Robust allograft tolerance in non-human primates

项目摘要 本申请是根据NOT-AI-19-040提交的。家长补助金旨在扩大 受体THBE，以提高在接受最低限度的，非THBE的猴子中实现的嵌合体的水平和持续时间。 清髓性预处理和异基因骨髓移植以诱导稳健的同种异体移植耐受。 基于小鼠间同种异体移植模型中的强有力结果，共移植小鼠源性 多克隆TclA是实现这一目标的有希望的方法。非人灵长类动物（NHP）模型研究 对于将这种方法最佳地转化为临床至关重要。我们努力将Treg方法应用于 NHPs已经导致嵌合体和耐受性的改善，但我们还没有实现永久性的治疗。 在小鼠模型中观察到嵌合体。我们已经探索了几种多克隆Treg扩增方案， 抗原呈递细胞来源，包括人工APC和来自多个供体的活化B细胞。而 额外的变化保证测试，需要相当多的时间和费用来测试NHP中的每一个。一 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 将有助于指导NHP研究。人源化免疫系统（HIS）小鼠，其通过将人 将胎肝CD 34+细胞移植至免疫缺陷小鼠，免疫缺陷小鼠在肾包膜下接受人胎胸腺组织 提供这样一个模型。在这些小鼠中产生的强大的人类免疫系统只允许短暂的 当在用以下物质预处理后施用同种异体人CD 34+细胞时， 方案基于猴模型和相关临床试验中使用的方案。生成了正常的T_i， 可以在体外扩增。因此，该HIS小鼠模型（称为HU/HU胸腺/HSC小鼠）提供了一种用于治疗肿瘤的方法。 一个测试扩增的受体THBE促进持久的混合同种异体移植的能力的极好平台。 嵌合诱导由于对该模型中人胎儿组织的可用性和使用的担忧，我们的目标是 修改以避免使用人胎组织，满足NOT-AI-19-040的要求。具体来说，我们的目标是 1）用猪胎儿胸腺和人脐带血CD 34+细胞产生HIS小鼠，并建立基线Treg 发育和扩增参数以及促进混合同种异体嵌合体的能力; 2） 用脐带血CD 34+细胞和含有人多能干细胞的猪“杂交”胸腺产生HIS小鼠- 来源的胸腺上皮细胞（hPSC-TEC）;确定hPSC-TEC对HLA-1阳性选择的影响。 限制TCR和Treg发育。Treg使用中的适应性来自混合免疫缺陷的HIS小鼠模型。 嵌合体诱导将直接应用于父母资助的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.