Donor CD8+ cell facilitates mixed chimerism in NOD mice

描述(申请人提供)：慢性免疫抑制的需要是人类胰岛移植作为治疗或可能治愈1型糖尿病的一个主要限制。因此，诱导胰岛移植免疫耐受仍然是一个重要的目标。目前，通过异基因骨髓移植诱导混合嵌合体是在NOD小鼠等实验动物中诱导免疫耐受的一种成功方案，NOD小鼠是人类1型糖尿病的最佳模型。然而，这一过程需要对受者进行全身照射(TBI)。TBI条件作用的毒性和移植物抗宿主病(GVHD)的可能性不能证明将其用于1型糖尿病治疗的合理性，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，在没有照射的情况下，联合注射大剂量供体骨髓细胞和供体CD8+T细胞可以诱导经抗CD3单抗预适应的糖尿病前期NOD小鼠的混合嵌合体。这允许供体特异性耐受性和在不诱发移植物抗宿主病的情况下逆转胰岛素炎。拟议的研究将探索在这一新方案中预防GVHD的机制。在我们的初步研究中，抗CD3条件受者在供者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条件的受者中，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者T细胞限制在宿主的淋巴造血系统。因此，供者CD8+T细胞促进供者干细胞在造血组织中的植入，而不会在上皮组织中引起移植物抗宿主病。我们进一步假设，在抗CD3条件的受者中，供者T细胞在宿主淋巴造血组织中的滞留是由于供者CD8+T细胞上缺乏趋化因子受体CCR5、CCR9、CCR10和CXCR3的上调所致；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抗体处理的NOD受者与单纯脑损伤患者相比，供者CD8+T细胞在淋巴组织、造血细胞和上皮组织中的迁移和扩增情况，并确定抗CD3条件受者中供者CD8+T细胞是否发生无能和/或凋亡；2)检测供者CD8+T细胞趋化因子受体(CCR5、CCR9、CCR10和CXCR3)的表达模式是否决定了供者CD8+T细胞在抗CD3或TBI条件受者中的迁移模式；3)检测宿主自然杀伤T(NKT)细胞产生的IL-4和IL-10是否在决定供者CD8+T的转运模式和预防抗CD3条件下的移植物抗宿主病中起关键作用。这些研究将为GVHD的预防机制提供新的见解，并促进开发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。

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.