TRANSPLANT IMMUNOLOGY OF IL2 IFNY DOUBLE KNOCKOUT MICE

约10，000例肾脏和1，500例胰腺移植（同种异体移植） 每年在美国举行。尽管积极免疫 由于免疫抑制，每年有数百个这样的器官因免疫抑制而丢失。 介导的排斥因为等待的个体数量 移植大大超过捐赠器官的供应，移植 排斥反应直接导致这些器官的短缺。移植肠 细胞因子白细胞介素2和干扰素γ（IFN-γ）的表达 已被发现是临床和实验性同种异体移植物的标志物 排斥反应尽管大多数临床免疫抑制方案， 移植患者可以降低IL-2和IFN-γ的水平， 介导的排斥仍然发生。目前，还没有实验模型 其中，当IL-2和IFN-γ都 完全不存在。这项提案的目标是创造一种转基因的 具有IL-2和IFN-γ的靶向基因破坏的小鼠模型 基因，并研究对这些移植问题的免疫反应， 小鼠 我们成功地培育了IL-2 IFN-γ双敲除小鼠（DKO）。的 小鼠通常是健康的，具有正常的窝，并且存活长达6 在无菌室中数月。我们的初步数据表明DKO小鼠 在IL-2中发现的自身免疫性结肠炎的形式要温和得多。 单基因敲除小鼠DKO小鼠可以安装一个强大的延迟型 超敏反应对外来蛋白抗原的反应。最后，DKO 小鼠在B细胞成熟方面存在严重缺陷。我们的具体目标 该建议是（1）表征DKO小鼠的能力， 细胞和体液免疫应答，以及（2）确定 在缺乏IL-2的情况下对胰腺同种异体移植物的免疫应答， IFN-γ（即排斥或耐受）。首先，我们将研究 DKO小鼠产生迟发型超敏反应， 组织相容性抗原（MHC），免疫后产生抗体， 并产生细胞毒性T淋巴细胞。其次，我们将使DKO小鼠 糖尿病患者使用链脲佐菌素，然后进行胰岛细胞 来自MHC不同供体的移植。同种异体移植物存活率 我们假设DKO小鼠会排斥胰岛同种异体移植物， 排斥反应的机制可能与IL-4或其他T细胞有关 生长因子和细胞毒性T细胞。细胞因子环境和细胞 将通过定量聚合酶链鉴定所涉及的表型 反应和荧光激活细胞分选。如果DKO 小鼠对同种异体胰岛移植物耐受，我们将通过以下方法确定其机制： 过继转移耐受诱导T淋巴细胞亚群。

Approximately, 10,000 renal and 1,500 pancreas transplants (allografts) are performed annually in the United States. Despite aggressive immune suppression, hundreds of these organs are lost each year to immune mediated rejection. Because the number of individuals waiting for a transplant greatly exceeds the supply of donated organs, transplant rejection directly contributes to the shortage of these organs. Intragraft expression of the cytokines interleukin 2 and interferon gamma (IFN-gamma) has been found to be markers for clinical and experimental allograft rejection. Although most clinical protocols for immune suppression in transplant patients can reduce the levels of IL-2 and IFN-gamma, immune mediated rejection still occurs. Currently, no experimental models exist in which allograft rejection can be studied when both IL-2 and IFN-gamma are completely absent. The goal of this proposal is to create a transgenic mouse model with targeted gene disruption of both the IL-2 and IFN-gamma genes, and to study the immune responses to transplanted issue in these mice. We have successfully bred IL-2 IFN-gamma double knockout mice (DKO). The mice are generally healthy, have normal litters, and survive as long as 6 months in sterile housing. Our preliminary data indicate that the DKO mice develop a much milder form of the autoimmune colitis seen in the IL-2 single knockout mice. DKO mice can mount a strong delayed-type hypersensitivity. response to a foreign protein antigen. Finally, the DKO mice have a profound defect in B cell maturation. Our specific goals in this proposal are to (1) characterize the ability of the DKO mice to mount cellular and humoral immune responses and (2) determine the outcome of the immune response to pancreatic allografts in the absence of IL-2 and IFN-gamma (i.e. rejection or tolerance). First, we will study the ability of DKO mice to generate delayed -type hypersensitivity responses to histocompatibility antigens (MHC), generate antibodies after immunization, and generate cytotoxic T-lymphocyte. Second, we will render DKO mice diabetic using streptozotocin, and then perform pancreatic islet cell transplantation from MHC disparate donors. Allograft survival will be monitored we hypothesize that the DKO mice will reject islet allografts, and that the mechanisms of rejection will involved IL-4 or other T cell growth factors and cytotoxic T cells. The cytokine milieu and cell phenotypes involved will be identified by quantitative polymerase chain reaction and fluorescence activated cell sorting. In contrast, if the DKO mice are tolerant to islet allografts, we will determine the mechanism by adoptive transfer of tolerance inducing T lymphocyte subsets.