Immune Development: Role of Milk Cell Transfer and Recipient Gender

描述（由申请人提供）： 我们已经研究了乳免疫细胞的跨上皮转移到哺乳幼仔，并检查了一旦受体成年后的转移结果。我们的新方法是用绿色荧光蛋白（GFP）转基因母鼠寄养非转基因小鼠幼仔。通过FACS分析、免疫组织化学和RT-PCR，在1、2、3和4周时跟踪幼鼠中GFP+免疫细胞的命运。尽管乳汁中存在大量GFP+ B细胞和巨噬细胞，但未观察到这些细胞发生跨上皮迁移。在第1周和第2周观察到的穿过幼仔小肠的GFP+细胞中，有80%可以被阳性鉴定为CD 8+或CD 4 + T细胞。这些T细胞归巢到脾脏和胸腺，在3-4周时达到最大积累，此后消失。由于T细胞被转移，我们研究了是否致敏母鼠与抗原，elevening一个T细胞介导的迟发型超敏反应（DTH）的反应，将导致致敏的成年后代。这一点并不明显。相反，由一个致敏的母鼠喂养会放大成年雌性后代随后的DTH反应（与非致敏母鼠喂养的雌性相比），但抑制了雄性后代随后的DTH反应（与非致敏母鼠喂养的雄性相比）。换句话说，细胞移植对DTH反应的长期影响取决于接受者的性别。从后来的时间点的分析来看，转移的细胞似乎不太可能成为受体免疫系统的长寿成分，而是信息从母体转移到了幼崽身上。因此，我们假设转移的母体细胞影响新生儿T细胞产生过程中T细胞的发育、选择和扩增。 本项目的长期目标是确定母乳细胞转移对后代免疫反应的重要性，特别是这种转移可能与a）一般T细胞发育，B）对免疫疾病易感性和免疫效力的两性异形，以及c）安全增强幼儿免疫力的可能性有关。 具体目的是：1）进一步表征与成年DTH反应相关的免疫信息从母体到幼仔的传递。在a）测试机制的工作模型和B）扩展研究以包括结核菌素作为对世界健康具有更大意义的疾病相关抗原，2）确定DTH反应性的调节是否是T细胞亚群平衡变化的结果，和3）使用新的四核心基因型小鼠模型的过程中，以确定性别效应是由性染色体补体还是由睾丸决定基因Sry引起的。提出了一种类似的生理护理方法来实现这些特定的目标，通过添加Rag 1-/-，IL-10 null，IL 17受体A null或四个核心基因型小鼠作为牛奶的寄养受体来增强机械询问。还将使用同源CD 45.1和CD 45.2母鼠和幼鼠对。 为了实现这些目标，组建了一个高级研究人员团队，他们具有催乳素内分泌学和免疫反应内分泌调节（PI）、免疫病理学（Muller）、粘膜免疫学和中枢耐受性（Lo）以及性别差异的生物学基础（Arnold）方面的专业知识。团队的所有成员都是高生产力和尊重他们的学科成员。

DESCRIPTION (provided by applicant): We have investigated the trans-epithelial transfer of milk immune cells to nursing pups and examined the result of transfer once the recipients were adult. Our novel approach used foster-nursing of non-transgenic mouse pups by green fluorescent protein (GFP) transgenic dams. The fate of GFP+ immune cells in the pups was followed at 1, 2, 3, and 4 weeks by FACS analysis, immunohistochemistry, and RT-PCR. Despite high numbers of GFP+ B cells and macrophages in milk, these cells were not observed undergoing trans-epithelial migration. Eighty percent of the GFP+ cells observed crossing the small intestine of the pup at weeks 1 and 2 could be positively identified as CD8+ or CD4+ T cells. These T cells homed to the spleen and thymus, with maximal accumulation at 3-4 weeks, and disappearance thereafter. Because T cells were transferred, we examined whether sensitizing dams with an antigen that elicits a T cell-mediated delayed-type-hypersensitivity (DTH) response, would result in sensitization of adult offspring. This was not apparent. Instead, nursing by a sensitized dam amplified a subsequent DTH response in adult female offspring (compared to females nursed by a non-sensitized dam) and yet suppressed a subsequent DTH response in males (compared to males nursed by a non-sensitized dam). In other words, there was a long-lived effect of cell transfer on the DTH response that was different depending on the sex of the recipient. From analysis at later time points, it seems unlikely that the transferred cells became long-lived components of the recipient's immune system, but rather that information was transferred from dam to pup. We therefore hypothesize that transferred maternal cells affect T cell development, selection and expansion during neonatal T cell production. The long term objectives of this project are to determine the importance of maternal milk cell transfer to immune responses in offspring, especially as such transfer might relate a) to T cell development in general, b) to sexual dimorphism both in susceptibility to immune disease and the efficacy of immunization, and c) to the possibility of safely enhancing immunity in young children. The specific aims are 1) to further characterize the transfer of immune information from dam to pup relevant to the adult DTH response. In the process to a) test a working model of the mechanism and b) expand the study to include tuberculin as a disease-related antigen of greater significance to world health, 2) to determine whether the modulation of DTH responsivity is the result of a change in the balance of T cell subsets, and 3) using the novel four core genotype mouse model, to determine whether the sex effect is caused by sex chromosome complement or by the testis-determining gene, Sry. A similar physiological nursing approach is proposed to achieve these specific aims, with mechanistic interrogation enhanced by the addition of Rag1-/-, IL-10 null, IL17 receptor A null, or four core genotype mice as foster recipients of the milk. Congenic CD45.1 and CD45.2 dam and pup pairs will also be used. A team of senior investigators with expertise in prolactin endocrinology and endocrine regulation of immune responses (PI), immunopathology (Muller), mucosal immunology and central tolerance (Lo), and the biological basis of sex differences (Arnold) has been assembled to accomplish these aims. All members of the team are highly productive and respected members of their disciplines.