与新生猪封装胰岛异种移植排斥相关的免疫机制。

Immune mechanisms associated with the rejection of encapsulated neonatal porcine islet xenografts.

作者信息

Kobayashi Tsunehiro, Harb George, Rajotte Ray V, Korbutt Gregory S, Mallett Aaron G, Arefanian Hossein, Mok Dereck, Rayat Gina R

机构信息

Department of Surgery, Dentistry Pharmacy Centre, Surgical-Medical Research Institute, University of Alberta, Edmonton, AB, Canada.

出版信息

Xenotransplantation. 2006 Nov;13(6):547-59. doi: 10.1111/j.1399-3089.2006.00349.x.

DOI:10.1111/j.1399-3089.2006.00349.x

PMID:17059582

Abstract

BACKGROUND

The immune mechanisms associated with the rejection of microencapsulated neonatal porcine islets (NPI) are not clearly understood. Therefore, in this study we characterized the immune cells and molecules that are involved in this process by examining the microencapsulated NPI xenografts at various time points post-transplantation in B6 mice.

METHODS

Microencapsulated NPI were transplanted into streptozotocin-induced diabetic immune-competent B6 and immune-deficient B6 rag-/- mice and blood glucose levels were monitored twice a week. Encapsulated NPI were then recovered from B6 mice at various time points post-transplantation to characterize the islets and immune response using immunohistochemical and RT-PCR analyses. To determine which T-cell subpopulation is important for the rejection of encapsulated NPI, B6 rag-/- mice with established microencapsulated NPI xenografts were reconstituted with either CD4(+) or CD8(+) T cells and a return to the diabetic state was noted. For controls, adoptive transfer experiments involved reconstitution of B6 rag-/- mice with established microencapsulated NPI with non-fractionated lymph node cells or non-reconstituted mice.

RESULTS

All B6 recipients of microencapsulated NPI remained diabetic throughout the study while B6 rag-/- recipients achieved normoglycemia and maintained normoglycemia for up to 100 days post-transplantation. Encapsulated NPI recovered from B6 mice at early time points (day 7 and day 14) post-transplantation were surrounded with very few layers of immune cells that increased with time post-transplantation. The extent of cellular overgrowth on the surface of encapsulated NPI has a significant correlation with islet cell death and the presence of CD4(+) T cells, B cells and macrophages. Mouse IgG antibody and complement as well as cytokines [gamma-interferon (IFN-gamma), interleukin10 (IL10)] and chemokines (monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha and beta) were detected within the microcapsules at several time points post-transplantation suggesting that these molecules can traverse the microcapsule. Mouse anti-porcine IgG antibodies in recipient sera were found to peak at 30 days post-transplantation indicating leakage of porcine xenoantigens. In contrast, microencapsulated NPI recovered from B6 rag-/- mice had no cellular overgrowth on the surface. Complement and cytokines (IL 10 but not IFN-gamma) including chemokines were detected within the microcapsules at several days post-transplantation. We also found that B6 rag-/- mice reconstituted with non-fractionated lymph node cells or CD4(+) T cells but not CD8(+) T cells became diabetic demonstrating that CD4(+) T cells are the necessary T-cell subtype for microencapsulated NPI rejection. In contrast, non-reconstituted B6 rag-/- mice remained normoglycemic for the entire duration of the study.

CONCLUSIONS

Our results demonstrate that CD4(+) T cells, B cells and macrophages are the immune cells recruited to and involved in the rejection of encapsulated NPI. Immune molecules secreted by these cells as well as complement can traverse the microcapsule membrane and are responsible for destroying the NPI cells. Treatment regimens which target these molecules may modify the rejection of encapsulated NPI and lead to prolonged islet xenograft survival.

摘要

背景

与微囊化新生猪胰岛（NPI）排斥相关的免疫机制尚不清楚。因此，在本研究中，我们通过在移植后的不同时间点检查B6小鼠体内的微囊化NPI异种移植物，对参与该过程的免疫细胞和分子进行了表征。

方法

将微囊化NPI移植到链脲佐菌素诱导的糖尿病免疫健全的B6和免疫缺陷的B6 rag - / - 小鼠中，每周监测两次血糖水平。然后在移植后的不同时间点从B6小鼠中回收包封的NPI，使用免疫组织化学和RT-PCR分析来表征胰岛和免疫反应。为了确定哪种T细胞亚群对包封的NPI排斥很重要，将具有已建立的微囊化NPI异种移植物的B6 rag - / - 小鼠用CD4(+)或CD8(+) T细胞进行重建，并观察其是否恢复到糖尿病状态。作为对照，过继转移实验包括用未分级的淋巴结细胞重建具有已建立的微囊化NPI的B6 rag - / - 小鼠或未重建的小鼠。

结果

在整个研究过程中，所有接受微囊化NPI的B6受体小鼠均保持糖尿病状态，而B6 rag - / - 受体小鼠实现了血糖正常，并在移植后长达100天维持血糖正常。移植后早期（第7天和第14天）从B6小鼠中回收的包封NPI周围仅有很少几层免疫细胞，随着移植后时间的推移而增加。包封NPI表面的细胞过度生长程度与胰岛细胞死亡以及CD4(+) T细胞、B细胞和巨噬细胞的存在显著相关。在移植后的几个时间点，在微囊内检测到小鼠IgG抗体、补体以及细胞因子[γ-干扰素（IFN-γ）、白细胞介素10（IL10）]和趋化因子（单核细胞趋化蛋白-1和巨噬细胞炎性蛋白-1α和β），表明这些分子可以穿过微囊。发现受体血清中的小鼠抗猪IgG抗体在移植后30天达到峰值，表明猪异种抗原发生渗漏。相比之下，从B6 rag - / - 小鼠中回收的微囊化NPI表面没有细胞过度生长。在移植后的几天内，在微囊内检测到补体和细胞因子（IL 10而非IFN-γ）包括趋化因子。我们还发现，用未分级的淋巴结细胞或CD4(+) T细胞而非CD8(+) T细胞重建的B6 rag - / - 小鼠变成了糖尿病，这表明CD4(+) T细胞是微囊化NPI排斥所必需的T细胞亚型。相比之下，未重建的B6 rag - / - 小鼠在整个研究期间保持血糖正常。