Highly differentiated IL5+ Th2 cells in food allergy and eosinophilic GI disease

嗜酸性粒细胞性胃肠疾病（EGID）是一组以胃肠道嗜酸性粒细胞炎症为特征的疾病。在过去的十年中，EGID的发病率急剧增加，特别是嗜酸性粒细胞性食管炎（EoE），但也有嗜酸性粒细胞性胃肠炎（EG）。EGID患者通常具有许多食物过敏，并且疾病随着基于氨基酸的元素饮食的建立而缓解。总之，这表明EGID是食物过敏原驱动的嗜酸性炎症性肠道疾病。 Omalizumab是一种人源化治疗性抗IgE单克隆抗体。抗IgE治疗降低循环游离IgE的浓度，阻断IgE与Fc RI和CD 23的结合，并下调肥大细胞、嗜碱性粒细胞和树突细胞上的表面Fc RI。由于抗IgE治疗的多种作用影响抗原呈递细胞（APC），因此推测通过抑制APC功能，抗IgE治疗可对T细胞具有免疫调节活性。我们假设两种不同的机制，抗IgE治疗可以抑制过敏原特异性Th 2反应。首先，抗IgE下调树突状细胞上的Fc RI并阻断CD 23介导的过敏原与APC的结合，从而抑制IgE促进的APC对Ag的捕获，这反过来可能导致过敏原特异性T细胞活化降低。第二，抗IgE的IgE信号传导抑制体内肥大细胞和嗜碱性粒细胞的活化，这可能降低IL-4和/或TSLP表达，其缺乏可能抑制Th 2细胞分化。 为了验证这一假设，我们在一项奥马珠单抗治疗过敏性嗜酸性粒细胞性胃肠炎的临床试验中评估了抗IgE对过敏原特异性T细胞应答的免疫调节作用。使用羧基荧光素琥珀酰亚胺酯（CFSE）染料稀释和流式细胞术测量四种过敏原特异性T细胞反应（最大增殖、增殖剂量反应EC 50、前体频率和细胞因子表达）。奥马珠单抗治疗前基线（10.0%）和奥马珠单抗治疗16周时间点（7.2%，p= 0.33）之间的过敏原特异性增殖（CFSE染料稀释）无显著差异。抗IgE治疗与过敏原增殖剂量反应的小但显著的左移（与假设相反）相关，因此基线EC 50是奥马珠单抗受试者的1.5倍。奥马珠单抗治疗前基线（4.0 x 10 e-4）和奥马珠单抗治疗16周时间点（6.5 x 10 e-4，p= 0.33）之间的过敏原特异性T细胞前体频率无显著差异。在IL-4：IFN-（基线0.81，奥马珠单抗0.63，p =0.15）或IL-5：IFN-（基线0.33，奥马珠单抗0.36，p=0.42）或Th 2细胞因子与TNF-的比率中未发现显著差异。与该假设相反，16周的抗IgE治疗没有降低任何过敏原特异性反应指数的效果。总之，使用T细胞功能的多个指标，该研究未能证明抗IgE治疗广泛或有效地抑制过敏原特异性T细胞应答。因此，这些数据不支持IgE促进的Ag呈递增强体内过敏原特异性T细胞应答的主要作用。 目前的工作正在进行中，以扩大这些发现肠道居民T细胞在嗜酸性胃肠道疾病患者。将获得GI活组织检查，并使用流式细胞术和细胞化学来确定是否存在与IL-5+ Th 2细胞的类似关联。另外的实验将鉴定在这些疾病状态中是否存在食物过敏原特异性Th 2细胞的其他特征，其可能特异性地促成过敏性与嗜酸性粒细胞炎性食物过敏。

Eosinophilic gastrointestinal disorders (EGIDs) are a group of diseases characterized by eosinophilic inflammation of the gastrointestinal tract. In the past decade, there has been a dramatic increase in the incidence of EGIDs, particularly eosinophilic esophagitis (EoE), but also eosinophilic gastroenteritis (EG). EGID patients often have numerous food hypersensitivities, and the disease goes into remission with the institution of an amino acid based elemental diet. In sum, this suggests that EGID is a food allergen driven eosinophilic inflammatory gut disease. Omalizumab is a humanized therapeutic anti-IgE monoclonal antibody. Anti-IgE therapy reduces the concentration of circulating free IgE, blocks IgE binding to both FcεRI and CD23, and down regulates surface FcεRI on mast cells, basophils and dendritic cells. Because of the multiple actions of anti-IgE therapy that affect antigen presenting cells (APCs), it has been postulated that by inhibiting APC function, anti-IgE therapy may have immunomodulatory activity on T cells. We hypothesized two distinct mechanisms whereby anti-IgE therapy could inhibit allergen specific Th2 responses. First, anti-IgE down regulates FcεRI on dendritic cells and blocks CD23 mediated allergen binding to APCs, thereby inhibiting IgE facilitated Ag capture by APCs, which in turn could result in decreased allergen specific T cell activation. Second, IgE signaling of anti-IgE inhibits mast cell and basophil activation in vivo, which may decrease IL-4 and/or TSLP expression, the lack of which could inhibit Th2 cell differentiation. To test this hypothesis, we assessed anti-IgE immunomodulation of allergen specific T cell responses during a clinical trial of omalizumab in subjects with allergic eosinophilic gastroenteritis. Four allergen specific T cell responses (maximal proliferation, proliferation dose response EC50, precursor frequency, and cytokine expression) were measured using carboxyfluorescein succinimidyl ester (CFSE) dye dilution and flow cytometry. There was no significant difference in allergen specific proliferation (CFSE dye dilution) between the pre-omalizumab baseline (10.0%) and the 16-week omalizumab time point (7.2%, p= 0.33). Anti-IgE therapy was associated with a small but significant left shift (opposite of the hypothesis) in the proliferative dose response to allergen, such that the EC50 at baseline was 1.5 times that of subjects on omalizumab. There was no significant difference in the precursor frequency of allergen specific T cells between the pre-omalizumab baseline (4.0 x 10e-4) and the 16 week omalizumab time point (6.5 x 10e-4, p= 0.33). No significant differences were found in the ratios of either IL-4:IFN-γ (baseline 0.81, omalizumab 0.63, p =0.15) or IL-5:IFN-γ (baseline 0.33, omalizumab 0.36, p=0.42) or of either Th2 cytokine to TNF-α. In contradistinction to the hypothesis, 16 weeks of anti-IgE therapy had no effect diminishing any index of allergen specific response. In sum, using multiple indices of T cell function, this study failed to demonstrate that anti-IgE therapy broadly or potently inhibits allergen specific T cell responses. As such, these data do not support a major role for IgE facilitated Ag presentation augmenting allergen specific T cell responses in vivo. Current work is underway to extend these findings to gut resident T cells in patients with eosinophilic GI disease. GI biopsies will be obtained and both flow cytometry and immunohistochemisty will be used to determine if a similar association with IL-5+ Th2 cells exists. Additional experiments will identify if there are other characteristics of food allergen specific Th2 cells in these disease states that may specifically contribute to anaphylactic vs. eosinophilic inflammatory food allergy.