Treatment with anti‐OX40L or anti‐TSLP does not alter the frequency of T regulatory cells in allergic asthmatics

OX40‐OX40L interactions and thymic stromal lymphopoietin (TSLP) are important in the induction and maintenance of Th2 responses in allergic disease, whereas T regulatory cells (Treg) have been shown to suppress pro‐inflammatory Th2 responses. Both OX40L and TSLP have been implicated in the negative regulation of Treg. The effect of anti‐asthma therapies on Treg is not well known. Our aim was to assess the effects of two monoclonal antibody therapies (anti‐OX40L and anti‐TSLP) on Treg frequency using a human model of allergic asthma. We hypothesized that the anti‐inflammatory effects of these therapies would result in an increase in circulating Treg (CD4⁺CD25⁺CD127^lowFoxp3⁺ cells) frequency. We measured Treg using flow cytometry, and our results showed that neither allergen challenge nor monoclonal antibody therapy altered circulating Treg frequency. These data highlight the need for assessment of airway Treg and for a more complete understanding of Treg biology so as to develop pharmacologics/biologics that modulate Treg for asthma therapy.     

Thymic Stromal Lymphopoietin: A Promising Therapeutic Target for Allergic Diseases

Thymic stromal lymphopoietin (TSLP), an interleukin 7-like cytokine, can trigger dendritic cell (DC)-mediated T-helper type 2 (Th2) inflammatory responses. Recent evidence demonstrates that cytokines TSLP and OX40 (CD134)/OX40 ligand seem to be important players in the maintenance of Th2 memory pool in the pathogenesis of asthma. Accumulating data reveal that the pathogenic T cells involved in asthma are likely to be inflammatory Th2 cells. TSLP is involved in the development of asthma through crosstalk with nuclear factor NF-?B. Progression of skin fibrosis in atopic dermatitis occurs via TSLP/TSLP receptor. TSLP-mediated dermal inflammation aggravates experimental allergic asthma. Also, TSLP polymorphisms are associated with susceptibility to asthma, atopic dermatitis, and eczema herpeticum. These findings suggest a master switch of TSLP in the initiation of allergic and adaptive inflammation through innate pathways at the epithelial cell-DC interface. The TSLP pathway is therefore a promising target for immunotherapy of allergic diseases.     

IL-33-activated dendritic cells are critical for allergic airway inflammation

IL-33, a new member of the IL-1 family cytokine, is involved in Th2-type responses in a wide range of diseases and signals through the ST2 receptor expressed on many immune cells. Since the effects of IL-33 on DCs remain controversial, we investigated the ability of IL-33 to modulate DC functions in vitro and in vivo. Here, we report that IL-33 activates myeloid DCs to produce IL-6, IL-1b, TNF, CCL17 and to express high levels of CD40, CD80 OX40L and CCR7. Importantly, IL-33-activated DCs prime naive lymphocytes to produce the Th2 cytokines IL-5 and IL-13, but not IL-4. In vivo, IL-33 exposure induces DC recruitment and activation in the lung. Using an OVA-induced allergic lung inflammation model, we demonstrate that the reduced airway inflammation in ST2-deficient mice correlates with the failure in DC activation and migration to the draining LN. Finally, we show that adoptive transfer of IL-33-activated DCs exacerbates lung inflammation in a DC-driven model of allergic airway inflammation. These data demonstrate for the first time that IL-33 activates DCs during antigen presentation and thereby drives a Th2-type response in allergic lung inflammation.     

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP. Objective Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation. Methods CD11c⁺DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4⁺T cells. Results CD11c⁺ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4⁺ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4⁺ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone. Conclusions These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.     

Thymic stromal lymphopoietin, OX40-ligand, and interleukin-25 in allergic responses

Allergic diseases are often triggered by environmental allergens that induce dominant type 2 immune responses, characterized by the infiltrated T‐helper type 2 (TH2) lymphocytes, eosinophils, and elevated TH2 cytokines. In addition to TH2 type immune responses, epithelial stress and injury linked to tissue remodelling are often observed, suggesting that epithelial cells may play important role in regulating allergic responses. Dendritic cells (DCs), the professional antigen‐presenting cells with the capabilities of sampling allergens, are considered as the key player on instructing TH2 immune responses. Whether inflamed epithelium can regulate innate immunity, such as macrophages and DCs, which in turn instructs adaptive immunity has long been hypothesized. Studies of thymic stromal lymphopoietin (TSLP), an epithelial cells‐derived cytokine, that can strongly activate DCs, provide important evidences that the epithelial barrier can trigger allergic diseases by regulating immune responses. The finding that OX40/OX40Ligand (OX40L) interactions are the molecular trigger responsible for the induction and maintenance of TH2 responses by TSLP‐activated DCs provides a plausible molecular explanation for TSLP‐mediated allergy. Recent progresses in characterizing the pro‐inflammatory IL‐17 cytokine family have added an additional layer of complexity on the regulation of allergic inflammation. TSLP–DCs can induce a robust expansion of TH2 memory cells and strengthen functional attributes by up‐regulating their surface expression of IL‐17RB (IL‐25R), the receptor for cytokine IL‐17E (IL‐25), a distinct member of IL‐17 cytokine family. IL‐17E (also known as IL‐25) produced by epithelial cells, and other innate cells, such as eosinphils, basophils, and mast cells, are shown to regulate adaptive immunity by enhancing TH2 cytokine productions. These exciting findings expand our knowledge of the complex immunological cascades that result in allergic inflammation and may provide novel therapeutic approaches for the treatment of allergic diseases.     

Transgenic expression of survivin compensates for OX40‐deficiency in driving Th2 development and allergic inflammation

Survivin, an inhibitor of apoptosis family molecule, has been proposed as a crucial intermediate in the signaling pathways leading to T‐cell development, proliferation, and expansion. However, the importance of survivin to T‐cell‐driven inflammatory responses has not been demonstrated. Here, we show that survivin transgenic mice exhibit an increased antigen‐driven Th2 lung inflammation and that constitutive expression of survivin reversed the defective lung inflammation even in the absence of OX40 costimulation. We found that OX40‐deficient mice were compromised in generating Th2 cells, airway eosinophilia, and IgE responses. In contrast, OX40‐deficient/survivin transgenic mice generated normal Th2 responses and exhibited strong lung inflammation. These results suggest that OX40 costimulation crucially engages survivin during antigen‐mediated Th2 responses. These findings also promote the notion that OX40 costimulation regulates allergic responses or lung inflammation by targeting survivin thereby enhancing T‐cell proliferation and resulting in more differentiated Th2 cells in the allergic inflammatory response.     

胸腺基质淋巴细胞生成素、OX40和OX40受体在过敏性腹泻小鼠大肠黏膜中表达的关系

目的 建立小鼠过敏性腹泻模型,检测其大肠黏膜中胸腺基质淋巴细胞生成素(TSLP)、OX40、OX40受体(OX40L)的表达和肠系膜淋巴结细胞培养上清中IL-4、IFN-γ的水平,分析其之间的关系,探讨TSLP、OX40、OX40L在过敏性腹泻中的作用.方法 雌性BALB/c小鼠20只随机分成两组即对照组和实验组.采用酶联免疫吸附法(ELISA)检测肠系膜淋巴结细胞培养上清中IL-4和IFN-γ的水平,取大肠组织HE染色观察大肠组织病理改变,SP免疫组织化学技术检测TSLP、OX40、OX40L的表达.结果 ①HE染色结果显示:实验组比对照组小鼠结肠黏膜组织非特异性炎症反应显著,上皮排列不规则,有大量炎性细胞浸润,固有层可见大量嗜酸性粒细胞浸润;②TSLP、OX40、OX40L在过敏性腹泻小鼠大肠黏膜中的表达水平高于对照组的表达水平(t=7.07,t =7.81,t =7.79,P均＜0.01);③Spearman等级相关分析发现TSLP、OX40、0X40L三者间表达强度存在正相关(r=0.889,r=0.932,r=0.943,P均＜0.01),同时三者与肠系膜淋巴结细胞培养上清中IL-4水平呈正相关(r=0.891,r =0.936,r=0.886,P均＜0.05),而与IFN-γ水平呈负相关(r=-0.829,r=-0.881,r=-0.937,P均＜0.05).结论 TSLP、OX40、OX40L三者间表达存在正相关,并诱导了Th1/Th2轴向Th2轴漂移,促进了过敏性腹泻的发生发展.     

Adoptive transfer of dendritic cells from allergic mice induces specific immunoglobulin E antibody in naïve recipients in absence of antigen challenge without altering the T helper 1/T helper 2 balance

Dendritic cells (DCs) are important in the regulation of immune responses and it has been proposed that these cells play an important role in asthma; however, their role in food allergy is still largely unknown. Our aim was to study specific immunoglobulin E (IgE) and immunoglobulin G (IgG) responses in naïve recipients following adoptive transfer of myeloid DCs from allergic and control mice. The phenotypic features and lymphokine production of DCs were also investigated. CD11c ^{+ /hi} B220^? DCs isolated from spleen and Peyer's patches (PP) of cow's milk (CM) allergic and control mice were transferred intravenously (i.v.) into naïve syngeneic recipients, and IgE‐ and IgG‐specific responses were evaluated. Experiments were also carried out to determine the levels of interferon‐γ (IFN‐γ) and interleukin (IL)‐4 produced by splenocytes from naïve recipients following the adoptive transfer, and CD40 ligand (CD40L)‐mediated IL‐10 production by DCs from allergic and control mice. DCs isolated from spleen and PP of allergic mice, but not control groups, induced CM‐specific IgG and IgE antibody production in naïve recipients in the absence of previous immunization, but did not modify the T helper 1 (Th1) and T helper 2 (Th2) balance. Furthermore, although no difference was observed in the expression of canonical DC surface markers, PP DCs from allergic mice produced less IL‐10 than DCs from controls. We interpret these data as showing that DCs play a pivotal role in allergen‐specific IgE responses and that a Th2‐skewed response may not be involved in the early phase of allergic responses. The identification of the mechanisms underlying these events may help to design novel strategies of therapeutic intervention in food allergy.     

Early production of thymic stromal lymphopoietin precedes infiltration of dendritic cells expressing its receptor in allergen-induced late phase cutaneous responses in atopic subjects

Background: Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)‐7‐like cytokine that triggers dendritic cell‐mediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL‐7 receptor alpha (IL‐7Rα) chain and the TSLP receptor (TSLPR), which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD4⁺ T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen‐induced late‐phase reaction (LPR) in atopic subjects. Methods: Skin biopsies were obtained from atopic subjects (n = 9) at various times after cutaneous allergen challenge. In situ hybridization and immunohistochemistry were used to determine TSLP mRNA expression and to measure infiltration of TSLPR⁺ DC in skin LPR. RT‐PCR and flow cytometry were employed to analyse TSLPR expression on isolated blood DC. Results: Allergen‐induced skin TSLP expression occurred as early as 1 h after allergen challenge, whereas TSLPR⁺ and CD11c⁺ cells infiltrated relatively late (24–48 h). The majority of TSLPR⁺ cells were DC co‐expressing blood DC antigen‐1 (BDCA‐1) or BDCA‐2. Freshly isolated blood DC expressed both TSLPR and IL‐7Rα chains. Maturation and stimulation with TSLP or polyriboinosinic–polyribocytidylic acid in vitro upregulated the expression of both TSLPR and IL‐7Rα chains in DC but not in chemoattractant receptor‐homologous molecule expressed on Th2 cells⁺ CD4⁺ T cells. Conclusion: The data suggest that TSLP plays a role in augmenting, through DC recruitment and activation, the development of Th2‐type T cells in allergic inflammation.     

Thymic stromal lymphopoietin and OX40 ligand pathway in the initiation of dendritic cell-mediated allergic inflammation

It was demonstrated 5 years ago that thymic stromal lymphopoietin (TSLP), a IL-7-like cytokine produced by epithelial cells, could strongly activate human myeloid dendritic cells to induce an inflammatory T(H)2 response characterized by high TNF-alpha and little IL-10 production, distinct from the regulatory T(H)2 responses characterized by low TNF-alpha and high IL-10 production. TSLP was found highly expressed by keratinocytes of skin lesions of atopic dermatitis and associated with dendritic cell activation in situ. This suggests for the first time that TSLP represents a master switch of allergic inflammation at the epithelial cell and dendritic cell interface. During the last several years, the evidence for the association of TSLP with human asthma was revealed. The direct link between TSLP expression with the pathogenesis of atopic dermatitis and asthma in vivo was demonstrated. In addition, OX40 ligand was found to be the TSLP-induced molecule on dendritic cells that triggers inflammatory T(H)2 differentiation in the absence of IL-12. TSLP was also demonstrated to direct the innate phase of allergic immune responses through activating mast cells. Therefore, TSLP and OX40 ligand may represent important targets for intervention of the initiation of allergic inflammatory responses.     

OX40 blockade inhibits house dust mite driven allergic lung inflammation in mice and in vitro allergic responses in humans

The costimulatory receptor OX40 is expressed on activated T cells and regulates T‐cell responses. Here, we show the efficacy and mechanism of action of an OX40 blocking antibody using the chronic house dust mite (HDM) mouse model of lung inflammation and in vitro HDM stimulation of cells from HDM allergic human donors. We have demonstrated that OX40 blockade leads to a reduction in the number of eosinophils and neutrophils in the lavage fluid and lung tissue of HDM sensitized mice. This was accompanied by a decrease in activated and memory CD4⁺ T cells in the lungs and further analysis revealed that both the Th2 and Th17 populations were inhibited. Improved lung function and decreased HDM‐specific antibody responses were also noted. Significantly, efficacy was observed even when anti‐OX40 treatment was delayed until after inflammation was established. OX40 blockade also inhibited the release of the Th2 cytokines IL‐5 and IL‐13 from cells isolated from HDM allergic human donors. Altogether, our data provide evidence of a role of the OX40/OX40L pathway in ongoing allergic lung inflammation and support clinical studies of a blocking OX40 antibody in Th2 high severe asthma patients.     

Cytokine‐dependent regulation of dendritic cell differentiation in the splenic microenvironment

The DC‐derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady‐state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α^?CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α‐galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α^? and CD8α⁺ splenic DC subsets and enhances cross‐presentation of exogenous antigens. Based on genome‐wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN‐γ‐mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN‐γR and are much less responsive to IFN‐γ. Under inflammatory conditions, particularly in the absence of IFN‐γ signaling in IFN‐γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM‐CSF in concert with IL‐4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression.     

Myeloid leukemia cells with a B7‐2+ subpopulation provoke Th‐cell responses and become immuno‐suppressive through the modulation of B7 ligands

Expression of the B7 family molecules in acute myeloid leukemia (AML) has been demonstrated by independent clinical studies. Intriguingly, the expression of the most potent costimulatory molecules B7‐2 (CD86) and B7‐H2 (ICOS Ligand) on AML cells has been associated with poor prognosis and disease severity. Here, this phenomenon was modeled in vitro with the myeloid leukemia cell line HL‐60, which is capable of differentiating through the FAB M2/M3 and M4/M5 immunophenotypes. These derivatives of HL‐60 harbored a B7‐2⁺ subpopulation and recapitulated the distribution of B7 ligands previously reported in primary AML cases. B7‐2⁺ AML cells significantly contributed to T‐cell responses. This costimulatory activity enabled helper (Th)‐cell activation, proliferation, and production of Th1‐associated cytokines. Conversely, even a short‐term incubation with stimulated T cells resulted in upregulation of inhibitory B7‐H1 (PD‐L1) and B7‐DC (PD‐L2), and downregulation of stimulatory B7‐H2 molecules on leukemia cells. Purified from iHL‐60‐T‐cell co‐cultures, these myeloid leukemia cells severely suppressed Th‐cell responses specifically through the PD‐1 pathway. In conclusion, Th‐cell responses can be directly supported by B7‐2⁺ leukemia subpopulations. However, this interaction can facilitate the acquisition of a suppressive character that may contribute to immune evasion in myeloid leukemia.     

T helper cell IL-4 drives intestinal Th2 priming to oral peanut antigen,under the control of OX40L and independent of innate-like lymphocytes

Intestinal T helper type 2 (Th2) immunity in food allergy results in IgG1 and IgE production, and antigen re-exposure elicits responses such as anaphylaxis and eosinophilic inflammation. Although interleukin-4 (IL-4) is critically required for allergic sensitization, the source and control of IL-4 during the initiation of Th2 immunity in vivo remains unclear. Non-intestinal and non-food allergy systems have suggested that natural killer-like T (NKT) or γδ T-cell innate lymphocytes can supply the IL-4 required to induce Th2 polarization. Group 2 innate lymphoid cells (ILCs) are a novel IL-4-competent population, but their contribution to initiating adaptive Th2 immunity is unclear. There are also reports of IL-4-independent Th2 responses. Here, we show that IL-4-dependent peanut allergic Th2 responses are completely intact in NKT-deficient, γδ T-deficient or ILC-deficient mice, including antigen-specific IgG1/IgE production, anaphylaxis, and cytokine production. Instead, IL-4 solely from CD4⁺ Th cells induces full Th2 immunity. Further, CD4⁺ Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule on dendritic cells (DCs) required for intestinal allergic priming. However, both Th2 cells and ILCs orchestrated IL-13-dependent eosinophilic inflammation. Thus, intestinal Th2 priming is initiated by an autocrine/paracrine acting CD4⁺ Th cell-intrinsic IL-4 program that is controlled by DC OX40L, and not by NKT, γδ T, or ILC cells.     

TSLP production by dendritic cells is modulated by IL‐1β and components of the endoplasmic reticulum stress response

Thymic stromal lymphopoietin (TSLP) produced by epithelial cells acts on dendritic cells (DCs) to drive differentiation of T_H2‐cells, and is therefore important in allergic disease pathogenesis. However, DCs themselves make significant amounts of TSLP in response to microbial products, but little is known about the key downstream signals that induce and modulate this TSLP secretion from human DCs. We show that human monocyte derived DC (mDC) secretion of TSLP in response to Candida albicans and β‐glucans requires dectin‐1, Syk, NF‐κB, and p38 MAPK signaling. In addition, TSLP production by mDCs is greatly enhanced by IL‐1β, but not TNF‐α, in contrast to epithelial cells. Furthermore, TSLP secretion is significantly increased by signals emanating from the endoplasmic reticulum (ER) stress response, specifically the unfolded protein response sensors, inositol‐requiring transmembrane kinase/endonuclease 1 and protein kinase R‐like ER kinase, which are activated by dectin‐1 stimulation. Thus, TSLP production by mDCs requires the integration of signals from dectin‐1, the IL‐1 receptor, and ER stress signaling pathways. Autocrine TSLP production is likely to play a role in mDC‐controlled immune responses at sites removed from epithelial cell production of the cytokine, such as lymphoid tissue.     

Dendritic cell-associated lectin 2 (DCAL2) defines a distinct CD8α- dendritic cell subset

CLRs on DCs play important roles in immunity and are expressed selectively on certain DC subsets. Murine DCAL2 (myeloid inhibitory C-type lectin/Clec12a) is a type-II CLR with an ITIM. Using a mouse DCAL2-specific mAb, we found that DCAL2 is expressed at relatively high levels on APCs and that DCAL2 expression can be used to divide CD8α- DCs into DCAL2+DCIR2- and DCAL2-DCIR2+ subpopulations. CD8α-DCAL2+ DC, CD8α-DCIR2+ DC, and CD8α+DCAL2+ DC subsets each express different levels of TLRs and respond to unique classes of TLR ligands by producing distinct sets of cytokines. Whereas CD8α-DCAL2+ DCs robustly produce cytokines, including IL-12, in response to CpG, CD8α-DCIR2+ DCs produce only TNF-α and IL-10 in modest amounts when stimulated with zymosan. However, CD8α-DCIR2+DCs, unlike the other DC subsets, strongly up-regulate OX40L when stimulated with bacterial flagellin. As predicted from their cytokine expression, CD8α-DCAL2+ DCs efficiently induced Th1 responses in the presence of CpG in vitro and in vivo, whereas CD8α-DCIR2+ DCs induced Th2 cells in response to flagellin. Thus, CD8α-DCAL2+ DCs comprise a distinct CD8α- DC subset capable of supporting Th1 responses. DCAL2 is a useful marker to identify a Th1-inducing CD8α- DC population.