Robust allograft tolerance in non-human primates

项目摘要 此申请是针对NOT-AI-19-040提交的。父赠款旨在使用扩展的 受体Tregs可提高嵌合体水平和嵌合体持续时间 清髓性调理和异基因骨髓移植可诱导强大的同种异体移植耐受。 基于在小鼠对小鼠的同种异体移植模型中的强劲结果，共同移植的受体来源 多克隆Tregs是实现这一目标的一种有前途的方法。非人灵长类(NHP)模型的研究 对于将这一方法最佳地转化到临床上是必不可少的。我们将Treg方法应用于 国家卫生保健计划促进了嵌合体和耐受性的改善，但我们还没有实现永久性的 在小鼠模型中可以看到嵌合体。我们已经探索了几种多克隆Treg扩展协议和 抗原提呈细胞来源，包括人工APC和来自多个捐赠者的激活的B细胞。而当 额外的变化需要测试，在NHP中测试每个变化都需要相当长的时间和费用。一个 更灵活的模型，反映了人类和猴子在实现持久混合嵌合体的挑战中的情况 将有助于指导NHP的研究。通过转移人产生人源化免疫系统(His)小鼠 人胎肝CD34+细胞移植给免疫缺陷小鼠肾包膜下移植人胎胸腺组织 提供这样一个模型。在这些小鼠身上产生的强大的人类免疫系统只允许短暂的 同种异体人CD34+细胞经A细胞处理后可实现嵌合体 方案以猴模型和相关临床试验中使用的方案为基础。生成正常树，并且 可以在体外扩增。因此，这种His小鼠模型(称为HU/HU胸腺/HSC小鼠)提供了一种 测试扩大受体Tregs促进持久混合同种异体移植能力的极佳平台 嵌合体诱导。由于对人类胎儿组织在这个模型中的可用性和使用的担忧，我们的目标是 对其进行修改，避免使用人类胎儿组织，满足NOT-AI-19-040的要求。具体来说，我们的目标是 目的：1)用猪胎胸腺和人脐血CD34+细胞制备His小鼠，建立基线Treg 发展和扩展参数以及促进混合同种异体嵌合体的能力；2) 用脐带血CD34+细胞和含有人多能干细胞的猪的杂交胸腺培育出他的小鼠-- 来源的胸腺上皮细胞(hPSC-TECs)；确定hPSC-TECs对人类白细胞抗原-1(HLA-1)阳性选择的影响 受限的TCR和Treg开发。MIXED His小鼠模型对Treg使用的适应性 嵌合体诱导将直接应用于父母赠款中的NHP模型，并最终应用于临床试验。

Project Abstract This application is being submitted in response to NOT-AI-19-040. The parent grant aims to use expanded recipient Tregs to enhance the level and duration of chimerism achieved in monkeys receiving minimal, non- myeloablative conditioning and allogeneic bone marrow transplantation to induce robust allograft tolerance. Based on strong results in a mouse-to-mouse allotransplantation model, co-transplantation of recipient-derived polyclonal Tregs is a promising approach to achieving this goal. Studies in the non-human primate (NHP) model are essential for the optimal translation of this approach to the clinic. Our efforts to apply the Treg approach to NHPs have led to improvements in chimerism and tolerance, but we have not yet achieved the permanent chimerism seen in the mouse model. We have explored several polyclonal Treg expansion protocols and antigen-presenting cell sources, including artificial APCs and activated B cells from multiple donors. While additional variations warrant testing, considerable time and expense is required to test each one in NHPs. A more nimble model that mirrors the human and monkey in the challenge of achieving durable mixed chimerism would help to guide the NHP studies. Humanized immune system (HIS) mice generated by transferring human fetal liver CD34+ cells to immunodeficient mice that receive human fetal thymus tissue under the kidney capsule provide such a model. The robust human immune systems generated in these mice permit only transient chimerism to be achieved when allogeneic human CD34+ cells are administered following conditioning with a regimen based on the one used in the monkey model and related clinical trials. Normal Tregs are generated and can be expanded in vitro. Therefore, this HIS mouse model (termed HU/HU thymus/HSC mice) provides an excellent platform for testing the ability of expanded recipient Tregs to promote durable mixed allogeneic chimerism induction. Due to concerns about the availability and use of human fetal tissue in this model, we aim to modify it to avoid using human fetal tissue, meeting the requirement for NOT-AI-19-040. Specifically, we aim to: 1) Generate HIS mice with pig fetal thymus and human cord blood CD34+ cells and establish baseline Treg development and expansion parameters as well as capacity to promote mixed allogeneic chimerism; 2) Generate HIS mice with cord blood CD34+ cells and pig “hybrid” thymus containing human pluripotent stem cell- derived thymic epithelial cells (hPSC-TECs); determine the impact of hPSC-TECs on positive selection of HLA- restricted TCRs and Treg development. Adaptations in Treg use resulting from the HIS mouse model of mixed chimerism induction will be directly applied to the NHP model in the parent grant and ultimately in clinical trials.