Understanding the Development and Function of T Follicular Helper Cells

REVIEW

Understanding the development and function of T follicular helper cells

Roza I Nurieva and Yeonseok Chung

A fundamental function of T helper (Th) cells is to regulate B-cell proliferation and immunoglobulin class switching, especially in the germinal centers. Th1 and Th2 lineages of CD41 T cells have long been considered to play an essential role in helping B cells by promoting the production immunoglobulin G2a (IgG2a) and IgG1/IgE, respectively. Recently, it has become clear that a subset CD41 T cells, named T follicular helper (Tfh) cells, is critical to B-cell response induction. In this review, we summarize the latest advances in our understanding of the regulation of Tfh cell differentiation, the relationship of Tfh cells to other CD41 T-cell lineages, and the role of Tfh cells in health and disease. Cellular & Molecular Immunology (2010) 7, 190–197; doi:10.1038/cmi.2010.24; published online 12 April 2010

Keywords: autoimmunity; Bcl6; costimulation; IL-21; T follicular helper (Tfh) cells

INTRODUCTION Consistent with this notion, CXCR5 has been shown to be important Upon encountering their cognate antigens on the surface of profes- for proper T- and B-cell localization in immune responses and during sional antigen-presenting cells (APCs), naive CD41 Thelper(Th)cells antibody production.9,10 Although activated T cells may transiently differentiate into various effector subsets, such as Th1, Th2, Th17 or express CXCR5, Tfh cells exhibit more stable expression of this che- regulatory T (Treg) cells. Th cell fate depends on microenvironmental mokine receptor. In addition to CXCR5, other Tfh cell markers have factors, including cytokine milieu (Figure 1). Each T-cell subset exerts been also reported, such as the costimulatory receptor—inducible cost- its unique effector functions by migrating to its target anatomic loca- imulatory molecule (ICOS), the cytokine IL-21 and the transcription tion, which is mediated by the expression of relevant chemokine recep- factor B-cell lymphoma 6 (Bcl6). ICOS is essential for generation and tors and production of signature cytokines.1–4 The development of maintenance of Tfh cells.11 Meanwhile, IL-21 produced by Tfh cells each of these Th cell subsets is driven by the following lineage-specific directly regulates B-cell proliferation and class switching, and IL-21R transcription factors: T-bet for Th1, GATA3 for Th2, retinoic acid- deficiency results in inadequate antibody responses and impaired ger- related orphan receptor-a (RORa) and RORc for Th17, and Forkhead minal-center formation.12 Moreover, sanroque mice, which have a box p3 (Foxp3) for Treg cells (Figure 1). The help that T cells provide mutation in the RING-type E3 ubiquitin ligase Roquin, developed to B cells is a critical component of normal immune responses. Th1 spontaneous autoantibody production and lupus-like autoimmunity, and Th2 cell lineages are essential to the regulation of B-cell prolifera- which is associated with greatly increased numbers of CXCR51CD41 tion and immunoglobulin class switching. For example, the Th1 cyto- T cells and enhanced IL-21 and ICOS expression.13 Despite the suc- kine interferon-c (IFN-c) regulates immunoglobulin G2a (IgG2a) cessful characterization of their surface markers and their potential production, while the Th2 cytokine interleukin-4 (IL-4) is critical to importance in humoral immunity and immunopathology, the devel- IgG1/IgE class switching5,6 and transforming growth factor-b (TGF-b) opmental regulation of Tfh cells is still being clarified. and IL-10 are implicated in the switch to IgA.7 Arecentstudyalso Over the past 2 to 3 years, there has been rapid and exciting progress suggested that IL-17 drives autoimmune responses by promoting the in our understanding of the developmental regulation of murine and spontaneous formation of germinal centers, implicating Th17 cells in human Tfh cells. In this review, we summarize the latest findings the regulation of autoreactive antibodies.8 regarding the origin of these cells, their developmental regulation by Recently, it has been elucidated that a subset of CD41 T cells known cytokines and transcription factors, and their relationship to other as T follicular helper (Tfh) cells, named for their localization in ger- effector CD41 T-cell subsets. Additionally, we review the function minal centers, are critical to germinal-center formation and function. of Tfh cells in the progression of autoimmune responses. In mice and humans, these cells are phenotypically different from other CD41 Th subsets. Differences include high-level expression of CXC IDENTIFICATION AND CHARACTERIZATION OF TFH CELLS receptor 5 (CXCR5), which leads to migration of Tfh cells to CXCL13- A fundamental function of Th cells is to provide help to B cells and to expressing B-cell follicles.6 These cells are then thought to regulate regulate their proliferation and immunoglobulin class switching, espe- humoral immunity and particularly germinal-center reactions. cially in the germinal centers. Follicle-associated CD41 T cells were

Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence: Dr RI Nurieva, Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. E-mail: [email protected] Received 5 February 2010; revised 12 March 2010; accepted 15 March 2010 Tfh cell generation and function RI Nurieva and Y Chung 191

Th1

Th2

Th17

Treg

Tfh

Figure 1 Effector Th cell differentiation. Upon encountering antigen presented by professional APCs, naive CD41 T cells differentiate into effector subsets (Th1, Th2, Th17, Treg and Tfh) that are characterized by their cytokine production, expression of transcription factors and chemokine receptors, and immune regulatory functions. APC, antigen-presenting cell; Bcl6, B-cell lymphoma 6; CCR, CC chemokine receptor; CXCR, CXC receptor; Foxp3, Forkhead box p3; IFN, interferon; ROR, retinoic acid-related orphan receptor; Tfh, T follicular helper; TGF, transforming growth factor; Th, T helper; Treg, regulatory T. first identified more than 20 years ago in the germinal centers of Tfh cells represent a distinct lineage.24 Genetic analysis further iden- human tonsil tissues.14 The best defining marker for human and tified molecules highly expressed by both mouse and human Tfh cells: mouse Tfh cells is CXCR5.15,16 Naive CD41 T cells do not express ICOS, CD40L, programmed death-1 (PD-1), B- and T-lymphocyte CXCR5 and use CC receptor 7 (CCR7) to enter secondary lymphoid attenuator, signaling lymphocytic activation molecule (SLAM)- organs and migrate into the T-cell zones. CXCR5 expression is then associated protein (SAP), IL-6R, IL-21R and Bcl6.6,23,24 Higher upregulated in CD41 T cells following activation. This upregulation is expression of these molecules was reported as necessary to both the dependent on costimulatory signals delivered by CD28, ICOS and development and maintenance of Tfh cells and their ability to provide OX40.11,17–19 However, CXCR5 expression by Th1 and Th2 cells is help to B cells. transient, so most fully differentiated Th cells do not express this For years, it was unclear whether Bcl6 was the master transcriptional receptor (Figure 2). A fraction of Th cells maintains CXCR5 express- regulator of Tfh cells. Recently, three groups including our own inde- ion and differentiates into Tfh cells, homes to the B-cell follicles, and pendently demonstrated that mice lacking Bcl6 are impaired in ger- provides help to B cells. In addition to sustained CXCR5 expression, minal-center formation and Tfh cell development.25–27 These studies downregulation of CCR7 may facilitate the migration of Tfh cells indicated that Bcl6 is necessary and sufficient for Tfh cell development. from the T-cell zones into the CXCL13-rich B-cell follicles.20,21 It was also posited that Bcl6 regulates Tfh cell differentiation by repres- Therefore, Tfh cells have been solely defined by their chemotaxis, sing alternative differentiation pathways, such as those of Th1-, Th2- dictated by CXCR5 expression, while their development is poorly and Th17-cell lineages. Taken together, these findings clearly identify characterized. Tfh cells as a distinct lineage of Th cells, with Bcl6 as a master regulator It has been suspected that a subset of Th1 and Th2 cells can function of their differentiation. as Tfh cells, rather than Tfh cells stemming from a specialized lineage for follicular helper function. However, gene expression analysis of DIFFERENTIATION OF TFH CELLS human Tfh cells (CD571CXCR5hi) revealed a unique expression pro- Naive T-cell activation and functional differentiation is triggered by file that is distinct from that of Th1 and Th2 cells.22,23 This finding was the innate immune system. In response to pathogens, dendritic cells further supported by the notion that in vivo generated mouse Tfh cells (DCs) upregulate costimulatory molecules to help prime antigen- do not express Th1-, Th2- and Th17-specific genes.24 More impor- specific T cells. In addition, APCs also secrete multiple cytokines that tantly, the generation of CXCR51 Tfh cells is independent of that of regulate T-cell terminal differentiation into effector T cells. Recent other Th cell lineages (Th1 (IFN-c, STAT1), Th2 (IL-4, STAT4), Th17 studies have established critical roles for costimulatory molecules (IL-17, IL-17F, RORa and RORc) and Treg (TGF-b)), suggesting that and certain cytokines in Tfh cell differentiation (Figure 2).

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Figure 2 Generation of Tfh cells. Activation of naive CD41 T cells by MHC-Ag and B7.1/B7.2 on APCs induces the expression of costimulatory molecules and the chemokine receptor CXCR5. Interaction of these cells with B cells facilitates further differentiation of Tfh cells. The interaction of B7h on B cells with ICOS on activated CXCR51 T cells leads to the upregulation of c-Maf on and subsequent IL-21 production by Tfh cells. IL-21 further sustains Tfh differentiation in an autocrine manner and upregulates the transcription factor Bcl6, which determines the final differentiation of these cells. APC, antigen-presenting cell; Bcl6, B-cell lymphoma 6; Blimp-1, B lymphocyte-induced maturation protein-1; CCR, CC chemokine receptor; CXCR5, CXC receptor 5; ICOS, inducible costimulatory molecule; MHC-Ag, major histocompatibility complex antigens; PD-1, programmed death-1; TCR, T-cell antigen receptor; Tfh, T follicular helper.

Costimulation humans and mice resulted in substantially reduced numbers of Tfh T-cell activation is regulated by costimulatory molecules. CD28, cells and profound defects in B-cell maturation and immunoglobulin which is expressed on naive T cells, is the most important costimula- isotype switching, indicating an essential role for ICOS in the differ- tory receptor involved in T-cell activation.28 In the absence of CD28 or entiation of Tfh cells.11,19 Interestingly, a mutation in the RING-type its ligands CD80 and CD86, Th cell differentiation into all lineages was E3 ubiquitin ligase Roquin in mice provoked spontaneous germinal- found significantly reduced. CD28 costimulation is also essential to center formation and enhanced autoantibody production, which is Tfh cell development, so CD28-deficient mice are linked with failed associated with greatly increased numbers of Tfh cells and enhanced upregulation of CXCR5 and OX40 on T cells and disrupted germinal- IL-21 and ICOS expression.13 center formation.17 However, CD28-mediated responses, the genera- Since B7h is constitutively expressed on B cells, we examined tion of Tfh cells and germinal-center formation become independent whether Tfh cell generation required cognate B–T-cell interaction.24 of CD28 and dependent on ICOS when the E3 ubiquitin ligase Roquin Analysis of a conditional knockout mouse deficient in B-cell express- is mutated.29 ion of B7h revealed that the absence of ICOS ligand from B cells leads ICOS belongs to the CD28 family and is expressed on activated T to a greatly reduced Tfh cell frequency. In addition to lower CXCR5 cells.30,31 while its ligand, B7h, is broadly expressed on APCs in non- expression, we found that IL-21 expression by T cells was greatly lymphoid tissues.32,33 The ICOS-B7h interaction is necessary for reduced in these mice. The ICOS-B7h interaction may thus regulate optimal T-cell activation as well as for certain effector functions, such Tfh cells via production of IL-21. We also observed that antigen- as IL-4 and IL-17 expression.34–38 In addition, ICOS plays an import- specific IgG production and Peanut agglutinin (PNA)1 germinal- ant role in the regulation of T cell-dependent antibody responses and center B cells were greatly reduced in the absence of B7h expression germinal-center reactions. Mice deficient in ICOS or B7h exhibit on B cells. Our data thus indicate that B7h expression on B cells is impaired germinal-center formation and isotype switching.34–37,39 necessary for the generation of CXCR51 Tfh cells, as well as for IL-21 Recently, this costimulatory pathway was also found to be important production and appropriate antibody responses, suggesting an for the generation and maintenance of CXCR51 Tfh cells. More spe- important in vivo function for B cells in the generation or maintenance cifically, ICOS-deficient mice showed impaired development of of Tfh cells. CXCR51 Tfh cells in response to primary or secondary immunization All of the above observations suggest that defects in Tfh cell with sheep red blood cells.6 Meanwhile, ablation of B7h on APCs development and germinal-center formation in ICOS-deficient mice resulted in decreased expression of IL-21 by T cells.24 It has also been and patients may be due in part to defective IL-21 production, which shown that ICOS is highly expressed by human tonsillar CXCR51 T usually acts as an autocrine factor that expands the Tfh cell population. cells within the light zone of germinal centers and efficiently supports In fact, Tfh cell numbers were reduced in IL-21-deficient mice. immunoglobulin production.40,41 Additionally, ICOS deficiency in A recent study presented evidence that ICOS promotes Tfh cell

Cellular & Molecular Immunology Tfh cell generation and function RI Nurieva and Y Chung 193 formation through upregulation of c-Maf expression, which enhanced reduction in CD41CXCR51 T cells, suggesting that IL-21 acts as an IL-21 expression in a dose-dependent manner.42 Thus, ICOS-induced autocrine fashion during Tfh cell development.24 In addition to Tfh c-Maf expression may support Tfh cell formation and maintenance via cells, significantly lowered numbers of germinal-center B cells and production of the autocrine growth factor IL-21. reduced IgG1 production were observed in immunized IL-21- SAP is expressed in T cells and mediates responses to members of deficient mice. Vogelzang et al. demonstrated that those defects were the SLAM family by binding to a conserved domain on those recep- T-cell, rather than B-cell intrinsic.55 tors. The SLAM family of receptors consists of seven members: It has been shown that IL-21 is induced by IL-6 in a STAT3- CD150/SLAM, CD48/SLAMF2, CD229/Ly9/SLAMF3, CD224/2B4/ dependent manner.24 However, unlike Th17 cells, Tfh cell generation SLAMF4, CD84/SLAMF5, NTB-A/Ly108/SLAMF6 and CD319/ was independent of TGF-b or the Th17-specific orphan nuclear recep- CRACC/SLAMF7. Mutation of SAP in humans, as well as studies tors RORa and RORc.24 Moreover, we have shown that IL-21- involving SAP-deficient mice, suggested a critical role for SAP in B- producing Th cells can be generated in vitro in the presence of IL-21 43 cell help and class switching in Th cells. Of note, SAP-deficient T cells but not TGF-b, and that these cells acquire the Tfh gene-expression exhibited delayed and decreased ICOS expression but elevated and profile.24 Transfer of these in vitro generated, IL-21-producing Tfh- 44 1 prolonged CD40L expression. SAP-deficient CD4 T cells also fail like cells into naive mice facilitated B-cell function, similar to Tfh cells. to stably interact with cognate B cells, therefore failing to induce B-cell Overall, these findings confirm the identity of Tfh cells as a separate Th 45 clonal expansion. However, these deficient T cells still express nor- lineage and suggest that IL-21 is important for the generation of Tfh mal levels of CXCR5. SAP thus plays a key role in Tfh cell-mediated cells and germinal-center reactions in mice. formation of germinal centers after encountering a cognate B cell, rather than regulating the Tfh cells’ migration. IL-12. Despite progress in characterization of the mechanisms under- CD84/SLAMF5 and Ly108/SLAMF6 are highly expressed in human 46,47 lying murine Tfh cell development, the developmental regulation of and mouse Tfh cells. Using CD84-deficient mice, Cannons et al. human Tfh cells remained unclear until recently. Two independent showed that CD84 is required for germinal-center formation and Tfh 47 studies have shown that IL-12 induces in vitro generation of human cell differentiation in vivo in a T cell-intrinsic manner. Therefore, the 1 46,56 Tfh-like cells from naive CD4 T cells. Tfh cells differentiating CD84-SAP axis is likely the canonical pathway in CD41 T cells that under the influence of IL-12 share key features with Tfh cells found in mediates Tfh cell development and function. the peripheral lymphoid tissues, including IL-21 production, sustained ICOS and CXCR5 expression, and the ability to assist B-cell Cytokines differentiation into immunoglobulin-secreting cells. Interestingly, Cytokines are major regulators of Th cell differentiation.1,3 Th1 dif- exposure of tonsillar, cord blood or adult peripheral blood naive T ferentiation is promoted by IL-12 through activation of STAT4. The cells to IL-12 (Th1 conditions) caused a significant increase in IL-21 IFN-c-STAT1 pathway in turn sustains Th1-cell development, leading expression, whereas IL-21, IL-6 and IL-23 induced IL-21 secretion to a to the induction of the transcription factor T-bet. Meanwhile, IL-4 is lesser extent.46 Schmitt et al. demonstrated that IL-12 triggers high secreted by activated T cells and drives Th2 polarization in a STAT6- levels of IL-21 production via STAT4 activation, while IL-21 and IL-23 dependent manner, resulting in activation of the transcription factor induce less IL-21 production in human CD41 T cells.56 It was also GATA3. Additionally, IL-6/IL-21, in combination with TGF-b, observed that IL-12-induced IL-21-producing cells included IFN-c1 induces Th17-cell differentiation via STAT3-dependent upregulation and IFN-c- subsets. of RORc. This cytokine-mediated Tfh cell differentiation, however, varies between mice and humans. It is unclear whether autocrine IL-21 and STAT3 activation play any role in the differentiation of IL-12-induced, IL-21-producing Th cells in humans. Interestingly, Ma et al. demonstrated that IFN-c1IL-212 IL-21. IL-21 plays a critical role in regulating immunoglobulin pro- 1 1 duction and germinal-center formation.12,48 This cytokine’s receptor and IFN-c IL-21 cells express high levels of T-bet, while generation of IFN-c2IL-211 cells is highly dependent on Bcl6 expression.46 belongs to the common c-chain family, which also includes the recep- 1 tors for IL-2, IL-4, IL-7, IL-9 and IL-15. IL-21 signals via STAT3 or Moreover, human naive CD4 T cells cultured in the presence of STAT5a to regulate the functions of a variety of cells types, such as T IL-12 express high levels of CXCR5, which may be important for the cells, B cells, natural killer cells and DCs, and is mainly expressed by localization of these cells to the B-cell follicles. In addition to CXCR5 CD41 T cells and natural killer T cells.49 Previously, it was also expression, IL-12 treatment also elevated levels of ICOS expression. reported that IL-21 is expressed at higher levels by Th2 than by Th1 More importantly, in vitro IL-12-primed, IL-21-secreting cells cells.50 In the latter case, the addition of IL-21 during differentiation induced significantly more immunoglobulin secretion by naive B cells. c 51 This effect was dependent on IL-21 and ICOS signaling but not on decreases IFN- production by repressing Eomes expression. It has 1 1 1 also been observed that IL-21 is induced by IL-6 by means of STAT3, IFN-c. Thus, human ICOS CXCR5 CD4 T cells generated in vitro and in combination with TGF-b, is necessary and sufficient for the in the presence of IL-12 share phenotypic and functional character- generation of Th17 cells via STAT3-dependent upregulation of istics with Tfh cells. IL-21 and IL-12 are therefore required for the RORc.52–54 Both IL-21- and IL-21R-deficient mice are defective in development of mouse and human Tfh cells, respectively. Th17 cell development, suggesting that IL-21 acts in an autocrine fashion, just as IFN-c does for Th1 cells and IL-4 does for Th2 cells. Type I IFNs. Type I IFNs represent a family of cytokines with antiviral Additionally, germinal-center development is impaired in IL-21- and immunomodulatory functions.57 Multiple subtypes of IFN-a and and IL-21R- deficient mice.12 In this context, as higher producers of a single IFN-b subtype are rapidly induced in response to infection and IL-21 than the Th1, Th2 and Th17 subsets, Tfh cells have a beneficial signal through a common IFN-a receptor.57 These IFNs have been impact on antibody class switch and germinal-center formation. Two shown to potently enhance the primary antibody response to a soluble groups have recently highlighted the role of IL-21 in the generation protein antigen, augmenting the production of all subclasses of of Tfh cells and germinal-center B cells.24,55 Immunization of IL-21- IgG.58,59 Additionally, type I IFNs promoted antibody responses when deficient mice with a T-cell-dependent antigen resulted in a dramatic wild-type DCs were transferred to IFN-a receptor-deficient mice,

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providing direct evidence that type I-IFN stimulation of DCs enhance cytokines.25 Bcl6 overexpression also leads to increased expression the in vivo immune response.58,59 of endogenous Bcl6 mRNA as well as IL-21R, IL-6R and CXCR5 A recent study revealed that type I-IFN signaling in DCs selectively mRNA, as is the case in cells treated with IL-6 or IL-21. Interes- stimulates Tfh cell development in response to antigen bound to Toll- tingly, IL-21 expression was not upregulated by Bcl6 overexpression. like receptor 3 or 4 agonists.60 Moreover, DC production of IL-6 is Bcl6 expression also was not required for the development of Th1, Th2 required for in vivo Tfh cell generation and antibody affinity matura- or Th17 cells, and in fact, Bcl6 overexpression repressed the production. Thus, type I IFNs enhance T cell-dependent antibody responses tion of Th1, Th2 and Th17 cytokines.25 by serving as a natural adjuvant for the generation of lymph node- Bcl6 function appears to be dependent on DNA binding.25,26 It resident CXCR51 Tfh cells. It remains to be determined what DC binds to the promoters of the transcriptional regulators T-bet and subset regulates the generation of Tfh cells. RORct, which determine Th1- and Th17-cell fate, respectively, result- Patients with systemic lupus erythematosus (SLE) have increased ing in repression of IFN-c and IL-17 production.26 Furthermore, Bcl6 IFN-a levels, which correlates with disease severity.61 In addition, suppresses expression of microRNAs that are thought to inhibit the studies in various lupus-prone mouse strains have revealed the critical Tfh cell signature, including miR-17-92, which represses CXCR5 role of type I IFNs in lupus-like pathology.62–64 An increased number expression. Thus, Bcl6 regulates Tfh cell development via repression of Tfh cells has also been observed in mouse models of SLE,6,65 where of Th1-, Th2- and Th17-specific transcription factors. Bcl6 deficiency they are required for lupus-like pathology.66 However, type I IFN- in T cells resulted in impaired Tfh cell development both in vitro and dependent generation of Tfh cells during an autoimmune response in vivo, and deficiency in both B and T cells is required for germinal- has not yet been addressed and requires further investigation. center reactions.25,26,28 Taken together, these results demonstrate that Bcl6 is both Transcription factors necessary and sufficient for Tfh cell development and provide further The differentiation of naive CD41 T cells into different lineages is evidence supporting the idea that Tfh cells are a distinct lineage of determined by cytokine signaling and subsequent activation of specific Th cells. transcription factors (Figure 1). For example, IFN-c/IL-12 signaling via STAT1/STAT4 regulates T-bet transcription in Th1 cells,67 while c-Maf. c-Maf was the first transcription factor identified to be pre- IL-4 signaling through STAT6 activates GATA3 expression in Th2 ferentially expressed in Th2 cells.75 It binds to the IL-4 proximal pro- cells.68 IL-6 and IL-21 both act via STAT3, leading to upregulation moter75 and serves as a Th2-specific factor. T cells deficient in c-Maf of the expression of the two Th17-specific orphan nuclear receptors were impaired in IL-4 expression during the effector phase, while the RORc and RORa, which ultimately determine Th17-cell terminal expression of other Th2 cytokines was either moderately reduced or differentiation.3 For Tfh cell differentiation it has been shown that unaffected.76 Previously, using ICOS- and B7h-deficient mice, Dong neither Th1-specific (STAT4, T-bet) nor Th2-selective (STAT6, et al. described mechanisms by which ICOS mediates IL-4 production GATA3) transcription factors are required.24 Despite the fact that both by regulating c-Maf expression in effector Th cells.36,77 Along these Th17 and Tfh lineages require IL-6/IL-21 and STAT3, Tfh cell lines, the phenotype of c-Maf knockout mice is similar to that of development does not require Th17-related transcription factors ICOS-deficient mice. Moreover, c-Maf overexpression restores IL-4 (RORa and RORc). Rather, Bcl6 is the master transcriptional regu- release by T cells activated in the absence of the ICOS-B7h interaction. lator of Tfh cell differentiation. In addition to defective IL-4 production, ICOS- and B7h-deficient mice are impaired in Tfh cell generation and IL-21 production.24 STAT3. IL-21 has been reported to be necessary for Tfh cell differ- Kuchroo et al. recently demonstrated that Tfh cells are characterized entiation24 and is induced in T cells by IL-6 in a STAT3-dependent by high c-Maf expression and that c-Maf enhanced IL-21 levels in a manner.52 To determine whether STAT3 signaling is required for Tfh dose-dependent manner.42 Meanwhile, deletion of c-Maf led to a cell development, Nurieva et al. analyzed Tfh cell generation in Stat3f/f reduced number of IL-21-expressing Tfh cells.42 ICOS thus regulates mice crossed with CD4-cre mice.24 Immunization of these mice with the differentiation of IL-21-producing Tfh cells via upregulation of keyhole limpet hemocyanin in complete Freund’s adjuvant revealed a c-Maf expression. great reduction in CXCR51 Tfh cells in the absence of STAT3. Moreover, STAT3 deficiency in T cells led to defective germinal-center IFN regulatory factor 4 (IRF4). IRF4 was originally described as a Th2- B-cell generation and reduced production of keyhole limpet hemo- specific transcription factor,78,79 in part since IRF4-deficient mice cyanin-specific IgG and IgM. Thus, STAT3 plays an important role in show significantly reduced levels of serum immunoglobulin.80 Tfh cell differentiation. However, recent studies revealed that this transcription factor is induced by IL-1 signaling and is necessary for Th17-cell differenti- Bcl6. The transcription factor Bcl6 is selectively expressed by mouse ation.81,82 IRF4-deficient T cells, on the other hand, fail to induce and human Tfh cells.69,70 It was previously shown to inhibit Th2 the Th17 lineage upon IL-21 and TGF-b stimulation.83 Moreover, T responses by blocking STAT6 binding to DNA,71,72 while Bcl6- cells lacking IRF4-binding protein, a natural antagonist of IRF4, deficient mice exhibit multi-organ inflammatory diseases, enhanced exhibit elevated IL-21 production.84 IRF4 is thus critical for the pro- IgE production, and defective germinal-center reactions.71,73 duction of IL-21 as well as for IL-21-mediated Th cell differentiation. However, it was unclear whether the germinal-center defect in these Given these findings, it seems that IRF4 may also play a role in Tfh mice was caused by a lack of proper T- and/or B-cell function since cell differentiation. Indeed, IRF4-deficient mice generate fewer germinal-center B cells also express Bcl6.74 Recently, our group and CXCR51ICOS1 Tfh cells upon immunization,85 indicating the others showed that the transcription factor Bcl6 is a master regulator importance of IRF4 during in vivo Tfh cell differentiation. It remains of Tfh cell differentiation.25–27 to be determined whether IRF4-dependent Tfh cell generation is T-cell Bcl6 expression is induced by IL-6 and IL-21 and overexpression of intrinsic and whether IRF4 regulates Bcl6 and B lymphocyte-induced Bcl6 promotes Tfh cell development in the absence of exogenous maturation protein-1 (Blimp-1) during Tfh lineage development.

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Negative regulation. Tfh cell differentiation is regulated by a program Foxp31 T cells into a CD3e2 recipient led to generation of Tfh-like that antagonizes the development of other Th lineages by suppressing cells, expressing CXCR5, Pde3b, IL-21 and Bcl6, in the Peyer’s patches. lineage-specific transcription factors (T-bet, GATA3 and RORc). These transferred Foxp31 T cells also induced germinal-center forma- However, it has not been reported whether these factors also negatively tion and IgA synthesis in the Peyer’s patches. By contrast, the same regulate Tfh cell differentiation. Blimp-1, which was previously cells could not differentiate into Tfh cells in secondary lymphoid described as antagonizing Bcl6 in B cells,86 is significantly reduced organs, suggesting that the gut microenvironment forces Treg cells in Tfh cells as compared with non-Tfh CD41 T cells.27 Overex- to redifferentiate into the Tfh lineage. The relationship between pression of Blimp-1 in CD41 T cells thus prevents Bcl6 expression Treg and Tfh cells and the plasticity of these two lineages remains to and significantly reduces the differentiation of Tfh cells, without be further investigated. affecting Th2-, Th17- and Treg-cell development. Moreover, Blimp- 1 1-deficient CD4 T cells showed enhanced differentiation into the Tfh TFH CELLS IN THE PATHOGENESIS OF AUTOIMMUNE 27 lineage. DISEASES Production of high-affinity pathogenic antibodies appears to be cent- TFH CELLS AND OTHER TH SUBSETS ral to most autoimmune disorders. Because normal high-affinity Despite the potential importance of Tfh cells in humoral immunity, antibodies arise from the germinal centers, aberrant selection of their relationship with other Th cell subsets is still unclear. Gene germinal-center B cells, caused by either failed negative selection or microarray analysis of human and mouse Tfh cells suggests that Tfh enhanced positive selection by Tfh cells, results in the production of cells have a unique expression profile, as they lack expression of most autoantibodies that cause autoimmunity. Indeed, the correlation cytokines and transcription factors associated with the Th1, Th2 and between an increased number of Tfh cells and autoimmunity has been Th17 lineages.23,24 Moreover, generation of mouse Tfh cells in vivo is described in a lupus model, suggesting that aberrant Tfh cell expansion independent of IFN-c, IL-4, IL-17A, IL-17F, TGF-b, STAT4, STAT6, can lead to the formation of autoantibodies and a lupus-like pheno- RORa and RORc.24 type.13,91 Mice homologous for the sanroque allele of Roquin, which Recent work from several groups indicates that CD41 T cells encodes the RING-type ubiquitin ligase, develop a lupus-like disease expressing markers specific to the Tfh cell subset are able to secrete accompanied by spontaneous development of germinal centers and an IL-4, IFN-c and IL-17. Two different groups have used IL-4 reporter excessive number of Tfh cells.13,66 Dysregulation of germinal-center mice to demonstrate a relationship between Th2 and Tfh cells during responses, leading to autoimmunity, in Roquinsan/san mice was caused Leishmania major and parasite infections, which induce a strong Th2- by excessive Tfh cell differentiation. Loss of one allele of Bcl6, the dominant host response.87,88 Specifically, Locksley et al. located IL-4- master transcriptional regulator of Tfh cells, significantly reduced expressing Th cells in the B-cell follicles and germinal centers during the number of germinal-center cells and minimized the lupus pheno- Leishmania infection. These cells predominantly expressed Tfh- type.66 In addition, SAP deficiency in Roquinsan/san mice abrogated specific markers, such as CXCR5, ICOS, SAP, IL-21 and Bcl6.87 Addi- spontaneous Tfh cell and germinal-center formation and reduced tionally, some IFN-c-producing Tfh cells were observed in the renal pathology.66 germinal centers after Leishmania infection.87 Using the Nippo- By contrast, adoptive transfer of Tfh cells from Roquinsan/san mice strongylus brasiliensis infection model, this group also showed that into wild-type recipient mice led to spontaneous germinal-center IL-4-secreting Tfh cells are distinct from conventional Th2 cells on formation. Interestingly, excessive Tfh cell generation and lupus the basis of phenotype, location and function. By contrast, some have pathogenesis in this model may be more dependent on ICOS than suggested that Tfh cells can differentiate from the Th2-cell lineage and on IL-21. Using the New Zealand black/New Zealand white mouse that this process is germinal center-dependent.88 Consistent with this model of SLE, Hu et al. demonstrated that blockade of ICOS-B7h hypothesis, after helminth infection the lymphoid organs of IL-4 signaling leads to decreased numbers of Tfh cells and germinal-center reporter mice contain an IL-4-expressing Tfh population, which dis- B cells and inhibition of autoimmunity.92 There is evidence that plays Tfh markers (IL-21, Bcl6, CXCR5, PD-1 and ICOS) as well as patients with SLE have increased numbers of circulating Th2 markers (GATA3).88 CXCR51ICOShiPD-1hiCD41 T cells in the peripheral blood, as com- In addition to evidence supporting the existence of a Tfh cell popu- pared to non-autoimmune individuals.93,94 This cell population was lation expressing IL-4 and IFN-c, several studies have shown that correlated with disease severity, indicated by higher titers of anti- during autoimmune disease, CD41 T cells with such Tfh markers as double-stranded DNA antibodies and more severe kidney damage. CXCR5 and ICOS are able to express IL-17.8,42,89 This cytokine accel- Thus, as in Roquinsan/san mice, dysregulation of Tfh cell formation erated germinal-center formation and antibody production. These may contribute to autoimmune pathology in SLE patients. studies suggest that Tfh cells can produce Th1-, Th2- and Th17- Overexpression of Tfh-specific cytokines has been shown to cause associated cytokines and are potentially derived from these lineages. autoimmunity. Previous work has demonstrated that BXSB-Yaa mice, Are there subsets of Tfh cells such as Th1-type, Th2-type or Th17-type a model of SLE, have elevated IL-21 DNA and serum protein levels, Tfh cells? Or are Th1, Th2 and Th17 cells able to redifferentiate into which correlates with more severe disease.95 Similarly, human studies Tfh cells while maintaining their cytokine profiles? At this stage, it is suggest that allelic variation in the IL-21 gene is a risk factor for SLE.96 difficult to explore these cells’ development due to the inadequacies of To determine whether IL-21 plays a significant role in the pathogenesis available reagents and a lack of distinct Tfh cell markers, since most of this disease, Bubier et al. compared IL-21R-deficient and IL-21R- Tfh markers also serve as activation markers of other Th lineages. Use competent BXCB-Yaa mice for multiple parameters of SLE-like auto- of Bcl6 reporter mice will help to distinct Tfh cell from other Th immune disease.97 IL-21 signaling appeared to be significant for the subsets and provide a clear answer to these critical questions. development of an SLE phenotype, as IL-21R-deficient mice lacked Interestingly, recent studies by Fagarasan et al. demonstrated that such SLE symptoms as autoantibody production and renal disease. Foxp31 T cells in the gut may be efficiently differentiated into cells Interestingly, in this model, the germinal-center Tfh cells and extra- with Tfh-like characteristics.90 More specifically, adoptive transfer of follicular population of ICOS1CD41 T cells produced IL-21. In the

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MRLlpr mouse model of lupus, the extrafollicular population of 17 Walker LS, Gulbranson-Judge A, Flynn S, Brocker T, Raykundalia C, Goodall M et al. hi 1 98 Compromised OX40 function in CD28-deficient mice is linked with failure to develop ICOS CD4 T cells is also a potent IL-21 producer. Thus, patho- CXC chemokine receptor 5-positive CD4 cells and germinal centers. J Exp Med 1999; genic IL-21 secretion is not restricted to germinal-center Tfh cells, but 190: 1115–1122. can also occur outside of the follicles. The IL-21 signaling pathway is 18 Brocker T, Gulbranson-Judge A, Flynn S, Riedinger M, Raykundalia C, Lane P. CD4 T cell traffic control: in vivo evidence that ligation of OX40 on CD4 T cells by OX40- therefore a promising target in the development of immunotherapy ligand expressed on dendritic cells leads to the accumulation of CD4 T cells in B against both Tfh- and Th17-mediated disorders. follicles. Eur J Immunol 1999; 29: 1610–1616. 19 Bossaller L, Burger J, Draeger R, Grimbacher B, Knoth R, Plebani A et al.ICOS deficiency is associated with a severe reduction of CXCR51CD4 germinal center Th CONCLUDING REMARKS cells. J Immunol 2006; 177: 4927–4932. Great advances in our understanding of Tfh cell differentiation and 20 Hardtke S, Ohl L, Forster R. Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is involvement in immune responses have been made over the past few essential for efficient B-cell help. Blood 2005; 106: 1924–1931. years. However, the signaling events required for commitment of 21 Haynes NM, Allen CD, Lesley R, Ansel KM, Killeen N, Cyster JG. Role of CXCR5 and CD41 Th cells to the Tfh subset are only beginning to be elucidated. CCR7 in follicular Th cell positioning and appearance of a programmed cell death gene-1high germinal center-associated subpopulation. J Immunol 2007; 179: Expression of IL-21 and Bcl6 is absolutely required for Tfh cell gen- 5099–5108. eration in both mice and humans, although the precise mechanisms 22 Kim CH, Lim HW, Kim JR, Rott L, Hillsamer P, Butcher EC. Unique gene expression controlling the expression of these major Tfh factors have yet to be program of human germinal center T helper cells. Blood 2004; 104: 1952–1960. 23 Chtanova T, Tangye SG, Newton R, Frank N, Hodge MR, Rolph MS et al. T follicular resolved. Additionally, the relationship of Tfh cells to other Th lineages helper cells express a distinctive transcriptional profile, reflecting their role as non- needs to be further characterized. Future identification and analysis of Th1/Th2 effector cells that provide help for B cells. J Immunol 2004; 173:68–78. new signaling components required for Tfh cell generation using both 24 Nurieva RI, Chung Y, Hwang D, Yang XO, Kang HS, Ma L et al. Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell traditional and new technologies, such as gene knockout and small lineages. Immunity 2008; 29: 138–149. interfering RNA-based gene knockdown, may advance our knowledge 25 Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD et al. Bcl6 of Tfh cell biology in health and disease. These technologies may also mediates the development of T follicular helper cells. Science 2009; 325: 1001–1005. provide a novel strategy for developing therapeutic agents that selec- 26 Yu D, Rao S, Tsai LM, Lee SK, He Y, Sutcliffe EL et al. The transcriptional repressor tively modulate Tfh cell function to treat autoimmunity. Bcl-6 directs T follicular helper cell lineage commitment. Immunity 2009; 31: 457–468. 27 Johnston RJ, Poholek AC, DiToro D, Yusuf I, Eto D, Barnett B et al. 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