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

嗜酸性胃肠道疾病(EGID)是一组以胃肠道嗜酸性炎症为特征的疾病。在过去的十年中，EGID的发病率急剧上升，尤其是嗜酸性食管炎(EoE)，但也有嗜酸性胃肠炎(EG)。EGID患者通常有许多食物过敏，随着以氨基酸为基础的元素饮食的建立，疾病进入缓解状态。综上所述，这表明EGID是一种由食物过敏原引起的嗜酸性炎症性肠病。 奥马珠单抗是一种人源化的治疗性抗IgE单抗。抗IgE治疗可降低循环中游离IgE的浓度，阻断IgE与Fc和CD23的结合，下调肥大细胞、嗜碱性粒细胞和树突状细胞表面Fc的表达。由于抗IgE治疗对抗原提呈细胞(APC)有多种作用，推测通过抑制APC功能，抗IgE治疗可能对T细胞具有免疫调节作用。我们假设了两种不同的机制，即抗IgE治疗可以抑制过敏原特异性Th2反应。首先，抗-IgE下调树突状细胞表面的Fc&RI，并阻断CD23介导的过敏原与APC的结合，从而抑制IgE促进APC捕获抗原，进而导致过敏原特异性T细胞活化减少。第二，抗IgE的IgE信号在体内抑制肥大细胞和嗜碱性粒细胞的激活，这可能会降低IL-4和/或TSLP的表达，而IL-4和/或TSLP的缺失可能会抑制Th2细胞的分化。 为了验证这一假设，我们在过敏性嗜酸性胃肠炎患者的临床试验中评估了奥马珠单抗对过敏原特异性T细胞反应的抗IgE免疫调节作用。用羧基荧光素琥珀酰亚胺酯(CFSE)染料稀释法和流式细胞术检测4种变应原特异性T细胞反应(最大增殖、增殖剂量反应EC50、前体频率和细胞因子表达)。在奥马珠单抗前基线(10.0%)和16周时相点(7.2%)之间，变应原特异性增殖(CFSE染料稀释度)没有显著差异(p=0.33)。抗IgE治疗与对变应原的增殖剂量反应有微小但显著的左移(与假设相反)，使得基线时的EC50是服用奥马珠单抗的受试者的1.5倍。奥马珠单抗前基线(4.0×10e-4)和16周时相点(6.5×10e-4，p=0.33)之间，变应原特异性T细胞前体频率无显著差异。IL-4：干扰素/干扰素比值(基线0.81，奥马珠单抗0.63，p=0.15)、IL-5：干扰素/干扰素比值(基线0.33，奥马利单抗0.36，p=0.42)和Th2细胞因子/肿瘤坏死因子/肿瘤坏死因子比值均无显著差异。与假设相反的是，16周的抗IgE治疗并没有降低任何过敏原特异性反应的指数。总之，使用T细胞功能的多个指标，这项研究未能证明抗IgE治疗广泛或有效地抑制过敏原特异性T细胞反应。因此，这些数据不支持免疫球蛋白促进的抗原提呈在体内增强变应原特异性T细胞反应的主要作用。 目前正在进行的工作是将这些发现扩展到嗜酸性胃肠道疾病患者的肠道驻留T细胞。将获得胃肠道活检，并将使用流式细胞术和免疫组织化学来确定是否存在与IL-5+Th2细胞类似的关联。额外的实验将确定在这些疾病状态下是否有食物过敏原特异性Th2细胞的其他特征，这些细胞可能特别导致过敏性和嗜酸性炎症性食物过敏。

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.