EFFECT OF CYCLOSPORIN A ON TCR ONTOGENY AND AUTOIMMUNE DISEASE

最近的观察表明，环孢菌素A（CsA）阻断TCR启动 信号转导，从而导致异常TCR库选择。 CsA治疗可导致两种不同的自身免疫性疾病， 实验模型 在这两种模型中，疾病的发展 需要存在来自CsA的自身反应性细胞 治疗胸腺和外周T细胞免疫调节异常。 初步数据表明，CsA抑制TCR 2的胸腺选择 （α TCR）库，但不是TCR 1的胸腺成熟 （γ δ TCR）亚系。 这些广泛的长期目标 研究旨在确定这些不同T细胞的生理功能， 亚系，并阐明外周免疫调节的性质， 与宽容有关的活动。 具体目标是测试三个 假设 第一个假设是CsA抑制了阳性和阴性 选择胸腺中的TCR 2库，但不影响胸腺 TCR 1细胞成熟。 这一假设将通过确定 利用一组识别单克隆抗体研究环孢素A对胸腺个体发育影响 不同的TCR和相关分子。 这些研究将涉及 小鼠和鸡T细胞发育的分析。 将侧重 在小鼠中选择表达特定Vbeta基因的细胞 和TCR 3亚群的成熟，一种新的类T细胞受体， 鸡 第二个假设是TCR 1库在外周血淋巴细胞中被选择。 组织. 这一假设将通过检查的发展进行检验 和表达特定V γ和T细胞的TCR 1细胞的组织归巢模式， Vdelta基因。 将使用两种实验方法：1）确定是否 CsA治疗导致特定组织归巢模式的抑制 这是利用某些V γ基因的TCR 1细胞的特征。（二） 胸腺内注射FITC，以鉴定近期胸腺迁移， 外周组织和确定这些组织中Vgamma使用的模式 细胞 第三个假设是在CsA诱导的自身免疫性疾病模型中， TCR 2细胞介导自身反应性，而TCR 1细胞调节疾病 活动 本研究采用两种不同的实验模型：1）新生儿CsA 加上胸腺切除术和2）用同系BM挽救的致死性辐照成人 移植加钙治疗。 将建立这些模型， 不同T细胞亚系的频率和组织学定位 将通过免疫组织化学染色和FACS分析进行表征。 在这两种模型中，“正常”T细胞的存在阻断了这种能力。 自身反应性T细胞会导致真正的组织损伤。 的表型 自身反应性效应细胞和“正常”调节细胞都将 可以使用磁激活细胞分选和过继细胞分选来确定。 传输协议。

Recent observations indicate that cyclosporin A (CsA) blocks TCR initiated signal transduction and thus leads to abnormal TCR repertoire selection. CsA treatment can lead to overt autoimmune disease in two different experimental models. In both these models, the development of disease requires both the presence of autoreactive cells derived from the CsA treated thymus and an immunoregulatory abnormality in peripheral T cells. Preliminary data suggest that CsA inhibits thymic selection of the TCR2 (alphabeta TCR) repertoire, but not thymic maturation of the TCR1 (gammadelta TCR) sublineage. The broad, long-term objectives of these studies are to determine the physiologic function of these different T cell sublineages and to elucidate the nature of the peripheral immunoregulatory activity involved in tolerance. The specific aims are to test three hypotheses. The first hypothesis is that CsA inhibits both positive and negative selection of the TCR2 repertoire in the thymus, but does not affect thymic TCR1 cell maturation. This hypothesis will be tested by determining the effect of CsA on thymic ontogeny utilizing a panel of mAb which recognize different TCRs and associated molecules. These studies will involve analysis of both murine and chicken T cell development. The focus will be on the selection of cells expressing particular Vbeta genes in the mouse and the maturation of the TCR3 subset, a novel alphabeta-like TCR, in the chicken. The second hypothesis is that the TCR1 repertoire is selected in peripheral tissue. This hypothesis will be tested by examination of the development and tissue homing pattern of TCR1 cells expressing particular Vgamma and Vdelta genes. Two experimental approaches will be used: 1) determining if CsA treatment results in inhibition of specific tissue homing patterns characteristic of TCR1 cells which utilize certain Vgamma genes. 2) intrathymic injection of FITC to identify recent thymic emigrants in peripheral tissues and the determining the pattern of Vgamma usage in these cells. The third hypothesis is that in models of CsA induced autoimmune disease, TCR2 cells mediate autoreactivity, while TCR1 cells regulate disease activity. Two different experimental models will studied: 1) neonatal CsA plus thymectomy and 2) lethally irradiated adults rescued with syngeneic BM transplant plus Ca treatment. These models will be established and the frequency and histologic localization of the different T cells sublineages will be characterized by immunohistochemical staining and FACS analysis. In both these models, the presence of "normal" T cells blocks the ability of autoreactive T cells induce actual tissue lesions. The phenotype of both the autoreactive effector cells and the "normal" regulatory cells will be determined using magnetic activated cell sorting and an adoptive transfer protocol.