Cell therapy for transient hematopoietic recovery

先前在犬模型中的研究表明，及时给予单一细胞因子G-CSF可以将犬从400 cGy的全身照射（TBI）中拯救出来，否则会致命。这种保护作用在500 cGy时丧失。类似地，不长期移植的MHC错配骨髓细胞也提供了保护作用，允许在450 cGy TBI后自体重建。在这种情况下，最大耐受辐射剂量是未知的，也是保护机制。假设，错配的细胞可以提供一个短暂的血细胞群体，这将减轻血细胞减少症，直到发生自体重建。可选择地， 它们可以通过同种异体反应性触发类似于G-CSF注射的“细胞因子风暴”。如果前者被证明是正确的，那么将生长因子与非移植细胞产品结合可能会产生额外的保护作用，从而提高可以拯救受害者的辐射剂量阈值。因此，拟定的研究旨在确定最大辐射剂量，在该剂量下，通过输注不匹配的造血细胞群体（添加或不添加生长因子），可以实现自体造血重建的生存。研究将使用犬辐照模型，该模型对人类患者的结局具有高度预测性。提出了三个具体目标：目标1;鉴于“最佳”细胞产物主要由定向髓系祖细胞组成的假设，将开发用于离体产生这些细胞的小规模方法。这包括开发用于扩增细胞产品质量控制的体外测定和优化冷冻保存技术。然后将使用良好生产规范（GMP）指南按比例扩大最佳离体扩增方案。在目标2中，将使用目标1中定义的最佳细胞产物 以确定在提供短暂造血支持时允许自体恢复的最大辐射剂量。使用VNTR分析将供体细胞与自体细胞区分开。然后将确定是否需要免疫抑制方案来防止宿主抗移植物反应，否则可能中和“拯救效应”。在目标3中，细胞因子将与骨髓祖细胞产物组合，旨在优化“拯救效果”。除G-CSF外，待检测的细胞因子将通过单独的支持物进行鉴别（RO 1 AI 66498 -01）。还将确定辐射暴露后的“机会窗口”，在此期间祖细胞产品的输注是有效的。本项目的总体目标是提供“现成”产品，这些产品可以促进自体重建或至少在确定适当的干细胞来源（在项目5和6中讨论）时提供一段时间的支持。

Previous studies in the canine model have shown that a single cytokine, G-CSF, given in a timely manner can rescue dogs from 400cGy total body irradiation (TBI), an otherwise lethal dose. This protective effect is lost at 500 cGy. Similarly, MHC-mismatched marrow cells that do not engraft long-term also provide a protective effect that allows for autologous reconstitution following 450 cGy TBI. In this case, the maximum tolerated radiation dose is not known, nor is the protective mechanism. Hypothetically, the mismatched cells could provide a transient population of blood cells that would mitigate the cytopenia until autologous reconstitution occurs. Alternatively, they could, through alloreactivity, trigger a 'cytokine storm' that mimics the injection of G-CSF. If the former proves true, combining growth factors with a non-engrafting cell product might have additive protective effects, increasing the radiation dose threshold from which victims can be rescued. Therefore, the proposed studies are designed to determine the maximum radiation dose at which survival, with autologous hematopoietic reconstitution, can be achieved by infusing a non-matched population of hematopoietic cells, with and without added growth factors. Studies will use the canine irradiation model that has been highly predictive for outcomes in human patients. Three Specific Aims are proposed: Aim 1; given the assumption that the "optimal" cell product consists primarily of committed myeloid progenitors, small-scale methods for ex vivo generation of these cells will be developed. This includes development of in vitro assays for quality control of expanded cell products and optimization of cryopreservation techniques. The optimal ex vivo expansion protocol will then be scaled-up using Good Manufacturing Practice (GMP) guidelines. In Aim 2, the optimal cell product as defined under Aim 1 will be used to determine the maximum radiation dose that allows for autologous recovery when transient hematopoietic support is provided. Donor cells will be distinguished from autologous cells using VNTR analysis. It will then be determined whether immunosuppressive regimens are needed to prevent host-versus-graft reactions that might otherwise neutralize the "rescue effect". In Aim 3, cytokines will be combined with the myeloid progenitor cell product aimed at optimizing the "rescue effect". The cytokines to be tested in addition to G-CSF will be identified through separate support (RO1 AI66498-01). The "window of opportunity" after radiation exposure, during which infusion of the progenitor cell product is effective, will also be determined. The overall goal of this project is to provide 'off-the-shelf' products that can facilitate autologous reconstitution or at least provide a period of support while an appropriate stem cell source (addressed in Projects 5 and 6) is identified.