gammadelta T cells condition dendritic cells in vivo for priming pulmonary CD8 T cell responses against Mycobacterium tuberculosis

gammadelta T cells and dendritic cells are quickly recruited to the lungs shortly after intranasal vaccination with BCG, but the functional in vivo interplay between these two cell populations and its role in the induction of adaptive immune responses is unclear. Using TCR-deficient mice and bone marrow chimeras, we show here that gammadelta T cells provide a non-redundant early source of IFN-gamma in vivo, which enhances IL-12 production by lung dendritic cells. The in vivo-conditioned dendritic cells, in turn, prime a more efficient lung CD8 T cell response against Mycobacterium tuberculosis. Thus, strategies exploiting gammadelta T cell function and IFN-gamma production could be valuable for the design and testing of mucosal vaccines.     

Prior elevation of IL-18 promotes rapid early IFN-gamma production during staphylococcal infection

Systemic Staphylococcus aureus infection is associated with significant morbidity and mortality arising from both bacterial and host immune factors. IL-18 is a pro-inflammatory cytokine of the IL-1 superfamily that exhibits broad functional effects in innate and acquired immune responses and which has been found in high levels in several chronic inflammatory and autoimmune diseases. Over-expression of IL-18 may promote early resolution of infection or could promote a detrimental exaggerated immune response. This was explored in a model of S. aureus infection. We report increased mortality in Swiss mice that were given recombinant IL-18 prior to inoculation with S. aureus LS-1. IL-18 administration prior to infection induced preferentially enhanced IFN-gamma mRNA expression in peripheral blood leukocytes and spleen, especially splenic NK cells. This correlated with increased IFN-gamma protein detection in serum, and leukocyte and spleen cultures at subsequent discrete time points. These data suggest that increased mortality following gram-positive infection in autoimmune diseases could in part reflect the impact of high levels of pleiotropic pro-inflammatory cytokines such as IL-18 present prior to the onset of infection.     

gammadelta-T cells expressing NK receptors predominate over NK cells and conventional T cells in the innate IFN-gamma response to Plasmodium falciparum malaria

Rapid production of interferon-gamma (IFN-gamma) in response to malaria by the innate immune system may determine resistance to infection, or inflammatory disease. However, conflicting reports exist regarding the identity of IFN-gamma-producing cells that rapidly respond to Plasmodium falciparum. To clarify this area, we undertook detailed phenotyping of IFN-gamma-producing cells across a panel of naive human donors following 24-h exposure to live schizont-infected red blood cells (iRBC). Here, we show that NK cells comprise only a small proportion of IFN-gamma-responding cells and that IFN-gamma production is unaffected by NK cell depletion. Instead, gammadelta-T cells represent the predominant source of innate IFN-gamma, with the majority of responding gammadelta-T cells expressing NK receptors. Malaria-responsive gammadelta-T cells more frequently expressed NKG2A compared to non-responding gammadelta-T cells, while non-responding gammadelta-T cells more frequently expressed CD158a/KIR2DL1. Unlike long-term gammadelta-T cell responses to iRBC, alphabeta-T cell help was not required for innate gammadelta-T cell responses. Diversity was observed among donors in total IFN-gamma output. This was positively associated with CD94 expression on IFN-gamma(+) NK-like gammadelta-T cells. Applied to longitudinal cohort studies in endemic regions, similar comparative phenotyping should allow assessment of the contribution of diverse cell populations and regulatory receptors to risk of infection and disease.     

Transient inhibition of Th1-type cytokine production by CD4 T cells in hepatitis B core antigen immunized mice is mediated by regulatory T cells

The non-cytopathic hepatitis B virus (HBV) can induce chronic infections characterized by weak and limited T cell responses against the virus. The factors contributing to the failure to clear HBV and subsequent development of chronic HBV infections are not clearly understood, but a strong interferon-gamma (IFN-gamma) response by CD4+ T cells against the nucleocapsid hepatitis B core antigen (HBcAg) of the virus appears to be important for viral clearance. The present study documents depressed numbers of CD4+ T cells secreting IFN-gamma and interleukin-2 (IL-2) in enzyme-linked immunospot assay (ELISPOT) assays restimulated for 24 hr with antigen following both primary and secondary immunizations of mice with recombinant hepatitis B core antigen (rHBcAg). The kinetics of these responses showed that the depression occurred following a peak response and lasted approximately 2 weeks before returning to the previous peak levels. The depression was abrogated by depletion of CD25+ cells prior to culture in the ELISPOT assay, suggesting inhibition by regulatory T cells. This inhibition of IFN-gamma and IL-2 production was also reversed by in vitro restimulation of the test cells for 48 hr rather than 24 hr in the assay. No such transient, reversible inhibition was detected in the production of IL-5, a Th2-type cytokine. The inhibition in cytokine production did not appear to correlate with the number of antibody-secreting cells or the isotypes produced. This delay by regulatory T cells of Th1-type cytokine production could contribute to viral persistence in chronic HBV infection by interfering with the critical role IFN-gamma plays in protection against viral infections.     

Yes T cells, but three different T cells (alphabeta, gammadelta and NK T cells), and also B-1 cells mediate contact sensitivity

Transfer of contact sensitivity (CS) responses by immune lymphoid cells was the first finding that distinguished cellular from humoral immunity. CS has remained the most studied T cell reaction in vivo, and is the prototype for a variety of delayed-type hypersensitivity (DTH) responses. DTH in essence is the recruitment of effector alphabeta-T cells out of vessels into peripheral tissues. The T cells then are activated by antigen presenting cells to produce pro-inflammatory cytokines. It has been assumed that the alphabeta-T cells alone are responsible, but recent studies show that three other lymphocyte subsets are involved: CS-inducing NK T cells, CS-initiating B-1 cells, and CS-assisting gammadelta-T cells. Therefore, the effector alphabeta-T cells are essential, but cannot be recruited into the tissues without the local action of IgM antibodies produced by B-1 cells rapidly (1 day) post-immunization. The IgM complexes with the challenge antigen to locally activate complement to lead to vascular activation required for T cell recruitment. This process occurs early (1-2 hours) in the elicitation phase, and is called CS-initiation. The essential CS-inducing NK T cells activate the B-1 cells by producing IL-4 rapidly (1 hour) after immunization, and gammadelta-T cells assist the local inflammatory function of the recruited CS-effector alphabeta-T cells. Thus, four lymphocyte subsets are required for elicitation of responses: CS-inducing NK T cells, CS-initiating B-1 cells, CS-assisting gammadelta-T cells, and finally the CS-effector alphabeta-T cells. Three of these four cell types are present in the immune lymphoid cell population that adoptively transfers CS: B-1 cells, gammadelta-T cells, and the alphabeta-T cells.     

Respiratory syncytial virus infection influences tight junction integrity

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air–liquid interface (ALI) and infected with RSV and ultraviolet (UV)‐irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV‐irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post‐infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down‐regulated claudin‐1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin‐1 and claudin‐7 at protein level.     

Respiratory syncytial virus enhances respiratory allergy in mice despite the inhibitory effect of virus-induced interferon-gamma

In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic Th2 immune response, characterised by production of interleukin (IL)-4, IL-5, and IL-13, and eosinophilic inflammation. This enhancement of the Th2 response occurs simultaneously with a strong RSV-induced Th1 cytokine response (IL-12 and IFN-gamma). The present study investigated whether IFN-gamma and IL-12 are critically involved in this RSV-enhanced OVA allergy. Therefore, IFN-gammaR- and IL-12-deficient mice (both on a 129/Sv/Ev background) were sensitised and challenged with ovalbumin (OVA) and infected with RSV during the OVA challenge period. Neither gene deletion affected the development of ovalbumin-induced allergic inflammation in mice. However, when OVA-allergic IFN-gammaR deficient mice were infected with RSV, an increased pulmonary eosinophilic infiltrate and increased IL-4 and IL-13 mRNA expression in lung tissue were observed compared with identically treated wild-type mice. In contrast, deficiency of IL-12 did not aggravate the Th2 immune and inflammatory response in OVA/RSV-treated mice, compared with wild-type. In conclusion, the virus-induced IFN-gamma response diminishes the Th2 inflammatory response during OVA allergy but fails to prevent totally the enhancement of the OVA allergy by RSV. In contrast, IL-12 is not involved in inhibiting nor increasing the RSV-enhanced allergy in 129/Sv/Ev mice.     

Role of IFN-alpha/beta and IL-12 in the activation of natural killer cells and interferon-gamma production during experimental infection with Trypanosoma cruzi

Control of Trypanosoma cruzi infection depends largely upon the production of interferon (IFN)-gamma. During experimental infection this cytokine is produced early, mainly by natural killer (NK) cells and later by T cells. As NK cells have been reported to participate in defence against T. cruzi, it is of importance to study the regulation of NK cell functions during infection with the parasite. Several innate cytokines regulate NK cell activity, among them being interferon (IFN)-alpha and IFN-beta (type 1 IFNs) and interleukin (IL)-12, which have all been reported to be involved in protection against T. cruzi. The role of these cytokines in regulation of NK cell functions and disease outcome were studied by infection of mutant mice lacking the IFN-alpha/beta receptor (IFNalpha/betaR-/-) or IL-12 (IL-12-/-) with T. cruzi. IFNalpha/betaR-/- mice were unable to activate the cytotoxic response but produced IFN-gamma, and were not more susceptible than controls. IL-12-/- mice were extremely susceptible and failed to produce T cell-derived IFN-gamma and nitric oxide (NO), although NK cytotoxicity was induced. The results indicate that IL-12 protects against T. cruzi by initiating T cell-mediated production of IFN-gamma, but that endogenous IFN-alpha/beta and NK cell cytotoxicity are not of major importance in defence.     

Respiratory syncytial virus replication is prolonged by a concomitant allergic response

Epidemiological studies show an association between early exposure to respiratory syncytial virus (RSV) and the development or exacerbation of asthma. This idea is supported by studies in mice that demonstrate worsened airway hyper-reactivity (AHR) when RSV-infected animals are exposed to allergen. The effect of allergen on RSV disease, however, has not been reported. Cotton rats (Sigmodon hispidus) that have been used as a model to study RSV pathogenesis were sensitized to extracts of Aspergillus fumigatus (Af), a common household mould. The allergic response to Af included eosinophilia, formation of granulomas and induction of Th2 type cytokines. RSV infection prior to allergen challenge resulted in exacerbation of the inflammatory response as well as increased airway responsiveness to methacholine. The exacerbated response was indeed dependent on virus replication. Virus replication in turn was influenced by the allergic response, with persistence in the noses for 2 days longer in animals challenged with allergen. This diminished clearance corresponded to decreased induction of mRNA for IFN-gamma, a Th1-type cytokine that is characteristic of viral infection. Treatment of RSV-infected Af-challenged animals with recombinant IFN-gamma reduced the allergic inflammatory response as well as the relative levels of Th1 and Th2 cytokine mRNA. However, this treatment did not reduce airway reactivity, showing that these pathologic and physiologic measures of exacerbated disease are independent. We speculate that the reciprocal effect of the allergic response on viral immunity may benefit the host by limiting exacerbation of physiologic responses that are IFN-gamma-dependent.     

Reciprocal stimulation of gammadelta T cells and dendritic cells during the anti-mycobacterial immune response

Gammadelta T cells and dendritic cells (DC) are two distinct cell types of innate immunity that participate in early phases of immune response against Mycobacterium tuberculosis infection. Here we show that a close functional relationship exists between these cell populations. Using an in vitro coculture system, Vgamma1 T cells from Tcrb(-/- )mice were found to be activated by DC infected in vitro with BCG, as indicated by the elevated CD69 expression, IFN-gamma secretion and cytotoxic activity. This activation process was due to a non-cognate mechanism since it required neither cell to cell contact nor interaction between the TCR and a specific antigen, but was mediated by DC-derived IL-12. Reciprocally, Vgamma1 T cells provided a key cytokine, IFN-gamma, which increased IL-12 production by BCG-infected DC. Moreover, exposure of BCG-infected DC to Vgamma1 T cells conditioned the former to prime a significantly stronger anti-mycobacterial CD8 T cell response. Consequently, stimulation of gammadelta T cells and their non-cognate interaction with DC could be applied as an immune adjuvant strategy to optimize vaccine-induced CD8 T cell immunity.     

Moderate local and systemic respiratory syncytial virus-specific T-cell responses upon mild or subclinical RSV infection

Respiratory syncytial virus (RSV) infections are a major cause of severe respiratory disease in infants. It has been shown that there is an increased frequency of childhood wheezing in ex-bronchiolitic preteen children. This was postulated to be mediated by a vigorous virus-specific Th2 response influencing the further development of the immune system. Little is known about the possible role of the immune response to clinically mild RSV infections in this respect. We have studied the RSV-specific cellular immune response in infants with a laboratory-confirmed RSV upper respiratory tract infection (URTI; n = 13, mean age 12 months, range 2-22 months) in comparison with infants with non-RSV mediated URTI (n = 9, mean age 9.3 months, range 4-18 months) or infants with severe RSV bronchiolitis (n = 11, mean age 2.3 months, range 1-6 months). RSV-specific cytokine-producing cells were enumerated using the ELISPOT method in peripheral blood mononuclear cells and nasal brush T-cells, collected during the acute and convalescent phase of the infection. Mixed Th1 (IFN-gamma) and Th2 (IL-4 and IL-13) responses were detected in all three groups. Frequencies of RSV-specific T-cells were lower in both URTI groups than in the RSV bronchiolitis group, and not significantly different between the RSV URTI and the non-RSV URTI group. The absence of vigorous virus-specific Th2 responses upon mild RSV infection does not support the hypothesis that these infections influence the development of the immune system and that they predispose for the development of atopic disease.     

TCR-beta chains derived from peripheral gammadelta T cells can take part in alphabeta T-cell development

Between 10 and 20% of the peripheral gammadelta T cells express cytoplasmic TCR-beta proteins, but whether such TCR-beta chains can partake in alphabeta T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3epsilon-deficient mice with Pax5-TCR-beta deficient proB cells expressing, via a retroviral vector, TCR-beta chains from either peripheral gammadelta or alphabeta T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (<15x10(6) cells), contained few gammadelta T but no alphabeta T cells. In contrast, thymi from mice receiving proB cells containing gammadelta or alphabeta T-cell-derived TCR-beta chains contained 80-130x10(6) cells, and showed a normal CD4, CD8 and alphabeta TCR expression pattern. However, regardless of the source of TCR-beta chain, reconstituted mice rapidly showed signs of autoimmunity dying 5-15 wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-beta chains from gammadelta T cells can efficiently take part in alphabeta T-cell development. The implications of these findings for gammadelta T-cell development will be discussed.     

Human T helper (Th) cell lineage commitment is not directly linked to the secretion of IFN-gamma or IL-4: characterization of Th cells isolated by FACS based on IFN-gamma and IL-4 secretion

Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN-gamma for Th1 and IL-4 for Th2, respectively). It is uncertain whether these IFN-gamma-/IL-4- cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN-gamma+, IL-4+ and IFN-gamma-/IL-4- cells isolated from Th cell cultures treated under Th1 or Th2 polarizing conditions by employing affinity matrix capture technology. RNA samples from the sorted cells were analyzed by real time RT-PCR and microarrays. The double negative cells from either Th1 or Th2 cultures expressed lower levels of Th1/Th2 marker cytokine genes (IFNgamma, IL4, and IL5). However, they were comparable with the IFN-gamma+ or IL-4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rbeta2). Most importantly, these double negative cells were already committed in their Th1/Th2 lineages. Gene expression profiling analysis showed that very few previously identified Th1/Th2 marker genes were differentially expressed between the IFN-gamma or IL-4 producers and the non-producers, further underscoring the similarity between these two groups.     

Respiratory syncytial virus protects against the subsequent development of ovalbumin‐induced allergic responses by inhibiting Th2‐type γδ T cells

Respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans still remain inconclusive. The association between RSV infection and allergic diseases may be dependent on an atopic background and previous history of RSV infection. It has been reported that RSV infection before sensitization to an allergen decreased the production of Th2‐like cytokines in the lung and the levels of allergen‐specific Th2‐type antibodies in the serum. However, the underlying mechanisms are largely unknown. In the present study, the role of pulmonary γδ T cells in RSV‐affected, allergen‐induced airway inflammation was investigated. BALB/c mice were sensitized to or challenged with ovalbumin (OVA) and infected with RSV either before or after the sensitization period. It became clear that sensitization and challenge of mice with OVA induced a large influx of γδ T cells to the lungs. However, prior RSV infection inhibited the infiltration of γδ T cells as well as activated γδ T cells, characterized by expression of CD40L or CD69 molecular in the cell surface. Moreover, prior RSV infection elevated the type 1 cytokine gene expression but suppressed type 2 cytokine expression in the lung γδ T cells. Adoptive transfer of γδ T cells from OVA‐sensitized and challenged mice increased airway inflammation, suggesting that γδ T cells may play a proinflammatory role in allergic responses. These results described here support the idea of an unknown γδ T cell‐dependent mechanism in the regulation of RSV‐affected, allergen‐induced allergic airway responses. J. Med. Virol. 85:149–156, 2012. © 2012 Wiley Periodicals, Inc.     

Regulation of LPS induced IL-12 production by IFN-gamma and IL-4 through intracellular glutathione status in human alveolar macrophages.

Interleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-gamma (IFN-gamma) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-gamma and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-gamma or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-gamma but inhibited by IL-4. These results suggest that IFN-gamma and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.     

Expansion of regulatory CD8+ CD25+ T cells after neonatal alloimmunization

Transplantation tolerance induced by neonatal injection of semi-allogeneic spleen cells is associated with a pathological syndrome caused by T helper type 2 (Th2) differentiation of donor-specific CD4(+) T lymphocytes. We have shown previously that this Th2-biased response is inhibited by host CD8(+) T cells. Herein, we demonstrate that upon neonatal immunization with (A/J × BALB/c)F(1) spleen cells, BALB/c mice expand a population of CD8(+) T cells expressing both CD25 and forkhead box P3 (FoxP3) markers. In this setting, CD8(+) CD25(+) T cells predominantly produce interferon (IFN)-γ and interleukin (IL)-10 and are efficient in controlling IL-4, IL-5 and IL-13 production by donor-specific CD4(+) T cells in vitro. CD8(+) FoxP3(-) T cells are single producers of IFN-γ or IL-10, whereas CD8(+) FoxP3(+) T cells are double producers of IFN-γ and IL-10. We further demonstrate that IFN-γ and IL-10 are two major cytokines produced by CD8(+) T cells involved in the in vivo regulation of Th2-type pathology. In this setting, we conclude that neonatal alloimmunization induces the expansion of several regulatory CD8(+) T cells which may control Th2 activities via IFN-γ and IL-10.     

Direct exosome stimulation of peripheral human T cells detected by ELISPOT

Exosomes from APC are nano-vesicles that can induce antigen-specific T cell responses and are presently explored as therapeutic tools in different clinical settings. Investigations of the capacity of exosomes to stimulate T cells in vitro have mostly been performed on T cell hybridomas, clones or lines. Whether exosomes can stimulate T cells directly or need the presence of dendritic cells (DC) is debated. We could detect exosome-induced antigen-specific CD8(+) T cell responses in peripheral blood from humans. Exosomes from monocyte-derived DC (MDDC) were loaded with a mix of 23 immunogenic peptides from EBV, CMV and influenza virus, and added to autologous peripheral CD8(+) T cells. IFN-gamma-producing cells were detected by enzyme-linked immunospot assay (ELISPOT). MDDC-exosomes induced IFN-gamma production in CD8(+) T cells without addition of DC. The response was exosome dose dependent, and dependent on exosomal MHC class I. Furthermore, we detected an enhanced T cell stimulatory capacity by exosomes from lipopolysaccharide-matured MDDC compared to exosomes from immature MDDC. Exosomes could also induce TNF-alpha production. These results show, for the first time, that exosomes can directly stimulate human peripheral CD8(+) T cells in an antigen-specific manner and that ELISPOT is a suitable method for detecting exosome-induced peripheral T cell responses. This system may provide a useful tool when developing exosomes as therapeutic agents.