Donor CD8+ cell facilitates mixed chimerism in NOD mice

描述（由申请人提供）：对慢性免疫抑制的需要代表了人类胰岛移植作为1型糖尿病治疗或可能的“治愈”的主要限制。因此，诱导对胰岛移植的免疫耐受仍然是一个重要的目标。通过异基因骨髓移植诱导混合嵌合体目前代表了在实验室动物如NOD小鼠中诱导免疫耐受的成功方案，NOD小鼠是人类1型糖尿病的最佳模型。然而，该过程需要接受者的全身照射（TBI）调节。TBI预处理的毒性和移植物抗宿主病（GVHD）的可能性并不能证明其用于治疗1型糖尿病是合理的，因此强调了开发成功的无辐射免疫耐受策略的必要性。我们最近发现，在没有辐射的情况下，注射高剂量的供体骨髓细胞与供体CD 8 + T细胞的组合诱导糖尿病前期NOD小鼠与抗CD 3单克隆抗体预处理的混合嵌合体。这允许供体特异性耐受和胰岛炎的逆转而不诱导GVHD。拟开展的研究将探讨这种新方案预防GVHD的机制。在我们的初步研究中，抗CD 3条件受体在供体CD 8 + T细胞注射后的早期表现出低水平的TNF-α产生和高水平的IL-4和IL-10产生。因此，我们推测，低水平的生产Th 1细胞因子和高水平的生产Th 2细胞因子在抗CD 3条件下的受者限制供体T细胞的宿主淋巴造血系统。因此，供体CD 8 + T细胞促进供体干细胞在造血组织中的植入，而不会引起上皮组织中的GVHD。我们进一步假设，在抗CD 3条件的受者中，供体T细胞在宿主淋巴造血组织中的保留是由于供体CD 8 + T细胞上的趋化因子受体（包括CCR 5、CCR 9、CCR 10和CXCR 3）缺乏上调;并且趋化因子受体的表达受Th 1细胞因子的调节。该研究将1）使用生物发光成像，与用TBI调节的受体相比，在用抗-CD 3调节的NOD受体的淋巴造血和上皮组织中显现供体CD 8 + T细胞迁移和扩增;并确定抗-CD 3调节的受体中的供体CD 8 + T细胞是否变得无反应性和/或凋亡; 2）检测趋化因子受体的表达模式是否供体CD 8 + T细胞的CCR 5、CCR 9、CCR 10和CXCR 3决定了供体CD 8 + T细胞在用抗CD 3或TBI调节的受体中的运输模式; 3）检测宿主自然杀伤T（NKT）细胞产生的IL-4和IL-10是否在决定供体CD 8 + T细胞的运输模式和在用抗CD 3调节的受体中预防GVHD中起关键作用。这些研究将为GVHD的预防机制提供新的见解，并促进适用于胰岛细胞移植和其他器官移植的无辐射耐受诱导方案的发展。

DESCRIPTION (provided by applicant): The need for chronic immunosuppression represents a major limitation for islet transplantation in humans as a treatment or possible "cure" for type-1 diabetes. Therefore, inducing immune tolerance to islet transplants remains an important goal. Induction of mixed chimerism via allogeneic bone marrow transplantation currently represents a successful regimen for induction of immune tolerance in laboratory animals such as NOD mice, the best model for human type-1 diabetes. However, this procedure requires whole body irradiation (TBI) conditioning of the recipients. The toxicity of TBI-conditioning and the potential for graft versus host disease (GVHD) does not justify its use in the treatment of type-1 diabetes, and thus underscores the need for the development of successful radiation-free immune tolerance strategies. We have recently shown that, in the absence of irradiation, injection of high doses of donor bone marrow cells in combination with donor CD8+ T cells induces a mixed chimerism in prediabetic NOD mice preconditioned with anti-CD3 mAb. This allows for donor-specific tolerance and reversal of insulitis without induction of GVHD. The proposed studies will explore the mechanisms of GVHD prevention in this novel regimen. In our preliminary studies, anti-CD3-conditioned recipients showed low-level production of TNF-alpha and high-level production of IL-4 and IL-10 in the early period following donor CD8+ T cell injection. Therefore, we hypothesize that low-level production of Th1 cytokines and high-level production of Th2 cytokines in anti-CD3-conditioned recipients confines donor T cells to the host lymphohematopoietic system. Thus, donor CD8+ T cells facilitate donor stem cell engraftment in hematopoietic tissues without causing GVHD in epithelial tissues. We further hypothesize that the retention of donor T cells in host lymphohematopoietic tissues in anti-CD3-conditioned recipients is due to the lack of up-regulation of chemokine receptors including CCR5, CCR9, CCR10 and CXCR3 on donor CD8+ T cells; and the expression of chemokine receptors is reciprocally regulated by Th1 cytokines. The studies will 1) visualize donor CD8+ T cell migration and expansion in the lymphohematopoietic and epithelial tissues of NOD recipients conditioned with anti-CD3 as compared to recipients conditioned with TBI, using bioluminescence imaging; and determine whether or not donor CD8+ T cells in anti-CD3 conditioned recipients become anergic and/or apoptotic; 2) examine whether the expression pattern of chemokine receptors (i.e. CCR5, CCR9, CCR10 and CXCR3) of donor CD8+ T cells determine the trafficking pattern of donor CD8+ T cells in recipients conditioned with anti-CD3 or TBI; 3) examine whether or not production of IL-4 and IL-10 by host natural killer T (NKT) cells play a critical role in determining the trafficking pattern of donor CD8+ T and preventing GVHD in recipients conditioned with anti-CD3. These studies will provide new insights into mechanisms of GVHD prevention as well as promote the development of a radiation-free tolerance induction regimen applicable for islet cell transplantation and other organ transplantation in humans.