 [51].In parasitic infectious diseases, ICOS is also necessary to produce high affinity protective antibodies [52].It is demonstrated that ICOS is crucial for the differentiation and maintenance of Tfh cells, the formation of functioning GC, and the production of high affinity antibodies.
ICOS expression is influenced by a variety of signaling pathways, cytokines and immune cells.Tfh cells stimulated by CD3 and ICOS signals enhance the activity of mTOR complex2.Lack of mTOR complex2 signal leads to a decrease in ICOS expression, and impaired differentiation of the Tfh cells [53].CD155 + L cell significantly promoted the proliferation of GC-Tfh, pre-Tfh and naïve CD4 + T cells, confirming that CD155 signaling acts as a synergistic stimulus in all tonsillar CD4 + T cell subsets.It was found that CD155 signaling, affected pre-Tfh cells proliferation by promoting IL-10, IL-21, ICOS and soluble CD40 ligand (sCD40L) expression.In turn, these expression upregulation was also dependent on CD226 signaling in naive and pre-Tfh cells [54].Thus, the CD155-CD226 axis drives the early to mid-stage of Tfh cells differentiation.IL-6 signaling is also necessary for the presence of ICOS expression in Tfh cells [55].IL-6 expression increased after co-culture of fibroblast-like synoviocytes and anti-CD3/CD28-stimulated peripheral blood mononuclear cell (PBMC) from RA patients, which promotes the ratios of CD4 + CXCR5 + ICOS + cells [56].The lack of E3 ubiquitin ligase Pellino1 (Peli1) promoted ICOS expression, and Peli1 mRNA expression was negatively correlated with ICOS expression on CD4 + T cells.It was revealed that enhanced ICOS expression in Peli1-deficient CD4 + T cells promoted Tfh cells differentiation [57].Tfr cells also express ICOS and more than Tfh cells, suggesting that Tfr cells competitively inhibit pre-Tfh cells from binding to ICOSL [58].MR2-1 is an anti-TNF-R2 antibody.ICOS and PD-1 expressed in MR2-1-stimulated Tfr cells, and Tfh cells differentiation was significantly inhibited [59].The special AT-rich sequence-binding protein-1 (SATB1) gene repressed the ICOS promoter, and silencing SATB1 in CD4 + T cells can derepress ICOS and impair Tfr cell differentiation, thus promoting the formation of antigen-specific Tfh cells and driving isotype-switching antibody response [60].E3 ubiquitin ligase Von Hippel-Lindau (VHL) deficiency leads to enhanced hypoxia-inducible factor 1α (HIF-1α)-mediated glycolytic activity, and it ultimately leads to reduced ICOS expression and thus inhibits Tfh cells differentiation [61].The results suggest that the VHL-HIF-1α axis plays a major role in Tfh cells development through glycolytic-epigenetic reprogramming.ICOS signaling is required for induction and maintenance of the CIA model, inhibition of glycolysis may ameliorate arthritis of CIA mice [62].ICOS-mediated T cell glycolytic pathway could be a potential therapeutic target for RA.Some medicines could affect Tfh cells differentiation by inhibiting ICOS expression.Berberine downregulates the expression of ICOS through calcium signaling and inhibits the differentiation of pre-Tfh cells into Tfh cells [63].Abatacept can also inhibit Tfh cells differentiation by reducing ICOS expression [64].Oral administration of high doses of cyclosporine A (10 mg/Kg/d) in rats resulted in reduced expression of IL-6, IL-21, PD-1 and ICOS, indicating that T-cell activation was inhibited and that cyclosporine A could inhibit Tfh cells differentiation through ICOS [65].
ICOS is essential for the targeted migration of Tfh cells, late Tfh cells development, maintenance of Tfh cells phenotype and GC B-cell maintenance.Multiple cytokines, transcription factors, Tfr cells and medicines can affect Tfh cells differentiation by acting on the ICOS/ICOSL signaling pathway.

PD-1 affects proliferation and function of the Tfh cells
PD-1-related research is currently a hot topic in oncology, which inhibits Tfh cells proliferation but positively regulates Tfh cells function and is an important marker of GC Tfh cells, while multiple cells, cytokines and signals affect Tfh cells differentiation by acting PD-1/PD-L1.In metastatic non-small cell lung cancer, It was demonstrated that regions with viable cancer cells were enriched for exhausted CD8 + T cells, Treg cells, and Tfh cells, consistent with a pan-cancer analysis [66,67].This recruitment was related to the capacity of transforming growth factor β (TGF-β) to drive chemokine C-X-C motif ligand 13 (CXCL13) expression, a chemoattractant of Tfh cells, by intratumor CD8 + T cells [68].And intratumoral ICOS + PD-1 + CD4 + Tfh cells preferentially recognize tumor-derived neoantigens compared with other CD4 + subsets [69].Tfh cells exert an antitumor immune effect in a CD8 + -dependent manner.The presence of Tfh cells is required for efficacy of anti-programmed cell death ligand-1 (anti-PD-L1) therapy.In patients treated with anti-PD-1 mAb, accumulation of Tfh cells and CD8 + at the tumor site is associated with outcome [68].
PD-1 is expressed at low levels on naïve CD4 + T cells, but a unique marker of GC Tfh cells that inhibits the activation of PI3K and migration of follicular T cells into the follicle.The inhibitory effect of PD-1 is mediated by PD-L1 expressed by bystander B cells [70].As previously shown, CXCR5 is up-regulated during the early stages of Tfh cells differentiation, and CXCR5 activates PI3K to drive T-cell migration into the follicle.ICOS/ICOSL signaling is required to activate PI3K when Tfh cells cross the T-B border to the follicle, and PD-1 ensures that T cells high in ICOS expression enter the next stage of maturation [71].PD-1 consistently suppresses ICOS-induced Tfh cells numbers by limiting the down-regulation of Klf2 in the GC response during TCR co-stimulation with ICOS [72].Tfh cells differentiation also requires migratory DC that transport antigen to the lymph node, and migratory cDC2 uniquely carries antigen to the T-B border of the lymph node where Tfh cells initiation occurs.Migrating CD11b + cDC2s express the appropriate chemotactic receptors to homing to the T-B border and induce Tfh cells-dependent antibody response [73].PD-1/PD-L1 interactions can inhibit Tfh cells differentiation.Studies have shown that loss of DC cell surface-specific PD-L1 results in a higher percentage of Tfh cells in the blood.Before entering B-cell follicles, PD-L1 on the surface of DC cells binds to pre-Tfh cells and controls Tfh cells differentiation and maintenance [74].
PD-1 deficiency significantly increased serum levels of monocyte chemoattractant protein-1 (MCP-1), IFN-γ, and IL-10 in infection treatment vaccine (ITV)-immunized mice.These elevated cytokines promoted the expansion of Plasmodium-specific Tfh cells and GC B cells in ITV-immunized PD-1 −/− mice [75].It was also observed that PD-1 signaling deficiency, although increasing the number of Tfh cells, impairs the function of Tfh cells by reducing the ability of important cytokines such as IL-4 and IL-21 [76].PD-1 is involved in the optimal concentrations of IL-21 product by Tfh cells, while PD-1 and PD-L1 can concentrate the Tfh cells from the follicle into the GC, and ensure the high-affinity B cells binding to mature Tfh cells.CXCR3 is expressed by T cells after stimulation of T-cell receptors and is the receptor for CXCL9 and CXCL10.CXCR3 is highly expressed outside the follicle, and PD-1 concentrates Tfh cells from the follicle into the GC by inhibiting CXCR3 expression.PD-1 correlates with the affinity of antibodies.It was shown that after anti-PD-1 immunotherapy, Tfh cells proliferated but antibody affinity decreased [77].
The OX40L-OX40 axis is of major importance for IL-21 + Tfh cells differentiation.Conventional DC2 (cDC2) and cDC1 subsets express similar concentrations of ICOSL, PDL1, but significantly increased expression of OX40L in cDC2 compared to cDC1.Blocking OX40L reduces the frequency of ICOS + PD-1 + Tfh cells [78].Exposure to the envelope glycoprotein gp120 induced a higher proportion of PD-1 + T cells and Tfh cells expressing PD-1 or ICOS but also resulted in poor B-cell repertoire development.The high proportion of memory B cells positively correlates with a lower proportion of PD-1 + CD4 + cells [79].Thus, PD-1 can be defined as an envelope glycoprotein-induced inhibitory receptor that attenuates and/or delays the recall antibody responses by negatively acting on Tfh/B cells interactions.
PD-1 abnormality is common in patients with autoimmune diseases, and regulation of PD-1 expression can be used to treat these diseases.The cell phenotype of elevated Tfh cells in SLE patients was mainly TCF1 − and TCF1 + for the Treg cells.CD62L, TCF1 and PD-1 jointly promote the production of IL-21 and participate in the physiological and pathological process of SLE [80].The level of Tfh cells in the spleen of CIA mice increased, and ICOS and PD-1 expression were up-regulated.After anti-TNFα and anti-IL-1β treated, the level of PD-1 and ICOS expression returned to normal and the number of Tfh cells decreased [81].This provides a theoretical basis for the use of TNFα inhibitors in RA.Ethanol and acetate prevent arthritis by decreasing PD-1 expression in Tfh cells and inhibiting IL-21 secretion and formation of Tfh: B-cell conjugates [82].
ICOS participates in Tfh cells differentiation through calcium signaling, PI3K, Klf2, and OX40 promote T-B cell interactions, maintain Tfh cells phenotype, and promote GC formation.PD-1 safeguards Tfh cells differentiation and function.Meanwhile, various cytokines, cells and signals act on ICOS and PD-1 to participate in Tfh cells differentiation, which eventually lead to various physiopathological processes and cure or cause diseases.

B cell-Tfh cell interactions
Pre-Tfh cells enter the follicle to interact with B cells.During the GC response, B-cell antigen binding affinity was positively correlated with the expression of C-C class chemokines 22 (CCL22).Under the stimulation of CD40, B cells in the GC up-regulate chemokines such as CCL22 and CCL17, which stimulate CC chemokine receptor 4 (CCR4) on Tfh cells to attract multiple helper cells from a distance and increase the chance of Th cell-B cell interactions.In the absence of affinity messages delivered to Tfh cells from CCL22 and CCL17, B cells would remain in the GC, suggesting that the CCR4-CCL17/22 axis promotes frequent contact between antigen-specific T cells and B cells and facilitates optimal GC formation [83].Tfh cells interact with DCs and B cells at the T-B boundary on a long-term basis, but interact transiently when in contact with GC B cells, facilitating their search for and preferential help to B cells expressing high levels of peptide-major histocompatibility complex II (pMHCII), as well as providing competition for other GC B cell.The increased size and duration of GC Tfh cells contact with B cells prolonged Ca 2+ signaling and improved the quality of the GC Tfh cell response, promoting the expression of IL-4 and IL-21 double-positive Tfh cells [84].
The transcription factor CR6-interacting factor 1 (CRIF1) is a multifunctional protein expressed in the nucleus and cytoplasm that regulates cell cycle and growth by regulating the expression of nerve growth factor IB, androgen receptor, STAT3 and others, and is also essential for mitochondrial function.The deletion of CRIF1 in B cells impairs mitochondrial oxidative function, increases STAT3 phosphorylation levels, expresses more ICOSL and intercellular adhesion molecule (ICAM) related to T-B cell interaction, and up-regulates the expression of Tfh cells characteristic genes such as signaling lymphocytic activation molecule family member 5 (SLAMF5), IFNG and C-X-C chemokine receptor 3 (CXCR3), thereby increasing the frequency of Tfh cells [85].B cells expressing PD-L1 and PD-L2 and T cells expressing PD-1 regulate the production of long-lived plasma cells and memory B cells through T-B cell interactions [76].IFN-γ, driven by TLR7 and produced by T cells, can control gene transcription for immune cell survival, proliferation, metabolism and autoimmunity through multiple cellular pathways.In vitro, IFN-αR and IFN-γR expression on B cells was elevated after TLR7 stimulation; in vivo, after IFN-γ signaling deletion, the renal pathological progression, GC size, Tfh cells response and antibody formation in TLR7-induced SLE-prone mice were attenuated, indicating that IFN-γ promotes the development of autoreactive B cells and the development of SLE [86].
During this stage of differentiation, pre-Tfh cells interact with antigen-specific homologous B cells to promote Tfh cells differentiation maturity which promote GC formation and secretion of specific antibodies.If we can specifically intervene in Tfh cells differentiation at this stage, it is of great significance to solve the clinical problem of treating disease without compromising autoimmunity.

IL-21 is produced by Tfh cells and regulates Tfh cells differentiation
IL-21 is mainly produced by Tfh cells.T-B cell interactions are critical for IL-21 production [87].In the GC, Tfh cells interact with B cells via ICOS/ICOSL to release large amounts of IL-21, and blocking the ICOS signaling pathway significantly reduces IL-21 production.Sustained TCR signaling is important for IL-21 production, and disruption of antigen presentation is detrimental to IL-21 production.Blockade of the CD40/CD40L pathway impairs the development of IL-21 + CD4 + T cells during lymphocytic choriomeningitis virus (LCMV) Cl13 infection, and the proportion of Tfh cells and GC B cells is significantly reduced, suggesting that CD40-CD40L interactions are a critical role in IL-21 production.When IL-21 was eliminated in CXCR5 + CD4 + T cells, LCMV-specific total IgG was reduced by approximately 30%, indicating that IL-21 produced by CXCR5 + CD4 + T cells contributes to antiviral humoral immunity.Studies have shown a 2-fold reduction in the proportion of virus-specific CD8 + T cells in mice in which IL-21 was eliminated [88].IL-21 production by Tfh cells is essential to promote CD8 + effector T-cell (Teff) differentiation, limit T-cell depletion and promote viral control.IL-21 is also important for maintaining Tfh cells homeostasis, promoting Tfh cells differentiation and inducing their self-expression in an autocrine manner [89].αIL-21R, a monoclonal antibody to the IL-21 receptor, does not have cytotoxic effects on CD4 + T cells but can inhibit Tfh cells differentiation.IL-21 decreases Treg cell frequency and can be reversed by αIL-21R, indicating that IL-21 regulates Tfh/Tfr cells homeostasis and has significant implications for T-and B-cell differentiation and antibody production [90].

Circulation Tfh cells classification and function
After interacting with B cells in the GC, Tfh cells have three outcomes: (i) reside in the primary GC, when CD90 expression of Tfh cells decreases; (ii) migrate to a new GC; (iii) down-regulate Bcl-6 to be converted into circulating memory Tfh cells, when memory Tfh cells express similar levels of Bcl-6 as naïve T cells [91][92][93].Upon secondary antigen exposure, circulating memory Tfh cells are rapidly recruited to the GC after up-regulation of CXCR5 or Bcl-6, producing an effector antibody response.
When co-cultured with naïve B cells, only Tfh2 and Tfh17 cells produce IL-21 and immunoglobulins.Tfh2 and Tfh17 cells support chronic humoral immunity such as antibody production in autoimmune diseases, whereas Tfh1 cells support antibody responses in infection and vaccines with relatively short duration [96].Activated circulating PD-1 + CXCR5 + Tfh cells expanded in IgG4-related sclerosing cholangitis/autoimmune pancreatitis and PD-1 expression was enhanced in all Tfh cells subsets, correlating with disease activity and driving IgG4-committed B cell class switch and proliferation [97].
It was found that the T cell immunoglobulin and immune receptor tyrosine-based inhibitory motif domain (TIGIT) expression was higher in peripheral Tfh cells than in other Th cell subsets, TIGIT + Tfh cells produced more IL-21 than TIGIT − Tfh cells [98].CD155 is a ligand for TIGIT.The activated cTfh cells express CD155, while memory B cells inhibit cTfh cells proliferation through the interaction of TIGIT on B cells and CD155 on cTfh cells [99].Purinergic 2X7 (P2X7) promotes caspase-mediated Tfh cells pyroptosis and controls the development of pathogenic ICOS + CD4 + T cells secreting IFN-γ.Circulating Tfh cells in SLE patients are hyporesponsive to P2X7 stimulation and resistant to P2X7-mediated cytokine-driven proliferation inhibition [100].Therefore, restoring P2X7 activity in SLE patients could help limit the increment of pathogenic autoantibodies and improve the patient's condition.IFN-α belongs to the type I interferon.It was found that after infection with foot-and-mouth disease virus (FMDV) recombinant adenovirus, IFN-α enhanced the production of memory Tfh cells subsets such as cTfh1 and cTfh2 cells with memory B cells, and also increased the expression level of Bcl-6 in memory Tfh cells.When the antigen attacked memory Tfh cells again, IFN-α enhanced the activation of memory Tfh cells and increased the expression levels of Bcl-6 and STAT1.It was hypothesized that IFN-α may enhance the differentiation of memory Tfh cells by regulating the transcription factors Bcl-6 and STAT1 [91].Compared with healthy donors, the proportion of CXCR3 + Tfh1 cells was significantly increased in PIK3CD Gain-of-function (GOF) patients, while the proportion of CCR6 + Tfh17 cells and CXCR3 − CCR6 − cTfh cells was reduced, indicating that the overactive PI3K signaling pathway has a selective effect on the differentiation of cTfh cells that biases them toward the Th1 phenotype and away from the Th17 phenotype [101].The frequency of cTfh2 and cTfh17 cells in CD4 + T cells of myasthenia gravis (MG) patients was significantly increased, and the levels of IL-21, IL-4, and IL-17a produced by ICOS high cTfh cells were significantly higher than those produced by ICOS low cTfh cells, indicating that high expression of ICOS in MG patients helps to exert the secretion function of Tfh2 and Tfh17 cells [102].

ICOS, PD-1, and cTfh cells proliferation and function
In MG patients, cTfh cells increased with elevated ICOS expression, and ICOS high cTfh cells produced significantly higher levels of IL-21, IL-4, and IL-17a than ICOS low cTfh cells.The proportion of cTfh cells in CD4 + T cells was correlated with disease severity.Immunotherapy may improve the abnormalities of cTfh cells [102].Patients with MG combined with diabetes mellitus had increased cTfh cells and expressed ICOS at high levels, and activated cTfh cells were positively correlated with plasmablasts.Further studies showed that hyperglycemia promoted the differentiation and activation of Tfh cells, causing abnormal plasma cell differentiation and antibody secretion through the mTOR signaling pathway [103].Tfh cells were increased and Tfr cells were decreased in the peripheral blood of RA patients.Tfr/Tfh cells ratio was negatively correlated with erythrocyte sedimentation rate, rheumatoid factor, hypersensitive C-reactive protein, IgG, DAS28 score, IL-21, CXCL13, and positively correlated with serum TGF-β level.Meanwhile, ICOS expression was higher in RA patients' blood and synovial membrane, suggesting that ICOS is also involved in RA pathogenesis [104].Iguratimod (IGU) is an antirheumatic drug that has been found to act directly on B cells and inhibit antibody production in RA patients.It was found that IGU also further inhibited RA cTfh cells expression and function by inhibiting the mTOR/hypoxia-inducible factor (hif1α) /hexokinase 2 (hk2)/glucose metabolism axis, significantly reducing the expression of ICOS [105].During the acute infection stage, human immunodeficiency virus type 1 (HIV-1) infection drives the expansion of cTfh cells, which in turn interferes with B-cell differentiation via the ICOS signaling pathway [106].
According to PD-1 expression, cTfh cells can be divided into resting and activated subsets, while cTfh cells with high PD-1 expression effectively promote antibody response [107].In ovarian cancer patients, PD-1 was expressed more in Tfh cells compared to non-Tfh CD4 + cells.Moreover, PD-1 + Tfh cells secreted more IL-21 and IL-10 and promoted immunoglobulin secretion than PD-1 − Tfh cells [108].Patients with multiple sclerosis exhibited significantly elevated PD1 + cTfh cells frequencies; 36 months after autologous haematopoietic stem cell transplantation (AHSCT), PD1 + cTfh cells frequencies were significantly reduced, consistent with normal subjects, suggesting that AHSCT can control the disease by modulating PD1 + cTfh cells expression in patients with multiple sclerosis [109].Patients with acute immune thrombotic thrombocytopenic purpura had decreased GC memory B cells, total cTfh cells, PD1 + cTfh cells, and increased frequency of circulating plasma cells, while dysregulation of B cell and cTfh cells homeostasis may be caused by hyperactivation of T and B cells leading to differentiation of memory B cells to antibody-producing plasmablast and migration of circulating Tfh cells into the GC [110].Peripheral blood PD-1 + ICOS + Tfh cells in patients with primary SS (pSS) correlate strongly and positively with disease activity [111].PD-1 expression in blood Tfh and Tfr cells of pSS patients was positively correlated with IL-21, and circulating CCR7 lo PD-1 hi Tfh cells suggest that patients are undergoing disease activity and glandular inflammation [112].During the active phase of ulcerative colitis (UC), ICOS + , PD-1 + and ICOS + PD-1 + Tfh cells levels were significantly elevated, and significantly decreased upon reaching clinical remission; activated ICOS + PD-1 + Tfh cells were positively correlated with serum C-reactive protein (CRP) and Mayo scores, and ICOS + PD-1 + Tfh cells also correlated significantly with circulating new memory B cells, plasmablasts, and serum IgG, IL-4, and IL-21, suggesting that ICOS and PD-1 are involved in UC pathogenesis [113].Mayo score is a criterion for evaluating the severity of UC patients' conditions, with higher scores indicating greater severity.The expression levels of ICOS and PD-1, the corresponding ligands ICOSL and PD-L1 in B cells and their soluble forms (sICOS, sPD-1, sICOSL and sPDL-1) in the plasma of patients with MG revealed that ICOS/ICOSL and PD-1/PD-L1 are involved in the MG pathological process.sICOSL and sPD-1 expression abnormalities may interfere with the normal signaling of ICOS and PD-1 on Tfh cells, leading to Tfh cells overactivation and promoting disease progression [114].Perinatally HIV-infected children have reduced peripheral Tfh cells levels and show high PD-1 expression, while the frequency of low-memory pTfh cells with this high PD-1 expression correlates with worsening disease status and activation of differentiated B-cell profiles, which may lead to an attenuated specific antibody response [115].
High expression of ICOS or PD-1 means that cTfh cells are in an activated state.Various cytokines, signals, cell surface markers, cells, etc. can affect cTfh cells expression through various pathways, thus worsening the disease state.

Other functions of cTfh cells
6-Sulfo LacNAc monocytes (slanMo) is a subset of monocytes that have a greater ability to produce IL-12 and induce CD4 + T cell proliferation compared with DCs.Comparing slanMo cells and cTfh cells from SSc patients and HC, SlanMo cells were found to be increased in SSc patients and positively correlated with cTfh cells, speculating that SlanMo cells may be involved in the activation of cTfh cells in SSc patients [116].After antigenic stimulation, cTfh cells express low or moderate levels of CD45RA.The number and function of antibodies decreased after vaccination in older adults compared to younger adults.After vaccination, cTfh cells was low in frequency despite an effector memory phenotype in older adults, while CCR7 + CD45RA + cTfh cells were transiently increased in the blood of younger adults.Upon antigen reencounter, CCR7 + CD45RA + cTfh cells showed higher levels of CD95, CD40L, CXCR3, and Bcl-6 [117].After COVID-19 infection, older adults had higher levels of pro-inflammatory cytokines, more circulating plasmablasts, and a lower proportion of cTfh cells with antibodies, demonstrating that poor prognosis in older adults is associated with a pro-inflammatory state in vivo and an inability to generate appropriate antiviral response [118].K/BxN mice is a kind of TCR transgenic autoimmune arthritis model, and anti-glucose-6-phosphate isomerase (gpi) autoantibodies serve as the disease index in the K/BxN model.The middle-aged K/BxN mice had greater ankle thickness and anti-gpi titers than young K/BxN mice; Tfh cells and GC responses significantly increased in the middle-aged group compared with the young group, regardless of the segmented filamentous bacteria status of induced Tfh cells; and it was demonstrated that the increased Tfh cells in middle-aged mice were due to effector Tfh cells accumulation rather than memory Tfh cells [119].The above studies suggest that Tfh cells are also involved in the immune aging process.It was also identified 'Tfh13' cells with an unusual cytokine profile (IL-13 hi IL-4 hi IL-5 hi IL-21 lo ) co-expressing the transcription factors Bcl-6 and GATA3.Tfh13 cells are required for the production of high-affinity IgE and subsequent allergen-induced anaphylaxis.Blockade of Tfh13 cells may be an alternative therapeutic target to ameliorate allergic responses [120].There is a super-functional Tfh-like cells subset in lupus-prone New Zealand Black x New Zealand White (NZBxW) mice, defined as IL-21 + IFN-γ high PD-1 low CD40L high CXCR5 − Bcl-6 − T cells, which express high levels of TNF-α and IL-2 and help B cells to produce IgG in an IL-21 and CD40L-dependent manner [121].
cTfh cells are currently a hot research topic, and in the future there will be different defined subsets of cTfh cells, each representing its different functions and characteristics.In immune-related diseases, cTfh cells play an important role, and it is hoped that there will be more ideas for disease diagnosis and treatment with Tfh cells as the starting point in the future.
The occurrence and development of AID also involve other cells, including Treg cells.Treg cells are essential for immunosuppressive, maintenance self-tolerance and suppressing autoimmunity through the production of anti-inflammatory cytokines, and a deficiency of Treg cells results in fatal AID in humans and mice [122].Increased plasticity of Tfh cells and CD4 + T cells polyfunctionality with enriched memory Treg cell responses was demonstrated in RA patient synovial tissue, identified the presence of a novel population of pathogenic polyfunctional T cells that are enriched in the RA joint [123].The administration of TGP had down-regulated the levels of Th1 cells and Th17 cells in CIA rats, and up-regulated the levels of Th2 cells and Treg cells.These results indicate that Treg can be AIDs' therapeutic target because of immunosuppressive functions [124].

Conclusion
Tfh cells have been the focus of research among T cells since their discovery in 2000.Numerous studies have demonstrated that Tfh cells' differentiation requires the participation of cells and cytokines such as DCs, Bcl-6, Ascl2, ICOS, PD-1, and B cells, as well as the assistance of signaling pathways such as OX40/OX40L, IFNs, STATs, and Ascl2/IκBNS.When Tfh cells interact with B cells in the GC, they either stay in the GC or enter the blood to become memory Tfh cells (Figure 1).Since Tfh cells help B cells to produce antibodies and promote the formation of GC, they can be https://doi.org/10.1042/BSR20231723found in various immune-related diseases.However, the targeting of Tfh cells is also a challenge because Tfh cells can promote the production of pathogenic antibodies by B cells and also participate in the production of protective antibodies, which requires a lot of future research to balance the therapeutic and pathogenic functions of Tfh cells.