Biology and Therapy of Lymphopenia

我们之前已经证明，年龄、疾病和治疗相关的胸腺功能影响是淋巴细胞耗竭后T细胞免疫重建减少的根本原因。因此，可以通过增强胸腺功能或增强胸腺独立免疫重建途径的效率来克服这一限制。我们专注于重组人IL-7的临床开发，这是一种有效的免疫恢复剂，主要通过增强胸腺非依赖性免疫重建而起作用。2011财年，我们发表了rhIL7在人体内的首次临床试验的临床结果，证明了对早期B细胞的有效作用，并记录了这种细胞因子的T1/2延长。这些结果令人惊讶，因为之前的工作已经证明细胞因子在体内的寿命非常短（Sportes等人，clincan Res 2010）。结果还清楚地记录了rhIL7的有效免疫恢复作用，并证明其毒性很小，从而为在各种情况下使用该药物奠定了基础，旨在增强慢性病毒感染情况下的免疫重建、疫苗反应和抗病毒反应。基于这项研究的结果，全世界正在进行20多项研究，利用IL7作为免疫调节剂，这种药物有望最终被批准用于各种临床环境。2011年，我们还在全球首屈一指的免疫学评论杂志（Mackall et al, Nat Rev immunology）上发表了一篇高影响力的总结文章，详细介绍了白细胞介素-7在临床应用中的前景。该项目在2012财年的第一个主要成就是完成了可溶性il - 7受体在调节il - 7生物活性中的重要作用的研究。我们启动了一个研究IL7受体生物学的项目，因为该基因的多态性与多发性硬化症的不同易感性有关。尽管有明确的遗传证据表明，IL7R在调节疾病易感性方面发挥着作用，但从本质上讲，人们对这些发现的生物学原因一无所知。通过这些研究，我们发现易发生自身免疫的IL7R基因变异导致该分子剪接率升高，缺乏跨膜结构域的mRNA（例如外显子6）拷贝数增加。这导致预测，易导致自身免疫的多态性将与较高水平的可溶性IL7R相关。事实上，通过对健康和多发性硬化患者的研究，我们发现自身免疫易感性多态性导致可溶性IL7Ra循环水平升高。此外，当可溶性IL7Ra存在时，IL7诱导短期“高爆发”信号减少，并在较长时间（如天）内增加IL-7信号。这是由于在可溶性IL7Ra存在的情况下，IL7消耗减少。我们还观察到，在可溶性受体存在或不存在的情况下，rhIL7诱导的下游事件存在质的差异，从而诱导负调节因子水平降低。总之，这导致了一种假设，即IL7R的多态性增加了增强IL-7生物活性的自身免疫风险，部分原因是减少了IL-7的消耗和增加了其可获得性。通过使用健康供体和多发性硬化症患者的临床样本，我们观察到可溶性IL7R水平受到IL7Ra基因多态性的严格调节，而在多发性硬化症患者中，IL-7水平本身也受到IL7Ra基因多态性的调节。最后，我们在自身免疫性脑炎小鼠模型中观察到，共同给药sIL7Ra显著增加IL-7诱导疾病的效能，这为可溶性IL7Ra水平与多发性硬化症易感性的关联提供了明确的基础。这些研究为IL7R在体内调节IL7生物活性中的重要作用提供了基本的见解，阐明了一个明确的基础，解释了当可溶性IL7R水平升高时，遗传多态性如何增加对多发性硬化症的易感性，并预测IL7加sIL7Ra联合治疗将增强IL7作为治疗剂的有效性。最后，这些数据表明，在正常情况下，人类的可溶性IL7Ra摩尔量远远超过IL7本身。这与发现的IL2和IL15的可溶性受体之间的关系有着根本的不同，并且可能解释了当IL7用作治疗剂时半衰期延长的原因。该项目在2012财年的第二个主要成就是将rhIL7作为一线治疗后免疫恢复的临床试验的一部分，应用于一大批癌症儿童。这是迄今为止儿童中唯一的rhIL7临床经验。我们证明了该药物在提高癌症强化化疗后免疫重建的速度和程度方面是安全有效的。此外，正如项目III中所讨论的，令人兴奋的临床结果表明，这种方法可能提高高危人群的生存率。

We previously demonstrated that age-, disease- and therapy-associated effects on thymic function are fundamental causes of diminished T cell immune reconstitution following lymphocyte depletion. Thus, one can overcome this limitation by either enhancing thymic function or enhancing the efficiency of thymic-independent pathways of immune reconstitution. We have focused our efforts on clinical development of recombinant human IL-7, which is a potent immunorestorative that works predominantly by enhancing thymic-independent immune reconstitution.In FY11 we published the clinical results of our first clinical trial of rhIL7 in humans, which demonstrated potent effects on early B cells and documented prolonged T1/2 of this cytokine agent. These results were surprising, as previous work had demonstrated cytokines to be very short lived in vivo (Sportes et al, Clin Can Res 2010). The results also clearly documented the potent immunorestorative effects of rhIL7 and demonstrated minimal toxicity, thus setting the stage for use of this agent in a variety of settings aimed at enhancing immune reconstitution, vaccine responses and antiviral responses in the setting of chronic viral infection. Based upon the results of this study, over 20 studies worldwide are underway utilizing IL7 as an immunomodulator and this agent holds great promise for eventual approval for use in a variety of clinical settings. In FY11 we also published a high impact summary article detailing the promising of interleukin-7 for clinical use in the premier immunology review journal worldwide (Mackall et al, Nat Rev Immunology).The first major accomplishment of this project during FY12 was completion of studies demonstrating an important role for soluble IL7 receptor in modulating bioactivity of IL7. We initiated a project studying the biology of IL7 receptor because polymorphisms in this gene have been linked to differential susceptibility to multiple sclerosis. Despite this clear genetic evidence of a role for IL7R in modulating susceptibility to disease, there was essentially no understanding of the biology responsible for these findings. Through these studies, we found that the genetic variation in IL7R that predisposes to autoimmunity results in a higher splicing rate of this molecule, with increased copies of the mRNA that is lacking the transmembrane domain (e.g. exon 6). This leads to the prediction that the polymorphisms predisposing to autoimmunity would be associated with higher levels of soluble IL7R. Indeed, using cohorts of healthy and MS-afflicted patients, we found that autoimmunity predisposing polymorphisms leads to higher circulating levels of soluble IL7Ra. Furthermore, when soluble IL7Ra is present, IL7 induces diminished short-term "high burst" signaling, and increased IL-7 signaling over the longer term (e.g. days). This is due to diminished IL7 consumption in the presence of soluble IL7Ra. We also observed qualitative differences in the downstream events induced by rhIL7 in the presence or absence of soluble receptor, such that diminished levels of negative regulators were induced. Together, this led to the hypothesis that polymorphisms in IL7R increase the risk of autoimmunity of enhancing the bioactivity of IL-7, in part by diminishing consumption and increasing availability. Using clinical samples from healthy donors and from patient with multiple sclerosis, we observed that soluble IL7R levels are tightly regulated by genetic polymorphisms in IL7Ra and that in patients with multiple sclerosis, IL-7 levels themselves are also modulated by polymorphisms in IL7Ra. Finally, we observed in a mouse model of autoimmune encephalitis, that co-administration of sIL7Ra significantly increases the potency of IL-7 induced disease, which provided a clear basis for implicating soluble IL7Ra levels in susceptibility to multiple sclerosis. These studies provide fundamental insights into the important role that IL7R plays in modulating IL7 bioactivity in vivo, elucidate a clear basis to explain how genetic polymorphisms increase susceptibility to multiple sclerosis when increased levels of soluble IL7R are present and lead to the prediction that co-treatment with IL7 plus sIL7Ra will enhance the potency of IL7 as a therapeutic agent. Finally, these data reveal that under normal circumstances, humans have a profound molar excess of soluble IL7Ra to IL7 itself. This is fundamentally different that the relationship between soluble receptor found for IL2 and IL15 and likely provided the explanation for the prolonged half-life of IL7 when used as a therapeutic agent.The second major accomplishment of this project in FY12 was administration of rhIL7 to a large cohort of children with cancer as part of a clinical trial of immune restoration after primary front line therapy. This represents the only clinical experience with rhIL7 in children thus far. We demonstrated the agent to be safe and highly effective at increasing the pace and degree of immune reconstitution following intensive chemotherapy for cancer. Furthermore, as discussed in Project III, exciting clinical results suggest that this approach may improve survival in this high-risk population.