Helper Cell Activity Research Articles

96: PD-L1hi B regulatory cells are critical for control of the humoral immune response

A defining feature of the majority of B regulatory (Breg) cells described in mouse and man is their production of IL-10. However, other subsets of Breg, which act in an IL-10 independent manner, also exist. Here, we describe a distinct subset of Breg, defined by high levels of PD-L1, which can suppress inflammation. We are able to describe the importance of PD-L1 expression on B cells in the context of humoral immunity and disease in both mouse and man. Previously, we have shown that PD-L1 on B cells is an important regulator of T follicular helper (TFH) cell activity and downstream antibody responses. Through the use of chimeric mice and transfer approaches of B cell populations with varying degrees of PD-L1 expression we now demonstrate that a PD-L1[hi] B cell subset dramatically suppressed humoral responses through attenuating the activation of T cells and antibody production, independent of other reported suppressor populations. We further describe how PD-L1 hi Breg are refractory to B cell depletion therapy ( α CD20 mAb treatment). The suppressive capacity of these Breg was demonstrated to be dependent on PD-L1. The retention of PD-L1 hi Breg, post B cell depletion therapy, was attributed to elevated expression of the B cell activating factor (BAFF) receptors BAFF-R, TACI and BCMA, which displayed enhanced uptake of BAFF compared to other B cell subsets. Blockade of BAFF-R rendered PD-L1 hi B cells sensitive to B cell depletion therapy. Together, our studies identify a previously unknown regulatory B cell that acts through the PD-1/PDL1 pathway in limiting the differentiation and function of TFH cells. It also demonstrates a novel mechanism by which B cell depletion therapy works, and thus provides insight into the dynamic control of humoral immune responses in both homeostasis and disease.

Early preservation of CXCR5+ PD-1+ helper T cells and B cell activation predict the breadth of neutralizing antibody responses in chronic HIV-1 infection.

Much is known about the characteristics of broadly neutralizing antibodies (bNAbs) generated during HIV-1 infection, but little is known about immunological mechanisms responsible for their development in only a minority of those infected by HIV-1. By monitoring longitudinally a cohort of HIV-1-infected subjects, we observed that the preservation of CXCR5(+) CD4(+) T helper cell frequencies and activation status of B cells during the first year of infection correlates with the maximum breadth of plasma neutralizing antibody responses during chronic infection independently of viral load. Although, during the first year of infection, no differences were observed in the abilities of peripheral CXCR5(+) CD4(+) T helper cells to induce antibody secretion by autologous naive B cells, higher frequencies of class-switched antibodies were detected in cocultures of CXCR5(+) CD4(+) T and B cells from the subjects who later developed broadly neutralizing antibody responses than those who did not. Furthermore, B cells from the former subjects had higher expression of AICDA than B cells from the latter subjects, and transcript levels correlated with the frequency of CXCR5(+) CD4(+) T cells. Thus, the early preservation of CXCR5(+) CD4(+) T cells and B cell function are central to the development of bNAbs. Our study provides a possible explanation for their infrequent generation during HIV-1 infection. Broadly neutralizing antibodies are developed by HIV-1-infected subjects, but so far (and despite intensive efforts over the past 3 decades) they have not been elicited by immunization. Understanding how bNAbs are generated during natural HIV-1 infection and why only some HIV-1-infected subjects generate such antibodies will assist our efforts to elicit bNAbs by immunization. CXCR5(+) PD-1(+) CD4(+) T cells are critical for the development of high-affinity antigen-specific antibody responses. In our study, we found that the HIV-1-infected subjects who develop bNAbs have a higher frequency of peripheral CXCR5(+) PD-1(+) CD4(+) T cells in early infection and also that this frequency mirrored what was observed in uninfected subjects and correlated with the level of B cell activation across subjects. Our study highlights the critical role helper T cell function has in the elicitation of broadly neutralizing antibody responses in the context of HIV infection.

Loss of Circulating CD4 T Cells with B Cell Helper Function during Chronic HIV Infection

The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7highCXCR5highCCR6highPD-1high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells.

Effects of Sustained Sleep Restriction on Mitogen-Stimulated Cytokines, Chemokines and T Helper 1/ T Helper 2 Balance in Humans

BackgroundRecent studies suggest that acute sleep deprivation disrupts cellular immune responses by shifting T helper (Th) cell activity towards a Th2 cytokine profile. Since little is known about more long-term effects, we investigated how five days of sleep restriction would affect pro-inflammatory, chemotactic, Th1- and Th2 cytokine secretion.MethodsNine healthy males participated in an experimental sleep protocol with two baseline sleep-wake cycles (sleep 23.00 – 07.00 h) followed by 5 days with restricted sleep (03.00 – 07.00 h). On the second baseline day and on the fifth day with restricted sleep, samples were drawn every third hour for determination of cytokines/chemokines (tumor necrosis factor alpha (TNF-α), interleukin (IL) -1β, IL-2, IL-4 and monocyte chemoattractant protein-1 (MCP-1)) after in vitro stimulation of whole blood samples with the mitogen phytohemagglutinin (PHA). Also leukocyte numbers, mononuclear cells and cortisol were analysed. Results5-days of sleep restriction affected PHA-induced immune responses in several ways. There was a general decrease of IL-2 production (p<.05). A shift in Th1/Th2 cytokine balance was also evident, as determined by a decrease in IL2/IL4 ratio. No other main effects of restricted sleep were shown. Two significant interactions showed that restricted sleep resulted in increased TNF-α and MCP-1 in the late evening and early night hours (p’s<.05). In addition, all variables varied across the 24 h day.Conclusions5-days of sleep restriction is characterized by a shift towards Th2 activity (i.e. lower 1L-2/IL-4 ratio) which is similar to the effects of acute sleep deprivation and psychological stress. This may have implications for people suffering from conditions characterized by excessive Th2 activity like in allergic disease, such as asthma, for whom restricted sleep could have negative consequences.

CXCR5+CD4+ follicular helper T cells accumulate in resting human lymph nodes and have superior B cell helper activity

Although many relevant immune reactions are initiated in the lymph nodes, this compartment has not been systematically studied in humans. Analyses have been performed on immune cells derived from tonsils, but as this tissue is most often inflamed, generalization of these data is difficult. Here, we analyzed the phenotype and function of the human CD4(+) T-cell subsets and lineages in paired resting lymph node and peripheral blood samples. Naive, central memory cells and effector memory cells as well as Th1, Th2, Th17 and Treg cells were equally represented in both compartments. On the other hand, cytotoxic CD4(+) T cells were strikingly absent in the lymph nodes. CXCR5(+)CD4(+) T cells, representing putative follicular Th (Tfh) cells were over-represented in lymph nodes and expressed higher levels of Tfh markers than their peripheral blood counterparts. Compared with the circulating pool, lymph-node-derived CXCR5(+)CD4(+) T cells were superior in providing help to B cells. Thus, functionally competent Tfh cells accumulate in resting human lymph nodes, providing a swift induction of naive and memory antibody responses upon antigenic challenge.

IgM autoantibodies: Roquin and Bob1ng to a different tune

Pathogenic autoantibodies directed against ubiquitous self-antigens such as immunoglobulin-G (IgG) anti-double-stranded DNA (anti-dsDNA) antibodies in systemic lupus erythematosus are produced by plasma cells that have undergone immunoglobulin isotype switching and affinity maturation in the germinal centre (GC).1 These autoantibodies form circulating immune complexes that are often deposited in the skin, kidneys and joints where they activate complement to cause tissue damage. In contrast, IgM (and IgA) natural autoantibodies are produced in the steady-state from birth by plasma cells derived from B1 cells, and are ‘hardwired’ to be polyreactive, low-affinity antibodies with relatively low pathogenic potential.2, 3 Nevertheless, IgM autoantibodies bound to antigen are still capable of activating the classical complement pathway and causing diseases such as autoimmune haemolytic anaemias due to cold agglutinins. In this issue, Corcoran and coworkers4 remind us of this by showing that sanroque mice harbouring mutations in Rc3h1/Roquin1 develop a GC-independent IgM-mediated autoimmune disease when crossed with mice deficient for the transcription factor Obf1/Ocab/Bob1 (Figure 1). These IgM anti-dsDNA antibodies are deposited in the kidneys where they cause glomerulonephritis. This remarkable observation adds to the growing list of insights into the regulation of autoantibody responses in the sanroque ENU-mutant mouse model, which is characterized by excessive follicular T helper (Tfh) cell activity and spontaneous GC development.5 Previous studies using this model have focussed on the role of specific genes and cellular interactions involved in the production of GC-dependent autoantibodies, such as the Bcl6, Sh2d1a, Cd28 and Icos.6, 7 However, the current study neatly turns this on its head by showing that sanroque mice develop an even more severe fatal autoimmunity when Obf1 is also deficient and they are unable to form GCs and secrete IgG autoantibodies. This unexpected finding is all the more intriguing given that Obf1 deficiency by itself does not cause autoimmunity, and can also prevent the GC-dependent development of lupus-like autoimmune diseases in aged Ailos−/− 8 and MRL-lpr mice.9

P055 HDAC dependent regulation of the IL-6/STAT3 pathway during T helper cell activation

P055 HDAC dependent regulation of the IL-6/STAT3 pathway during T helper cell activation

New Perspectives on Ancient Granulomas

EDITORIAL article Front. Immunol., 15 November 2013Sec. Inflammation Volume 4 - 2013 | https://doi.org/10.3389/fimmu.2013.00345

AIRE expressing marginal zone dendritic cells balances adaptive immunity and T-follicular helper cell recruitment

Autoimmune polyendocrine syndrome Type I (APS I) results in multiple endocrine organ destruction and is caused by mutations in the Autoimmune regulator gene (AIRE). In the thymic stroma, cells expressing the AIRE gene dictate T cell education and central tolerance. Although this function is the most studied, AIRE is also expressed in the periphery in DCs and stromal cells. Still, how AIRE regulated transcription modifies cell behaviour in the periphery is largely unknown. Here we show that AIRE is specifically expressed by 33D1+ DCs and dictates the fate of antibody secreting cell movement within the spleen. We also found that AIRE expressing 33D1+ DCs expresses self-antigens as exemplified by the hallmark gene insulin. Also, as evidence for a regulatory function, absence of Aire in 33D1+ DCs led to reduced levels of the chemokine CXCL12 and increased co-stimulatory properties. This resulted in altered activation and recruitment of T-follicular helper cells and germinal centre B cells. The altered balance leads to a change of the early response to a T cell-dependent antigen in Aire−/− mice. These findings add to the understanding of how specific DC subtypes regulate the early responses during T cell-dependent antibody responses within the spleen and further define the role of AIRE in the periphery as regulator of self-antigen expression and lymphocyte migration.

Fluorescent target array T helper assay: A multiplex flow cytometry assay to measure antigen-specific CD4+ T cell-mediated B cell help in vivo

CD4+ T cells play a central role in regulating the immune response. Their effector function is commonly assessed by their capacity to secrete cytokines detected by ELISPOT and intracellular cytokine staining. However, one aspect of their effector function that is often overlooked is their ability to help activation of cognate B cells directly, a process that is initiated through the engagement of their T cell-receptor (TCR) with cognate peptide presented on major histocompatibility complex class II (MHC-II) molecules by B cells. Here we report a method to monitor CD4+ T cell-mediated B cell help in vivo using a multiplex high throughput assay. This assay utilizes a fluorescent target array (FTA), which is composed of lymphocytes labeled with numerous (>200) unique fluorescence signatures that can be delineated in a single recipient animal based on combination labeling with the three vital dyes carboxyfluorescein diacetate succinimidyl ester (CFSE), CellTrace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD). By pulsing different B cell populations in a FTA with titrated amounts of cognate MHC-II binding peptides, CD4+ T cell help could be assessed by measuring induction of the B cell activation markers CD69 and CD44 by antibody labeling and flow cytometry. We call this the “FTA T helper assay”, and have found it to be a robust and sensitive assay to measure CD4+ T cell helper activity across a multitude of peptide-pulsed B “target” cells in real time in vivo. Furthermore, the technique can be used simultaneously with the FTA killing assay that measures cytotoxic T cell function, to provide a comprehensive tool for measuring both CD4+ and CD8+ T cell activity during an immune response in vivo.

IL-21 Regulates the Differentiation of a Human γδ T Cell Subset Equipped with B Cell Helper Activity

Vγ9Vδ2 T lymphocytes recognize nonpeptidic antigens without presentation by MHC molecules and display pleiotropic features. Here we report that coculture of Vγ9Vδ2 cells with phosphoantigen and IL-21 leads to selective expression of the transcription repressor Bcl-6 and polarization toward a lymphocyte subset displaying features of follicular B-helper T (TFH) cells. TFH-like Vγ9Vδ2 cells have a predominant central memory (CD27+CD45RA−) phenotype and express ICOS, CD40L and CXCR5. Upon antigen activation, they secrete IL-4, IL-10 and CXCL13, and provide B-cell help for antibody production in vitro. Our findings delineate a subset of human Vγ9Vδ2 lymphocytes, which, upon interaction with IL-21-producing CD4 TFH cells and B cells in secondary lymphoid organs, is implicated in the production of high affinity antibodies against microbial pathogens.

De-regulated expression of microRNAs contributes to augmented T helper (TH) cell activity in experimental asthma

Background: Bronchial asthma is a heterogeneous chronic inflammatory disease, which is orchestrated by antigen-specific TH cells. By secreting cytokines, these cells shape the augmented immune response, which finally leads to structural changes of chronic inflamed airways. MicroRNA (miRNA), as small non-coding RNAs, are recognized as key regulatory elements in gene expression and have been linked with a number of complex human chronic inflammatory diseases. Yet, their implication in augmented TH cell activity in asthma has not been thoroughly investigated. Methods: T helper cell were isolated from murine lungs in the progression of ovalbumin- induced allergic airway inflammation and profiled by FACS, microarray and real-time RT-PCR. Results: Effector T helper cell subsets were FACS-sorted and characterized based on gene, protein and miRNA expression. These data were employed to identify TH- cell subset specific gene and miRNA expression levels. In addition, changes in gene and miRNA levels in the progression of experimental asthma were identified. Conclusion: The generated data provide evidence for TH cell subset specific miRNA expression as well as augmented expression of miRNA in the progression of experimental asthma. Furthermore, they permit the identification of miRNA-dependent and independent signalling pathways in TH cell subset important for disease development.

Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling

BackgroundT lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells.ResultsCalcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells.ConclusionsThis study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.

Cytokine profiles and salivary IgA and hormonal responses to an acute bout of strenuous endurance exercise

Introduction: Strenuous endurance exercise suppresses some immune variables such as T helper (Th) cell activities during the post-exercise recovery period. Changes in the Th1/Th2 response to exercise have been reported previously; however, earlier reports have not investigated cytokine production by phytohemagglutinin (PHA)-stimulated Th cells following strenuous endurance exercise. Changes in plasma cortisol and salivary IgA concentrations have been linked to the exercise-induced immunosuppression explained by the ‘open window’ theory.

A High-Affinity T-Helper Epitope Enhances Peptide-Pulsed Dendritic Cell–Based Vaccine

The NV epitope, a dominant helper determinant from the circumsporozoite antigen of Plasmodium falciparum, is strongly immunogenic and can provide help for cytotoxic T-lymphocyte (CTL) activation. In this study, we evaluated whether the addition of NV peptide can augment the efficacy of peptide-pulsed dendritic cell (DC) immunization in vivo. Using B16 melanoma as tumor model, we demonstrated that DCs pulsed with both NV and gp100 (a melanoma-specific antigen) peptide enhanced immune priming and protection from tumor challenge in vivo. Further, we showed the mechanisms of the NV epitope that help CTL activation; MHC-II-restricted NV peptide induced dramatically more effective helper cells, with a higher level of CD40L expression and IFN-γ production, which, in turn, more effectively conditioned DCs for CTL activation. The improved helper cells also induced greater IL-12 production by DCs, accounting for the reciprocal T-helper polarization to Th1, and increased the expression of costimulatory molecules. Collectively, these findings demonstrate that NV peptide in addition to tumor antigen-pulsed DC immunizations augment helper cell activation, which in turn promotes maturation of DC, and enhance in vivo antitumor activity.

&lt;em&gt;Trichuris muris&lt;/em&gt; Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that infect humans and livestock. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment. This model is a powerful tool to examine factors that control CD4(+) T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated cytokines (IL-12, IL-18, IFN-γ) promote chronic infections in genetically susceptible AKR/J mice. Th2 cytokines promote physiological changes in the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle contraction, all of which have been implicated in worm expulsion. Here we detail a protocol for propagating Trichuris muris eggs which can be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in the gastrointestinal tract as well as immune effector functions of IECs.

Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements

T helper cell differentiation and activation require specific transcriptional programs accompanied by changes in chromatin structure. However, little is known about the chromatin remodeling enzymes responsible. We performed genome-wide analysis to determine the general principles of BRG1 binding, followed by analysis of specific genes to determine whether these general rules were typical of key T cell genes. We found that binding of the remodeling protein BRG1 was programmed by both lineage and activation signals. BRG1 binding positively correlated with gene activity at protein-coding and microRNA (miRNA) genes. BRG1 binding was found at promoters and distal regions, including both novel and previously validated distal regulatory elements. Distal BRG1 binding correlated with expression, and novel distal sites in the Gata3 locus possessed enhancer-like activity, suggesting a general role for BRG1 in long-distance gene regulation. BRG1 recruitment to distal sites in Gata3 was impaired in cells lacking STAT6, a transcription factor that regulates lineage-specific genes. Together, these findings suggest that BRG1 interprets both differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate. Our findings suggest that BRG1 binding is a useful marker for identifying active cis-regulatory regions in protein-coding and miRNA genes.

Antigen Presentation and MHC Class II Expression by Human Esophageal Epithelial Cells: Role in Eosinophilic Esophagitis

Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis.

Grand Challenges in Molecular Antigen-presenting Cell Biology

SPECIALTY GRAND CHALLENGE article Front. Immunol., 21 March 2011Sec. Antigen Presenting Cell Biology volume 2 - 2011 | https://doi.org/10.3389/fimmu.2011.00008

The influence of regulatory T cells and diurnal hormone rhythms on T helper cell activity.

Symptoms of diseases such as rheumatoid arthritis, which is T helper 1 (Th1) dependent, and asthma, which is T helper 2 (Th2) dependent, are influenced by diurnal rhythms and natural regulatory T cells (nTreg). However, the mechanisms responsible for the diurnal rhythm of disease activity have not been identified and it is unclear whether nTreg activity is diurnal rhythm-dependent. We therefore investigated whether a 24-hr diurnal cycle affected the ability of various helper T-cell populations to generate immunomodulatory and pro-inflammatory cytokines, as well as its suppression by nTreg cells. Using a within-subject crossover design, sleep versus continuous wakefulness was compared over a 24-hr period in healthy young volunteers under defined environmental conditions. Venous blood was drawn periodically every 5 hr and the function of T cells was explored in vitro. We demonstrated that interleukin (IL)-2, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and IL-10 secretion by naïve CD4(+) T cells follows a diurnal rhythm. Furthermore, multiple regression analysis, as well as subsequent in vitro experiments, suggested that serum levels of cortisol and prolactin are part of the underlying mechanism. Additionally, we observed that nTreg suppressed the secretion of IFN-γ, IL-2 and TNF-α, but not the secretion of IL-4, IL-6, IL-10 and IL-17A. However, the abrogation of IL-2 release was reversed upon inhibiting CD25 on nTreg. Highly purified nTreg secreted IL-6, IL-10 and IL-17A, but not IL-2, IL-4, IFN-γ or TNF-α. Taken together, our results demonstrate that hormones and nTreg modulate the diurnal rhythm of T helper cell activity.