PHENOTYPIC ANALYSES OF RECENT THYMIC EMIGRANTS IN MICE

描述（由申请人提供）：胸腺是T细胞成熟的主要部位，因此，在动物的整个生命周期中，胸腺在建立和维持外周T细胞库中起着重要作用。在没有事先实验操作的情况下测量胸腺输出量是确定胸腺输出细胞数量和质量的年龄依赖性的关键。在RAG2启动子的控制下，利用转基因小鼠的绿色荧光蛋白（GFP），设计了一种新的标记新近胸腺移植物（rte）的系统。虽然这些小鼠的胸腺细胞在预期的发育阶段开始表达GFP，但在RAG2表达消失后，GFP仍在这些细胞中存在。由此产生的GFP（hi）外周T细胞群是rte，因为它们在胸腺切除术后一周内消失。对来自RAG2pGFP转基因小鼠的GFP（hi）外周T细胞的初步分析表明，rte在到达淋巴样外周后的几周内经历了表型和功能成熟。与成熟的外周血T细胞相比，新外周血T细胞的CD4:CD8比值更高，CD24表达更高，Qa-2表达更低。CD8+ rte含有大约一半的预期细胞溶解前体，并且没有外源性IL-2， CD4+ rte在T细胞受体交联时增殖不良。当前提案的一个目标是完成来自未操作的年轻成年小鼠的rte的功能和表型特征，并将这些分析扩展到年轻和老年个体。小鼠rte的年龄依赖性定量及其功能和表面抗原表型的表征将有助于确定从渐进式器官迁移的程度。第二个目的是研究当RTE细胞群被纳入年轻成年小鼠的成熟外周T细胞池时，CD4:CD8比值下降的途径。无论这种调整是由CD4+ rte的丢失，CD8+ rte的优先增殖，还是这些因素的某种组合驱动的，我们将分析这种调整的年龄依赖性。最终目的是分析不同年龄小鼠RTE的功能异质性，以确定RTE室的组成并测量抗原受体库。这项工作的总体目标是了解胸腺输出和胸腺后成熟如何随年龄变化，着眼于调节免疫衰老和淋巴消融治疗和疾病的恢复。

DESCRIPTION (provided by applicant): The thymus is the primary site of T cell maturation, and as such, it plays a major role in the establishment and maintenance of the peripheral T cell pool throughout the lifespan of the animal. Measuring thymic output without prior experimental manipulation is key to determining the age-dependence of the quantity and quality of cells exported by the thymus. A novel system has been devised for marking recent thymic emigrants (RTEs), using mice transgenic for green fluorescent protein (GFP) under the control of the RAG2 promoter. While thymocytes from these mice begin expressing GFP at the expected developmental stage, GFP lingers in these cells after RAG2 expression is extinguished. The resulting population of GFP(hi) peripheral T cells are RTEs because they disappear within one week of thymectomy. Preliminary analyses of GFP(hi) peripheral T cells from RAG2pGFP transgenic mice indicate that RTEs undergo a phenotypic and functional maturation in the weeks after they reach the lymphoid periphery. Within this population of new peripheral immigrants, the CD4:CD8 ratio is higher, CD24 expression is higher, and Qa-2 expression is lower than found on more mature peripheral T cells. CD8+ RTEs contain approximately half the expected cytolytic precursors, and without exogenous IL-2, CD4+ RTEs proliferate poorly upon T cell receptor crosslinking. One aim of the current proposal is to complete this functional and phenotypic characterization of RTEs from unmanipulated young adult mice and to extend these analyses to younger and older individuals. Age-dependent quantitation of murine RTEs and characterization of their function and surface antigen phenotype will help determine the extent of emigration from the involuting organ. A second aim is to investigate the means by which the CD4:CD8 ratio declines as the RTE population becomes incorporated into the pool of mature peripheral T cells in the young adult mouse. Whether this adjustment is driven by the loss of CD4+ RTEs, preferential proliferation of CD8+ RTEs, or some combination of these factors, the age-dependence of this adjustment will be analyzed. The final aim is to analyze the functional heterogeneity of RTEs in mice of various ages to determine the composition of the RTE compartment and to measure the antigen receptor repertoire. The overall goal of this work is to understand how thymic output and post thymic maturation vary with age, with an eye toward modulating immune senescence and recovery from lymphoablative therapies and diseases.