Airway hyperresponsiveness through synergy of gammadelta} T cells and NKT cells

Mice sensitized and challenged with OVA were used to investigate the role of innate T cells in the development of allergic airway hyperresponsiveness (AHR). AHR, but not eosinophilic airway inflammation, was induced in T cell-deficient mice by small numbers of cotransferred gammadelta T cells and invariant NKT cells, whereas either cell type alone was not effective. Only Vgamma1+Vdelta5+ gammadelta T cells enhanced AHR. Surprisingly, OVA-specific alphabeta T cells were not required, revealing a pathway of AHR development mediated entirely by innate T cells. The data suggest that lymphocytic synergism, which is key to the Ag-specific adaptive immune response, is also intrinsic to T cell-dependent innate responses.     

Cutting edge: invariant V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity in an experimental asthma model

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jalpha18(-/-) mice, which are exclusively deficient in the invariant Valpha14(+) (iValpha14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iValpha14 NKT cells fully reconstitutes the capacity of Jalpha18(-/-) mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69(+)) iValpha14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iValpha14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iValpha14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.     

IL-13 induces airways hyperreactivity independently of the IL-4R alpha chain in the allergic lung

The potent spasmogenic properties of IL-13 have identified this molecule as a potential regulator of airways hyperreactivity (AHR) in asthma. Although IL-13 is thought to primarily signal through the IL-13Ralpha1-IL-4Ralpha complex, the cellular and molecular components employed by this cytokine to induce AHR in the allergic lung have not been identified. By transferring OVA-specific CD4(+) T cells that were wild type (IL-13(+/+) T cells) or deficient in IL-13 (IL-13(-/-) T cells) to nonsensitized mice that were then challenged with OVA aerosol, we show that T cell-derived IL-13 plays a key role in regulating AHR, mucus hypersecretion, eotaxin production, and eosinophilia in the allergic lung. Moreover, IL-13(+/+) T cells induce these features (except mucus production) of allergic disease independently of the IL-4Ralpha chain. By contrast, IL-13(+/+) T cells did not induce disease in STAT6-deficient mice. This shows that IL-13 employs a novel component of the IL-13 receptor signaling system that involves STAT6, independently of the IL-4Ralpha chain, to modulate pathogenesis. We show that this novel pathway for IL-13 signaling is dependent on T cell activation in the lung and is critically linked to downstream effector pathways regulated by eotaxin and STAT6.     

ICOS Mediates the Generation and Function of CD4+CD25+Foxp3+ Regulatory T Cells Conveying Respiratory Tolerance

Costimulatory molecules like ICOS are crucial in mediating T cell differentiation and function after allergen contact and thereby strongly affect the immunologic decision between tolerance or allergy development. In this study, we show in two independent approaches that interruption of the ICOS signaling pathway by application of a blocking anti-ICOSL mAb in wild-type (WT) mice and in ICOS(-/-) mice inhibited respiratory tolerance development leading to eosinophilic airway inflammation, mucus hypersecretion, and Th2 cytokine production in response to OVA sensitization. Respiratory Ag application almost doubled the number of CD4(+)Foxp3(+) regulatory T cells (Tregs) in the lung of WT mice with 77% of lung-derived Tregs expressing ICOS. In contrast, in ICOS(-/-) mice the number of CD4(+)CD25(+)Foxp3(+) Tregs did not increase after respiratory Ag application, and ICOS(-/-) Tregs produced significantly lower amounts of IL-10 than those of WT Tregs. Most importantly, in contrast to WT Tregs, ICOS(-/-) Tregs did not convey protection when transferred to "asthmatic" recipients demonstrating a strongly impaired Treg function in the absence of ICOS signaling. Our findings demonstrate a crucial role of ICOS for the generation and suppressive function of Tregs conveying respiratory tolerance and support the importance of ICOS as a target for primary prevention strategies.     

Kinetics and activation requirements of contact-dependent immune suppression by human regulatory T cells

Naturally occurring regulatory T cells (Tregs) maintain self tolerance by dominant suppression of potentially self-reactive T cells in peripheral tissues. However, the activation requirements, the temporal aspects of the suppressive activity, and mode of action of human Tregs are subjects of controversy. In this study, we show that Tregs display significant variability in the suppressive activity ex vivo as 54% of healthy blood donors examined had fully suppressive Tregs spontaneously, whereas in the remaining donors, anti-CD3/CD2/CD28 stimulation was required for Treg suppressive activity. Furthermore, anti-CD3/CD2/CD28 stimulation for 6 h and subsequent fixation in paraformaldehyde rendered the Tregs fully suppressive in all donors. The fixation-resistant suppressive activity of Tregs operated in a contact-dependent manner that was not dependent on APCs, but could be fully obliterated by trypsin treatment, indicating that a cell surface protein is directly involved. By add-back of active, fixed Tregs at different time points after activation of responding T cells, the responder cells were susceptible to Treg-mediated immune suppression up to 24 h after stimulation. This defines a time window in which effector T cells are susceptible to Treg-mediated immune suppression. Lastly, we examined the effect of a set of signaling inhibitors that perturb effector T cell activation and found that none of the examined inhibitors affected Treg activation, indicating pathway redundancy or that Treg activation proceeds by signaling mechanisms distinct from those of effector T cells.     

Leukotriene B4 receptor-1 is essential for allergen-mediated recruitment of CD8+ T cells and airway hyperresponsiveness

Recent studies in both human and rodents have indicated that in addition to CD4+ T cells, CD8+ T cells play an important role in allergic inflammation. We previously demonstrated that allergen-sensitized and -challenged CD8-deficient (CD8-/-) mice develop significantly lower airway hyperresponsiveness (AHR), eosinophilic inflammation, and IL-13 levels in bronchoalveolar lavage fluid compared with wild-type mice, and that all these responses were restored by adoptive transfer of in vivo-primed CD8+ T cells or in vitro-generated effector CD8+ T cells (T(EFF)). Recently, leukotriene B4 and its high affinity receptor, BLT1, have been shown to mediate in vitro-generated T(EFF) recruitment into inflamed tissues. In this study we investigated whether BLT1 is essential for the development of CD8+ T cell-mediated allergic AHR and inflammation. Adoptive transfer of in vivo-primed BLT1+/+, but not BLT1-/-, CD8+ T cells into sensitized and challenged CD8-/- mice restored AHR, eosinophilic inflammation, and IL-13 levels. Moreover, when adoptively transferred into sensitized CD8-/- mice, in vitro-generated BLT1+/+, but not BLT1-/-, T(EFF) accumulated in the lung and mediated these altered airway responses to allergen challenge. These data are the first to show both a functional and an essential role for BLT1 in allergen-mediated CD8+ T(EFF) recruitment into the lung and development of AHR and airway inflammation.     

Regulatory T cell expression of herpesvirus entry mediator suppresses the function of B and T lymphocyte attenuator-positive effector T cells

The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA(-/-) CD4(+)CD25(+) Tregs had normal suppressive activity, whereas BTLA(-/-) Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM(-/-) Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM gene expression, and adoptively transferred wild-type but not HVEM(-/-) Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory ligand HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo.     

The interaction between regulatory T cells and NKT cells in the liver: a CD1d bridge links innate and adaptive immunity

Background/Aims

Regulatory T cells (Tregs) and natural killer T (NKT) cells are two distinct lymphocyte subsets that independently regulate hepatic adaptive and innate immunity, respectively. In the current study, we examine the interaction between Tregs and NKT cells to understand the mechanisms of cross immune regulation by these cells.

Methods

The frequency and function of Tregs were evaluated in wild type and NKT cell deficient (CD1dko) mice. In vitro lymphocyte proliferation and apoptosis assays were performed with NKT cells co-cultured with Tregs. The ability of Tregs to inhibit NKT cells in vivo was examined by adoptive transfer of Tregs in a model of NKT cell mediated hepatitis.

Results

CD1dko mice have a significant reduction in hepatic Tregs. Although, the Tregs from CD1dko mice remain functional and can suppress conventional T cells, their ability to suppress activation induced NKT cell proliferation and to promote NKT cell apoptosis is greatly diminished. These effects are CD1d dependent and require cell to cell contact. Adoptive transfer of Tregs inhibits NKT cell-mediated liver injury.

Conclusions

NKT cells promote Tregs, and Tregs inhibit NKT cells in a CD1d dependent manner requiring cell to cell contact. These cross-talk immune regulations provide a linkage between innate and adaptive immunity.     

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.     

Activation of nonclassical CD1d-restricted NK T cells induces airway hyperreactivity in beta 2-microglobulin-deficient mice

Allergic asthma is characterized by Th2-driven eosinophilic airway inflammation and by a central feature called airway hyperreactivity (AHR), development of which requires the presence of classical type I invariant NK T (iNKT) cells. Allergen-induced AHR, however, develops in beta(2)-microglobulin (beta(2)m)(-/-) mice, which lack classical iNKT cells, suggesting that in some situations iNKT cells may be dispensable for the development of AHR. In contrast, our studies now suggest that a CD1d-restricted, NK1.1(+) noninvariant TCR NKT cell population is present in beta(2)m(-/-) mice and is responsible for the development of AHR but not for Th2 responses. Furthermore, treatment of beta(2)m(-/-) mice with anti-CD1d mAb or anti-NK1.1 mAb unexpectedly abolished allergen-induced AHR. The CD1-restricted NKT cells in these mice, which failed to respond to alpha-galactosylceramide and which therefore were not classical type I iNKT cells, appear to represent an NKT cell subset restricted by a beta(2)m-independent form of CD1d. These results indicate that, although classical type I iNKT cells are normally required for the development of AHR, under different circumstances other NKT cell subsets, including nonclassical NKT cells, may substitute for classical iNKT cells and induce AHR.     

Suppression of HIV-specific and allogeneic T cell activation by human regulatory T cells is dependent on the strength of signals

Regulatory T cells (Tregs) suppress immune responses against both self and non-self antigens. Tregs require activation through the T cell receptor (TCR) and IL-2 to exert their suppressive functions. However, how strength of TCR signals modulate the potency of Treg-mediated suppression of antigen-specific T cell activation remain unclear. We found that both strength of TCR signals and ratios of Tregs to target cells, either through superantigen, allogeneic antigens or HIV-specific peptides, modified the suppressive ability of Tregs. While human Tregs were able to mediate suppression in the presence of only autologous antigen-presenting cells, this was much less efficient as compared to when Tregs were activated by allogeneic dendritic cells. In another physiologically relevant system, we show that the strength of peptide stimulation, high frequency of responder CD8+ T cells or presence of high IL-2 can override the suppression of HIV-specific CD8+ T cells by Tregs. These findings suggest that ratios and TCR activation of human Tregs, are important parameters to overcome robust immune responses to pathogens or allogeneic antigens. Modulating the strength of T cell signals and selective enhancement or depletion of antigen-specific Tregs thus may have implications for designing potent vaccines and regulating immune responses during allogeneic transplantation and chronic infections.     

Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients

CD4+CD25+FOXP3+ T regulatory cells (Tregs) are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. Rapamycin has been shown to promote tolerance in experimental models and to favor CD4+CD25+ Treg-dependent suppression. We recently reported that rapamycin allows in vitro expansion of murine CD4+CD25+FoxP3+ Tregs, which preserve their suppressive function. In the current study, we show that activation of human CD4+ T cells from healthy subjects in the presence of rapamycin leads to growth of CD4+CD25+FOXP3+ Tregs and to selective depletion of CD4+CD25- T effector cells, which are highly sensitive to the antiproliferative effect of the compound. The rapamycin-expanded Tregs suppress proliferation of both syngeneic and allogeneic CD4+ and CD8+ T cells. Interestingly, rapamycin promotes expansion of functional CD4+CD25+FOXP3+ Tregs also in type 1 diabetic patients, in whom a defect in freshly isolated CD4+CD25+ Tregs has been reported. The capacity of rapamycin to allow growth of functional CD4+CD25+FOXP3+ Tregs, but also to deplete T effector cells, can be exploited for the design of novel and safe in vitro protocols for cellular immunotherapy in T cell-mediated diseases.     

Gammadelta T cells promote anterior chamber-associated immune deviation and immune privilege through their production of IL-10

Anterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance that is induced by introducing Ags into the anterior chamber (AC) of the eye, and is maintained by Ag-specific regulatory T cells (Tregs). ACAID regulates harmful immune responses that can lead to irreparable injury to innocent bystander cells that are incapable of regeneration. This form of immune privilege in the eye is mediated through Tregs and is a product of complex cellular interactions. These involve F4/80+ ocular APCs, B cells, NKT cells, CD4+CD25+ Tregs, and CD8+ Tregs. gammadelta T cells are crucial for the generation of ACAID and for corneal allograft survival. However, the functions of gammadelta T cells in ACAID are unknown. Several hypotheses were proposed for determining the functions of gammadelta T cells in ACAID. The results indicate that gammadelta T cells do not cause direct suppression of delayed-type hypersensitivity nor do they act as tolerogenic APCs. In contrast, gammadelta T cells were shown to secrete IL-10 and facilitate the generation of ACAID Tregs. Moreover, the contribution of gammadelta T cells ACAID generation could be replaced by adding exogenous recombinant mouse IL-10 to ACAID spleen cell cultures lacking gammadelta T cells.     

Dietary lycopene supplementation suppresses Th2 responses and lung eosinophilia in a mouse model of allergic asthma

Allergic airways disease (AAD) is associated with an increased influx of eosinophils to the lungs, mucus hypersecretion and Th2 cytokine production. Dietary antioxidant supplementation may alter cytokine responses and thus allergic inflammation. Lycopene is a potent dietary antioxidant. The objective of this study was to investigate the effects of lycopene, on allergic inflammation, in a mouse model of AAD. BALB/c mice receiving lycopene supplement or control were intraperitoneally sensitised and intranasally challenged with ovalbumin (OVA) to induce AAD. The effect of supplementation on inflammatory cell influx into bronchoalveolar lavage fluid, lung tissue and blood, mucus-secreting cell numbers in the airways, draining lymph node OVA-specific cytokine release, serum IgG1 levels and lung function in AAD was assessed. Supplementation reduced eosinophilic infiltrates in the bronchoalveolar lavage fluid, lung tissue and blood, and mucus-secreting cell numbers in the airways. The OVA-specific release of Th2-associated cytokines IL-4 and IL-5 was also reduced. We conclude that supplementation with lycopene reduces allergic inflammation both in the lungs and systemically, by decreasing Th2 cytokine responses. Thus, lycopene supplementation may have a protective effect against asthma.     

APL1, an altered peptide ligand derived from human heat-shock protein 60, increases the frequency of Tregs and its suppressive capacity against antigen responding effector CD4 + T cells from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by a chronic relapsing-remitting joint inflammation. Perturbations in the balance between CD4?+?T cells producing IL-17 and CD4?+?CD25^highFoxP3?+?Tregs correlate with irreversible bone and cartilage destruction in RA. APL1 is an altered peptide ligand derived from a CD4+ T-cell epitope of human HSP60, an autoantigen expressed in the inflamed synovium, which increases the frequency of CD4?+?CD25^highFoxP3+ Tregs in peripheral blood mononuclear cells from RA patients. The aim of this study was to evaluate the suppressive capacity of Tregs induced by APL1 on proliferation of effector CD4+ T cells using co-culture experiments. Enhanced Treg-mediated suppression was observed in APL1-treated cultures compared with cells cultured only with media. Subsequent analyses using autologous cross-over experiments showed that the enhanced Treg suppression in APL1-treated cultures could reflect increased suppressive function of Tregs against APL1-responsive T cells. On the other hand, APL1-treatment had a significant effect reducing IL-17 levels produced by effector CD4+ T cells. Hence, this peptide has the ability to increase the frequency of Tregs and their suppressive properties whereas effector T cells produce less IL-17. Thus, we propose that APL1 therapy could help to ameliorate the pathogenic Th17/Treg balance in RA patients.     

Cyclophosphamide-induced type-1 diabetes in the NOD mouse is associated with a reduction of CD4+CD25+Foxp3+ regulatory T cells

Regulatory T cells (Tregs) have been implicated as key players in immune tolerance as well as suppression of antitumor responses. The chemotherapeutic alkylating agent cyclophosphamide (CY) is widely used in the treatment of tumors and some autoimmune conditions. Although previous data has demonstrated that Tregs may be preferentially affected by CY, its relevance in promoting autoimmune conditions has not been addressed. The nonobese diabetic mouse spontaneously develops type-1 diabetes (T1D). We demonstrate in this study that CY targets CD4+CD25+Foxp3+ Tregs in vivo. CD4+CD25+ T cells isolated from CY-treated mice display reduced suppressive activity in vitro and increased expression of apoptotic markers. Although Treg numbers rapidly recovered to pretreatment levels in the peripheral lymphoid tissues, Tregs failed to recover proportionally within pancreatic infiltrates. T1D progression was effectively prevented by adoptive transfer of a small number of islet Ag-specific CD4+CD25+ Tregs to CY-treated recipients. Prevention of T1D was associated with reduced T cell activation and higher Treg proportions in the pancreas. We conclude that acceleration of T1D by CY is associated with a reduction in CD4+CD25+Foxp3+ Tregs and can be prevented by transfer of CD4+CD25+ Tregs.     

B7+ iris pigment epithelium induce CD8+ T regulatory cells; both suppress CTLA-4+ T cells

Ocular pigment epithelia contribute to immune privilege by suppressing T cell activation and converting T cells into regulatory T regulatory cells (Tregs) that inhibit bystander T cell activation. Iris pigment epithelium (IPE) does so through direct cell-cell contact with naive T cells, and this suppressive contact is via interactions between B7 expressed constitutively on IPE cells and CTLA-4 expressed on a subpopulation of CD8+ T cells. We have now examined whether TGFbeta is required in this process. We report that IPE produces both soluble and membrane-bound active TGFbeta, but that only the latter is actually delivered to CD8+ T cells. In turn, these T cells become IPE Tregs by up-regulating their own expression of B7-1/B7-2 and soluble and membrane-bound TGFbeta. IPE Tregs through their expression of B7 are able to engage CTLA-4+ bystander T cells, and thus precisely, target delivery of membrane-bound TGFbeta. We propose that this mechanism of suppression via TGFbeta ensures that soluble active TGFbeta is not released into the ocular microenvironment where it can have unregulated and deleterious effects, including elevation of intraocular pressure and development of glaucoma.     

Regulatory T cells prevent control of experimental African trypanosomiasis

African trypanosomes are single-cell, extra-cellular blood parasites causing profound immunosuppression. Susceptible BALB/c mice infected s.c. into a footpad with 10(4) Trypanosoma congolense die with fulminating parasitemia within 10 days. We injected BALB/c mice 2 days before such an infection with different doses of a depleting mAb specific for CD25, a surface marker of regulatory T cells (Tregs). Pretreatment with a low, optimal dose of anti-CD25 resulted in a dramatic effect, in that the infected mice did not develop parasitemia, as well as eliminated all parasites and showed no signs of disease. Their spleens showed a 100% reduction of CD4(+)CD25(high) T cells and overall a 70% reduction of CD4(+)CD25(+)Foxp3(+) T cells 7 days postinfection. The protective effect of treatment with an optimal dose of anti-CD25 could be reversed by administration of l-N6-(1-imminoethyl) lysine, a specific inhibitor of inducible NO synthase or administration of anti-CD8 Ab. Analysis of the cytokine patterns and cell surface marker in infected mice pretreated with anti-CD25 Abs pointed to a potential NKT cell response. We then conducted infections in CD1d(-/-) mice. From our observations, we conclude that CD4(+)CD25(high)Foxp3(+) Tregs prevent, in normal infected susceptible mice, an early protective response mediated by CD8(+) NKT cell-dependent activation of macrophages to kill parasites by production of NO. Our results also indicate that different populations of NKT cells have protective or suppressive effects. Our observations lead us to propose a hypothesis of cross-regulation of NKT cells and Tregs in trypanosome infections.