Expansion of follicular helper T cells in the absence of Treg cells: Implications for loss of B‐cell anergy

The maintenance of B‐cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B‐cell extrinsic mechanisms that regulate B‐cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B‐cell anergy. We now show that in Treg‐cell‐deficient mice, helper T cells are necessary and sufficient for loss of B‐cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4⁺ cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC‐T_FH) cells. These GC‐T_FH cells, but not those from Treg‐cell‐sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of T_FH cells, especially GC‐T_FH cells, which support autoantibody production.     

Clonal composition and persistence of antigen-specific circulating T follicular helper cells

T follicular helper (T_FH) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4⁺ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC T_FH cells. However, the antigen specificity and relationship of these circulating T_FH (cT_FH) cells with other memory CD4⁺ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cT_FH and non-cT_FH subsets, although with different frequencies and effector functions. Interestingly, cT_FH and non-cT_FH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu-specific cT_FH and non-cT_FH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1⁺ cT_FH cells had a “GC T_FH-like” phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cT_FH and non-cT_FH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cT_FH with non-cT_FH cells and on the heterogeneity and persistence of antigen-specific human cT_FH cells.     

Identification of novel markers for mouse CD4+ T follicular helper cells

CD4⁺ T follicular helper (T_FH) cells are central for generation of long‐term B‐cell immunity. A defining phenotypic attribute of T_FH cells is the expression of the chemokine R CXCR5, and T_FH cells are typically identified by co‐expression of CXCR5 together with other markers such as PD‐1, ICOS, and Bcl‐6. Herein, we report high‐level expression of the nutrient transporter folate R 4 (FR4) on T_FH cells in acute viral infection. Distinct from the expression profile of conventional T_FH markers, FR4 was highly expressed by naive CD4⁺ T cells, was downregulated after activation and subsequently re‐expressed on T_FH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory T_FH cells. Comparative gene expression profiling of FR4^hi versus FR4^lo Ag‐specific CD4⁺ effector T cells revealed a molecular signature consistent with T_FH and T_H1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto‐enzyme CD73, were enriched in T_FH cells compared with T_H1 cells, and phenotypic analysis confirmed expression of CD73 on T_FH cells. As there is now considerable interest in developing vaccines that would induce optimal T_FH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of T_FH cells following vaccination and infection.     

The hippo kinase MST1 negatively regulates the differentiation of follicular helper T cells

Follicular helper T (T_FH) cells are essential for inducing germinal centre (GC) reactions to mediate humoral adaptive immunity and antiviral effects, but the mechanisms of T_FH cell differentiation remain unclear. Here, we found that the hippo kinase MST1 is critical for T_FH cell differentiation, GC formation, and antibody production under steady-state conditions and viral infection. MST1 deficiency intrinsically enhanced T_FH cell differentiation and GC reactions in vivo and in vitro. Mechanistically, mTOR and HIF1α signalling is involved in glucose metabolism and increased glycolysis and decreased OXPHOS, which are critically required for MST1 deficiency-directed T_FH cell differentiation. Moreover, upregulated Foxo3 expression is critically responsible for T_FH cell differentiation induced by Mst1^−/−. Thus, our findings identify a previously unrecognized relationship between hippo kinase MST1 signalling and mTOR-HIF1α-metabolic reprogramming coupled with Foxo3 signalling in reprogramming T_FH cell differentiation.     

TFH cells in bystander and cognate interactions with B cells

Follicular T‐helper (T_FH) cells play a crucial role in three aspects of the germinal center (GC) response. They promote GC formation, arbitrate competition among GC B cells to determine the outcome of affinity maturation, and regulate GC output of memory and plasma cells to shape the long‐lived humoral immune memory. Of fundamental importance are dynamic physical interactions between T_FH and B cells, which are the main platform for T_FH cells to deliver “help” factors to B cells and also for reciprocal signaling from B cells to maintain the helper state of T_FH cells. Recent work has significantly expanded our understanding of how T‐B interactions are spatiotemporally regulated and molecularly orchestrated to fulfill those T_FH functions. In this review, we elaborate two modes of T‐B interactions, the antigen‐specific or cognate mode in which T_FH cells engage individual antigen‐presenting B cells and the antigen nonspecific bystander mode in which T_FH cells are engaged with the ensemble of follicular B cells. We discuss findings that indicate how short‐lived cognate T‐B contacts coupled with an intercellular positive feedback drive affinity‐based selection and how bystander interactions between T and B cells regulate follicular T‐cell recruitment and maintenance of an appropriate helper state. We argue that this combination of bystander and cognate interactions with B cells constantly shapes the internal state of T_FH cells and provides the platform to execute their helper functions.     

Galectin‐3 negatively regulates the frequency and function of CD4+CD25+Foxp3+ regulatory T cells and influences the course of Leishmania major infection

Galectin‐3, an endogenous glycan‐binding protein, plays essential roles during microbial infection by modulating innate and adaptive immunity. However, the role of galectin‐3 within the CD4⁺CD25⁺Foxp3⁺ T regulatory (T_REG) cell compartment has not yet been explored. Here, we found, in a model of Leishmania major infection, that galectin‐3 deficiency increases the frequency of peripheral T_REG cells both in draining lymph nodes (LNs) and sites of infection. These observations correlated with an increased severity of the disease, as shown by increased footpad swelling and parasite burden. Galectin‐3‐deficient (Lgals3^?/?) T_REG cells displayed higher CD103 expression, showed greater suppressive capacity, and synthesized higher amounts of IL‐10 compared with their wild‐type (WT) counterpart. Furthermore, both T_REG cells and T effector (T_EFF) cells from Lgals3^?/? mice showed higher expression of Notch1 and the Notch target gene Hes‐1. Interestingly, Notch signaling components were also altered in both T_REG and T_EFF cells from uninfected Lgals3^?/? mice. Thus, endogenous galectin‐3 regulates the frequency and function of CD4⁺CD25⁺Foxp3⁺ T_REG cells and alters the course of L. major infection.     

T follicular‐like helper cells in the peripheral blood of patients with primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by exocrine gland dysfunction, mainly causing sicca symptoms. B cells have a prominent role in SS, and the T follicular helper (T_FH) cells provide B cells with survival and specialization signals in germinal centres. Here, we investigate peripheral T_FH cells in pSS. Sixteen pSS patients and healthy controls were enrolled in the study, with 13 women and 3 men in each group. Whole blood was collected and separated into PBMC and plasma, followed by cryopreservation. Plasma samples were analysed for Ro52, Ro60 and La48 autoantibodies by indirect ELISA. For flow cytometric analysis, we defined 4 subsets of TFH‐like cells within the CD3⁺CD4⁺CXCR5⁺ population, namely the ICOS⁻PD‐1⁻, ICOS⁻PD‐1⁺, ICOS⁺PD‐1⁻ and ICOS⁺PD‐1⁺ (“TFH”) cells. We also investigated 4 CD19⁺ B cell subsets, the CD20⁺CD27⁺CD38⁻ memory B cells, CD20⁺CD27⁺CD38⁺ memory B cells, CD20⁻CD27⁺CD38⁺⁺CD138⁻ plasmablasts and CD20⁻CD27⁺CD38⁺⁺CD138⁺ plasma cells. We observed higher fractions of ICOS⁺PD‐1⁻ cells, ICOS⁺PD‐1⁺ (“T_FH”) cells and plasmablasts in pSS patients compared to controls, and lower frequencies of both types of memory B cells. The number of T_FH cells correlated positively with the levels of plasmablasts and plasma cells in the pSS patients, but not in the controls. The pSS patients were stratified according to Ro52/Ro60/La48 serology, and a positive association was found between autoantibody levels and increased level of T_FH cells, plasmablasts and plasma cells and lowered levels of memory B cells. We observed a higher response to Ro/La stimulation in pSS patients compared to controls of the memory B cells, although only significantly for the CD38⁻ memory B cells. Overall, a pathological relation between the ICOS⁺ T follicular‐like helper cells and B cells in pSS was observed, but further work should be conducted to explore their overall impact upon disease progression.     

Human pregnancy levels of estrogen and progesterone contribute to humoral immunity by activating TFH/B cell axis

Circulating T_FH (cT_FH) cells express CXCR5, PD-1, and, when activated, ICOS, and release IL-21. According to the production of IFN-γ, IL-4, and IL-17 and expression of FoxP3, these cells are also classified as cT_FH1, cT_FH2, cT_FH17, and cT_FR cells, respectively. This CD4⁺T-cell subset is pivotal to efficient humoral immunity, and pregnancy appears to favor IgG production. Here, not only pregnancy amplified the in vivo production of anti-HBsAg IgG in HBV immunized women, but the frequency of cT_FH cells was directly correlated with estradiol levels. In vitro, pregnancy-related dose of 17-β-estradiol (E2) directly increased the percentage of different cT_FH subsets. While E2 and progesterone (P4) increased the proportion of differentiated T_FH cells derived from naïve CD4⁺T-cells, only E2 amplified the release of IL-21 in those cell cultures. In addition, E2 and P4 increased the proportion of memory B cells and plasma cells, respectively. In SEB-activated B/T_FH cell co-cultures, E2, in the presence of P4, increased the production of total IgG. Finally, among the hormones, P4 was stronger in upregulating the percentage of IL-10⁺T_FR cells. Collectively, our findings suggested that E2 and P4 cooperate in the humoral immune response by favoring the expansion of different cT_FH and B cell subsets.     

Abundance of follicular helper T cells in Peyer's patches is modulated by CD155

The secondary humoral immune response is characterized by plasma B cells secreting isotype‐switched and affinity‐matured antibodies. The efficient generation of plasma B cells in the GC depends on the presence of follicular helper T (T_FH) cells, a cell type thought to arise from naive CD4‐positive T cells by a hitherto unresolved differentiation pathway. Mice deficient for CD155, an adhesion receptor of the immunoglobulin superfamily, are impaired to mount a secondary humoral immune response upon oral administration of antigen, while the primary IgM response is unaffected. Here, we show that mice lacking CD155 harbor significantly reduced numbers of T_FH cells in their Peyer's patches. This was paralleled by a decreased frequency of T_FH cells in the GC. Moreover, the CD155 ligand CD226, which is involved in T‐cell activation, is down‐regulated during T_FH cell differentiation, resulting in a complete absence of CD226 on those T_FH cells residing in the GC. Concurrently, the expression of TIGIT/WUCAM, a newly discovered CD155 ligand, is induced in T_FH cells. Thus, these cells replace an activating by a putative inhibitory CD155‐binding partner during their differentiation.     

Roles of follicular helper and regulatory T cells in allergic diseases and allergen immunotherapy

Allergic diseases are characterized by overactive type 2 immune responses to allergens and immunoglobulin E (IgE)-mediated hypersensitivity. Emerging evidence suggests that follicular helper T (T_FH) cells, rather than type 2 T-helper (T_H2) cells, play a crucial role in controlling IgE production. However, follicular regulatory T (T_FR) cells, a specialized subset of regulatory T (T_REG) cells resident in B-cell follicles, restricts T_FH cell-mediated help in extrafollicular antibody production, germinal center (GC) formation, immunoglobulin affinity maturation, and long-lived, high-affinity plasma and memory B-cell differentiation. In mouse models of allergic asthma and food allergy, CXCR5⁺ T_FH cells, not CXCR5⁻ conventional T_H2 cells, are needed to support IgE production, otherwise exacerbated by CXCR5⁺ T_FR cell deletion. Upregulation of T_FH cell activities, including a skewing toward type 2 T_FH (T_FH2) and IL-13 producing T_FH (T_FH13) phenotypes, and defects in T_FR cells have been identified in patients with allergic diseases. Allergen immunotherapy (AIT) reinstates the balance between T_FH and T_FR cells in patients with allergic diseases, resulting in clinical benefits. Collectively, further understanding of T_FH and T_FR cells and their role in the immunopathogenesis of allergic diseases creates opportunities to develop novel therapeutic approaches.     

Accumulation of somatic hypermutation and antigen-driven selection in rapidly cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during the primary antihapten response in mice

Well-developed germinal centers (GC) contain rapidly dividing surface immunoglobulin-negative (sIg^-) B cells (centroblasts), and most of their progeny are sIg⁺ B cells (centrocytes) in a resting state. It has been predicted that somatic hypermutation occurs in centroblasts, whereas antigen-driven selection takes place in centrocytes. The present analysis indicates that murine GC B cells bearing sIg with specificity for an immunizing antigen are in a rapidly cycling state and increase exponentially in number to occupy spleen GC at high frequency during the 1st week after primary immunization; however, the number of these cells is significantly reduced in the 2nd week of immunization. During that period, these proliferating sIg⁺ GC B cells accumulate somatic hypermutations with nucleotide exchanges indicative of affinity maturation. These sIg⁺ GC B cells co-express B7-2, ICAM-1, and LFA-1, and have potent antigen-presenting activity which results in T cell activation in vitro. These observations indicate that the sIg⁺ GC B cells accumulate somatic hypermutations and undergo antigen-driven selection through proliferation, probably upon activation by T cells. This sIg⁺ GC B cell population may represent cell cycling centrocytes; however, the possibility that these may represent centroblasts undergoing re-expression of sIg could not be excluded.     

Tracking antigen‐specific CD4+ T cells throughout the course of chronic Leishmania major infection in resistant mice

Primary Leishmania major infection typically produces cutaneous lesions that not only heal but also harbor persistent parasites. While the opposing roles of CD4⁺ T‐cell‐derived IFN‐γ and IL‐10 in promoting parasite killing and persistence have been well established, how these responses develop from naïve precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex class II (pMHCII) tetramers to investigate the endogenous, parasite‐specific primary CD4⁺ T‐cell response to L. major in mice resistant to infection. Maximal frequencies of IFN‐γ⁺ CD4⁺ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL‐10⁺ CD4⁺ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type‐1 (Th1) cells, not Foxp3⁺ regulatory T cells, were the chief producers of IL‐10 and were not exhausted. Therefore, tracking antigen‐specific CD4⁺ T cells revealed that IL‐10 production by Th1 cells is not due to persistent T‐cell antigen receptor stimulation, but rather driven by early antigen encounter at the site of infection.     

Glucocorticoid treatment restores the impaired suppressive function of regulatory T cells in patients with relapsing–remitting multiple sclerosis

Patients relapsing from multiple sclerosis (MS) are treated with high‐dose, short‐term intravenous injection of glucocorticoid (GC), although its mechanism of action remains only partly understood. We evaluated the ex vivo and in vitro effects of GC on regulatory T cell (T_reg) function in 14 relapsing–remitting MS (RR‐MS) patients in acute phase and 20 healthy controls (HC). T_reg function was enhanced significantly after 5 days of GC treatment. Furthermore, there was a trend towards increasing proportions of CD4⁺CD25⁺forkhead box P3⁺ T cells and interleukin‐10 secretion with GC treatment when compared with HC. In conclusion, GC treatment restores the impaired T_reg function in patients with RR‐MS in its acute phase.     

T cell suppression in the bone marrow of visceral leishmaniasis patients: impact of parasite load

Visceral leishmaniasis (VL) is a disseminated and lethal disease of reticulo‐endothelial system caused by protozoan parasites Leishmania donovani and L. infantum, which are known to induce host T cell suppression. To understand the impact of parasite load on T cell function, the present was focused on parasite load with T cell function in bone marrow of 26 VL patients. We observed significant enrichment of forkhead box protein 3 (FoxP3)⁺ (P = 0·0003) and interleukin (IL)‐10⁺ FoxP3⁺ regulatory T cells (T_reg) (P = 0·004) in the bone marrow (BM) of patients with high parasite load (HPL) compared with low parasite load (LPL). Concordantly, T effector cells producing interferon (IFN)‐γ (P = 0·005) and IL‐17A (P = 0·002) were reduced in the BM of HPL. Blocking of T_reg‐cell derived suppressive cytokines [(IL‐10 and transforming growth factor (TGF)‐β] rescued the effector T cells and their functions. However, it was observed that TGF‐β levels were dominant, favouring T_reg cell differentiation. Furthermore, the low ratio of IL‐6/TGF‐β favours the suppressive milieu in HPL patients. Here we show the change in levels of various cytokines with the parasitic load during active VL, which could be helpful in devising newer immunotherapeutic strategies against this disease.     

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells

We previously reported that Cd3e‐deficient mice adoptively transferred with CD4⁺ T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B‐cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation‐induced cytidine deaminase. Furthermore, GC B cells from Cd3e^–/– mice accumulate fewer somatic mutations as compared with GC B cells from wild‐type mice, and exhibit impaired affinity maturation and reduced differentiation into long‐lived plasma cells. Reconstitution of Cd3e^–/– mice with regulatory T (Treg) cells restored Tfh‐cell numbers, GC B‐cell numbers and B‐cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh‐cell numbers and GC B‐cell numbers and dynamics were also restored by pre‐reconstitution of Cd3e^–/– mice with Cxcr5^–/– Treg cells or non‐regulatory, memory CD4⁺ T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh‐cell response for an efficient and long‐lasting serological response.     

Early parasite containment is decisive for resistance to Leishmania major infection

We investigated the early spread of Leishmania major in various mouse strains. In BALB/c mice, which are extremely vulnerable to L. major infection, the parasites disseminated within 10-24 h from the site of subcutaneous footpad infection in to the popliteal lymph node, spleen, lung, liver and bone marrow. Application of recombinant (r)IL-12 prior to infection prevented the early dissemination of parasites into visceral organs and the animals healed the infection. In three mouse strains tested, C57BL/6, CBA/J and C3H/HeJ, which are all resistant to L. major infection, the parasites remained localized in the footpad and in the draining LN for 3 days without evidence of dissemination. In C57BL/6 mice, depletion of NK1.1⁺ cells or neutralization of interferon (IFN)-γ prior to infection led to rapid parasite spreading with kinetics similar to those seen in susceptible animals. Depletion of either CD4⁺ or CD8⁺ T cells in vivo prior to infection did not alter the kinetics of dissemination in any mouse strain tested. Experiments with severe-combined immunodeficient mice provided further evidence that parasite containment depends on natural killer cells and IFN-γ, but is independent of T cells. The finding that all resistant mouse strains restrict the spread of the parasites within the first 24 h after infection strongly suggests that early parasite containment is closely associated with a resistant phenotype. The data show that local restriction of parasites in the pre-T cell phase of the infection is mediated by the innate immune system and suggest that this function plays an important role in the development of a protective T cell response.     

Skin resident memory CD8+ T cells are phenotypically and functionally distinct from circulating populations and lack immediate cytotoxic function

The in‐depth understanding of skin resident memory CD8⁺ T lymphocytes (T_RM) may help to uncover strategies for their manipulation during disease. We investigated isolated T_RM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8⁺ T cell populations from the same donors. There were significantly increased proportions of CD8⁺CD45RA^–CD27^– T cells in the skin that expressed low levels of killer cell lectin‐like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8⁺ T_RM in skin were therefore phenotypically distinct from circulating CD8⁺CD45RA^–CD27^– T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8⁺ T_RM with T cell receptor (TCR)/CD28 or interleukin (IL)‐2 or IL‐15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)‐1 further boosted granzyme B expression. A unique feature of some CD8⁺ T_RM cells was their ability to secrete high levels of tumour necrosis factor (TNF)‐α and IL‐2, a cytokine combination that was not seen frequently in circulating CD8⁺ T cells. The cutaneous CD8⁺ T_RM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous T_RM appears to be stringently controlled by environmental signals in situ .