Follicular helper T cell in immunity and autoimmunity

Introduction

The production of high-affinity class-switched antibodies is necessary for the clearanceof pathogens after infection, for the establishment of long-term humoral immunity andfor the effectiveness of vaccines (1). Follicularhelper T (Tfh) cells have been recently shown to play a crucial role in instructing Bcells to form a repertoire of antibody producing cells that provide life-long supply ofhigh affinity, pathogen-specific antibodies (2).

Adaptive immune responses are regulated by the fine-tuning of the functional activity ofseveral T cell subsets through a complex mechanism that integrates signals from innateimmune cells and the cytokine milieu acting over naive T and B cells. SeveralCD4⁺ T cell subsets functionally distinct from the traditional Th1 and Th2subsets have been described, displaying either effector (Tfh, Th17 and Th9) orregulatory functions (natural and inducible regulatory T cells, Tr1 and Th3) (3). It is a consensus that, during immune responses,many cell subsets are directly involved as effector cells in the inflammatory process.Tfh cells are described as non-polarized CD4⁺ T cells that express thehighest levels of the chemokine receptor CXCR5, which is critical for their homing andfunction. Other distinguishing features of Tfh cells include the expression of thesurface receptors inducible T cell co-stimulator (ICOS) and programmed cell deathprotein 1 (PD1; also known as PDCD1) as well as the nuclear transcriptional repressor Bcell lymphoma 6 (bcl-6) (Figure 1). Tfh cellsexpress high levels of IL-21 and other cytokines that influence B cell differentiationand antibody production. Also, they have down-regulated the T cell zone-homing receptorCC-chemokine receptor 7 (CCR7) and IL-7 receptor-α (IL-7Rα) (4). Due to this profile of receptors and cytokines, Tfh cells havethe unique ability to home to B cell follicles and to induce antibody production duringco-culture with B cells (5).

Figure 1

Follicular helper T cells (Tfh) lymphocytes are critically involved in theformation of germinal centers and in the development of T cell-dependent B cellresponse in secondary lymphoid tissues. The figure represents Tfh cells with theirmost important membrane molecules, transcription factor and soluble effectormolecules.

The B cell maturation process requires cognate help provided by CD4⁺ T cellsin the T cell-rich extra follicular areas of secondary lymphoid organs. These arestructures within the B cell follicles of lymphoid organs that support intense B cellproliferation and differentiation, somatic hypermutation, selection of high-affinity Bcells, and class switching of immunoglobulin genes. B cells ultimately aredifferentiated into memory B cells and long-lived plasma cells that secretehigh-affinity antibodies (6). In fact, withoutTfh cells, germinal center do not develop, long-lived plasma cells are not generated,and long-term antibody responses are impaired (7). Memory B cells mediate long-term protective immunity due to their capacityto generate secondary humoral responses that surpass naive B cell responses by rapidlydifferentiating into high-affinity plasma cells (PC). Of interest, novel B cell receptorspecificities, including autoreactive ones, arise continuously during the ongoingprocesses inside germinal centers. Antibody secreting cells that emerge from thegerminal centers need to be tightly controlled due to their longevity and high-affinityantibody production. Autoantibody production indicates a profound breakdown in humoraltolerance mechanisms and B cell hyperactivity caused by either B cell-intrinsicabnormalities or immune-regulatory defects in other cell types.

In the last few years, significant progress has been made in the study of Tfh cells andthere has been a surge of research activity aimed at understanding the function anddifferentiation of these important cells (1),examining the biochemistry of transcription factors involved in programming Tfh celldifferentiation, and exploring the cellular biology of Tfh cell-mediated selection ofgerminal center B cells (8). Given the importanceof Tfh in practically all T cell-dependent humoral responses, this review will addressthe most important biological aspects of this "new" lymphocyte subset in normal immuneresponses against infectious agents, and discuss its relevance on deregulated autoimmuneprocesses.

Tfh: An overview

In the early 2000's, a number of prominent studies in mice and in humans led to theidentification of B follicular helper T cells, a subset of CD4 T cells localized in thetonsils and characterized by high expression of the master regulator bcl-6, whichrepresses the expression of other T-cell subset-specific transcription factors andpromotes the sustained expression of chemokine receptor CXCR5, which is essential forthe migration of T cells into the B-cell follicular zones (5). Within the follicle, crosstalk occurs between B cells and Tfhcells, leading to class switch, recombination and affinity maturation (2).

Tfh cell differentiation is a multistage, multifactorial process with significantheterogeneity involving a variety of cytokines, surface molecules and transcriptionfactors (4,8). Following immunization or infection, a cohort of naive CD4 T cells in theT cell zone acquire characteristics of pre-Tfh cells after interacting with dendriticcells (DCs) (9). The transformation ofCD4⁺ T cells into Tfh lineage seems to be determined early during T-DCinteraction by means of an increase in bcl-6 expression and downregulation of itsantagonist Blimp-1 under the influence of a combination of elements, including IL-6,IL-21(mice) or IL-12 (humans), IL-2, inducible co-stimulator (ICOS), T cell receptor(TCR) and likely CD28 (8,), as depicted in Figure 2A.

Figure 2

Schematic view of dentritic cell (DC) participation in the naiveCD4⁺ T cells differentiation into follicular helper T cells (Tfh)lineage by means of an increase in Bcl-6 expression and downregulation of itsantagonist Blimp-1 under the influence of a combination of elements, includingIL-21, inducible co-stimulator (ICOS), T cell receptor (TCR) and likely CD28(A). Since T cells are primed during interaction with DC inthe T cell zone and B cells reside in the B cell follicle, antigen-specific Tcells and their cognate B cells must migrate towards a secondary lymphoid organ tomeet each other and promote the generation of germinal centers by differentiationof primed B cells (B).

CD4⁺ T cells bearing high affinity TCRs seem to preferentially suffer pre-Tfhdifferentiation over other effector T helper cells (13). Fazilleau et al. (14) showed thatafter the transfer of CD4⁺T cells into immunized mice, those T cellsharboring TCRs with the highest affinity to peptide-MHC class II complexes and the mostlimited TCR diversity were selected into the Tfh cell pool. CD28 and ICOS also have animportant role in the induction of Tfh cells differentiation and germinal centerformation; CD28 appears to play a major role in the early phase of Tfh generation whileICOS is critical for generation of pre-Tfh cells as well as in later stages for Tfhdifferentiation (12).

The ability of activated CD4⁺ T cells to undergo differentiation into Tfh orinto polarized effector T cells is dictated by the balance of cytokines that stimulateor prevent Tfh differentiation. For example, IL-6 and IL-21 cooperate to induce Tfh cellformation by activating STAT3, which in turn promotes bcl-6 and CXCR5 expression. Incontrast, IL-2 signaling avoids Tfh cell development via STAT5 activation (7).

Several studies over the last few years have provided insights into the roles of thesecytokines in Tfh cell commitment. Using different animal models of viral infection,researchers found varying and transient degrees of impairment in Tfh cell numbers in theabsence of IL-6. Choi et al. (15) found thatearly bcl-6(+) CXCR5(+) Tfh differentiation was severely suppressed in the absence ofIL-6; however, STAT3 deficiency failed to recapitulate that defect. IL-6R signalingactivates the transcription factor STAT1 specifically in CD4⁺ T cells.Furthermore, IL-6-mediated STAT3 activation is important for downregulation of IL-2Rα,which contributes to limit Th1 cell differentiation in an acute viral infection. Thus,IL-6 signaling is an early inducer of the Tfh differentiation program mediated by bothSTAT3 and STAT1 transcription factors (15).According with this, Karnowski et al. (16) foundthat IL-6 production in follicular B cells in the draining lymph node was an importantearly event during antiviral response and that B cell-derived IL-6 was necessary toinduce IL-21 from CD4⁺ T cells in vitro and to support Tfhcell development in vivo.

The requirement of IL-21 for Tfh cell differentiation from naive T cells and formationof the germinal center has been consistently demonstrated by several groups (11,17,18). Furthermore, recent studies convincingly showedthat IL-12 can also drive Tfh cell differentiation by inducing IL-21 in aSTAT3-dependent manner both in mice and in humans (10,19). Interestingly, a recent studyprovided evidence for a pivotal role of IL-7 in Tfh generation and germinal centerformation in vivo, as treatment with anti-IL-7 neutralizing antibody,which markedly impaired the development of Tfh cells and IgG responses. Moreover,co-delivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation ofgerminal center B cells as well as Tfh cells but not of other lineages of T helpercells, including Th1, Th2, and Th17 cells (6).

IL-2 is considered a canonical growth factor for CD4⁺ and CD8⁺ Tcells (7). IL-2 signaling probably disturbs thedifferentiation of Tfh by stimulating Blimp-1 expression via STAT5 or by inducing theexpression of T-bet, which forms complexes with bcl-6 masking the DNA-binding domain ofbcl-6 and preventing bcl-6 from repressing the expression of Blimp-1 (20). Early studies found that the transcriptionrepressor bcl-6 is necessary (when ectopically overexpressed) for programming Tfh cells,including CXCR5 expression. CD4⁺ T cells from bcl6^−/− mice are impaired in the production of CXCR5⁺ Tfh cellsin vivo whereas the differentiation of other CD4⁺ T cellsubsets is relatively unaffected by the loss of bcl-6. This transcription factor acts inpart by repressing the transcription of Tbx21 [encoding T-box expressed in T cells(T-bet)] and Rorc [encoding retinoic acid-related orphan receptor γt (RORγt)] or bydirect binding to GATA-bind protein 3 (GATA3) (11,18). However, a study conducted byLiu et al. (21), using bcl-6-RFP reporter miceand phenotypic, functional and genome-wide transcriptome analysis of Tfh cells generatedin vivo, found that the initial up-regulation of CXCR5 was notdependent on bcl-6, but once bcl-6 is highly expressed, Tfh cells can persist invivo and some of them develop into memory cells.

Recently, Liu et al. (22) showed that theexpression of transcription factor achaete-scute homologue 2 (Ascl2) is selectivelyupregulated in Tfh cells. Ectopic expression of Ascl2 upregulates CXCR5but not bcl-6, and down regulates CCR7 expression in T cells in vitro,as well as accelerates T-cell migration to the follicles and Tfh cell developmentin vivo in mice. Furthermore, studies indicate that Ascl2 directlyregulates Tfh-related genes and inhibits the expression of Th1 and Th17 signature genes.Deletion of Ascl2, as well as blockade of its function with the Id3 protein inCD4⁺ T cells, results in impaired Tfh cell development and germinal centerresponse (22). In addition to bcl-6, Ascl-2 andSTAT3, other transcription factors are also known to be crucial for Tfh celldevelopment, such as the basic leucine zipper transcription factor (BATF) (23) and the IFN regulatory factor 4 (IRF4) (24). It is interesting to note that STAT3, BATF, andIRF4 are also needed for differentiation of the Th17 cell lineage.

Since T cells are primed during interaction with DC in the T cell zone and B cellsreside in the B cell follicle, antigen-specific T cells and their cognate B cells mustmigrate towards a secondary lymphoid organ to meet each other. This process is requiredfor the generation of germinal centers and the differentiation of primed B cells alongboth germinal centers and extra follicular pathways (Figure 2B).

Tfh cells have a high ability to stimulate naive B-lymphocytes present in the folliclegerminal center of secondary lymphoid organs by engaging B cells through co-stimulatormolecules like CD40L, ICOS and SAP, and by producing important cytokines to humoralresponse as IL-10 and IL-21. Tfh cells produce also a diversity of cytokines, such asINF-γ and IL-4, which direct B cells antibody isotype commitment (25), and IL-17, a pro-inflammatory cytokine, recently reported as animportant B cell factor, directly influencing its survival, proliferation anddifferentiation (26). IL-4-producing Tfh cellsinduce B cell IgG1 switch, and IFN-γ-producing Tfh cells induce B cell IgG2a switch.Interestingly, high-affinity IgG1 antibodies could only be induced by IL-4 produced byTfh cells (25).

A cluster of microRNAs (miRNAs) known as miR17-92 has been recently reported to have aregulatory role on Tfh cell differentiation and in germinal center reaction. Initially,bcl-6 was proposed to repress the miR17-92 inhibiting effect over Tfh cell development(18). However, more recent studies show thatmiR17-92 cluster acts as a positive regulator of Tfh cell differentiation since micewith T cell-specific deletion of miR17-92 cluster (tKO mice) exhibit severelycompromised Tfh differentiation, germinal center formation and antibody responses (27).

The inducible co-stimulator (ICOS) is another highly expressed molecule in Tfh cells andis essential for both Tfh differentiation and its effector function over B cells. Theimportance of ICOS is highlighted by the multiple ways in which ICOS signaling isregulated. Roquin inhibits ICOS, and combined loss of Roquin 1 and Roquin 2 results inspontaneous Tfh cell and germinal center development (28). A study suggested that ICOS is also essential for Th17 cell development(29); however, it has been shown that itsimportance for these cells is mostly associated with cell survival and to its functionby regulating IL-21 production, which contributes to the expression and maintenance ofIL-23R. In addition to the dependency to ICOS, Tfh and Th17 cells have more features incommon. Both subsets produce IL-21 and IL-17, express IL-23R and are dependent of thetranscription factors c-Maf and Stat3 to expand and produce IL-21. However, Th17 cellsexpress the transcription factor RORy that is neither expressed by Tfh cells nornecessary for its development. Tfh and Th17 cells also differ in the ability to home todifferent immune microenvironments; while most Tfh cells are CXCR5⁺ andmigrate to the secondary lymphoid tissue B cell areas, Th17 cells, when activated, downregulate CCR7 and up regulate CCR6, migrating to the target organs where they exerttheir effector functions. However, one may not exclude the possibility that some Th17cells, with high expression of ICOS and IL-23R, may down regulate CCR7 and up regulateCXCR5 becoming part of the heterogeneous Tfh cell pool that migrates to the follicles.When activated, Tfh cells also express other non-specific markers such as CD69, CD95 andCD40L, but none of them characterizes these cells as a distinct subset (29).

As mentioned before, it was recently shown that Tfh cells develop preferentially fromnaive T cells with high avidity TCR. In the same study the authors proposed theexistence of three Tfh compartments based on their migratory properties and molecularcharacteristics (14). Evidence indicates thatdepending on the cytokine microenvironment and the nature of the APC-activating antigen,naive CD4⁺ T cells acquire specific chemokine receptors that dictate theirmigration to a specific environment and their fate as specific Th subset. In fact,recent studies in mice and humans show that Tfh lineage cells are composed of subsetsthat differ in their localization, phenotype and function. The compartment ofcirculating Tfh memory cells in human blood contains heterogeneous subsets that differin phenotype and function (30). Growing evidencehas demonstrated that dysfunction of Tfh cells results in abnormal positive selection ofautoreactive B cells, which contributes to the development of autoimmune diseases.

Tfh and autoimmune diseases

The avoidance of autoimmunity is heavily dependent of T and B lymphocyte tolerancemechanisms. It is postulated that progressive breakdown in the tolerance mechanism wouldinvolve an increasing variety of cells. The classical example is shown by murine modelsfor studying CD4⁺ T cells tolerance, in which a single disturb in CD4 helperresponses can result in both cellular and humoral mediated immune responses againstself-antigens (31). This occurs because most Bcell responses depend on T cell help. The absence of T cell help during B cell priming,a mechanism of peripheral tolerance, leads to apoptosis or anergy; B cells lose theiraccess to lymphoid follicles and the chance to differentiate into germinal center cellsor plasma cells. How auto-reactive B cells avoid central tolerance in bone marrow andhow they evade peripheral tolerance to access follicles remains an area of activeinvestigation. The exclusion of self-reactive B cells from germinal center has beenshown to be defective in systemic lupus erythematosus (SLE) patients, and spontaneousgerminal center organization has been observed in different murine models of lupus(32). Another possibility is that B cellsbecome auto-reactive after gaining access to the follicle. In this situation, afterforeign antigen recognition in germinal centers, B cells undergoing affinity maturation,which improves the receptor affinity by somatic mutations, accidentally would give riseto auto-reactive cells. In SLE patients and murine models of lupus, auto-reactive Bcells become competent to produce autoantibodies, mostly high avidity IgG (33).

Whether and how Tfh cells collaborate to the pathogenesis of human autoimmunity is notclear. Recent advance in understanding the biology of peripheral memory Tfh cells hasrendered the analysis of human Tfh responses in the context of autoimmunity feasible(30). Different groups have studied thepresence of circulating Tfh cells as a potential biomarker of disease in variousautoimmune conditions, including myasthenia gravis (MG), autoimmune thyroiditis,Sjögren’s syndrome (SS), rheumatoid arthritis (RA), multiple sclerosis (MS), systemiclupus erythematosus, ulcerative colitis, Crohn's disease, ankylosing spondylitis, type 1diabetes mellitus (T1D), autoimmune hepatitis, primary biliary cirrhosis (Table 1) and juvenile dermatomyositis.

MG is an organ-specific autoimmune disease characterized by the T cell-dependentproduction of anti-acetylcholine receptor (AChR) antibodies. Patients with MG show asignificantly higher frequency of CXCR5⁺ CD4⁺ T cells in theperipheral blood, which correlates with disease severity (34). Furthermore serum CXCL13 was found to be increased in MGpatients and high CXCL13 serum level was associated with severe clinical stages (35). Interaction between CXCR5 and CXCL13 isespecially required for B-cell architectural organization regulatingcompartmentalization of B- and T-cells in secondary lymphoid organs. Accordingly,Meraouna et al. (36) reported that CXCL13expression was also increased in the thymus as well as in sera of MG patients notreceiving glucocorticoid therapy and that CXCL13 level decreased with glucocorticoidtreatment, in correlation with clinical improvement. Taken together, these resultssuggest dysregulation of blood CXCR5⁺ CD4⁺ T cells in MG patientsand that serum CXCL13 reflects the general status of MG severity.

Also, in Graves' disease, the affected thyroid tissue showed a positive correlation ofCXCR5 and CXCL13 mRNA expression with the number of lymphocytic infiltrates and ectopicgerminal centers (37). Recent studies detectedincreased percentages of circulating Tfh cells in patients with autoimmune thyroiddisease as well as a positive correlation between the percentages of circulating Tfhcells and the serum concentrations of antibodies against TSH receptor, thyroperoxidaseand thyroglobulin (38).

Patients with juvenile dermatomyositis show a strong skewing of blood CXCR5⁺Th cell subsets toward Th2 and Th17 phenotypes. Importantly, this skewing correlatedwith disease activity and the frequency of blood plasma blasts (39). In light of this observation, circulating CD4⁺CXCR5⁺ T cells from patients with SS were recently re-examined withrespect to the Th phenotypes. A positive correlation was found between the levels ofserum autoantibodies and the numbers of circulating CD4⁺ CXCR5⁺ Tcells, particularly with regard to those that also expressed CCR6. The frequency ofTh17-like subsets (CD4⁺ CXCR5⁺ CCR6⁺) in SS patientswas found to be significantly higher than in healthy controls. Functional assays showedthat activated Th17-like subtypes in the peripheral blood display the key features ofTfh cells, including invariably co-expressed PD-1, ICOS, CD40L and IL-21. Th17 subsetswere found to highly express bcl-6 protein in contrast to Th1 and Th2 cells that do notexpress these markers (40).

RA is another autoimmune disorder that has been recently studied with regard to Tfh celldysregulation. Increased frequency of CD4⁺ CXCR5⁺ICOS^high circulating Tfh cells was detected in the peripheral blood of RApatients, and this was positively correlated with high levels of serum anti-CCPantibody. Furthermore, increased expression of bcl-6 mRNA and plasma IL-21concentrations was observed in these patients (41). Increased serum IL-21 levels in RA patients correlate with disease activityscore, anti-CCP antibody titer and the frequency of circulating Tfh-like cells (42). In addition, Jang et al. (2009) reported thatIL-21 receptor-deficient K/BxN mice have less severe RA with reduced Tfh cell populationin draining lymph nodes (43). Platt et al. (44) found increased Tfh cells and antibodyproduction in an OVA-induced RA mouse model.

The involvement of activated Tfh cells in MS was recently demonstrated by Christensen etal. (45). This study was the first to reportprominent Tfh, Th17 and B-cell activation in the peripheral blood from patients withprogressive MS, and these findings parallel recent pathology studies. Tfh and B cellactivation correlated with disease progression and Tfh activation marker IL-21 wasdecreased in MS patients treated with mitoxantrone. Furthermore, there was increasedexpression of genes associated with Tfh and B cell activation in the cerebral-spinalfluid cells from MS patients. These findings emphasize an association between thesystemic immune compartment and disease progression in the protected central nervoussystem environment.

Dysregulated activation of both T and B lymphocytes with overt production ofauto-reactive antibodies is a hallmark of SLE. This prototypic systemic autoimmunedisease is characterized by various immunologic abnormalities, including the presence ofantibodies against double stranded DNA (dsDNA). Previous studies demonstrated that Bcell chemokine CXCL13 is highly expressed in the thymus and kidneys in murine models forSLE (46). These results are in accordance withthe study by Wong et al. (47), who showed thatthe significant increase in plasma concentration of CXCL13 in SLE patients correlatedsignificantly with SLE disease severity. Recently, Le Coz et al. (48) found an increased proportion of Tfh cells in SLE patients withactive disease. This increase was associated with key biological SLE parameters (totalimmunoglobulin serum levels and anti-dsDNA antibodies), B cell subset alterations andthe presence of high IgE levels (49).

The relationship between Tfh cells and autoantibody production is evident in severalmouse strains over or under expressing important Tfh cell-associated molecules such asICOS, CD40L, SAP and IL-21 (50,51). In mice homozygous to Roquin gene mutation(Rc3h1 mice), both naive and activated T cells express abnormally high levels of ICOS,which apparently contributes to the development of SLE-like disease and early-onsetdiabetes (52). The disruption of ICOS-ICOSLsignaling prevents autoantibody formation and organ inflammation in Rc3h1 mice and othermurine models of autoimmune diseases, such as SLE-like disease, collagen-inducedarthritis, and myasthenia gravis (53-56).

In addition, three murine lupus models (NZB\NZW, C57BL/6J (B6) and BXSB) and onearthritis model (collagen-induced arthritis) are known to be dependent on themaintenance of the ICOS pathway. They are characterized by a Tfh cell-liketranscriptome, with excessive numbers of Tfh cells and germinal centers (51,57).Humans who are deficient in ICOS develop common variable immunodeficiency, in whichthere is impairment in the development of memory B cells and immunoglobulin class switchdoes not occur (58), highlighting the importanceof the ICOS-ICOSL interaction for the development of an effective humoral response. Ithas been reported that ICOS signaling can stimulate Tfh cells to produce IL-10, whichhas been implicated (at high-levels) in the terminal differentiation of germinal centerB cells into plasma cells (19). In this context,it is relevant that circulating CD4 and CD8 T cells from SLE and rheumatoid arthritis(RA) patients and synovial fluid cells from RA patients show increased ICOS expression(49,59).

However, Tfh cells are not the single effector T cell subtype that determine thedominant pathway of high-affinity isotype-switched autoantibody production in murinemodels of lupus. In the MRL/MpJ-Fas lpr murine model of lupus, a subset ofCD162^lowCXCR4⁺ T cells, localized in extra-follicular sites,has been shown to mediate IgG production through IL-21 and CD40L.CD162^lowCXCR4⁺ T cells are abundant in other autoimmune murinemodels and can exhibit either a follicular or an extra-follicular phenotype (60). However, although isotype-switched autoantibodyproduction may occur outside germinal centers, the process is far less efficient (48).

Similarly to ICOS, CD40L and SAP are essential for B cell effective help, and deficiencyof either one results in a poor germinal-center humoral response with defectivedifferentiation of both long-lived effector memory and plasma cells (61,62). Inhuman patients and in mouse models of autoimmune diseases, an imbalanced expression ofthese molecules has been reported to contribute to the immune pathology, leading tomaturation and differentiation of long-term antibody-secreting cells and autoantibodyproduction (63). Some studies observed that theblockade of CD40L/CD40 interaction might be an efficient therapeutic approach toalleviate immunoglobulin secretion, autoantibody production and disease activity inlupus patients with proliferative glomerulonephritis (64,65). It was also reported that SAPdeficiency prevents the development of experimentally induced SLE-like disease. Anexcessive Tfh number in Roquin^san/san (sanroque) mice is associated withspontaneous germinal center development, autoantibody production and lupus-likeautoimmunity (66). In these mice, SAP deletioncaused a substantial reduction in Tfh frequency, IL-21 levels, as well as reduced ICOSexpression by Tfh cells. SAP deficiency also abrogated formation of germinal centers,autoantibody production and renal pathology in sanroque mice (67). Interestingly, the adoptive transfer of sanroque Tfh cellscaused spontaneous germinal center formation in wild type mice. Altogether, thesefindings indicate a noticeable causative link between Tfh dysfunction and systemicautoimmune pathways (66).

IL-21 implication

IL-21 has arisen as a powerful inducer of human B cell differentiation (68,69). Ithas been considered the most potent human T cell-derived cytokine for the induction of Bcell proliferation (69). In mice, this cytokineis largely produced by Th2, Th17, NKT and Tfh CD4 cells, however, in humans the T celltypes responsible for IL-21 production are less well characterized (70,71), andTfh cells are still considered one of the most important sources (70).

In mouse models, Tfh cells are a central source of IL-21 in germinal centers ofsecondary lymphoid organs, providing cognate help to B cells in the germinal centerdynamic microenvironment, acting specially on naive B cells to induce isotype switch toIgG and IgA (68). IL-21 also acts on B cells ofcord blood and peripheral blood inducing plasma cell differentiation (69), whereas IL-10, a well-known mediator of human Bcell differentiation, has the same effect on terminally differentiated B cells (72). The ability of IL-21 to induce differentiationof naive B cells into plasma cells suggests that IL-21 may have a major role in primaryresponses to antigens (73). One study withIL-21-transgenic mice and using hydrodynamic injection of IL-21 plasmid-basedmethodologies into wild-type mice showed that IL-21 induced apoptosis just in a subsetof mature B cells, but increased the number of immature and post switch B cells (74). Thus, it appears that IL-21 differentiallyinfluences B cell fate depending on the signaling context and B cell differentiationstage. This would explain how IL-21 can be pro-apoptotic for B cells in some invitro experiments and yet critical for Ag-specific immunoglobulin productionin vivo. In transgenic mice, IL-21 overexpression promotes thedifferentiation of activated B cells into plasma cells and unexpectedly inducesexpression of both Blimp-1 and bcl-6, indicating mechanisms by which IL-21 can serve asa complex regulator of B cell maturation and terminal differentiation. BXSB-Yaa mice,which develop SLE-like disease, have an increased serum expression of IL-21, suggestinga possible role for IL-21 in the development of the autoimmune disease in this animalmodel (74). Accordingly, other animal studieshave indicated that the production of autoantibodies and systemic autoimmunity isassociated with elevated production of IL-21, Tfh dysfunction within germinal centersand aberrant positive selection of germinal center B cells (50,66).

In human autoimmune diseases, IL-21 appears to have the potential to exacerbate cellularprocesses that determine the course of autoimmune response. In patients with RA, IL-21Ris overexpressed in the inflamed synovial membrane and in leukocytes from peripheralblood (PB) and synovial fluid (SF). In addition, PB and SF T cells from RA patients,when stimulated with IL-21 and anti-CD3 monoclonal antibody, secreted markedly higherlevels of TNF-α and IFN-γ than those from controls, indicating that IL-21 enhances localT-cell activation and pro-inflammatory cytokine secretion (75). The blockage of IL-21R signaling pathway may have a therapeuticpotential in RA patients. In fact, blockade of IL-21 and IL-15, cytokines belonging tothe common γ-chain receptor family, is effective to inhibit the release ofpro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in RA synovial cell cultures (76). On the other hand, another study showed thatIL-21R expression by fibroblasts and macrophages in RA synovium did not correlate withthe destruction of articular cartilage and bone (77).

IL-21R mRNA was up-regulated in keratinocytes and dermal fibroblasts in biopsy specimensfrom patients with systemic sclerosis (SSc; scleroderma). In addition, insitu hybridization and immunohistochemical analysis showed up-regulation ofIL-21R in samples of epidermis from SSc patients (78).

Polymorphism of IL-21 gene has also been reported to be associated with SLE (79), however it is not known whether thispolymorphism is functional. IL-21 implication in human SLE remains to be moreextensively investigated since in several murine models of SLE, IL-21 has been eitherdirectly or indirectly shown to be a contributing factor to disease (51,53).Recently, Dolff et al. (80) published the firststudy demonstrating increased proportions of circulating IL-21+ T-cells in SLE patients.Elevated plasma IL-21 in SLE is probably a result of Tfh cell activity in the formationof germinal centers (17).

In both Crohn's disease (CD) and ulcerative colitis (UC), the major forms ofinflammatory bowel diseases (IBD) in humans, high IL-21 production was related to thepathological process. High levels of IL-21 were observed in the inflamed colon of mostpatients with UC. In addition, IL-21 stimulated gut fibroblasts to secrete extracellularmatrix degrading enzymes and was involved in recruiting T cells to the inflamed gut byinducing MIP-3 α production by epithelial cells. Altogether, these data denote thatIL-21 is an important mediator of the chronic inflammatory response in CD and UC, andmight be a potential therapeutic target in IBD (81).

Concluding remarks

In vivo and in vitro experiments present considerableevidence that Tfh cells interact with B cells and play a critical role in the formationof germinal centers and in the development of T cell-dependent B cell response insecondary lymphoid tissues. Exaggerated expansion of Tfh cells results in excessivegerminal center reaction, self-reactive B cell proliferation, and excess long-livedplasma cells differentiation, as well as overproduction of high-affinity pathogenicautoantibodies. The pathological abundance of Tfh cells could provide a crucial help forthe cognate self-reactive B cells survival and escape from the tolerance checkpoints atthe germinal center. These observations suggest an important role for Tfh cells in humanautoimmunity. Alteration of Tfh cells have been reported in patients with variousautoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus andautoimmune thyroid diseases, where Tfh cells are present at increased frequency and showpositive correlation with serum autoantibody titer. Therefore, a better understanding ofthe biology and roles of Tfh cells is expected to contribute in designing tools toabrogate the inappropriate activity of these cells. The intervention by agentsselectively targeting specific signature molecules of Tfh cells, such as ICOS and IL-21,may prove to be therapeutically effective.