Developmental Immunotherapeutics of Allergic Diseases

嗜酸性胃肠道疾病(EGID)是以胃肠道嗜酸性炎症为特征的一系列疾病。在过去的十年中，EGID的发病率急剧增加，尤其是嗜酸性食管炎(EoE)。EGID包括嗜酸性胃肠炎(EG)和EoE，通常与食物和空气过敏原过敏有关。大多数EGID患者有许多食物过敏，在许多患者中，以氨基酸为基础的元素饮食是一种有效的治疗方法。这表明EGID的发病机制是由于食物过敏原引起的嗜酸性炎症。 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)的多种作用，推测抗-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，我们在上述临床试验中评估了奥马珠单抗对嗜酸性胃肠炎和食物过敏受试者过敏原特异性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，我们没有发现证据支持抗IgE治疗对变应原特异性Th2细胞反应有免疫调节或抑制作用的观点。因此，这些数据不支持IgE介导的Ag聚焦在体内增强变应原特异性T细胞反应中的主要作用。 我们使用了类似的技术来研究小鼠肺部炎症模型中T细胞细胞因子的产生，使用的是呼吸道病原体小鼠肺炎病毒(PVM)。PVM感染导致局部产生促炎趋化因子CCL3，并且在CCL3-/-小鼠中，响应PVM感染的中性粒细胞募集显著减少。在这项工作中，我们证明了在IFNGamma受体基因缺失的小鼠中，CCL3介导的中性粒细胞募集减少。同样，在没有PVM感染的情况下，CCL3的过度表达在没有IFNGamma的情况下不能单独诱导中性粒细胞募集。这些发现揭示了迄今为止未知的IFNGamma和CCL3之间的相互作用，这表明IFNGamma在CCL3介导的中性粒细胞体内募集中是至关重要的。

Eosinophilic gastrointestinal diseases (EGIDs) are a spectrum 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). EGIDs, including eosinophilic gastroenteritis (EG) and EoE, are commonly associated with food and aeroallergen hypersensitivity. Most EGID patients have numerous food allergies, and in many patients an amino acid based elemental diet is an effective treatment. This suggests that EGID pathogenesis is due to food allergen driven eosinophilic inflammation. 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 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 the above clinical trial of omalizumab in subjects with eosinophilic gastroenteritis and food allergy. 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, we found no evidence to support the concept that anti-IgE therapy has an immunomodulatory or inhibitory effect on allergen specific Th2 cell responses. As such, these data do not support a major role for IgE mediated Ag focusing in augmenting allergen specific T cell responses in vivo. We have used similar techniques to study T cell cytokine production in mouse models of lung inflammation, using the respiratory pathogen, pneumonia virus of mice (PVM). PVM infection results in local production of the proinflammatory chemokine, CCL3, and that neutrophil recruitment in response to PVM infection is reduced dramatically in CCL3 -/- mice. In this work, we demonstrate that CCL3-mediated neutrophil recruitment is diminished in IFNgamma receptor gene-deleted mice. Similarly, in the absence of PVM infection, CCL3 overexpression alone could not elicit neutrophil recruitment in the absence of IFNgamma. These findings reveal a heretofore unrecognized interaction between the IFNgamma and CCL3, which demonstrate that IFNgamma is crucial for CCL3-mediated neutrophil recruitment in vivo.