Assessment of immunogenicity and antigenicity of different human cell types in natural and 3D-printed allografts

7.项目总结/摘要 同种异体移植排斥反应仍然是影响同种异体移植长期成功的一个问题， 影响组织工程移植物的功能和寿命， 缺乏可用的器官。在同种异体识别的直接途径中，排斥反应通常由激活启动 与非自身MHC/肽复合物交叉反应的静息宿主T效应记忆细胞， 需要移植细胞提供额外的信号，如共刺激因子和细胞因子。移植细胞能够 这样做是“免疫原性的”。这样的T细胞然后分化成能够杀死细胞的效应细胞 表达相同的非自身MHC/肽复合物。表达相同同种异体抗原的移植细胞类型 并且可以被效应T细胞识别和杀死，但不能启动静息T细胞的分化。 效应记忆进入效应T细胞被认为是“抗原性的”而不是免疫原性的。两个免疫原性 与引发排斥有关的人类细胞类型是移植物树突细胞（DC）和移植物内皮细胞 （EC）。虽然DC（和其他乘客白细胞）可以从天然移植物中清除或从移植物中排除，但它们可以被移植物中清除。 在生物工程移植物中，EC是维持移植物活力的血管衬里所必需的。我们有 提出了通过限制移植物的免疫原性以补充当前的免疫原性来减少排斥反应的策略。 抑制宿主免疫反应的做法。目前还不清楚去除DC和改变EC是否是非- 免疫原性将足以保护移植物免受排斥。在这里，我们建议回答这个问题。 解决这个问题需要用人类EC进行，因为通常使用的啮齿动物EC 不显示出与它们的人类对应物相同的免疫原性能力。为此，我们将 5项新技术：a）。对EC进行基因工程改造以使其具有非免疫原性和非抗原性; B）. 纳米药物以调节EC的免疫原性能力; c）. 3D打印人体皮肤， 多种不同的细胞类型作为排斥的靶标; d）.一种最先进的人类免疫系统小鼠， 移植受体;和e）.尖端的高维系列免疫荧光，以确定我们的影响， 对拒绝过程的干预。我们将首先测试我们的模型小鼠模型，并优化我们的检测方法。 使用自然的人类皮肤（目标1），然后继续测试我们的3D皮肤结构（目标2），我们将 修改细胞类型以确定除EC和DC以外的人类细胞的作用（如果有的话）。我们承认 合并以前没有组合的多种技术的提议存在一些风险 因此，我们选择使用R21探索机制来支持这项研究。但 该项目的成功实施不仅将为人类皮肤的一个重要问题提供初步答案， 细胞，但也将为进一步研究更复杂的人体组织的同种异体移植创造一个平台 并为替代器官的生物工程提供有价值的见解。

7. PROJECT SUMMARY/ABSTRACT Allograft rejection remains an issue in long term success of allotransplantation and is also likely to negatively impact the function and longevity of tissue engineered grafts being explored as a possible solution to the shortage of available organs. In the direct pathway of allorecognition, rejection is typically initiated by activation of resting host T effector memory cells that cross-react with non-self MHC/peptide complexes, a process that requires graft cells to provide additional signals such as co-stimulators and cytokines. Graft cells capable of doing this are “immunogenic”. Such T cells then differentiate into effector cells that are capable of killing cells that express the same non-self MHC/peptide complexes. Graft cell types that express the same alloantigens and can be recognized and killed by effector T cells but are unable to initiate the differentiation of resting effector memory into effector T cells are said to be “antigenic” rather than immunogenic. Two immunogenic human cell types implicated in initiating rejection are graft dendritic cells (DCs) and graft endothelial cells (ECs). While DCs (and other passenger leukocytes) can be purged from a natural graft or left out of a bioengineered graft, ECs are essential for lining the blood vessels that sustain graft viability. We have proposed strategies to reduce rejection by limiting the immunogenicity of the graft to complement the current practice of suppressing the host immune response. It is unclear if removing DCs and altering ECs to be non- immunogenic will be sufficient to protect a graft from rejection. Here we propose to answer this question. Addressing this question requires that it be performed with human ECs because commonly used rodent ECs do not exhibit the same immunogenic capabilities as their human counterparts. To do so, we will bring to bear 5 novel technologies: a). genetic engineering of ECs to render them non-immunogenic and non-antigenic; b). nanomedicine to modulate the immunogenic capacity of ECs; c). 3D printing of a human skin composed of multiple different cell types as a target for rejection; d). a state-of-the-art human immune system mouse as a graft recipient; and e). cutting edge high dimensional serial immunofluorescence to determine the effects of our interventions on the rejection process. We will initially test our model mouse model and optimize our assays using natural human skin (aim 1) and then proceed to test our 3D skin constructs (aim 2) in which we will modify cell types to determine the role, if any, of human cells other than ECs and DCs. We acknowledge that there is some risk in a proposal that merges multiple technologies which have not been previously combined and therefore have chosen to use the R21 exploratory mechanism to support this research. However, the successful conduct of this project will not only provide an initial answer to an important question for human skin cells, but will also create a platform for further studies of allotransplantation with more complex human tissues and provide valuable insights for bioengineering of replacement organs.