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求是人类胰岛移植作为治疗或可能“治愈”1型糖尿病的主要限制。因此，诱导对胰岛移植的免疫耐受仍然是一个重要的目标。通过同种异体骨髓移植诱导混合嵌合目前是一种成功的方案，用于诱导实验动物（如NOD小鼠）的免疫耐受，NOD小鼠是人类1型糖尿病的最佳模型。然而，该程序需要对受者进行全身照射（TBI）调理。tbi调节的毒性和移植物抗宿主病（GVHD）的潜力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。我们最近的研究表明，在没有照射的情况下，注射高剂量的供体骨髓细胞与供体CD8+ T细胞结合，可在抗cd3单抗预处理的糖尿病前期NOD小鼠中诱导混合嵌合。这允许供体特异性耐受性和逆转胰岛素炎而不诱导GVHD。提出的研究将探索这种新方案预防GVHD的机制。在我们的初步研究中，抗cd3条件受体在供体CD8+ 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，我们假设，在抗cd3条件受体中，Th1细胞因子的低水平产生和Th2细胞因子的高水平产生将供体T细胞限制在宿主淋巴造血系统。因此，供体CD8+ T细胞促进供体干细胞植入造血组织，而不会引起上皮组织的GVHD。我们进一步假设，在抗cd3条件受体中，供体T细胞在宿主淋巴造血组织中的滞留是由于趋化因子受体（包括CCR5、CCR9、CCR10和CXCR3）在供体CD8+ T细胞上缺乏上调；趋化因子受体的表达受Th1细胞因子的相互调节。这些研究将1)利用生物发光成像技术，观察抗cd3治疗NOD受者与TBI受者在淋巴造血和上皮组织中的供体CD8+ T细胞迁移和扩增情况；并确定抗cd3条件受体的供体CD8+ T细胞是否发生失能和/或凋亡；2)检测供体CD8+ T细胞趋化因子受体（即CCR5、CCR9、CCR10和CXCR3）的表达模式是否决定了抗cd3或TBI受体中供体CD8+ T细胞的转运模式；3)研究宿主自然杀伤T细胞（NKT）产生的IL-4和IL-10是否在决定供体CD8+ T的运输模式和抗cd3受体的GVHD预防中起关键作用。这些研究将为GVHD的预防机制提供新的见解，并促进适用于人类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.