Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4⁺ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4⁺ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4⁺ T‐cell subsets. Proportions of the other double or single CD4⁺ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4⁺ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4⁺ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.     

Dendritic cells from human mesenteric lymph nodes in inflammatory and non‐inflammatory bowel diseases: subsets and function of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDC) in mesenteric lymph nodes (MLN) may be important regulators of both inflammatory and non‐inflammatory mucosal immune responses but human studies are rare. Here we compare pDC from human MLN and peripheral blood (PB) by phenotype and function. MLN from patients with or without inflammatory bowel disease (IBD) undergoing colon surgery and PB from patients with IBD and from controls were used to isolate mononuclear cells. The pDC were analysed by flow cytometry for the expression of CD40, CD80, CD83, CD86, CCR6, CCR7, CX3CR1, CD103 and HLA‐DR. Purified pDC from MLN and PB were stimulated with staphylococcus enterotoxin B (SEB), CpG‐A, interleukin‐3 (IL‐3), SEB + IL‐3, CpG‐A + IL‐3 or left unstimulated, and cultured alone or with purified allogeneic CD4⁺ CD45RA⁺ HLA‐DR‐ T cells. Subsequently, concentrations of IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐17, interferon‐α (IFN‐α), IFN‐γ and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by multiplex bead array. The PB pDC from IBD patients exhibited an activated and matured phenotype whereas MLN pDC and control PB pDC were less activated. CpG‐A and CpG‐A + IL‐3‐stimulated MLN pDC secreted less IL‐6 and TNF‐α compared with PB pDC from controls. Compared with co‐cultures of naive CD4 T cells with PB pDC, co‐cultures with MLN pDC contained more IL‐2, IL‐10 and IFN‐γ when stimulated with SEB and SEB + IL‐3, and less IFN‐α when stimulated with CpG‐A. MLN pDC differ phenotypically from PB pDC and their pattern of cytokine secretion and may contribute to specific outcomes of mucosal immune reactions.     

Combination use of immune complexes and a Ca2+ channel blocker azelnidipine enhances interleukin‐12 p40 secretion without T helper type 17 cytokine secretion in human monocyte‐derived dendritic cells

Immune complexes (ICs) improve the capacity of priming specific CD8⁺ cytotoxic T cell responses of dendritic cells (DCs). ICs induce phosphorylation of mitogen‐activated protein kinases (MAPK) and calcium influx, although the precise regulating mechanism still remains unclear. In the present study, we investigated the effect of a Ca2⁺ channel blocker on the phosphorylation of p38 MAPK and extracellular signal‐regulated kinase (ERK) in immature monocyte‐derived DCs stimulated with lipopolysaccharide (LPS) or LPS‐ICs, and the production of interleukin (IL)‐12 family members (p40, p70, IL‐23), T helper type 17 (Th17) cytokines (IL‐6 and IL‐23), tumour necrosis factor (TNF)‐α and IL‐10 were also investigated. In comparison with LPS stimulation, LPS‐ICs stimulation enhanced p38 MAPK phosphorylation significantly, which was associated with an increase in IL‐12 p40 monomer/homodimer secretion. LPS‐ICs also enhanced TNF‐α and IL‐6 secretion, but suppressed IL‐23 secretion. The use of azelnidipine (Aze), a long‐acting L‐type Ca2⁺ channel blocker with a high lipid solubility, suppressed p38 MAPK phosphorylation stimulated with LPS or LPS‐ICs, but surprisingly enhanced IL‐12 p40 monomer/homodimer secretion stimulated with LPS‐ICs. This IL‐12 p40 secretion‐enhancing effect was not accompanied by IL‐10 or IL‐23 production, but was associated with ERK phosphorylation. The use of Aze did not affect IL‐12 p70 production. These results suggest that the use of Aze enhances ICs‐mediated IL‐12 p40 secretion without additional IL‐23 secretion. Therefore, the use of Aze and ICs could be a new therapeutic approach to immunomolecular therapy, as it does not cause Th17 differentiation which induces autoimmunity or reduces anti‐tumour immunity.     

Epstein–Barr virus‐specific CD8+ T lymphocytes from diffuse large B cell lymphoma patients are functionally impaired

Epstein–Barr virus (EBV) is a persistent virus with oncogenic capacity that has been implicated in the development of aggressive B cell lymphomas, primarily in immunosuppressed individuals, although it can be present in immunocompetent individuals. Changes in the function and clonal diversity of T lymphocytes might be implied by viral persistence and lymphoma development. The aim of the present study was to evaluate the frequency, phenotype, function and clonotypical distribution of EBV‐specific T cells after peripheral blood stimulation with a virus lysate in newly diagnosed patients with diffuse large B cell lymphoma (DLBCL) aged more than 50 years without prior histories of clinical immunosuppression compared with healthy controls. Our results showed impaired EBV‐specific immune responses among DLBCL patients that were associated primarily with decreased numbers of central and effector memory CD8⁺ T lymphocytes. In contrast to healthy controls, only a minority of the patients showed CD4⁺/tumour necrosis factor (TNF)‐α⁺ T cells expressing T cell receptor (TCR)‐Vβ17 and CD8⁺/TNF‐α⁺ T cells with TCR‐Vβ5·2, Vβ9 and Vβ18 in response to EBV. Notably, the production of TNF‐α was undetectable among TCR‐Vβ5·3⁺, Vβ11⁺, Vβ12⁺, Vβ16⁺ and Vβ23⁺ CD8⁺ T cells. In addition, we observed decreased numbers of CD4⁺/TNF‐α⁺ and CD8⁺/TNF‐α⁺, CD8⁺/interleukin (IL)‐2⁺ and CD8⁺/TNF‐α⁺/IL‐2⁺ T lymphocytes in the absence of T cells capable of producing TNF‐α, IL‐2 and IFN‐γ after EBV stimulation simultaneously. Moreover, DLBCL patients displayed higher IL‐10 levels both under baseline conditions and after EBV stimulation. These findings were also observed in patients with positive EBV viral loads. Prospective studies including a large number of patients are needed to confirm these findings.     

Signals required for programming effector and memory development by CD8+ T cells

Summary: Stimulation of naïve CD8⁺ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin‐12 (IL‐12) or interferon‐α (IFN‐α). CD4⁺ T cells condition dendritic cells (DCs) to effectively present antigen to CD8⁺ T cells, and this conditioning involves, at least in part, CD40‐dependent upregulation of the production of these signal 3 cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation‐induced non‐responsiveness (AINR), a form of split anergy characterized by an inability to produce IL‐2 to support continued expansion. If antigen remains present, IL‐2 provided by CD4⁺ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL‐2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8⁺ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations.     

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4+ T cells

Human type I interferons (IFNs) include IFN‐β and 12 subtypes of IFN‐α. During viral infection, infiltrating memory CD4 ⁺ T cells are exposed to IFNs, but their impact on memory T‐cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN‐α8 and ‐α10 most potently enhanced expression of IFN‐γ, IL‐2, and IL‐4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL‐2 and IL‐4 correlated with the time of preincubation with type I IFN, IFN‐γ production was enhanced best when IFN‐α was added immediately preceding or simultaneously with T‐cell stimulation. Comparison of T‐cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN‐α best enhanced cytokine expression when CD4 ⁺ T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T‐cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4 ⁺ T‐cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.     

Activated γδ T cells inhibit osteoclast differentiation and resorptive activity in vitro

Extensive evidence suggests that the immune system exerts powerful effects on bone cells, particularly in chronic disease pathologies such as rheumatoid arthritis (RA). The chronic inflammatory state in RA, particularly the excessive production of T cell‐derived proinflammatory cytokines such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐17, triggers bone erosions through the increased stimulation of osteoclast formation and activity. While evidence supports a role for IL‐17 and TNF‐α secreted by conventional CD4⁺ T cells in RA, recent evidence in animal models of RA have implicated γδ T cells as a major producer of pathogenic IL‐17. However, the capacity of γδ T cells to influence osteoclast formation and activity in humans has not yet been investigated widely. To address this issue we investigated the effects of γδ T cells on osteoclast differentiation and resorptive activity. We have demonstrated that anti‐CD3/CD28‐stimulated γδ T cells or CD4⁺ T cells inhibit human osteoclast formation and resorptive activity in vitro. Furthermore, we assessed cytokine production by CD3/CD28‐stimulated γδ T cells and observed a lack of IL‐17 production, with activated γδ T cells producing abundant interferon (IFN)‐γ. The neutralization of IFN‐γ markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN‐γ is the major factor responsible for the inhibitory role of activated γδ T cells on osteoclastogenesis and resorptive activity of mature osteoclasts. Our work therefore provides new insights on the interactions between γδ T cells and osteoclasts in humans.     

Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses

An adequate effector response against pathogens and its subsequent inactivation after pathogen clearance are critical for the maintenance of immune homeostasis. This process involves an initial phase of T‐cell effector (Teff) activation followed by the expansion of regulatory T cells (Tregs), a unique cell population that limits Teff functions. However, significant questions remain unanswered about the mechanisms that regulate the balance between these cell populations. Using an in vitro system to mimic T‐cell activation in human peripheral blood mononuclear cells (PBMC), we analysed the patterns of Treg and Teff activation, with special attention to the role of type I interferon (IFN‐I). Interestingly, we found that IFN‐α, either exogenously added or endogenously induced, suppressed the generation of CD4⁺ FoxP3^HI IFN‐γ^Neg activated Tregs (aTregs) while simultaneously promoting propagation of CD4⁺ FoxP3^Low/Neg IFN‐γ^Pos activated Teffs (aTeffs). We also showed that IFN‐α‐mediated inhibition of interleukin (IL)‐2 production may play an essential role in IFN‐α‐induced suppression of aTregs. In order to test our findings in a disease state with chronically elevated IFN‐α, we investigated systemic lupus erythematosus (SLE). Plasma from patients with SLE was found to contain IFN‐I activity that suppressed aTreg generation. Furthermore, anti‐CD3 activated SLE PBMCs exhibited preferential expansion of aTeffs with a very limited increase in aTreg numbers. Together, these observations support a model whereby a transient production of IFN‐α (such as is seen in an early antiviral response) may promote CD4 effector functions by delaying aTreg generation, but a chronic elevation of IFN‐α may tip the aTeff:aTreg balance towards aTeffs and autoimmunity.     

Selective effects of Lactobacillus casei Shirota on T cell activation, natural killer cell activity and cytokine production

Modulation of host immunity is an important potential mechanism by which probiotics confer health benefits. This study was designed to investigate the effects of a probiotic strain, Lactobacillus casei Shirota (LcS), on immune function using human peripheral blood mononuclear cells (PBMC) in vitro. In addition, the role of monocytes in LcS‐induced immunity was also explored. LcS promoted natural killer (NK) cell activity and preferentially induced expression of CD69 and CD25 on CD8 ⁺ and CD56 ⁺ subsets in the absence of any other stimulus. LcS also induced production of interleukin (IL)‐1β, IL‐6, tumour necrosis factor (TNF)‐α, IL‐12 and IL‐10 in the absence of lipopolysaccharide (LPS). In the presence of LPS, LcS enhanced IL‐1β production but inhibited LPS‐induced IL‐10 and IL‐6 production, and had no further effect on TNF‐α and IL‐12 production. Monocyte depletion reduced significantly the impact of LcS on lymphocyte activation, cytokine production and natural killer (NK) cell activity. In conclusion, LcS activated cytotoxic lymphocytes preferentially in both the innate and specific immune systems, which suggests that LcS could potentiate the destruction of infected cells in the body. LcS also induced both proinflammatory and anti‐inflammatory cytokine production in the absence of LPS, but in some cases inhibited LPS‐induced cytokine production. Monocytes play an important role in LcS‐induced immunological responses.     

Endogenous interleukin‐6 amplifies interleukin‐17 production and corticoid‐resistance in peripheral T cells from patients with multiple sclerosis

Interleukin‐6 (IL‐6) has been implicated in the induction of pathogenic IL‐17‐producing T cells in autoimmune diseases, and studies evaluating the role of this cytokine in T‐cell function in patients with multiple sclerosis (MS) are lacking. Our objective was to evaluate the role of IL‐6 receptor (IL‐6R) signalling on in vitro functional status of T cells from patients with relapsing–remitting MS during clinical remission. Our results demonstrated that, even during the remission phase, activated T cells from patients produce higher levels of IL‐17, and this cytokine was positively correlated with disease severity, as determined by Expanded Disability Status Scale score. In the MS group, the blockade of IL‐6R signalling by anti‐IL‐6R monoclonal antibody reduced IL‐17 production and elevated IL‐10 release by activated CD4⁺ T cells, but it did not alter the production of these cytokines by activated CD8⁺ T cells. Blockade of IL‐6R signalling also reduced the ability of monocytes to up‐regulate T helper type 17 phenotype in patients with MS. Finally, both cell proliferation and IL‐17 release by CD4⁺ and, mainly, CD8⁺ T cells from patients with MS were less sensitive to hydrocortisone inhibition than control group. Interestingly, IL‐6R signalling blockade restored the ability of hydrocortisone to inhibit both T‐cell proliferation and IL‐17 production. Collectively, these results suggest that IL‐6 might be involved in MS pathogenesis by enhancing IL‐17 production and reducing corticoid inhibitory effects on activated T cells.     

Functional deficits of pertussis-specific CD4(+) T cells in infants compared to adults following DTaP vaccination

Understanding the immune responses that explain why infants require multiple doses of pertussis vaccine to achieve protection against infection is a high priority. The objective of this study was to compare the function and phenotypes of antigen‐specific CD4⁺ T cells in adults (n = 12), compared to infants (n = 20), following vaccination with acellular pertussis (DTaP) vaccine. Peripheral blood mononuclear cells (PBMCs) were stimulated with pertussis toxoid (PT), pertactin (PRN) and filamentous haemagglutinin (FHA). Multi‐parameter flow cytometry was used to delineate CD4⁺ T cell populations and phenotypes producing interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐α and IL‐4. Based on surface CD69 expression, infants demonstrated activation of vaccine antigen‐specific CD4⁺ T cells similar to adults. However, among infants, Boolean combinations of gates suggested that type 1 (Th‐1) CD4⁺ T cell responses were confined largely to TNF‐α⁺IL‐2⁺IFN‐γ^‐ or TNF‐α⁺IL‐2^‐IFN‐γ^‐. A significantly lower percentage of polyfunctional T helper type 1 (Th1) responses (TNF‐α⁺IFN‐γ⁺IL‐2⁺) and type 2 (Th2) responses (IL‐4) were present in the infants compared to adults. Moreover, a significantly higher percentage of infants' functional CD4⁺ T cells were restricted to CD45RA^‐CCR7⁺CD27⁺ phenotype, consistent with early‐stage differentiated pertussis‐specific memory CD4⁺ T cells. We show for the first time that DTaP vaccination‐induced CD4⁺ T cells in infants are functionally and phenotypically dissimilar from those of adults.     

Effector T‐cell differentiation during viral and bacterial infections: Role of direct IL‐12 signals for cell fate decision of CD8+ T cells

To study the role of IL‐12 as a third signal for T‐cell activation and differentiation in vivo, direct IL‐12 signaling to CD8⁺ T cells was analyzed in bacterial and viral infections using the P14 T‐cell adoptive transfer model with CD8⁺ T cells that lack the IL‐12 receptor. Results indicate that CD8⁺ T cells deficient in IL‐12 signaling were impaired in clonal expansion after Listeria monocytogenes infection but not after infection with lymphocytic choriomeningitis virus, vaccinia virus or vesicular stomatitis virus. Although limited in clonal expansion after Listeria infection, CD8⁺ T cells deficient in IL‐12 signaling exhibited normal degranulation activity, cytolytic functions, and secretion of IFN‐γ and TNF‐α. However, CD8⁺ T cells lacking IL‐12 signaling failed to up‐regulate KLRG1 and to down‐regulate CD127 in the context of Listeria but not viral infections. Thus, direct IL‐12 signaling to CD8⁺ T cells determines the cell fate decision between short‐lived effector cells and memory precursor effector cells, which is dependent on pathogen‐induced local cytokine milieu.     

Blockade of PD‐1 or p38 MAP kinase signaling enhances senescent human CD8+ T‐cell proliferation by distinct pathways

Immune enhancement is desirable in situations where decreased immunity results in increased morbidity. We investigated whether blocking the surface inhibitory receptor PD‐1 and/or p38 MAP kinase could enhance the proliferation of the effector memory CD8⁺ T‐cell subset that re‐expresses CD45RA (EMRA) and exhibits characteristics of senescence, which include decreased proliferation and telomerase activity but increased expression of the DNA damage response related protein γH2AX. Blocking of both PD‐1 and p38 MAPK signaling in these cells enhanced proliferation and the increase was additive when both pathways were inhibited simultaneously in both young and old human subjects. In contrast, telomerase activity in EMRA CD8⁺ T cells was only enhanced by blocking the p38 but not the PD‐1 signaling pathway, further indicating that nonoverlapping signaling pathways were involved. Although blocking p38 MAPK inhibits TNF‐α secretion in the EMRA population, this decrease was counteracted by the simultaneous inhibition of PD‐1 signaling in these cells. Therefore, end‐stage characteristics of EMRA CD8⁺ T cells are stringently controlled by distinct and reversible cell signaling events. In addition, the inhibition of PD‐1 and p38 signaling pathways together may enable the enhancement of proliferation of EMRA CD8⁺ T cells without compromising their capacity for cytokine secretion.     

Treatment of collagen‐induced arthritis by Natura‐α via regulation of Th‐1/Th‐17 responses

Cytokines and CD4⁺ Th cells play a crucial role in the pathogenesis of rheumatoid arthritis. Among the Th populations, Th‐1 and Th‐17 have been described as pathogenic in collagen‐induced arthritis (CIA) whereas Th‐2 and Treg were found to have protective effects. The objective of this study was to examine the affect of Natura‐α, a newly developed cytokine regulator, on CIA and on Th cell development. Natura‐α treatment was administered before or during arthritis induction. Anti‐type II collagen antibodies and cytokine expression were evaluated by ELISA. Emergence of CD4⁺CD25⁺Foxp3⁺ T cells was assessed by flow cytometry. Th‐17 differentiation of naive CD4 T cells was assessed in cultures with anti‐CD3 and anti‐CD28. We showed that Natura‐α both prevented and treated CIA. We further demonstrated that in vivo treatment with Natura‐α inhibited IL‐17 production and anti‐type II collagen IgG development. We showed in vitro, using an APC‐free system, that Natura‐α acted directly on differentiating T cells and inhibiting the formation of Th‐1 and Th‐17 cells but did not affect Th‐2 cells. Since Natura‐α inhibits a large spectrum of important pathogenic factors in CIA, it may provide a new and powerful approach to the treatment of rheumatoid arthritis and other inflammatory diseases.     

ICOS cooperates with CD28, IL-2, and IFN-gamma and modulates activation of human naïve CD4+ T cells

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4⁺ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti‐CD3 mAb. ICOS strikingly potentiated secretion of IL‐2, IFN‐γ, IL‐10, and TNF‐α, but not IL‐4, promoted by optimal stimulation of CD3+CD28, and it was the key switching‐factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL‐2. In these conditions, blockade of IL‐2 and IFN‐γ showed that ICOS builds up a positive feedback loop with IFN‐γ, which required IL‐2 and was inhibited by IL‐4. By contrast, in the absence of CD28 triggering or exogenous IL‐2, ICOS‐induced costimulation mainly supported expression of TGF‐β1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4⁺ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL‐2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.     

Age‐associated Epstein–Barr virus‐specific T cell responses in seropositive healthy adults

Epstein–Barr virus (EBV) is present in 95% of the world's adult population. The immune response participates in immune vigilance and persistent infection control, and this condition is maintained by both a good quality (functionality) and quantity of specific T cells throughout life. In the present study, we evaluated EBV‐specific CD4⁺ and CD8⁺ T lymphocyte responses in seropositive healthy individuals younger and older than 50 years of age. The assessment comprised the frequency, phenotype, functionality and clonotypic distribution of T lymphocytes. We found that in both age groups a similar EBV‐specific T cell response was found, with overlapping numbers of tumour necrosis factor (TNF)‐α⁺ T lymphocytes (CD4⁺ and CD8⁺) within the memory and effector cell compartments, in addition to monofunctional and multi‐functional T cells producing interleukin (IL)‐2 and/or interferon (IFN)‐γ. However, individuals aged more than 50 years showed significantly higher frequencies of IL‐2‐producing CD4⁺ T lymphocytes in association with greater production of soluble IFN‐γ, TNF‐α and IL‐6 than subjects younger than 50 years. A polyclonal T cell receptor (TCR)‐variable beta region (Vβ) repertoire exists in both age groups under basal conditions and in response to EBV; the major TCR families found in TNF‐α⁺/CD4⁺ T lymphocytes were Vβ1, Vβ2, Vβ17 and Vβ22 in both age groups, and the major TCR family in TNF‐α⁺/CD8⁺ T cells was Vβ13·1 for individuals younger than 50 years and Vβ9 for individuals aged more than 50 years. Our findings suggest that the EBV‐specific T cell response (using a polyclonal stimulation model) is distributed throughout several T cell differentiation compartments in an age‐independent manner and includes both monofunctional and multi‐functional T lymphocytes.     

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.