Human mast cells costimulate T cells through a CD28‐independent interaction

Mast cells are innate immune cells usually residing in peripheral tissues, where they are likely to activate T‐cell responses. Similar to other myeloid immune cells, mast cells can function as antigen‐presenting cells. However, little is known about the capacity of human mast cells to costimulate CD4⁺ T cells. Here, we studied the T‐cell stimulatory potential of human mast cells. Peripheral blood derived mast cells were generated and cocultured with isolated CD4⁺ T cells. In the presence of T‐cell receptor triggering using anti‐CD3, mast cells promoted strong proliferation of T cells, which was two‐ to fivefold stronger than the “T‐cell promoting capacity” of monocytes. The interplay between mast cells and T cells was dependent on cell–cell contact, suggesting that costimulatory molecules on the mast cell surface are responsible for the effect. However, in contrast to monocytes, the T‐cell costimulation by mast cells was independent of the classical costimulatory molecule CD28, or that of OX40L, ICOSL, or LIGHT. Our data show that mast cells can costimulate human CD4⁺ T cells to induce strong T‐cell proliferation, but that therapies aiming at disrupting the interaction of CD28 and B7 molecules do not inhibit mast cell mediated T‐cell activation.     

Local and systemic effects of co‐stimulatory blockade using cytotoxic T lymphocyte antigen‐4‐immunoglobulin in dinitrofluorobenzene‐ and oxazolone‐induced contact hypersensitivity in mice

Cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4)‐immunoglobulin (Ig) has immunosuppressive properties both in vivo and in vitro, but much is still unknown about the mechanisms by which CTLA‐4‐Ig exerts its immunosuppressive activities in vivo. The aim of this study was to investigate the effect of CTLA‐4‐Ig in a mouse model of contact hypersensitivity (CHS). The inflammatory response in the presence or absence of CTLA‐4‐Ig was evaluated by measuring the increase in ear thickness in sensitized animals after challenge. We observed a dose‐dependent suppression of the ear swelling in both dinitrofluorobenzene (DNFB)‐ and oxazolone‐induced CHS. The suppressive effect was still present 3 weeks after administration, even in the absence of circulating levels of CTLA‐4‐Ig. It was further shown that CTLA‐4‐Ig inhibits activation of T cells in the draining lymph node after sensitization and affects the maturation level of both dendritic cells and B cells. Furthermore, CTLA‐4‐Ig reduces infiltration of activated CD8⁺ T cells into the inflamed ear tissue and suppresses both local and systemic inflammation, as illustrated by reduced expression of cytokines and chemokines in the inflamed ear and a reduced level of acute‐phase proteins in circulation. Finally, our results suggest that CTLA‐4‐Ig has a mainly immunosuppressive effect during the sensitization phase. We conclude that CTLA‐4‐Ig induces long‐term immunosuppression of both DNFB‐ and oxazolone‐induced inflammation and our data are the first to compare the effect of this compound in both DNFB‐ and oxazolone‐induced CHS and to show that CTLA‐4‐Ig exerts an immunosuppressive effect on both local and systemic inflammatory mediators which is mediated principally during the sensitization phase.     

The effect of CTLA-4Ig, a CD28/B7 antagonist, on the lung inflammation and T cell subset profile during murine hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease characterized by an influx of activated T cells to the lung, in which the CD28/B7 costimulatory signals are essential for the T cell activation and the outcome of the inflammatory response. In this study, we investigated the effect of the CD28/B7 antagonist, CTLA-4Ig, on the lung inflammation and the T cell subset profile in experimental Saccharopolyspora recivirgula (SR)-induced HP. C57BL/6 mice were treated with SR or saline during two and three weeks and in addition of CTLA-4Ig was administrated after either the second or third week and mice were sacrificed seven days later. The extent of the lung inflammation was quantified by histopathology and the lung T cell subsets (Treg, Th17, γδT and NKT) were analyzed by flow cytometry. Mice treated with CTLA-4Ig showed a significant decrease in the extent of lung damage (p < 0.05), and exhibited a decreased number of inflammatory cells in the bronchoalveolar lavage (BAL) with diminished CD4/CD8 T cell ratio. Also, a significant increase in the percentage of lung γδT (p < 0.01) and NKT (p < 0.05) cells was observed in two weeks SR-treated mice with the administration of CTLA-4Ig/SR. At 3 weeks, SR-treated mice showed an increased percentage of regulatory T cells but no significantly differences were found in the percentage of Th17 cells when compared with CTLA-4Ig/SR-treated mice. Our findings suggest that the treatment with CTLA-4Ig affects the HP progression and the lung T cell subset kinetics in mice.     

Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist

CD4(+)CD25(+) T cells play a central role in the suppression of autoimmunity and inflammation, making their in vivo expansion a highly attractive therapeutic target. By phenotyping with a novel rat CTL antigen-4 (CTLA-4)-specific monoclonal antibody (mAb) and functional in vitro assays, we here first establish that rat CD4(+)CD25(+) T cells correspond to the regulatory T cells (Treg cells) described in mice and humans: they constitutively express CTLA-4, produce IL-10 but not IL-2, and are able to suppress the proliferation of costimulated CD25-negative indicator cells. Furthermore, we show that rat Treg cells respond less well than CD25(-) T cells to conventional costimulation, but are readily expanded in vitro with "superagonistic" CD28-specific mAb which are potent mitogens for all T cells without the need for TCR engagement. In vivo, functional Treg cells are preferentially expanded by CD28 stimulation over other T cell subsets, leading to a 20-fold increase within 3 days in response to a single antibody dose. These data suggest that CD28-driven activation of Treg cells may be highly effective in the treatment of inflammatory and autoimmune diseases.     

Immature dendritic cells convert anergic nonregulatory T cells into Foxp3−IL‐10+ regulatory T cells by engaging CD28 and CTLA‐4

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide‐specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL‐10^?Foxp3^?CTLA‐4⁺CD25^lowEgr2⁺ T cells could be converted into suppressive IL‐10⁺Foxp3^?CTLA‐4⁺CD25^highEgr2⁺ cells resembling type‐1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF‐β during anergy induction favored Foxp3⁺ Treg‐cell induction, while TGF‐β had little effect when added to the second stimulation. Expression of both CD28 and CTLA‐4 molecules on anergic T cells was required to allow their conversion into Tr1‐like cells. Suppressor activity was enabled via CD28‐mediated CD25 upregulation, acting as an IL‐2 sink, together with a CTLA‐4‐mediated inhibition of NFATc1/α activation to shut down IL‐2‐mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1‐like cells.     

CD28/CTLA‐4/B7 costimulatory pathway blockade affects regulatory T‐cell function in autoimmunity

Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA‐4 on effector T (Teff) cells, its immune‐modulatory effects are potentially more complex. Here, we used the mouse model of multiple sclerosis (MS), EAE, to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation, and therefore we injected CTLA‐4Ig at day 7 and 9 after immunization, when myelin‐reactive T cells have been primed and start migrating toward the CNS. Surprisingly, B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination, and was associated with an enhanced production of the inflammatory cytokines IL‐17 and IFN‐γ. Importantly, CTLA‐4Ig treatment resulted in a transient reduction of Ki67 and CTLA‐4 expression and function of peripheral Treg cells. Taken together, B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells, leading to a more severe disease.     

IL‐12 and IL‐4 activate a CD39‐dependent intrinsic peripheral tolerance mechanism in CD8+ T cells

Immune responses to protein antigens involve CD4⁺ and CD8⁺ T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T‐cell (CTL) activity after T‐cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL‐12 and IL‐4. Cytokine‐induced CD8⁺ regulatory T (Treg) cells are one of several Treg‐cell phenotypes and are Foxp3^? IL‐10⁺ with contact‐dependent suppressive capacity. Here, we show they also express high level CD39, an ecto‐nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8⁺ T cells during peripheral tolerance induction in vivo, accompanied by release of IL‐12 and IL‐10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor‐infiltrating CD8⁺ T cells. Production of IL‐10 and expression of CD39 by CD8⁺ T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.     

Abatacept (cytotoxic T lymphocyte antigen 4‐immunoglobulin) improves B cell function and regulatory T cell inhibitory capacity in rheumatoid arthritis patients non‐responding to anti‐tumour necrosis factor‐α agents

The use of biological agents combined with methotrexate (MTX) in rheumatoid arthritis (RA) patients has strongly improved disease outcome. In this study, the effects of abatacept on the size and function of circulating B and T cells in RA patients not responding to anti‐tumour necrosis factor (TNF)‐α have been analysed, with the aim of identifying immunological parameters helpful to choosing suitable tailored therapies. We analysed the frequency of peripheral B and T cell subsets, B cell function and T regulatory cell (T_reg) inhibitory function in 20 moderate/severe RA patients, according to the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) criteria, primary non‐responders to one TNF‐α blocking agent, who received abatacept + MTX. Patients were studied before and 6 months after therapy. We found that abatacept therapy significantly reduced disease activity score on 44 joints (DAS)/erythrocyte sedimentation rate (ESR) values without causing severe side effects. The size of the circulating B and T cell compartments in RA patients was not significantly different from healthy donors, but B cell proliferation and plasma cell differentiation was impaired before therapy and restored by abatacept. While T_reg cell frequency was normal, its inhibitory function was absent before therapy and was partially recovered 6 months after abatacept. B and T_reg cell function is impaired in RA patients not responding to the first anti‐TNF‐α agent. Abatacept therapy was able to rescue immune function and led to an effective and safe clinical outcome, suggesting that RA patients, in whom anti‐TNF‐α failed, are immunologically prone to benefit from an agent targeting a different pathway.     

V‐set and Ig domain‐containing 4 (VSIG4)‐expressing hepatic F4/80+ cells regulate oral antigen‐specific responses in mouse

Oral tolerance can prevent unnecessary immune responses against dietary antigens. Members of the B7 protein family play critical roles in the positive and/or negative regulation of T cell responses to interactions between APCs and T cells. V‐set and Ig domain‐containing 4 (VSIG4), a B7‐related co‐signaling molecule, has been known to act as a co‐inhibitory ligand and may be critical in establishing immune tolerance. Therefore, we investigated the regulation of VSIG4 signaling in a food allergy and experimental oral tolerance murine models. We analyzed the contributions of the two main sites involved in oral tolerance, the mesenteric lymph node (MLN) and the liver, in VSIG4‐mediated oral tolerance induction. Through the comparative analysis of major APCs, dendritic cells (DCs) and macrophages, we found that Kupffer cells play a critical role in inducing regulatory T cells (Tregs) and establishing immune tolerance against oral antigens via VSIG4 signaling. Taken together, these results suggest the possibility of VSIG4 signaling‐based regulation of orally administered antigens.     

Bacillus‐derived poly‐γ‐glutamic acid attenuates allergic airway inflammation through a Toll‐like receptor‐4‐dependent pathway in a murine model of asthma

Background Asthma is an inflammatory disease of the airways that is mediated by Th2 responses. Poly‐γ‐glutamic acid (γ‐PGA) is an extracellular polymeric compound that is synthesized by Bacillus cells. Previously, we found that γ‐PGA promoted Th1 cell development in a manner dependent on antigen‐presenting cells, but inhibited Th2 cell development. Objective To investigate the effect of γ‐PGA on dendritic cells (DCs), and its potential for treating Th2‐mediated allergic asthma. Methods Wild‐type, Toll‐like receptor (TLR)‐2 deficient, and TLR‐4‐defective mice were used. DCs derived from the bone marrow and extracted from the lung were stimulated with γ‐PGA and assayed for the expression of signalling molecules, costimulatory molecules, and cytokines. Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. They were repeatedly injected intranasally with γ‐PGA before and during the challenge period, and inflammation and structural remodelling of the airways were examined. Results γ‐PGA selectively signalled conventional DCs to activate NF‐κB and mitogen‐activated protein kinase, leading to the up‐regulation of CD86, CD40, and IL‐12, but not IL‐10 and IL‐6. These effects of γ‐PGA were dependent on TLR‐4 and independent of TLR‐2. Importantly, the intranasal administration of γ‐PGA to OVA‐sensitized/challenged mice reduced the airway hyperresponsiveness and allergic inflammation such as leucocyte influx, goblet cell hyperplasia, eosinophilia, and Th2 cytokine production. In addition to lowered IgE titres, the treatment of mice with γ‐PGA significantly reduced the multiplication and Th2 polarization of mediastinal lymph node T cells upon allergen‐specific restimulation. These anti‐asthmatic effects of γ‐PGA were also abolished in TLR‐4‐defective mice. Conclusions and Clinical Relevance Our data indicate that γ‐PGA activates DCs to favour Th1 cell induction through a TLR‐4‐dependent pathway and alleviates pathologic symptoms in a Th2‐biased asthmatic model. These findings highlight the potential of γ‐PGA for the treatment of asthma and other allergic disease in which Th2 polarization plays an important role. Cite this as: K. Lee, S.‐H. Kim, H. J. Yoon, D. J. Paik, J. M. Kim and J. Youn, Clinical & Experimental Allergy, 2011 (41) 1143–1156.     

Thymic stromal lymphopoietin is a critical mediator of IL‐13‐driven allergic inflammation

Both thymic stromal lymphopoietin (TSLP) and IL‐13 are essential cytokines for the development of allergic inflammation. However, a causal link between TSLP and IL‐13 has not yet been fully elucidated. This study aimed to investigate whether IL‐13 induces TSLP expression and whether the induction contributes to the development of allergic inflammation. We found that IL‐13 induced TSLP expression in mouse nasal tissue specimens in a Stat6‐dependent manner. In addition, intranasal challenge of mice with IL‐13 induced TSLP expression in the nasal epithelium. Importantly, intranasal IL‐13 challenge induced eosinophilia and goblet cell hyperplasia in the nasal mucosa in mice, which was inhibited by the blockade of TSLP activity with anti‐TSLP Ab. These findings suggest that TSLP is an important mediator of IL‐13‐driven allergic inflammation in the nasal mucosa. Taken together with the recent findings that IL‐13 is a critical downstream element for TSLP‐driven allergic inflammation, TSLP may function both upstream and downstream of IL‐13, thus providing an additional rationale as to why TSLP plays such a central role in the development of allergic inflammation.     

Heme oxygenase‐1 inhibits basophil maturation and activation but promotes its apoptosis in T helper type 2‐mediated allergic airway inflammation

The anti‐inflammatory role of heme oxygenase‐1 (HO‐1) has been studied extensively in many disease models including asthma. Many cell types are anti‐inflammatory targets of HO‐1, such as dendritic cells and regulatory T cells. In contrast to previous reports that HO‐1 had limited effects on basophils, which participate in T helper type 2 immune responses and antigen‐induced allergic airway inflammation, we demonstrated in this study, for the first time, that the up‐regulation of HO‐1 significantly suppressed the maturation of mouse basophils, decreased the expression of CD40, CD80, MHC‐II and activation marker CD200R on basophils, blocked DQ‐ovalbumin uptake and promoted basophil apoptosis both in vitro and in vivo, leading to the inhibition of T helper type 2 polarization. These effects of HO‐1 were mimicked by exogenous carbon monoxide, which is one of the catalytic products of HO‐1. Furthermore, adoptive transfer of HO‐1‐modified basophils reduced ovalbumin‐induced allergic airway inflammation. The above effects of HO‐1 can be reversed by the HO‐1 inhibitor Sn‐protoporphyrin IX. Moreover, conditional depletion of basophils accompanying hemin treatment further attenuated airway inflammation compared with the hemin group, indicating that the protective role of HO‐1 may involve multiple immune cells. Collectively, our findings demonstrated that HO‐1 exerted its anti‐inflammatory function through suppression of basophil maturation and activation, but promotion of basophil apoptosis, providing a possible novel therapeutic target in allergic asthma.     

Cytotoxic T lymphocyte antigen 4‐immunoglobulin G is a potent adjuvant for experimental allergen immunotherapy

Allergen‐specific immunotherapy (SIT) is the only treatment for allergic diseases that targets allergen‐specific T helper type 2 (Th2) cells, which are the cause of the disease. There is an unmet requirement for adjuvants that increase the clinical efficacy of SIT allowing application of lower doses of the allergen, thereby reducing the risk of anaphylactic reactions. Cytotoxic T lymphocyte antigen 4–immunoglobulin (CTLA‐4–Ig) has been shown to induce immunological tolerance in autoimmunity and allograft transplantation by blocking T cell co‐stimulation and induction of the immunoregulatory enzyme indoleamine 2,3 dioxygenase (IDO). Previously, we showed that CTLA‐4–Ig treatment at the time of allergen inhalation induced tolerance to subsequent allergen exposure in a mouse model of asthma. In this study, we test the hypothesis that CTLA‐4–Ig acts as an adjuvant for experimental SIT. We evaluated the adjuvant effects of CTLA‐4–Ig on SIT in a mouse model of ovalbumin‐driven asthma. We used both wild‐type and IDO‐deficient mice to assess the role of IDO in the adjuvant effects of CTLA‐4–Ig. Co‐administration of CTLA‐4–Ig strongly increased SIT‐induced suppression of airway hyperreactivity (AHR), specific IgE in serum, airway eosinophilia and Th2 cytokine levels. Moreover, we found that CTLA‐4–Ig, as an adjuvant for SIT, is equally effective in IDO‐deficient and wild‐type mice, demonstrating that the effect of CTLA‐4–Ig is independent of IDO expression. We show that CTLA‐4–Ig acts as a potent adjuvant to augment the therapeutic effects of SIT. As the adjuvant activity of CTLA‐4–Ig is independent of IDO, we conclude that it acts by blocking CD28‐mediated T cell co‐stimulation.     

‐318C/T polymorphism of the CTLA‐4 gene is an independent risk factor for RBC alloimmunization among sickle cell disease patients

Cytotoxic T‐lymphocyte‐associated antigen 4 (CTLA‐4) molecule is expressed on T‐lymphocyte membrane and negatively influences the antigen‐presenting process. Reduced expression of CTLA‐4 due to gene polymorphisms is associated with increased risk of autoimmune disorders, whose physiopathology is similar to that of post‐transfusion red blood cell (RBC) alloimmunization. Our goal was to evaluate if polymorphisms of CTLA‐4 gene that affect protein expression are associated with RBC alloimmunization. This was a case–control study in which 134 sickle cell disease (SCD) patients and 253 non‐SCD patients were included. All patients were genotyped for the polymorphisms 49A/G and ‐318C/T of CTLA‐4 gene. The genotype frequency of ‐318C/T differed significantly between alloimmunized and nonalloimmunized SCD patients, irrespective of clinical confounders (p = .016). SCD patients heterozygous for ‐318T allele presented higher risk of alloantibody development (OR: 5.4, CI: 1.15–25.6). In conclusion, the polymorphism ‐318C/T of CTLA‐4 gene is associated with RBC alloimmunization among SCD patients. This highlights the role played by CTLA‐4 on post‐transfusion alloantibody development.     

4‐Thiouridine induces dose‐dependent reduction of oedema,leucocyte influx and tumour necrosis factor in lung inflammation

Recent reports demonstrate a role for nucleotides as inflammatory modulators. Uridine, for example, reduces oedema formation and leucocyte infiltration in a Sephadex‐induced lung inflammation model. Tumour necrosis factor (TNF) concentration was also reduced. Previous in vivo observations indicated that 4‐thiouridine might have similar effects on leucocyte infiltration and TNF release. The aim of this study was thus to investigate the effects of 4‐thiouridine in greater detail. We used a Sephadex‐induced acute lung inflammation model in Sprague–Dawley rats. The dextran beads were instilled intratracheally into the lungs, which were excised and examined after 24 h. Sephadex alone led to massive oedema formation and infiltration of macrophages, neutrophils and eosinophils. Microgranulomas with giant cell formations were clearly visible around the partially degraded beads. A significant increase in bronchoalveolar lavage fluid (BALF) content of TNF and leukotrienes was also seen. 4‐Thiouridine co‐administration affected all variables investigated in this model, i.e. oedema, microscopic and macroscopic appearance of lung tissue, total leucocyte and differential leucocyte counts in BALF, TNF and leukotrienes C₄ (LTC₄), LTD₄ and LTE₄ in BALF, indicating a reproducible anti‐inflammatory effect. In conclusion, we have demonstrated that 4‐thiouridine has anti‐inflammatory effects similar to those of uridine. To our knowledge, this is the first demonstration of pharmacological 4‐thiouridine effects in vivo. The results suggest nucleoside/nucleotide involvement in inflammatory processes, warranting further studies on nucleoside analogues as attractive new alternatives in the treatment of inflammatory diseases.