Immune Development: Role of Milk Cell Transfer and Recipient Gender

描述(由申请人提供)： 我们研究了乳汁免疫细胞经上皮细胞向哺乳仔猪的转移，并检测了受体成年后的转移结果。我们的新方法是利用绿色荧光蛋白(GFP)转基因小鼠对非转基因小鼠进行寄养。通过流式细胞仪分析、免疫组织化学和RT-PCR观察1、2、3和4周时GFP免疫细胞在仔鼠体内的命运。尽管牛奶中有大量的GFP B细胞和巨噬细胞，但没有观察到这些细胞进行跨上皮迁移。在第1周和第2周观察到的穿过幼崽小肠的GFP细胞中，80%可以被阳性鉴定为CD8或CD4T细胞。这些T细胞归巢于脾和胸腺，在3-4周达到最大积聚，之后消失。因为T细胞被转移了，所以我们研究了用一种能引起T细胞介导的迟发型超敏反应(DTH)的抗原致敏DAM是否会导致成年后代致敏。这一点并不明显。取而代之的是，在成年雌性后代中，由致敏的母狗哺育增强了随后的DTH反应(与非致敏的母羊哺育的雌性相比)，而在雄性后代中则抑制了随后的DTH反应(与非致敏的母坝哺育的雄性相比)。换句话说，细胞转移对DTH反应有一个长期的影响，这种影响因接受者的性别而异。从后来的时间点分析，被转移的细胞似乎不太可能成为接受者免疫系统的长期组成部分，而是信息从母犬传递到幼崽。因此，我们假设被转移的母体细胞在新生儿T细胞产生过程中影响T细胞的发育、选择和扩增。该项目的长期目标是确定母体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)总体T细胞发育有关，b)与免疫性疾病的易感性和免疫效果有关，c)与安全增强幼儿免疫力的可能性有关。其具体目的是：1)进一步表征与成体DTH反应相关的免疫信息从母犬到幼犬的传递。在这个过程中，a)测试机制的工作模型，b)扩大研究，将结核菌素作为对世界健康更重要的疾病相关抗原，2)确定DTH响应性的调节是否是T细胞亚群平衡改变的结果，以及3)使用新的四核心基因小鼠模型，以确定性别效应是由性染色体互补引起的，还是由睾丸决定基因Sry引起的。为了达到这些特定的目的，提出了一种类似的生理护理方法，通过添加Rag1-/-、IL-10缺失、IL17受体A缺失或四个核心基因型鼠作为寄养接受者来增强机械性询问。也将使用同源CD45.1和CD45.2母犬和幼崽对。为了实现这些目标，一支由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(LO)以及性别差异的生物学基础(Arnold)方面的专业知识的高级研究人员组成的团队已经成立。团队的所有成员都是高生产率的，并在各自的学科中受到尊重。

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.