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

7.项目摘要/摘要 同种异体移植排斥反应仍然是同种异体移植长期成功的一个问题，也有可能产生负面影响。 影响组织工程移植物的功能和寿命作为一种可能的解决方案 可用器官短缺。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/肽复合体交叉反应的宿主T效应记忆细胞，这一过程 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/多肽复合体。表达相同同种异体抗原的移植物细胞类型 能被效应T细胞识别和杀伤，但不能启动静息状态的分化 进入效应器T细胞的效应器记忆被认为是抗原性的，而不是免疫原性的。两种免疫原性 与启动排斥反应有关的人类细胞类型是移植物树突状细胞(DC)和移植物内皮细胞 (ECS)。虽然DC(和其他乘客白细胞)可以从自然移植物中清除或从移植物中排除 经过生物工程处理的移植物，内皮细胞对于维持移植物活力的血管衬里是必不可少的。我们有 建议的策略，通过限制移植物的免疫原性来补充目前的排斥反应 抑制宿主免疫反应的做法。目前尚不清楚移除DC和将EC更改为非 免疫原性将足以保护移植物免受排斥反应。在这里，我们建议回答这个问题。 解决这个问题需要用人类内皮细胞进行，因为常用的啮齿动物内皮细胞 表现出与人类同行不同的免疫原性。为此，我们将采取行动 5项新技术：a)。对内皮细胞进行基因工程，使其不具有免疫原性和抗原性；b) 调节内皮细胞免疫原性的纳米药物；c)。人体皮肤的3D打印 多种不同类型的细胞作为排斥反应的靶点；一种最先进的人类免疫系统小鼠作为 移植物接受者；和e)。尖端高维系列免疫荧光法确定我们的 对拒绝程序的干预。我们将首先测试我们的模型小鼠模型，并优化我们的分析 使用天然人类皮肤(目标1)，然后继续测试我们的3D皮肤结构(目标2)，在其中我们将 修改细胞类型以确定除内皮细胞和树突状细胞以外的人类细胞的角色(如果有)。我们承认 合并以前未合并过的多项技术的提案存在一些风险 并因此选择使用R21探索机制来支持这项研究。然而， 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 为替代器官的生物工程提供了有价值的见解。

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.