Phenotypic analysis of T follicular helper and T follicular regulatory cells in primary selective IgM deficiency

Selective IgM deficiency (SIgMD) is a rare immunodeficiency characterized by serum IgM below two standard of mean, and normal IgG and IgA levels. Both in human and mice with selective IgM deficiency, germinal centers cells are decreased. The development of germinal center and humoral immunity are regulated in part by follicular helper T (T_FH) and follicular regulatory T (T_FR) cells. However, the analysis of circulating T_FH (cT_FH) and T_FR (cT_FR) cells in the pathogenesis of SIgMD has not been explored. We observed lower percentage of cT_FR cells in SIgMD patients than in control group. However, we did not observe any significant difference in the percentage of cT_FH cells and their subsets between both experimental groups. When data were analyzed according to specific antibody response to pneumococcal polysaccharide, we observed a higher percentage of cT_FH cells in SIgMD patients with specific antibody deficiency than in SIgMD patients with normal specific antibody response. Our results suggest that cT_FH cells and their subsets are preserved in SIgMD patients. However, the role of lower percentage of cT_FR cells in the pathogenesis of this immunodeficiency is not clear.     

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.     

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 emerging role of T follicular helper (TFH) cells in aging: Influence on the immune frailty

The world population is undergoing a rapid expansion of older adults. Aging is associated with numerous changes that affect all organs and systems, including every component of the immune system. Immunosenescence is a multifaceted process characterized by poor response to vaccine and higher incidence of bacterial and viral infections, cancer, cardiovascular and autoimmune diseases. Immunosenescence has been associated with chronic low-grade inflammation referred to as inflammaging, whose underlying mechanisms remain incompletely elucidated, including age-related changes affecting components of the innate and adaptive immune system. T follicular helper (T_FH) cells, present in lymphoid organs and in peripheral blood, are specialized in providing cognate help to B cells and are required for the production of immunoglobulins. Several subsets of T_FH cells have been identified in humans and mice and modifications in T_FH cell phenotype and function progressively occur with age. Dysfunctional T_FH cells play a role in cancer, autoimmune and cardiovascular diseases, all conditions particularly prevalent in elderly subjects. A specialized population of Treg cells, named T follicular regulatory (T_FR) cells, present in lymphoid organs and in peripheral blood, exerts opposing roles to T_FH cells in regulating immunity. Indeed, changes in T_FH/T_FR cell ratio constitute a relevant feature of aging. Herein we discuss the cellular and molecular changes in both T_FH cells and T_FR cells that occur in aging and recent findings suggesting that T_FH cells and/or their subsets could be involved in atherosclerosis, cancer, and autoimmunity.     

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.     

Langerhans cells promote early germinal center formation in response to Leishmania‐derived cutaneous antigens

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen‐primed CD4⁺ follicular helper T cells (T_FH). A tight and stable formation of T_FH/B cell conjugates is required for cytokine‐driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of T_FH/B cell conjugates is crucial for B‐cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for T_FH‐cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin⁺ DC subsets in vivo, we show that the functionality of T_FH/B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC‐depleted mice show a reduction in somatic hypermutation in B cells isolated from T_FH/B cell conjugates and markedly reduced GC reactions within skin‐draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B‐cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.     

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.     

Aire deficient dendritic cells promote the T follicular helper cells differentiation

Autoimmune regulator (Aire), primarily expressed in medullary thymic epithelial cells (mTECs), maintains central immune tolerance through the clearance of self-reactive T cells. Aire can also be expressed in dendritic cells (DCs), and DCs can mediate T follicular helper (T_FH) cell differentiation and self-reactive B cell activation through inducible costimulator molecule ligand (ICOSL) and interleukin 6 (IL-6), which can cause autoimmune diseases. To confirm whether Aire in DCs affects T_FH cell differentiation and to determine the role of Aire in the maintenance of peripheral immune tolerance, this study observed the effects of Aire deficiency on T_FH cells using Aire knockout mice. The results showed that Aire deficiency caused increased number of T_FH cells, both in vivo and in vitro. Further studies showed that Aire deficiency promoted T_FH differentiation through the upregulation of ICOSL and IL-6 in DCs. Thus Aire could suppress the expression of ICOSL and IL-6 to inhibit T_FH cell differentiation.     

Identification of the association of CD28+CD244+ Tc17/IFN-γ cells with chronic hepatitis C virus infection

CD8⁺ T cells play multiple and complex immunological roles including antiviral, regulatory, and exhaustive effects in hepatitis C virus (HCV) infected patients. Some CD8⁺ T-cell subsets were confirmed to be closely related to HCV infection such as T_CM, T_EM, T_EMRA, Tc17, and CD8⁺ Treg. Herein, we report a new subset of interleukin (IL)-17/interferon (IFN)-γ producing CD8⁺ T (Tc17/IFN-γ) cells that markedly correlate with CD28⁺CD244⁺ cells, IL-17 levels, and HCV RNA in HCV patients. During early treatment with peg-IFN-a2a plus ribavirin, the imbalance of these Tc17/IFN-γ cells could be partially restored, together with normalized serum alanine aminotransferase but not aspartate transaminase. Also, we analyzed the dynamic change of the percentage of this T cells subset in patients with different outcome after 4-week course of treatment with peg-IFN-a2a plus ribavirin and found that the percentage of CD8⁺CD28⁺CD244⁺ T cells significantly decreased in recovered patients but not in nonrecovered patients. In vitro, CD28⁺CD244⁺ T cells were the only CD8⁺ T-cell group that secreted both IL-17 and IFN-γ in this axis and blockade with anti-CD244 antibodies significantly reduced cytokine production. Taken together, this study demonstrates that the frequency and regulatory functions of CD28⁺CD244⁺ Tc17/IFN-γ cells may play an important role in persistent HCV infection.     

Divergent chemokine receptor expression and the consequence for human IgG4 B cell responses

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of T_FH/T_H2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20⁺ B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.     

Heparin affects the induction of regulatory T cells independent of anti-coagulant activity and suppresses allogeneic immune responses

Heparin is a widely used anti-coagulant that enhances anti-thrombin (AT) activity. However, heparin also suppresses immune and inflammatory responses in various rodent models and clinical trials, respectively. The mechanism by which heparin suppresses immune responses is unclear. The effect of heparin on regulatory T cells (T_regs) in allogeneic immune responses was analysed using an acute graft-versus-host disease (aGVHD) mouse model and mixed lymphocyte reactions (MLRs). In-vitro culture systems were utilized to study the effects of heparin on T_regs. Heparin administration reduced mortality rates and increased the proportion of T_regs in the early post-transplantation period of aGVHD mice. In both murine and human MLRs, heparin increased T_regs and inhibited responder T cell proliferation. Heparin promoted functional CD4⁺CD25⁺forkhead box protein 3 (FoxP3)⁺ T_reg generation from naive CD4⁺ T cells, increased interleukin (IL)-2 production and enhanced the activation of pre-existing T_regs with IL-2. Heparin-induced T_reg increases were not associated with anti-coagulant activity through AT, but required negatively charged sulphation of heparin. Importantly, N-acetyl heparin, a chemically modified heparin without anti-coagulant activity, induced T_regs and decreased mortality in aGVHD mice. Our results indicate that heparin contributes to T_reg-mediated immunosuppression through IL-2 production and suggest that heparin derivatives may be useful for immunopathological control by efficient T_reg induction.     

Short‐term anti‐CD25 monoclonal antibody administration down‐regulated CD25 expression without eliminating the neogenetic functional regulatory T cells in kidney transplantation

CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺regulatory T (T_reg) cells are generated and play a key role in the induction and maintenance of transplant tolerance in organ recipients. It has been proposed that interleukin (IL)-2/IL-2 receptor (IL-2R) signalling was essential for the development and proliferation of antigen-activated T cells that included both effector T cells and T_reg cells. Basiliximab (Simulect™), a chimeric monoclonal antibody directed against the α-chain of the IL-2R (CD25), can be expected to not only affect alloreactive effector T cells, but also reduce the number and function of T_reg cells. We therefore examined the effect of basiliximab induction therapy on the number and function of the T_reg cells in renal recipients. Basiliximab decreased the percentage of CD4⁺CD25⁺T cells, but failed to influence the percentage of CD4⁺FoxP3⁺ T_reg cells. The cellular CD25 expression was decreased significantly by basiliximab injection, but CD4⁺CD25⁺ T cells was not depleted from the circulating pool through monoclonal antibody activation-associated apoptosis. Functional analysis revealed that inhibitory function of T_reg cells from recipients with basiliximab injection was not significantly different from recipients without injection. These data indicate that the functional T_reg population may not be influenced by short-term basiliximab treatment.     

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.     

The relative contribution of IL-4 and IL-13 to human IgE synthesis induced by activated CD4 or CD8 T cells

The relative contribution of IL-4 and IL-13 to the regulation of IgE synthesis has remained relatively poorly characterized, partially because of lack of suitable animal models. We have studied the roles of IL-4 and IL-13 in human IgE synthesis induced by supernatants derived from activated CD4⁺⁺ or CD8⁺ T cell clones. Neutralizing anti–IL-4 and anti–IL-13 monoclonal antibodies (mAbs) inhibited IgE synthesis induced by anti-CD40 mAbs and supernatants from CD4⁺ T cells by an average 61% and 42%, respectively (n = 25). Recombinant IL-13 had additive effects on IL-4-induced IgE synthesis, but only when IL-4 was present at low concentrations. Accordingly, IL-4 was the dominant IgE synthesis–inducing cytokine derived from highly polarized T helper (TH)₂ cells. However, anti–IL-13 mAbs also significantly inhibited IgE synthesis induced by two of three supernatants derived from allergen-specific T_H2-like cell lines generated from the skin of patients with atopic dermatitis. Furthermore, anti–IL-13 mAbs almost completely inhibited IgE synthesis induced by supernatants from T_H1 cells or CD8⁺ T cell clones. Taken together, these data indicate that IL-13, in addition to IL-4, contributes to IgE synthesis induced by all T helper cell subsets, including allergen-specific T_H2 cells. Moreover, IL-13 appears to be the major IgE synthesis–inducing cytokine derived from T_H1 cells or CD8⁺ T cells. (J Allergy Clin Immunol 1997;100:792-801.)     

Regulation of IL-10 and IL-17 mediated experimental autoimmune encephalomyelitis by S-nitrosoglutathione

In this study, we investigated IL-10 and IL-17 specific immunomodulatory potential of S-nitrosoglutathione (GSNO), a physiological nitric oxide carrier molecule, in experimental autoimmune encephalomyelitis (EAE). In active EAE model, GSNO treatment attenuated EAE severity and splenic CD4⁺ T cells isolated from these mice exhibited decreased IL-17 expression without affecting the IFN-γ expression compared to the cells from untreated EAE mice. Similarly, adoptive transfer of these cells to nave mice resulted in reduction in IL-17 expression in the spinal cords of recipient mice with milder EAE severity. CD4⁺ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, still expressed lower levels of IL-17 under T_H17 skewing conditions, but expressed similar levels of IFN-γ under T_H1 skewing condition. Interestingly, under both T_H17 and T_H1 skewing condition, CD4⁺ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, expressed higher levels of IL-10 and adoptive transfer of these T_H17 and T_H1 skewed cells seemingly exhibited milder EAE disease. In addition, adoptive transfer of CD4⁺ T cells from GSNO treated EAE mice to active EAE mice also ameliorated EAE disease with induction of spinal cord expression of IL-10 and reduction in of IL-17, thus suggesting the participation of IL-10 mechanism in GSNO mediated immunomodulation. GSNO treatment of mice passively immunized with CD4⁺ T cells either from GSNO treated EAE mice or untreated mice further ameliorated EAE disease, supporting efficacy of GSNO for prophylaxis and therapy in EAE. Overall, these data document a modulatory role of GSNO in IL-17/IL-10 axis of EAE and other autoimmune diseases.