Tfh Cell Development Research Articles

MiR-155 Promotes T Follicular Helper Cell Accumulation during Chronic, Low-Grade Inflammation

Chronic inflammation is a contributing factor to most life-shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood, making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a(-/-) mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice, we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells, and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155(fl/fl) mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counterregulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.

Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity.

T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells

Follicular helper T cells (T(FH) cells) are responsible for effective B cell-mediated immunity, and Bcl-6 is a central factor for the differentiation of T(FH) cells. However, the molecular mechanisms that regulate the induction of T(FH) cells remain unclear. Here we found that the E3 ubiquitin ligase Itch was essential for the differentiation of T(FH) cells, germinal center responses and immunoglobulin G (IgG) responses to acute viral infection. Itch acted intrinsically in CD4(+) T cells at early stages of T(FH) cell development. Itch seemed to act upstream of Bcl-6 expression, as Bcl-6 expression was substantially impaired in Itch(-/-) cells, and the differentiation of Itch(-/-) T cells into T(FH) cells was restored by enforced expression of Bcl-6. Itch associated with the transcription factor Foxo1 and promoted its ubiquitination and degradation. The defective T(FH) differentiation of Itch(-/-) T cells was rectified by deletion of Foxo1. Thus, our results indicate that Itch acts as an essential positive regulator in the differentiation of T(FH) cells.

Transcription factor Ascl2 limits Tfh cell differentiation and function (IRC8P.493)

Abstract Follicular helper T (Tfh) cells promote germinal center (GC) formation and subsequent B cell maturation through the secretion of cytokines such as IL-21 and IL-4. Achaete-scute complex homologue 2 (Ascl2), a transcription factor previously shown to have a role in stem cell maintenance, is highly upregulated in the Tfh-cell subset of CD4+ T cells. However, its role in Tfh-cell development and function has not been established. We generated mice with conditional deletion of Ascl2 in the CD4 T cell compartment to assess whether this protein has a functional role in Tfh cells. Tfh cells deficient in Ascl2 promote a more robust germinal center response, with larger germinal centers and an increased percentage of GC B cells after immunization. This phenotype was coupled with augmented class-switched antibody production in the Ascl2-deficient mice. The expansion of GCs and class-switched antibody production is likely a result of increased IL-4 production by Tfh cells in the Ascl2-deficient mice. These data suggest that Ascl2 may play a role in limiting the functionality of Tfh cells by preventing their secretion of IL-4 and repressing their potential as cell helpers.

Basic leucine zipper transcription factor, ATF-like (BATF) is essential for the development of protective immunity against parasitic helminth infection (MPF1P.779)

Abstract Parasitic helminth infections afflict roughly a quarter of the world’s population. Immunity against parasitic helminth infection relies on the induction of allergic inflammation. Major hallmarks of allergic inflammation, such as IgE, eosinophilia, and mucus production, depend on the cytokines interleukin-4 (IL-4) and IL-13. In mice, IL-4 from CD4+ follicular T helper (Tfh) cells is required for IgE production, while IL-13 from CD4+ T-helper 2 (Th2) cells is required for worm clearance and mucus production. The basic leucine zipper transcription factor, ATF-like (BATF) is critical for Tfh function, however its necessity in Th2 cell development and IL-4 production remains unclear. To investigate the role of BATF in the context of allergic inflammation, we infected wild-type and BATF-deficient mice with the helminth Nippostrongylus brasiliensis to compare the resultant immune responses. In wild-type mice, N. brasiliensis infection induced a robust response as indicated by lung eosinophilia, high serum IgE, and worm expulsion. In contrast, BATF-deficient mice exhibited dramatically reduced eosinophilia, no IgE, and were unable to clear parasites from the intestine. Studies with IL-4-reporter mice revealed that these defects in allergic immunity were attributed to impaired Tfh and Th2 cell development. These findings suggest that BATF plays a central role in protection against helminths, and BATF is critical for the development of both Th2 and Tfh cells during allergic responses.

A novel population of interleukin 21-producing pre-follicular T helper cells develop spontaneously in young naïve mice (LYM3P.743)

Abstract Interleukin 21 is a cytokine vital for driving the proliferation of antigen-stimulated B cells within the germinal center and their subsequent differentiation into antibody-secreting plasmablasts and plasma cells. IL21 is predominately produced by a subset of activated CD4+ T cells known as T follicular helper cells. These cells develop at the T cell-B cell border and express ICOS, PD1, CD44, CXCR5 and BCL6. While this cell type has been identified, the processes that ultimately result in TFH differentiation are poorly understood. Here we use a novel IL21-venus fluorescent protein reporter mouse to investigate the signals and pathways involved in the earliest stages of development of TFH cells and production of IL21. We identify and characterize a naturally occurring population of ICOShi CD44hi CD4+ T cells that produce IL21 and are present within 4 weeks of age in naïve healthy C57BL/6J mice. These T cells show characteristics of TFH cells, including the upregulation of Bcl6 and Sostdc1, but lack surface staining for CXCR5 and PD1 and, surprisingly, develop in the absence of B cells. Therefore, these spontaneously arising cells actively producing IL21 appear to identify the earliest precursor to the mature TFH state.

T follicular helper cells and HIV/SIV-specific antibody responses

Here, we describe recent data on the characterization of follicular helper CD4 T cells (Tfh) and the dynamics of Tfh-B-cell interactions in HIV and simian immunodeficiency virus (SIV) infection and discuss important aspects of these interactions that need to be addressed in order to design more effective vaccines that elicit broadly neutralizing antibodies. Mouse, nonhuman primate (NHP) and human Tfh cells share phenotypic, functional and molecular programs, which are regulated by local signals and spatiotemporal parameters. Chronic HIV/SIV infection results in accumulation of Tfh, germinal center B cells and circulating virus-specific immunoglobulins in some individuals. However, most HIV/SIV-infected individuals do not mount broadly neutralizing antibodies, pointing to functional defects in Tfh cells in chronic HIV/SIV infection. The susceptibility of particular CD4 T-cell populations to HIV/SIV infection within lymph nodes notably impacts upon the dynamics of Tfh-germinal center B-cell interactions. Some circulating CD4 T cells share certain characteristics with Tfh cells, however, their direct origin from germinal center Tfh cells is not clear. There are many ways in which HIV and SIV influence the complex signals and mechanisms regulating the development of Tfh cells and their interactions with germinal center B cells. Understanding the biology of Tfh cells will be necessary to appropriately recruit these cells during vaccination with the goal of stimulating a more broad and potent neutralizing antibody response.

B Cells in T Follicular Helper Cell Development and Function: Separable Roles in Delivery of ICOS Ligand and Antigen

B cells are required for follicular Th (Tfh) cell development, as is the ICOS ligand (ICOS-L); however, the separable contributions of Ag and ICOS-L delivery by cognate B cells to Tfh cell development and function are unknown. We find that Tfh cell and germinal center differentiation are dependent on cognate B cell display of ICOS-L, but only when Ag presentation by the latter is limiting, with the requirement for B cell expression of ICOS-L overcome by robust Ag delivery. These findings demonstrate that Ag-specific B cells provide different, yet compensatory, signals for Tfh cell differentiation, while reconciling conflicting data indicating a requirement for ICOS-L expression on cognate B cells for Tfh cell development with those demonstrating that the latter requirement could be bypassed in lieu of that tendered by noncognate B cells. Our findings clarify the separable roles of delivery of Ag and ICOS-L by cognate B cells for Tfh cell maturation and function, and have implications for using therapeutic ICOS blockade in settings of abundantly available Ag, such as in systemic autoimmunity.

Follicular Helper T Cells Mediate IgE Antibody Production and Allergic Immune Responses in Mice

Follicular helper T (Tfh) cells develop when animals are infected with helminths. However, little is known about the roles for Tfh cells in allergic airway diseases. Mice were exposed intranasally to endotoxin-free ovalbumin (OVA) with or without IL-33 or IL-1β. Adaptive immune responses to OVA were analyzed by using IL-4 reporter mice, gene-deficient animals, and adoptive transfer approaches. Airway exposure of naïve mice to OVA plus IL-33 induced OVA-specific IgE and IgG1; when challenged with OVA, these mice developed robust Th2 cytokine response and airway eosinophilia. Exposure of naïve mice to OVA plus IL-1β induced comparable levels of anti-OVA IgE/IgG1 and IL-4, but minimal IL-5, IL-13 and eosinophilia. Isolation of IL-4-positive CD4+ T cells and microarray analysis showed that IL-33 induces antigen-specific Th2 cells and Tfh cells and that IL-1β induces mainly Tfh cells. Mice deficient in ICOS (Icos-/-), which is critical for development of Tfh cells, developed Th2 cells and anti-IgE/IgG1 antibodies normally when exposed to OVA plus IL-33. In contrast, when exposed to OVA plus IL-1β, Icos-/-mice showed decreased numbers of Tfh cells and reduced levels of anti-OVA IgE/IgG1. Finally, CD4-deficient animals recovered production of anti-OVA IgE/IgG1 antibodies when they were reconstituted with Tfh cells. IL-1-family cytokines regulate development of antigen-specific Th2 cells and Tfh cells in the airway. Tfh cells mediate IgE antibody production and modest airway inflammation, while Th2 cells mediate both antibody production and inflammation. The roles for Tfh cells in human allergic diseases need to be investigated.

Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development

In immune responses, activated T cells migrate to B cell follicles and develop to T follicular helper (Tfh) cells, a new subset of CD4+ T cells specialized in providing help to B lymphocytes in the induction of germinal centers 1,2. Although Bcl6 has been shown to be essential in Tfh cell function, it may not regulate the initial migration of T cells 3 or the induction of Tfh program as exampled by C-X-C chemokine receptor type 5 (CXCR5) upregulation 4. Here, we show that Achaete-Scute homologue 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor 5, is selectively upregulated in its expression in Tfh cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6 and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro and accelerates T cell migration to the follicles and Tfh cell development in vivo. Genome-wide analysis indicates that Ascl2 directly regulates Tfh-related genes while inhibits expression of Th1 and Th17 genes. Acute deletion of Ascl2 as well as blockade of its function with the Id3 protein in CD4+ T cells results in impaired Tfh cell development and the germinal center response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances Tfh cell generation. Thus, Ascl2 directly initiates Tfh cell development.

Notch Signaling Regulates Follicular Helper T Cell Differentiation

Follicular helper T (TFH) cells are specialized in providing help for B cell differentiation and Ab secretion. Several positive and negative regulators of TFH cell differentiation have been described but their control is not fully understood. In this study, we show that Notch signaling in T cells is a major player in the development and function of TFH cells. T cell-specific gene ablation of Notch1 and Notch2 impaired differentiation of TFH cells in draining lymph nodes of mice immunized with T-dependent Ags or infected with parasites. Impaired TFH cell differentiation correlated with deficient germinal center development and the absence of high-affinity Abs. The impact of loss of Notch on TFH cell differentiation was largely independent of its effect on IL-4. These results show a previously unknown role for Notch in the regulation of TFH cell differentiation and function with implications for the control of this T cell population.

IL-17RA Is Essential for Optimal Localization of Follicular Th Cells in the Germinal Center Light Zone To Promote Autoantibody-Producing B Cells

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

The role of TOX in peripheral T cell subsets (P1121)

Abstract TOX (Thymocyte-selection associated HMG-box) is a DNA-binding protein identified in our laboratory that is required for development of the CD4 T cell lineage, including canonical CD4 T cells, NKT cells and T regulatory (Treg) cells. In addition, NK cell and lymphoid tissue inducer cell development requires TOX, the latter resulting in failure of lymph node development in TOX-deficient mice. Here we turn our attention to investigation of TOX in mature CD4 T cell subsets, facilitated by a knock-in Tox reporter (tdTomato) strain of mouse. The reporter faithfully mirrors TOX expression in the thymus and NK cells. We find that TOX (Tomato) is also upregulated in natural Treg and at highest levels in T follicular helper (Tfh) cells. Interestingly, both T cell subsets show heterogeneity in reporter expression, with cell populations of high, intermediate and low expression evident. In Treg, varied expression of TOX is independent of FOXP3 expression, as demonstrated by analysis of FOXP3 and TOX dual reporter mice. Among Tfh cells (CXCR5hiPD1hi), Toxhi cells are Bcl6hiCCR7-CD62Llo and likely represent mature follicular effector cells. To study the functional role of TOX, we expressed Cre in CD4 T cells from OTII Tox conditional knockout mice, and analyzed the development of phenotypically-defined Tfh in response to antigen upon adoptive transfer. Loss of TOX prevented Tfh cell development in this system, indicating a critical role for TOX in this peripheral T cell subset.

Environmental and T Cell–Intrinsic Factors Limit the Expansion of Neonatal Follicular T Helper Cells but May Be Circumvented by Specific Adjuvants

Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses.

B and T cells collaborate in antiviral responses via IL-6, IL-21, and transcriptional activator and coactivator, Oct2 and OBF-1

A strong humoral response to infection requires the collaboration of several hematopoietic cell types that communicate via antigen presentation, surface coreceptors and their ligands, and secreted factors. The proinflammatory cytokine IL-6 has been shown to promote the differentiation of activated CD4(+) T cells into T follicular helper cells (T(FH) cells) during an immune response. T(FH) cells collaborate with B cells in the formation of germinal centers (GCs) during T cell-dependent antibody responses, in part through secretion of critical cytokines such as IL-21. In this study, we demonstrate that loss of either IL-6 or IL-21 has marginal effects on the generation of T(FH) cells and on the formation of GCs during the response to acute viral infection. However, mice lacking both IL-6 and IL-21 were unable to generate a robust T(FH) cell-dependent immune response. We found that IL-6 production in follicular B cells in the draining lymph node was an important early event during the antiviral response and that B cell-derived IL-6 was necessary and sufficient to induce IL-21 from CD4(+) T cells in vitro and to support T(FH) cell development in vivo. Finally, the transcriptional activator Oct2 and its cofactor OBF-1 were identified as regulators of Il6 expression in B cells.

LAPCs promote follicular helper T cell differentiation of Ag-primed CD4+ T cells during respiratory virus infection

The humoral immune response to most respiratory virus infections plays a prominent role in virus clearance and is essential for resistance to reinfection. T follicular helper (Tfh) cells are believed to support the development both of a potent primary antibody response and of the germinal center response critical for memory B cell development. Using a model of primary murine influenza A virus (IAV) infection, we demonstrate that a novel late activator antigen-presenting cell (LAPC) promotes the Tfh response in the draining lymph nodes (dLNs) of the IAV-infected lungs. LAPCs migrate from the infected lungs to the dLN "late," i.e., 6 d after infection, which is concomitant with Tfh differentiation. LAPC migration is CXCR3-dependent, and LAPC triggering of Tfh cell development requires ICOS-ICOSL-dependent signaling. LAPCs appear to play a pivotal role in driving Tfh differentiation of Ag-primed CD4(+) T cells and antiviral antibody responses.

Follicular helper T cells in immune homeostasis

Follicular helper T cells in immune homeostasis

The origins, function, and regulation of T follicular helper cells

The generation of high-affinity antibodies (Abs) plays a critical role in the neutralization and clearance of pathogens and subsequent host survival after natural infection with a variety of microorganisms. Most currently available vaccines rely on the induction of long-lived protective humoral immune responses by memory B cells and plasma cells, underscoring the importance of Abs in host protection. Ab responses against most antigens (Ags) require interactions between B cells and CD4+ T helper cells, and it is now well recognized that T follicular helper cells (Tfh) specialize in providing cognate help to B cells and are fundamentally required for the generation of T cell–dependent B cell responses. Perturbations in the development and/or function of Tfh cells can manifest as immunopathologies, such as immunodeficiency, autoimmunity, and malignancy. Unraveling the cellular and molecular requirements underlying Tfh cell formation and maintenance will help to identify molecules that could be targeted for the treatment of immunological diseases that are characterized by insufficient or excessive Ab responses.

The development and function of follicular helper T cells in immune responses

Follicular helper T cells (Tfh) have been referred as a lineage that provides a help for B cells to proliferate and undergo antibody affinity maturation in the germinal center. Evidence has supported that Tfh subset development, like other lineages, is dependent on microenvironment where a particular transcriptional program is initiated. It has been shown that Bcl-6 and IL-21 act as master regulators for the development and function of Tfh cells. Tfh dysregulation is involved in the development of autoimmune pathologies, such as systemic lupus erythematosus, rheumatoid arthritis and other autoimmune diseases. The present review highlights the recent advances in the field of Tfh cells and focus on their development and function.

STAT5 controls Tfh development (121.19)

Abstract Recent work has identified a new subset of CD4+ T cells named as T follicular helper (Tfh) cells that are localized in germinal centers and critical in germinal-center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and Bcl6 is specifically expressed in Tfh cells and required for their lineage specification. In current study, we characterized the role of STAT5 in developmental regulation of Tfh cells. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors CXCR5, c-Maf, Bcl6, Batf and IL-21, whereas STAT5 deficiency greatly enhanced Tfh differentiation. In addition, deletion of STAT5 in CD4+ T cells in vivo resulted in enhanced development of Tfh cells and germinal center B cells, and led to an impairment of B cell tolerance, suggesting that STAT5 signaling controls the humoral immunity. This knowledge may help us to find ways to treat antibody-mediated autoimmune diseases.