Bcl6-Mediated Transcriptional Regulation of Follicular Helper T cells (TFH)

B cell help, afforded by T cells, is an essential process of adaptive immunity.The transcription factor B cell lymphoma 6 (Bcl6), by serving as an apex of a repressor-of-repressors network, can provide further insights into follicular helper T cell (TFH) regulation in mice and humans.Bcl6 is essential for TFH differentiation and harbors various significant activities, including inhibition of Prdm1 expression.We posit that Bcl6 can control non-TFH and TFH genes by at least two modes of action: direct repression and repression-of-repressor mechanisms. Follicular helper T cells (TFH) are essential B cell-help providers in the formation of germinal centers (GCs), affinity maturation of GC B cells, differentiation of high-affinity antibody-producing plasma cells, and production of memory B cells. The transcription factor (TF) B cell lymphoma 6 (Bcl6) is at the center of gene regulation in TFH biology, including differentiation and function, but how Bcl6 does this, and what additional TFs contribute, remain complex questions. This review focuses on advances in our understanding of Bcl6-mediated gene regulation of TFH functions, and the modulation of TFH by other TFs. These advances may have important implications in deciphering how repressor TFs can regulate many immunological cell types. An improved understanding of TFH biology will likely provide insights into biomedically relevant diseases. Follicular helper T cells (TFH) are essential B cell-help providers in the formation of germinal centers (GCs), affinity maturation of GC B cells, differentiation of high-affinity antibody-producing plasma cells, and production of memory B cells. The transcription factor (TF) B cell lymphoma 6 (Bcl6) is at the center of gene regulation in TFH biology, including differentiation and function, but how Bcl6 does this, and what additional TFs contribute, remain complex questions. This review focuses on advances in our understanding of Bcl6-mediated gene regulation of TFH functions, and the modulation of TFH by other TFs. These advances may have important implications in deciphering how repressor TFs can regulate many immunological cell types. An improved understanding of TFH biology will likely provide insights into biomedically relevant diseases. Vaccines are among the most cost-effective treatments in modern times [1.Piot P. et al.Immunization: vital progress, unfinished agenda.Nature. 2019; 575: 119-129Crossref PubMed Scopus (32) Google Scholar], and most current human vaccines function by eliciting protective antibody (Ab) responses. Indeed, a key aspect of adaptive immunity to many pathogens and vaccines is the B cell help provided by T cells. As such, follicular helper T cells (TFH) are specialized B cell-help facilitators [2.Crotty S. Follicular helper CD4 T cells (TFH).Annu. Rev. Immunol. 2011; 29: 621-663Crossref PubMed Scopus (1698) Google Scholar]. In humans and mouse models, there is compelling evidence that TFH cells frequently limit the magnitude of germinal center (GC, see Glossary) responses and, hence, limit the production of GC-derived high-affinity Abs, memory B cells, and long-lived plasma cells, which constitute the basis of long-lived protective humoral immunity [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. Therefore, an improved understanding of TFH cell biology can enhance our understanding of antipathogen immune responses and vaccine-elicited humoral immunity, as well as enable strategies to mitigate autoimmunity in numerous contexts [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. B cell lymphoma 6 (Bcl6), initially identified as the master regulator of GC B cell differentiation [4.Basso K. Favera R.D. Roles of BCL6 in normal and transformed germinal center B cells.Immunol. Rev. 2012; 247: 172-183Crossref PubMed Scopus (237) Google Scholar], is now recognized as the lineage-defining transcription factor (TF) for TFH differentiation [5.Johnston R.J. et al.Bcl6 and Blimp1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (0) Google Scholar, 6.Nurieva R.I. et al.Bcl6 mediates the development of T follicular helper cells.Science. 2009; 325: 1001-1005Crossref PubMed Scopus (926) Google Scholar, 7.Yu D. et al.The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.Immunity. 2009; 31: 457-468Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar]. Work by several laboratories established that the differentiation and function of TFH cells depend on the expression of the transcriptional repressor Bcl6 [5.Johnston R.J. et al.Bcl6 and Blimp1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (0) Google Scholar, 6.Nurieva R.I. et al.Bcl6 mediates the development of T follicular helper cells.Science. 2009; 325: 1001-1005Crossref PubMed Scopus (926) Google Scholar, 7.Yu D. et al.The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.Immunity. 2009; 31: 457-468Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar]. The generation of TFH is completely impaired in mouse Bcl6−/− CD4+ T cells [5.Johnston R.J. et al.Bcl6 and Blimp1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (0) Google Scholar, 6.Nurieva R.I. et al.Bcl6 mediates the development of T follicular helper cells.Science. 2009; 325: 1001-1005Crossref PubMed Scopus (926) Google Scholar, 7.Yu D. et al.The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.Immunity. 2009; 31: 457-468Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar], and enforced Bcl6 expression by retroviral transduction in CD4+ T cells elicits robust TFH differentiation in mice [5.Johnston R.J. et al.Bcl6 and Blimp1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (0) Google Scholar]. Recent work showed that tamoxifen-mediated temporal Bcl6 ablation in CD4+ T cells (Bcl6fl/fl CD4-CreERT2) resulted in the transition of already established TFH cells into T helper type 1 (TH1) cells during acute lymphocytic choriomeningitis virus (LCMV) infection (Armstrong strain) in mice [8.Alterauge D. et al.Continued Bcl6 expression prevents the transdifferentiation of established Tfh cells into Th1 cells during acute viral infection.Cell Rep. 2020; 33: 108232Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]; this suggested that Bcl6 is essential not only for the development of TFH cells, but also for the maintenance of TFH integrity [8.Alterauge D. et al.Continued Bcl6 expression prevents the transdifferentiation of established Tfh cells into Th1 cells during acute viral infection.Cell Rep. 2020; 33: 108232Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. This is important because continuous help signals from TFH are crucial for the maintenance of GCs [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. While TFH cells have been extensively studied, how Bcl6 accomplishes its functional task as a lineage-defining TF has remained a longstanding knowledge gap. Elucidating immunobiology underlying the differentiation of TFH and the process of generating protective antiviral antibody responses may facilitate future vaccine development. In this review, we discuss recent advances concerning Bcl6-mediated transcriptional regulation of TFH cells, including the demonstration that genes positively associated with a cell type can be upregulated downstream of a lineage-defining TF located at the apex of a repressor-of-repressors network. The up-to-date TF network in TFH differentiation is also summarized, aiming to increase our understanding of the gene regulatory circuitry of TFH cells. TFH differentiation is a multifactorial and multistage process [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar,9.Vinuesa C.G. et al.Follicular helper T cells.Annu. Rev. Immunol. 2016; 34: 335-368Crossref PubMed Scopus (441) Google Scholar]. Our group, and others, have shown that TFH differentiation begins at the time of dendritic cell (DC) priming in mice [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. It initiates with activation of a naïve CD4+ T cell receiving signals in the form of peptide:MHC, co-stimulatory molecules, and cytokines provided by an antigen-presenting cell (APC) such as a DC (Figure 1). Inducible T cell co-stimulator (ICOS) and interleukin-6 receptor (IL-6R) provide signals for early TFH differentiation in mice, both of which contribute to Bcl6 expression [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar,9.Vinuesa C.G. et al.Follicular helper T cells.Annu. Rev. Immunol. 2016; 34: 335-368Crossref PubMed Scopus (441) Google Scholar]. Helper T cell fate decision in vivo is determined as early as the second cell division, as cells become either Bcl6+ CD4+ T cells (further differentiate to TFH) or Blimp1+ CD4+ T cells (further differentiate to a non-TFH fate) [10.Choi Y.S. et al.ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor Bcl6.Immunity. 2011; 34: 932-946Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 11.Johnston R.J. et al.STAT5 is a potent negative regulator of TFH cell differentiation.J. Exp. Med. 2012; 209: 243-250Crossref PubMed Scopus (314) Google Scholar, 12.Baumjohann D. et al.Cutting edge: distinct waves of BCL6 expression during T follicular helper cell development.J. Immunol. 2011; 187: 2089-2092Crossref PubMed Scopus (151) Google Scholar, 13.Choi Y.S. et al.Bcl6 expressing follicular helper CD4 T cells are fate committed early and have the capacity to form memory.J. Immunol. (Baltimore, Md. : 1950). 2013; 190: 4014-4026Crossref PubMed Scopus (0) Google Scholar]. There is a reciprocal antagonistic relationship between Bcl6 and Blimp1 (encoded by Prdm1); Bcl6 represses Prdm1, and Blimp1 represses Bcl6 [5.Johnston R.J. et al.Bcl6 and Blimp1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (0) Google Scholar,14.Crotty S. et al.Effectors and memories: Bcl6 and Blimp1 in T and B lymphocyte differentiation.Nat. Immunol. 2010; 11: 114-120Crossref PubMed Scopus (0) Google Scholar]. TFH cells accomplish the inhibition of Prdm1 by Bcl6 in coordination with transcription factor 1 (Tcf1; encoded by Tcf7) and additional TFs [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar,15.Choi Y.S. et al.LEF-1 and TCF-1 orchestrate TFH differentiation by regulating differentiation circuits upstream of the transcriptional repressor Bcl6.Nat. Immunol. 2015; 16: 980-990Crossref PubMed Scopus (153) Google Scholar, 16.Xu L. et al.The transcription factor TCF-1 initiates the differentiation of TFH cells during acute viral infection.Nat. Immunol. 2015; 16: 1-11Crossref PubMed Scopus (110) Google Scholar, 17.Wu T. et al.TCF1 is required for the T follicular helper cell response to viral infection.Cell Rep. 2015; 12: 2099-2110Abstract Full Text Full Text PDF PubMed Google Scholar], as described later. Cognate B cells interact with TFH cells through peptide:MHC, CD40L, CD80/CD86, and SLAM receptors, and provide additional signals to TFH cells to drive GC-TFH differentiation [3.Crotty S. T follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. GC-TFH cells can be identified by several markers, including C-X-C motif chemokine receptor 5 (CXCR5)hi, Bcl6hi, programmed cell death protein-1 (PD-1)hi, SLAM-associated protein (SAP)hi, ICOS+ B and T lymphocyte-associated protein (BTLA)hi, and CD200hi in humans and mice (Figure 1) [2.Crotty S. Follicular helper CD4 T cells (TFH).Annu. Rev. Immunol. 2011; 29: 621-663Crossref PubMed Scopus (1698) Google Scholar]. Almost all of these core signature markers of GC-TFH cells are conserved across species [18.Weinstein J.S. et al.Global transcriptome analysis and enhancer landscape of human primary T follicular helper and T effector lymphocytes.Blood. 2014; 124: 3719-3729Crossref PubMed Scopus (33) Google Scholar, 19.Havenar-Daughton C. et al.CXCL13 is a plasma biomarker of germinal center activity.Proc. Natl. Acad. Sci. U. 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GC-TFH cells are commonly limiting for GCs [5.Johnston R.J. et al.Bcl6 and Blimp1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (0) Google Scholar,23.Victora G.D. et al.Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter.Cell. 2010; 143: 592-605Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 24.Rolf J. et al.Phosphoinositide 3-kinase activity in T cells regulates the magnitude of the germinal center reaction.J. Immunol. Baltim Md 1950. 2010; 185: 4042-4052Google Scholar, 25.Baumjohann D. et al.Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype.Immunity. 2013; 38: 596-605Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar], indicating that focusing on TFH biology may be an effective way to enhance vaccine-elicited humoral immunity. In addition, TFs are central controllers of T cell differentiation and function, as described later. Multiple models of TFH differentiation have been suggested [2.Crotty S. Follicular helper CD4 T cells (TFH).Annu. Rev. Immunol. 2011; 29: 621-663Crossref PubMed Scopus (1698) Google Scholar]. One proposed model suggested that TFH fate is the default differentiation pathway of activated CD4+ T cells [2.Crotty S. Follicular helper CD4 T cells (TFH).Annu. Rev. Immunol. 2011; 29: 621-663Crossref PubMed Scopus (1698) Google Scholar]. In this model, Blimp1+ CD4+ T cells potentially differentiate into non-TFH effector cells, TH1, TH2, or TH17, or regulatory T cells (TREG), through repression of the default TFH pathway by Blimp1. The primary role of Bcl6 in that model would be to repress Blimp1, blocking non-TFH differentiation pathways and, thus, releasing the default TFH differentiation pathway [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. This hypothesis was recently tested using multiple genetically modified mice [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. If TFH were the default pathway of CD4+ T cell differentiation, CD4+ T cells might be presumed to differentiate into TFH cells in the absence of both Bcl6 and Prdm1 [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. However, TFH differentiation did not occur when Bcl6/Prdm1 double-deficient CD4+ T cells (Bcl6fl/fl Prdm1fl/fl CD4-Cre) were primed in mouse LCMV infection and KLH-gp61 immunization models [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. Furthermore, GCs did not develop in CD4-specific Bcl6-deficient mice (Bcl6fl/fl CD4-Cre, devoid of endogenous TFH development) when receiving Bcl6fl/fl Prdm1fl/fl CD4-Cre CD4+ T cells, indicating that Bcl6/Prdm1 double-deficient CD4+ T cells cannot differentiate into functional TFH cells. Similar observations were made using Bcl6fl/fl Prdm1fl/fl CD4-Cre mice in a sheep red blood cells (SRBC) injection model [27.Xie M.M. et al.Bcl6 promotes follicular helper T-cell differentiation and PD-1 expression in a Blimp1-independent manner in mice.Eur. J. Immunol. 2017; 47: 1136-1141Crossref PubMed Scopus (19) Google Scholar]. These results demonstrated that TFH was not the default fate of activated CD4+ T cells [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. This was also relevant because it indicated that Bcl6 had important activities beyond inhibiting Prdm1 to drive TFH differentiation [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. How does Bcl6 regulate TFH differentiation and function? Bcl6, as an obligate repressor in mouse B cells [28.Mendez L.M. et al.CtBP is an essential corepressor for BCL6 autoregulation.Mol. Cell. Biol. 2008; 28: 2175-2186Crossref PubMed Scopus (46) Google Scholar, 29.Hatzi K. et al.A hybrid mechanism of action for BCL6 in B cells defined by formation of functionally distinct complexes at enhancers and promoters.Cell Rep. 2013; 4: 578-588Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 30.Huang C. et al.Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms.Nat. Immunol. 2013; 14: 380-388Crossref PubMed Scopus (74) Google Scholar, 31.Pasqualucci L. et al.Mutations of the BCL6 proto-oncogene disrupt its negative autoregulation in diffuse large B-cell lymphoma.Blood. 2003; 101: 2914-2923Crossref PubMed Scopus (214) Google Scholar], might control non-TFH and TFH genes by at least two repressor modes of action: (i) direct repression; and (ii) repression-of-repressor mechanisms. Genes downregulated in TFH cells (and genes upregulated in non-TFH cells) are likely controlled by the direct repression mechanism because Bcl6 is highly expressed in TFH cells. This category of genes could include lineage-defining TFs for alternative cell fates, cytokines, and cytokine receptors expressed by non-TFH cells, as well as migration-associated genes that are downregulated in TFH cells. Genes upregulated in TFH cells (and genes downregulated in non-TFH cells) might be controlled by the indirect repression-of-repressor mechanism because their expression amounts positively correlate with Bcl6 expression in TFH cells. This category of genes might include molecules positively involved in TFH differentiation and function (Figure 2A ) [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. Bcl6 directly binds and represses a set of TFs important for alternative cell fates (Figure 2A). Prdm1, Tbx21, Gata3, Rora, Rorc, and signal transducers and activators of transcription 5 (Stat5) are all directly repressed by Bcl6 in human GC-TFH cells and mouse CD4+ T cells [6.Nurieva R.I. et al.Bcl6 mediates the development of T follicular helper cells.Science. 2009; 325: 1001-1005Crossref PubMed Scopus (926) Google Scholar,7.Yu D. et al.The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.Immunity. 2009; 31: 457-468Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar,32.Kusam S. et al.Inhibition of Th2 differentiation and GATA-3 expression by BCL-6.J. Immunol. 2003; 170: 2435-2441Crossref PubMed Scopus (136) Google Scholar, 33.Mondal A. et al.Transcriptional repressor BCL6 controls Th17 responses by controlling gene expression in both T cells and macrophages.J. Immunol. Baltim Md 1950. 2010; 184: 4123-4132Google Scholar, 34.Hatzi K. et al.BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.J. Exp. Med. 2015; 212: 539-553Crossref PubMed Scopus (119) Google Scholar, 35.Liu X. et al.Genome-wide analysis identifies Bcl6-controlled regulatory networks during T follicular helper cell differentiation.Cell Rep. 2016; 14: 1735-1747Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. Moreover, Bcl6 inhibits non-TFH differentiation fates by repressing Prdm1 in mice [5.Johnston R.J. et al.Bcl6 and Blimp1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (0) Google Scholar,9.Vinuesa C.G. et al.Follicular helper T cells.Annu. Rev. Immunol. 2016; 34: 335-368Crossref PubMed Scopus (441) Google Scholar,11.Johnston R.J. et al.STAT5 is a potent negative regulator of TFH cell differentiation.J. Exp. Med. 2012; 209: 243-250Crossref PubMed Scopus (314) Google Scholar]. In human TFH, BCL6 inhibits Runt-related transcription factor 3 (RUNX3) [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar,34.Hatzi K. et al.BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.J. Exp. Med. 2015; 212: 539-553Crossref PubMed Scopus (119) Google Scholar]. Enforced expression of Runx3 by retroviral transduction disrupts TFH differentiation and increases the formation of a TH1 population in mouse LCMV infection [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar]. From another angle, Bcl6 repression of T-bet, GATA-3, and RORγt in the context of TFH differentiation has been somewhat controversial, given that some TFs are co-expressed with Bcl6 [36.Gowthaman U. et al.Identification of a T follicular helper cell subset that drives anaphylactic IgE.Science. 2019; 365eaaw6433Crossref PubMed Scopus (79) Google Scholar,37.Kim C.J. et al.The transcription factor Ets1 suppresses T follicular helper type 2 cell differentiation to halt the onset of systemic lupus erythematosus.Immunity. 2018; 49: 1034-1048Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. For example, T-bet co-expression with Bcl6 is observed at early TFH/TH1 differentiation in mouse LCMV infection [38.Oestreich K.J. et al.Molecular mechanisms that control the expression and activity of Bcl-6 in TH1 cells to regulate flexibility with a TFH-like gene profile.Nat. Immunol. 2012; 13: 405-411Crossref PubMed Scopus (216) Google Scholar,39.Weinstein J.S. et al.STAT4 and T-bet control follicular helper T cell development in viral infections.J. Exp. Med. 2017; 215: 337-355Crossref PubMed Scopus (34) Google Scholar]. Another example at steady state is GATA-3, which is co-expressed with Bcl6 in TFH cells in Alternaria extract-immunized mice [36.Gowthaman U. et al.Identification of a T follicular helper cell subset that drives anaphylactic IgE.Science. 2019; 365eaaw6433Crossref PubMed Scopus (79) Google Scholar]. Simultaneous expression of GATA-3 and Bcl6 is also observed in TFH cells in Ets1-deficient (Ets1fl/fl CD4-Cre) mice harboring systemic lupus erythematosus (SLE)-like disease [37.Kim C.J. et al.The transcription factor Ets1 suppresses T follicular helper type 2 cell differentiation to halt the onset of systemic lupus erythematosus.Immunity. 2018; 49: 1034-1048Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. It appears that alternative-fate TFs might be expressed in the presence of Bcl6 under particular pathological conditions and drive different gene expression programs, including signature genes, such as those upregulated in TFH and TH2 cells. However, direct evidence of Bcl6 inhibition via T-bet, GATA-3, and RORγt will require experiments assessing the selective disruption of Bcl6-mediated inhibition under normal versus pathological conditions. Bcl6 can also directly bind and repress key cytokines, receptors, and migration genes that are expressed by non-TFH cells (Figure 2A). For instance, Bcl6 binds the IL17A and IL17F loci in human GC-TFH cells [34.Hatzi K. et al.BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.J. Exp. Med. 2015; 212: 539-553Crossref PubMed Scopus (119) Google Scholar] and represses the expression of Il17a in mouse CD4+ T cells [6.Nurieva R.I. et al.Bcl6 mediates the development of T follicular helper cells.Science. 2009; 325: 1001-1005Crossref PubMed Scopus (926) Google Scholar]. Moreover, cytokine receptors essential for signals supporting TH1 (IL-12R and IL-23R), TH2 (IL-4R), TH17 [IL-23R and transforming growth factor (TGF)βR], and TREG (TGFβR) are direct targets of Bcl6 [34.Hatzi K. et al.BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.J. Exp. Med. 2015; 212: 539-553Crossref PubMed Scopus (119) Google Scholar]. The activation of Blimp1 and Stat5 by IL-2/IL-2R signaling is a potent inhibitory mechanism of TFH differentiation [11.Johnston R.J. et al.STAT5 is a potent negative regulator of TFH cell differentiation.J. Exp. Med. 2012; 209: 243-250Crossref PubMed Scopus (314) Google Scholar,40.Ballesteros-Tato A. et al.Interleukin-2 inhibits germinal center formation by limiting T follicular helper cell differentiation.Immunity. 2012; 36: 847-856Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar,41.DiToro D. et al.Differential IL-2 expression defines developmental fates of follicular versus nonfollicular helper T cells.Science. 2018; 361eaao2933Crossref PubMed Scopus (0) Google Scholar]. IL-7-mediated activation of STAT5 can also contribute to repression of TFH-associated genes, including Bcl6 [35.Liu X. et al.Genome-wide analysis identifies Bcl6-controlled regulatory networks during T follicular helper cell differentiation.Cell Rep. 2016; 14: 1735-1747Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,42.McDonald P.W. et al.IL-7 signalling represses Bcl-6 and the TFH gene program.Nat. Commun. 2016; 7: 10285Crossref PubMed Google Scholar]. Bcl6 also binds Il2r and Il7r and represses their expression [34.Hatzi K. et al.BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.J. Exp. Med. 2015; 212: 539-553Crossref PubMed Scopus (119) Google Scholar,35.Liu X. et al.Genome-wide analysis identifies Bcl6-controlled regulatory networks during T follicular helper cell differentiation.Cell Rep. 2016; 14: 1735-1747Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. Furthermore, migration of TFH cells to the T:B border and further localization to the GC is accomplished by upregulation of CXCR5 and downregulation of C-C motif chemokine receptor 7 (CCR7), P-selectin glycoprotein ligand-1 (PSGL1; encoded by Selplg), and Epstein–Barr virus (EBV)-induced G-protein-coupled receptor 2 (EBI2; encoded by Gpr183) [43.Crotty S. T follicular helper cell differentiation, function, and roles in disease.Immunity. 2014; 41: 529-542Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar,44.Suan D. et al.T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses.Immunity. 2015; 42: 704-718Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. EBI2 is required for positioning of TFH cells at the T:B border, and later, EBI2 expression is downregulated to complete further migration of TFH cells to GCs [44.Suan D. et al.T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses.Immunity. 2015; 42: 704-718Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,45.Li J. et al.EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells.Nature. 2016; 533: 110-114Crossref PubMed Scopus (173) Google Scholar]. Indeed, Bcl6 binds to Ccr7, Selplg, and Gpr183 and downregulates their expression, thereby preventing chemokine receptor-dependent migration to the T cell zone and enabling proper localization of GC-TFH cells to GCs [26.Choi J. et al.Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.Nat. Immunol. 2020; 21: 777-789Crossref PubMed Scopus (4) Google Scholar,34.Hatzi K. et