Regulators of Tfh Cell Differentiation

BackgroundFollicular helper T (Tfh) cells are specialized in helping B lymphocytes, which play a central role in autoimmune diseases that have a major B cell component, such as in rheumatoid arthritis (RA). Follicular regulatory T (Tfr) cells control the over-activation of Tfh and B cells in germinal centers. Dysregulation of Tfh cells and Tfr cells has been reported to be involved in the pathogenesis of some autoimmune diseases. However, the balance of Tfh and Tfr cells, and their roles in the development and progression of RA are still not clear.MethodsIn this study, we enrolled 44 patients with RA (20 patients with active RA and 24 patients with inactive RA) and 20 healthy controls, and analyzed the frequencies of circulating Tfh and Tfr cells, expression of programmed death-1 (PD-1), inducible co-stimulator (ICOS), intracellular IL-21, and pSTAT3 in Tfh cells, and serum levels of IL-6. The correlation among these parameters and that of Tfh or Tfr cells with disease activity were also analyzed.ResultsPatients with RA (especially active RA) had higher frequencies of Tfh cells, but lower percentages of Tfr cells, thereby resulting in elevated ratios of Tfh/Tfr. Expression levels of PD-1 and IL-21 in Tfh cells were higher in patients with RA than in healthy subjects, while no difference in ICOS expression was observed between patients and controls. Both pSTAT3 expression and serum IL-6 levels increased in patients with RA, and positive correlation between them was observed. Additionally, pSTAT3 expression was positively correlated with Tfh cell frequency. The Disease Activity Score in 28 joints based on C-reactive protein (DAS28-CRP) was negatively correlated with Tfr cell frequency, but was positively correlated with both Tfh/Tfr ratio and PD-1 expression.ConclusionsResults demonstrated that enhanced IL-6/pSTAT3 signaling may contribute to promotion of Tfh cells, consequently skewing the ratio of Tfh to Tfr cells, which may be crucial for disease progression in RA.

Toll-like receptor 7 agonist imiquimod prevents the progression of SLE in MRL/lpr mice via inhibiting the differentiation of T follicular helper cells

The generation of Immunglobulin (Ig) E and high-affinity IgG1 by B cells is a hallmark of allergic inflammation. These hallmarks are heavily reliant on interactions with T follicular helper (Tfh) cells. A critical cytokine provided by Tfh cells to the germinal center B cells is Interleukin-4 (IL-4). The mechanisms controlling IL-4 production by Tfh cells remain unknown. Given that Notch signaling is required for the differentiation of Tfh cells and it is known to influence cytokine production in T cells, we hypothesized that Notch also plays an important role in regulating the function of Tfh cells. Here, we use intestinal helminth infection to characterize whether persistent Notch signaling is required to maintain Tfh cell fate and function during allergic immunity. Genetic deletion of Notch receptors in infected mice results in reduced IL-4 production by Tfh and Th2 cells. Conversely, over-expression of Notch signaling leads to increased IL-4 production by Tfh cells. However, increased Notch signaling also appears to alter Tfh cell fate and phenotype. In support, chemical inhibition of Notch signaling after Tfh differentiation results in decreased expression of the Tfh chemokine receptor, CXCR5 among IL-4 producing T cells in the lymph node. These findings suggest that Notch signaling is not only important for Tfh differentiation, but that strength of Notch signaling is important for regulating Tfh cell fate, function, and maintenance.

The gut microbiota plays an essential role in mucosal immunity, with secretory immunoglobulin A (IgA) acting as a key effector in neutralizing pathogens and maintaining host-microbiota homeostasis. IgA production occurs via T cell-dependent (TD) and -independent pathways, with T follicular helper (Tfh) cells driving high-affinity, antigen-specific IgA responses. However, the specific microbial taxa and metabolites that regulate Tfh-mediated IgA responses under steady-state conditions remain poorly understood. This study investigated how gut microbiota-derived signals shape Tfh responses and IgA production, with implications for enhancing mucosal vaccine efficacy. We demonstrate that Peyer's patches (PP)-derived Tfh cells exhibit superior IgA-inducing capacity compared to splenic Tfh cells. RNA sequencing revealed distinct transcriptional profiles in PP-Tfh cells, including upregulation of the genes associated with Tfh differentiation and activation (Bcl6, Cd40lg, Maf), T-B cell interactions (Il21, Sh2d1a, Fyn), and migration (Ccr6, Cxcr5). Functionally, PP-Tfh cells formed larger T-B cell contact areas and induced significantly higher IgA secretion in co-culture than their splenic counterparts. Microbiota depletion experiments revealed that eliminating neomycin-depleted bacteria reduced fecal IgA levels and diminished PP-Tfh cell frequencies. Fecal microbiota transplantation from neomycin-treated mice restored both IgA production and Tfh responses in germ-free (GF) mice. Bioinformatic analysis (PICRUSt2 and LEfSe) identified butyrate-producing Lachnospiraceae and Ruminococcaceae as key drivers of the Tfh-IgA axis. Butyrate supplementation enhanced Tfh differentiation and IgA⁺ germinal center B cell development in vitro and increased fecal IgA levels in vivo. Mechanistically, butyrate promoted IgA production via GPR43 signaling, as its effect was lost in co-cultures with Gpr43⁻/⁻ Tfh cells. Moreover, treatment with tributyrin, a butyrate prodrug, enhanced vaccine-induced IgA and protected mice against Salmonella Typhimurium infection, reducing bacterial burden and tissue damage. These findings define a functional microbiota-Tfh-IgA axis sustained by neomycin-depleted, butyrate-producing bacteria. Our study underscores the crucial role of the gut microbiota, particularly neomycin-depleted butyrate producing taxa, in regulating PP-Tfh cell function and IgA production. Butyrate emerges as a metabolite linking microbial metabolism to Tfh differentiation and IgA class switching. Together, these findings establish a microbiota-metabolite-Tfh cell axis essential for mucosal immune homeostasis and suggest novel strategies for enhancing vaccine efficacy and protection against enteric infections. Video Abstract.

Human and mouse studies performed over the last decade have established that follicular helper T (Tfh) cells are a CD4(+) helper subset specialized in the provision of help to B cells. Tfh differentiation is driven by expression of the transcriptional repressor B-cell lymphoma-6 (Bcl-6), which turns on a program that guides T cells close to B-cell areas where Tfh cells first provide help to B cells. Sustained Bcl-6 expression promotes the entry of Tfh cells into follicles and modulates their cytokine expression profile so they can support and select germinal center B cells that have acquired affinity-enhancing mutations in their immunoglobulin genes. Forkhead box 3 protein (Foxp3)(+) regulatory T cells and invariant natural killer T (NKT) cells can also co-opt the Bcl-6-dependent follicular differentiation pathway to migrate into B-cell follicles and regulate antibody responses. The resulting NKT follicular helper cells drive a distinctive type of T-dependent B-cell response to lipid-containing antigens, whereas FoxP3(+) follicular regulatory (Tfr) cells exert a suppressive function on germinal centers. Elucidating how Tfr cells are functionally and numerically regulated and the factors that control the balance between Tfh and Tfr cells is likely to be critical for improved understanding of the pathogenesis and progression of autoimmunity and lymphomas of germinal center origin, and generation of effective vaccines.

Tfh cells with NLRP3 inflammasome activation are essential for high-affinity antibody generation, germinal centre formation and autoimmunity

Germinal centers (GCs) are confined anatomic regions where rapidly proliferating B cells undergo somatic mutation and selection and eventual differentiation into memory B cells or long-lived plasma cells. GCs are also the origin of malignancy, namely follicular lymphoma (FL), GC B cell-diffuse large B cell lymphoma (GCB-DLBCL), and Burkitt lymphoma (BL). GC B cell lymphomas maintain their GC transcriptional signatures and sustain many features of the GC microenvironment, including CD4+ T follicular helper (Tfh) cells. Tfh cells are essential for the formation and maintenance of GCs, providing critical helper signals such as CD40L. Large-scale sequencing efforts have led to new insights about the tightly regulated selection mechanisms that are commonly targeted during GC B cell lymphomagenesis. For instance, HVEM, a frequently mutated surface molecule in GC-derived lymphomas, engages the inhibitory receptor BTLA on Tfh cells and loss of HVEM leads to exaggerated T cell help. Here, we review current understanding of how Tfh cells contribute to the selection of GC B cells, with a particular emphasis on how Tfh cell signals may contribute to lymphomagenesis. The possibility of targeting Tfh cells for the treatment of GC-derived lymphomas is discussed.

SIRT1, an NAD+-dependent deacetylase, is upregulated in CD4+ T cells from SLE patients and MRL/lpr lupus-like mice. This study aimed to explore the role of SIRT1 in follicular helper T (Tfh) cell expansion and its potential value as a therapeutic target for SLE. Frequencies of CD4+CXCR5+PD-1+ Tfh cells in peripheral blood from SLE patients and their expression of SIRT1 and B cell lymphoma 6 (BCL-6) were determined with flow cytometry. Naïve CD4+ T cells were transfected with SIRT1-expressing lentivirus and small interfering RNA (siRNA) targeting SIRT1, respectively, and then cultured under Tfh-polarizing conditions to study the impact of SIRT1 on Tfh cell differentiation. This impact was also evaluated in both CD4+ T cells and naïve CD4+ T cells by treatment with SIRT1 inhibitors (EX527 and nicotinamide) in vitro. MRL/lpr mice and pristane-induced lupus mice were treated with continuous daily intake of nicotinamide, and their lupus phenotypes (including skin rash, arthritis, proteinuria and serum anti-dsDNA autoantibodies) were compared with those of controls. Expression of SIRT1 was elevated in Tfh cells from SLE patients and was positively correlated with Tfh cell frequencies. SIRT1 expression gradually increased during Tfh cell differentiation. Overexpression of SIRT1 by lentiviral vectors significantly promoted Tfh cell differentiation/proliferation. Reciprocally, suppressing expression of SIRT1 by siRNA and inhibiting SIRT1 activity by EX-527 or nicotinamide hindered Tfh cell expansion. Continuous daily intake of nicotinamide alleviated lupus-like phenotypes and decreased serum CXCL13 in the two mouse models. SIRT1 overexpression contributed to the expansion of Tfh cells in SLE and may serve as a potential target for treatment.

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.

T follicular helper cells in autoimmune diseases

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.

The importance of extracellular ATP for cell-to-cell communication in the nervous and vascular systems has been deeply studied for years, while less characterized is its role in regulating immune responses. Extracellular ATP is sensed by a class of ubiquitously expressed plasma membrane receptors termed P2 purinergic receptors (P2Rs), whose activation elicits different responses. Furthermore, extracellular ATP is rapidly degraded by ectonucleotidases such as CD39 and CD73 into adenosine, which in turn exerts additional regulatory effects through its own P1 receptors. In the first part of my PhD I addressed the role of ATP-gated ion channel P2X7 receptor in regulating T follicular helper (Tfh) cells functions. Tfh cells support affinity maturation of B cells in germinal centers (GCs) and differentiation to IgA-secreting plasma cells in gut-associated lymphoid tissues (GALTs). This function is critical in maintaining intestinal homeostasis and efficient mucosal defense. We have shown that P2X7 receptor is selectively and robustly upregulated in Tfh cells isolated from chronically stimulated secondary lymphoid organs, such as Peyer’s patches (PPs) and mesenteric lymph nodes. Deletion of P2rx7 in Tfh cells resulted in resistance to cell death, enhanced GC reaction in PPs, increased IgA binding to commensals, and reduction of mucosal bacteria. Our results unravel P2X7 as a regulator of the adaptive IgA response in the small intestine to allow commensalism and systemic stimulation of the immune system. In the second part of my PhD I addressed the role of the ectonucleotidases CD73 in the human B cell memory generation. It was previously shown from my host lab that peripheral human B cells co-expressing CD73 and CD39 have a more prompt capacity to expand and differentiate into antibody secreting cells in vitro. This property has been linked to the hydrolyzing enzymatic function of CD39 and CD73, which in tandem generates adenosine starting from extracellular ATP. We developed a method to functionally dissect antibody repertoire in different human B cell subsets differentiated according to CD73 expression and identified a novel subset of antigen-experienced B cells. We have shown for the first time that human peripheral IgM+IgD+CD27− B cells lacking expression of CD73 carry somatically mutated variable region genes, in contrast to CD73+ counterpart. Furthermore, IgM+IgD+CD27−CD73− B cells were less prone to perform class switch recombination in vitro and showed comparable functional properties with GC-independent memory B cells. Collectively, these results reveal CD73 as a novel human B cell marker that identifies functional distinct populations with different genetic hallmarks and a distinct differentiation fate.

16: MIR-17 ∼ 92 promotes T follicular helper cell differentiation and represses subset-inappropriate gene expression

Follicular helper T (Tfh) cells are a subtype of helper T cells critical for antibody responses, particularly germinal centre (GC) responses in the lymphoid organs. Tfh cells can become memory Tfh cells, which play important roles in memory antibody responses. Memory Tfh cells, after exiting the lymphoid organs into the circulation, possibly induce memory responses in lymphoid organs distal from the vaccination site. Circulating memory Tfh cells may also contribute to ectopic GC responses in inflamed nonlymphoid organs. Tfh cells and memory Tfh cells seem to be also involved in the development of autoimmune diseases. Human blood memory Tfh cells have begun to be characterised in detail to understand their function in protective or pathological antibody responses. Mouse studies, especially those using live imaging techniques, have illustrated Tfh cell dynamics within the lymphoid organs and identified important molecules that regulate them. In addition, imaging studies using photoactivatable or photoconvertible fluorescent probes have started to reveal how Tfh cells exit the lymphoid organs to become circulating memory cells. In future studies, in order to investigate directly the involvement of circulating memory Tfh cells in memory responses and inflammatory diseases, it is important to develop new methods to genetically and/or optogenetically manipulate Tfh cells for longitudinal tracking of their cell fate.