Bystander activation of CD4+ T cells

Bystander activation of T cells, i.e. the stimulation of unrelated (heterologous) T cells by cytokines during an Ag‐specific T‐cell response, has been best described for CD8⁺ T cells. In the CD8⁺ compartment, the release of IFN and IFN‐inducers leads to the production of IL‐15, which mediates the proliferation of CD8⁺ T cells, notably memory‐phenotype CD8⁺ T cells. CD4⁺ T cells also undergo bystander activation, however, the signals inducing this Ag‐nonspecific stimulation of CD4⁺ T cells are less well known. A study in this issue of the European Journal of Immunology sheds light on this aspect, suggesting that common γ‐chain cytokines including IL‐2 might be involved in bystander activation of CD4⁺ T cells.     

Intravenous immunoglobulin modulates the expansion and cytotoxicity of CD8+ T cells

Intravenous immunoglobulin (IVIg) is successfully used in the treatment of autoimmune diseases involving self‐reactive CD8⁺ T cells. However, its direct influence on the cytotoxic response remains unknown. Using an antigen cross‐presentation assay and a mouse model of ovalbumin (OVA) immunization, we showed that IVIg decreases the in vitro activation, proliferation and cytokine secretion of OVA‐specific CD8⁺ T cells (OT‐I), as well as the in vivo generation of OVA‐specific CD8⁺ T cells. In addition, IVIg significantly decreases the proportion of perforin‐ and CD107a‐expressing CD8⁺ T cells, and inhibits the cytotoxic activity of OVA‐activated OT‐I cells. The interference of IVIg with the CD8⁺ T‐cell response is associated with T‐cell receptor blockade, therefore reducing the interaction between effector and target cells. A similar blockade is observed on human CD8⁺ T cells, suggesting that the observations reported here could apply to the IVIg‐mediated improvement of CD8⁺ T‐cell‐mediated autoimmune conditions in human patients.     

Innate profiles of cytokines implicated on oral tolerance correlate with low- or high-suppression of humoral response

Oral tolerance (OT) is being studied with great interest because of its therapeutic potential in allergy and autoimmunity. In the present study, two mouse strains with extreme phenotypes of OT susceptibility (TS) or resistance (TR) to ovalbumin (OVA) were used to demonstrate whether the tr and ts genes, cumulated during 18 generations of bi‐directional genetic selection, influence expression of immunobiological traits in naive or antigen‐gavaged TR/TS mice. The difference in anti‐OVA titres was 2048‐fold between OVA‐gavaged TS and TR mice. Tolerance susceptibility to OVA gavage in individuals from a (TS × TR)F₂ population was 24% high‐susceptibility, 62% low‐susceptibility and 14% non‐tolerant. Different antigens, unrelated to OVA, were tested by gavage and TS mice were generally susceptible while TR mice were resistant. The stability of TS and TR phenotypes was not affected by the use of strict protocols of intraperitoneal immunization or feeding over 30 consecutive days. The levels of interleukin‐2 (IL‐2), IL‐4, interferon‐γ and IL‐10 cytokines evaluated in concanavalin A‐stimulated spleen cells from naive mice and in OVA‐stimulated spleen cells from OVA‐gavaged mice were higher in TS mice. Interleukin‐10 was up‐regulated in OVA‐gavaged TS mice and down‐regulated in TR mice. In naive mice, the percentage of CD4⁺ CD25⁺ and CD4⁺ Foxp3⁺ spleen cells and IL‐10 expression by CD4⁺ cells was significantly higher in TS mice. These results indicate that regulation of IL‐10 expression could be an important factor contributing to the mechanisms controlling OT susceptibility, and that the OT responses of TR and TS individuals strongly correlate with their innate potential to secrete this cytokine.     

Hyaluronan binding identifies the most proliferative activated and memory T cells

CD44 is expressed on T cells where its ability to bind hyaluronan is tightly regulated. Here, we investigated when T cells bind hyaluronan during an immune response. We found that naïve, murine T cells do not bind fluoresceinated hyaluronan but are induced to bind upon antigen‐induced T‐cell activation in vitro and in vivo. Hyaluronan binding occurred on proliferating T cells and the percentage of hyaluronan‐binding cells correlated with the strength of the activation stimulus. A small percentage of hyaluronan‐binding cells persisted after in vitro activation and had a memory phenotype (CD122⁺CD44^hi). This hyaluronan‐binding population increased after culture with IL‐7 or IL‐15 and proliferated more rapidly than nonbinding cells. In vivo, approximately 20–30% of antigen‐specific OT‐I CD8⁺ memory T cells in the spleen and BM bound hyaluronan. Hyaluronan binding identified memory cells that proliferated faster in IL‐7 and IL‐15, and enriched for CD62L⁺ central memory cells. In vivo homeostatic proliferation induced hyaluronan binding on a small percentage of the most rapidly dividing cells after several cell divisions. This study demonstrates that hyaluronan binding is induced upon antigen‐induced T‐cell activation and occurs on a percentage of the most proliferative activated and memory T cells.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma

Background Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. Objective To examine the role of Tregs in tolerance induction in a murine model of asthma. Methods Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25⁺ T cells by anti‐CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4⁺CD25⁺ and CD4⁺CD25^? T cells, both T cell subsets were transferred from tolerized or non‐tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. Results Intranasal treatment with OVA led to increased levels of IL‐10, TGF‐β and IL‐17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4⁺CD25⁺Foxp3⁺ T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA‐specific T cell responses. Depletion of CD25⁺ cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4⁺CD25^? T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4⁺CD25⁺ T cells. As compared with control mice, a significantly higher proportion of CD4⁺CD25^? T cells from OVA treated mice expressed mTGF‐β. Conclusion Both CD4⁺CD25⁺ and CD4⁺CD25^? T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.     

TLR2 engagement on memory CD8+ T cells improves their cytokine‐mediated proliferation and IFN‐γ secretion in the absence of Ag

Persistence of memory CD8⁺ T cells is known to be largely controlled by common gamma chain cytokines, such as IL‐2, IL‐7 and IL‐15. However, other molecules may be involved in this phenomenon. We show here that TLR2^?/? mice have a decreased frequency of memory phenotype CD8⁺ T cells when compared with WT mice. This prompted us to investigate the role of TLR2 in the homeostasis of memory CD8⁺ T cells. We describe here a new TLR2‐dependent mechanism which, in the absence of specific antigen, directly controls memory CD8⁺ T‐cell proliferation and IFN‐γ secretion. We demonstrate that TLR2 engagement on memory CD8⁺ T cells increases their proliferation and expansion induced by IL‐7 both in vitro and in vivo. We also show that TLR2 ligands act in synergy with IL‐2 to induce IFN‐γ secretion in vitro. Both conclusions are obtained with spontaneously arising memory phenotype and antigen‐specific memory CD8⁺ T cells. Altogether, our data support the idea that continuous TLR2 signaling in response to microbial stimuli or endogenous danger signals might directly contribute to the maintenance of the diversity memory CD8⁺ T cells in the organism.     

IL‐7 is superior to IL‐2 for ex vivo expansion of tumour‐specific CD4+ T cells

It is well established that tumours hinder both natural and vaccine‐induced tumour‐specific CD4⁺ T‐cell responses. Adoptive T‐cell therapy has the potential to circumvent functional tolerance and enhance anti‐tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8⁺ T cells are currently available, data on tumour‐specific CD4⁺ T cells remain scarce. We report here that CD4⁺ T cells sensitized to tumour‐associated Ag in vivo, proliferate in vitro in response to IL‐7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory‐like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour‐sensitized T cells among other memory and naive lymphocytes following exposure to IL‐7. Also IL‐2, previously used to expand anti‐tumour CTL, promotes tumour‐specific CD4⁺ T‐cell accumulation. However, IL‐7 is superior to IL‐2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4⁺ T cells to confer therapeutic efficacy upon transplantation in tumour‐bearing hosts. Together our data support a unique role for IL‐7 in retrieving memory‐like CD4⁺ T cells suitable for adoptive T‐cell therapy.     

IL‐27 enhances the survival of tumor antigen‐specific CD8+ T cells and programs them into IL‐10‐producing,memory precursor‐like effector cells

IL‐27 is a member of the IL‐12 family of cytokines that is comprised of an IL‐12 p40‐related protein subunit, EBV‐induced gene 3, and a p35‐related subunit, p28. IL‐27 functions through IL‐27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8⁺ T‐cell responses. However, the exact mechanisms of how IL‐27 enhances antitumor CD8⁺ T‐cell responses remain unclear. Here we show that IL‐27 significantly enhances the survival of activated tumor antigen‐specific CD8⁺ T cells in vitro and in vivo, and programs tumor antigen‐specific CD8⁺ T cells into memory precursor‐like effector cells, characterized by upregulation of Bcl‐6, SOCS3, Sca‐1, and IL‐10. While STAT3 activation and the CTL survival‐enhancing effects can be independent of CTL IL‐10 production, we show here that IL‐27‐induced CTL IL‐10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL‐27 enhances antitumor CTL responses via programming tumor antigen‐specific CD8⁺ T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.     

UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells

Background In human asthma, and experimental allergic airways disease in mice, antigen‐presenting cells and CD4⁺ effector cells at the airway mucosa orchestrate, and CD4⁺CD25⁺ regulatory T cells attenuate, allergen immunity. UV irradiation of skin before sensitization with ovalbumin (OVA) causes significantly reduced asthma‐like responses in respiratory tissues. Objective To determine whether UV‐induced changes in CD11c⁺ cells, CD4⁺CD25⁺ effector cells or CD4⁺CD25⁺ regulatory cells in the trachea and airway draining lymph nodes (ADLNs) were responsible for reduced allergic airways disease. Methods The phenotype and function of CD11c⁺ cells and CD4⁺CD25⁺ cells in the trachea and ADLNs of UV‐ and non‐irradiated, OVA‐sensitized mice was examined 24 h after a single exposure to aerosolized OVA. Results No changes in the function of CD11c⁺ cells from UV‐irradiated mice were observed. CD4⁺CD25⁺ cells from UV‐irradiated, OVA‐sensitized mice harvested 24 h after OVA aerosol proliferated less in response to OVA in vitro and were unable to suppress the proliferation of OVA‐sensitized responder cells. This result suggested reduced activation of effector T cells in the airway mucosa of UV‐irradiated, OVA‐sensitized mice. To exclude regulatory cells of any type, there was similar proliferation in vivo to aerosolized OVA by CFSE‐loaded, OVA‐TCR‐specific CD4⁺ cells adoptively transferred into UV‐ and non‐irradiated, OVA‐sensitized mice. In addition, there was no difference in the expression of regulatory T cell markers (Foxp3, IL‐10, TGF‐β mRNA). To examine effector T cells, ADLN cells from UV‐irradiated, OVA‐sensitized and ‐challenged mice were cultured with OVA. There was reduced expression of the early activation marker CD69 by CD4⁺CD25⁺ cells, and reduced proliferation in the absence of the regulatory cytokine, IL‐10. Conclusion Reduced allergic airways disease in UV‐irradiated mice is due to fewer effector CD4⁺CD25⁺ cells in the trachea and ADLNs, and not due to UV‐induced regulatory cells. Cite this as: J. P. McGlade, D. H. Strickland, M. J. M. Lambert, S. Gorman, J. A. Thomas, M. A. Judge, J. T. Burchell, G. R. Zosky and P. H. Hart, Clinical & Experimental Allergy, 2010 (40) 772–785.     

Mycobacterium tuberculosis PstS1 amplifies IFN‐γ and induces IL‐17/IL‐22 responses by unrelated memory CD4+ T cells via dendritic cell activation

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4⁺ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4⁺ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4⁺ and CD8⁺ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α^? subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.     

EGF receptor activation during allergic sensitization affects IL‐6‐induced T‐cell influx to airways in a rat model of asthma

EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA‐specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4⁺ T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL‐6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA‐specific IgE and IL‐4 mRNA expression in CD4⁺ T cells were not affected by AG1478. BALF from OVA‐sensitized/challenged rats induced CD4⁺ T‐cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL‐6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4⁺ T cells to airways, mainly mediated through IL‐6.     

Atopic asthma: differential activation phenotypes among memory T helper cells

Background T cells have been implicated in the pathogenesis of atopic asthma. We have previously shown that memory T helper cells (CD4⁺CD45RO⁺) are preferentially activated relative to naïve T helper cells (CD4⁺CD45RA⁺) after bronchial allergen challenge. However, specific T helper subpopulations that are activated in atopy and/or asthma remain undefined. Objective To determine the T helper subpopulations and activation phenotypes relevant to acute and stable asthma that may be common with or distinct from atopy. Methods Two groups of atopic asthmatics (ten acute and nine stable asthmatics) and two non‐asthmatic groups (14 non‐asthmatic atopics and eight normal non‐atopic controls) were analysed. Ten acute asthmatics were assessed in the emergency room during an acute episode (FEV₁ 43.6% ± 18.4). Nine stable asthmatics were assessed during a symptom‐free period (FEV₁ 85% ± 6). Using multiple colour flow cytometry we analysed T cell subpopulations and the expression of IL‐2‐receptor (IL‐2R) and MHC‐class II antigens (MHC II) on naïve and memory T helper cells in the peripheral blood of asthmatic and non‐asthmatic groups. Results Atopic asthmatics (acute and stable) had an increased percentage of memory T helper cells expressing IL‐2R compared with normal non‐atopics (mean SD 16.1 ± 6%, 12.4 ± 2% and 7.7 ± 1.8%, P < 0.05) but not compared with non‐asthmatic atopics (10 ± 3.5%). Naïve T helper cells had low expression of IL‐2R and MHC II in all four groups. MHC II antigen expression was increased in memory T helper cells of asthmatics (acute and stable) compared with normal non‐atopics (13.9 ± 7.5, 10.6 ± 5 and 4.9 ± 2.5, P < 0.05) but not compared with non‐asthmatic atopics (7.92 4). A novel finding was that IL‐2R and the MHC II molecules were mainly expressed in non‐overlapping populations and coexpression was found predominantly on memory T helper cells. Asthmatics (acute and stable) had higher proportion of double positive memory T helper cells (IL‐2R⁺MHC II⁺) compared with both non‐asthmatic groups (P < 0.05). Conclusions We demonstrate a differential expression of IL‐2R⁺ and MCH II⁺ on CD45RO⁺ T helper cells that would suggest that there are three subsets of activated memory T helper cells in asthmatics. Two non‐overlapping IL‐2R⁺ or MHC II⁺ CD45RO⁺ T helper cells and a third subpopulation of activated cells that coexpress IL‐2R and MHC II (double positives). This latter subpopulation is significantly higher in asthmatics (acute or stable) compared with both non‐asthmatic groups, suggesting a specific T helper activation phenotype distinct to atopic asthmatics as compared with atopic non‐asthmatics.     

Predominance of activated,clonally expanded T helper type 17 cells within the CD4+ T cell population in psoriatic lesions

Recent evidence points to the T helper type 17 (Th17) subset as key in the pathogenesis of psoriasis, but cells of this type in lesions remain to be fully characterized. Here we isolated, enumerated, functionally tested and clonotyped the CD4⁺ Th cell population ex vivo from lesional biopsies and paired peripheral blood samples from psoriasis patients. Th17 cells were over‐represented dramatically in lesions from all patients, representing 49–93% of CD4⁺ Th cells compared with 3–18% in blood. Most lesional Th17 cells produced interleukin (IL)‐17A ex vivo without further stimulation and expressed the CD45RO⁺ phenotype characteristic of activated or memory cells. There was no increase in ‘natural’ [CD25^hiforkhead box protein 3 (FoxP3⁺)] regulatory T cells in lesions versus peripheral blood, but there was enrichment of ‘induced’ IL‐10⁺ regulatory T cell numbers in biopsies from some patients. The lesional Th17 cells exhibited a bias in T cell receptor Vβ chain usage, suggestive of specific expansion by antigen. The therapeutic challenge is to overcome the dominance of overwhelming numbers of such antigen‐specific Th17 cells in psoriatic lesions.     

Early simultaneous production of intranodal CD4 Th2 effectors and recirculating rapidly responding central‐memory‐like CD4 T cells

This study characterizes the diversity of CD4 Th cells produced during a Th2 response in vivo. Kinetics of effector and memory cell differentiation by mouse OVA‐specific CD4 T cells was followed during primary responses to alum‐precipitated OVA. The complexity of the CD4 T response was assessed in nodes draining and distant from the site of immunization for phenotype, location and function. By 3 days IL‐4‐producing effector CD4 T cells developed in the draining node and a proportion of the responding cells had migrated to B‐cell follicles, while yet others had left the draining node. Some of these early migrant cells were recirculating cells with a central memory phenotype. These had divided four or more times in the draining node before migrating to distant nodes not exposed to antigen. We questioned the responsiveness of these early central‐memory‐like cells on secondary antigen challenge at sites distant to the primary immunization. They re‐entered cell cycle and migrated to B‐cell follicles, much more rapidly than naïve CD4 T cells and could still be induced to produce IL‐4. Their production and survival were independent of the starting frequency of antigen‐specific CD4 T cells. Thus intranodal effector cells and recirculating, rapidly responding central‐memory‐like cells emerged simultaneously from the third day of a primary Th2 response.     

DRB1*0402 may influence arthritis by promoting naive CD4+ T‐cell differentiation in to regulatory T cells

HLA‐DRB1*0401 expression in humans has been associated with a predisposition to developing rheumatoid arthritis (RA) and collagen‐induced arthritis (CIA), while HLA‐DRB1*0402 is not associated with susceptibility. Here, we determined if mice transgenic (Tg) for human *0401 have a CD4⁺ T‐cell repertoire that predetermines proinflammatory cytokine production. The data show that both *0401 and *0402 Tg mice can produce TH1/TH17 cytokines, although the kinetics of response may be different. However, in the context of antigen‐specific responses in a CIA model, *0402 Tg mice generate a TH2 response that may explain their resistance to developing arthritis. In addition, a significant subset of naïve CD4⁺ T cells from *0402 Tg mice can be activated in polarizing conditions to differentiate into Treg cells that produce IFN‐γ. *0401 Tg mice harbor memory CD4⁺ T cells that differentiate into IL‐17⁺ cells in various polarizing conditions. Our data suggest that *0401 Tg mice generate a strong immune response to lipopolysaccharide and may be efficient in clearing infection, and may *0401 have been evolutionarily selected for this ability. Autoimmunity, such as RA, could likely be a bystander effect of the cytokine storm that, along with the presence of low Treg‐cell numbers in *0401 Tg mice, causes immune dysregulation.     

Influenza infection results in local expansion of memory CD8+ T cells with antigen non‐specific phenotype and function

Primary viral infections induce activation of CD8⁺ T cells responsible for effective resistance. We sought to characterize the nature of the CD8⁺ T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8⁺ T cells exhibiting a unique bystander phenotype with significant up‐regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44^highCD8⁺ T cells, suggesting an antigen non‐specific phenotype. We further confirmed the non‐specificity of this phenotype on ovalbumin‐specific (OT‐I) CD8⁺ T cells, which are not specific to influenza. These non‐specific CD8⁺ T cells also displayed increased lytic capabilities and were observed primarily in the lung. Thus, influenza infection was shown to induce a rapid, antigen non‐specific memory T cell expansion which is restricted to the specific site of inflammation. In contrast, CD8⁺ T cells of a similar phenotype could be observed in other organs following administration of systemic agonistic anti‐CD40 and interleukin‐2 immunotherapy, demonstrating that bystander expansion in multiple sites is possible depending on whether the nature of activation is either acute or systemic. Finally, intranasal blockade of NKG2D resulted in a significant increase in viral replication early during the course of infection, suggesting that NKG2D is a critical mediator of anti‐influenza responses prior to the initiation of adaptive immunity. These results characterize further the local bystander expansion of tissue‐resident, memory CD8⁺ T cells which, due to their early induction, may play an important NKG2D‐mediated, antigen non‐specific role during the early stages of viral infection.     

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.     

Human peripheral blood and bone marrow Epstein–Barr virus‐specific T‐cell repertoire in latent infection reveals distinct memory T‐cell subsets

EBV infection leads to life‐long viral persistence. Although EBV infection can result in chronic disease and malignant transformation, most carriers remain disease‐free as a result of effective control by T cells. EBV‐specific IFN‐γ‐producing T cells could be demonstrated in acute and chronic infection as well as during latency. Recent studies, however, provide evidence that assessing IFN‐γ alone is insufficient to assess the quantity and quality of a T‐cell response. Using overlapping peptide pools of latent EBV nuclear antigen 1 and lytic BZLF‐1 protein and multicolor flow cytometry, we demonstrate that the majority of ex vivo EBV‐reactive T cells in healthy virus carriers are indeed IL‐2‐ and/or TNF‐producing memory cells, the latter being significantly more frequent in BM. After in vitro expansion, a substantial number of EBV‐specific CD4⁺ and CD8⁺ T cells retained a CC‐chemokine receptor 7 (CCR7)‐positive memory phenotype. Based on their cytokine profiles, six different EBV‐specific T‐cell subsets could be distinguished with TNF‐single or TNF/IL‐2‐double producing cells expressing the highest CCR7 levels resembling early‐differentiated memory T cells. Our study delineates the memory T‐cell profile of a protective immune response and provides a basis for analyzing T‐cell responses in EBV‐associated diseases.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.