EVALUATING THE ENGINEERING OF CYTOMEGALOVIRUS-SPECIFIC CD8+ T CELL CLONES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 巨细胞病毒(CMV)的重新激活是CD8+T细胞免疫缺陷导致的异基因造血干细胞和实体器官移植免疫抑制受者发病和死亡的主要原因。抗原特异性T细胞克隆的过继免疫治疗已被证明可以安全地纠正允许病毒感染进展的T细胞数量或质量上的缺陷。然而，T细胞治疗未能恢复分别接受免疫抑制皮质类固醇治疗移植物抗宿主病或移植物排斥反应的患者的保护性T细胞免疫。为了克服这一障碍，我们研究了一种创新的策略，利用锌指核酸内切酶(ZFN)选择性干扰糖皮质激素受体(GR)信号，在成熟T细胞中定向缺失GR基因。我们已经建立了一种过继转移CMV特异性T细胞克隆的猕猴模型，并利用该模型来研究ZFN靶向缺失后缺乏GR基因(GR-/-)的CMV特异性T细胞免疫治疗的安全性和有效性。我们鉴定了在外周血中可检测到CMV特异性CD8+T细胞反应的免疫活性猕猴，并使用复制缺陷Ad5/35载体递送针对猕猴T细胞中GR基因的ZFN。然后用有限稀释克隆法分离CMV特异性GR-/-CD8+T细胞克隆。GR-/-T细胞克隆表现出与CMV特异性Grwt克隆相当的CMV特异性细胞杀伤活性。然而，与Grwt T细胞相比，GR-/-T细胞在体外对皮质类固醇的淋巴毒性作用具有抵抗力，这与GR基因的破坏和功能GR蛋白的缺乏一致。我们目前正在从2只猕猴中产生自体GR-/-CMV特异性CD8+T细胞克隆，可用于过继转移研究，以评估过继转移带有编辑的GR基因的CD8+CMV特异性T细胞克隆的安全性和有效性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Reactivation of cytomegalovirus (CMV) represents a major cause of morbidity and mortality in immunosuppressed recipients of allogeneic hematopoietic stem cell and solid organ transplant as a consequence of deficient CD8+ T cell immunity. Adoptive immunotherapy with antigen-specific T cell clones has been shown to safely correct quantitative or qualitative deficiencies of T cells that permit progression of viral infection. However, T cell therapy has failed to restore protective T cell immunity in the subset of patients that receive immunosuppressive corticosteroids to treat graft versus host disease or graft rejection, respectively. To overcome this obstacle, we have investigated an innovative strategy for selectively interfering with glucocorticoid receptor (GR) signaling using zinc-finger endonucleases (ZFNs) for targeted deletion of the GR gene in mature T cells. We have developed a macaque model for adoptive transfer of CMV-specific T cell clones and employ this model to investigate the safety and efficacy of immunotherapy with CMV-specific T cells that lack the GR gene (GR-/-) after ZFN targeted deletion. We identified immunocompetent macaques with detectable CMV-specific CD8+ T cell responses in the peripheral blood and used a replication defective Ad5/35 vector to deliver ZFNs that target the GR gene in macaque T cells. CMV-specific GR-/- CD8+ T cell clones were then isolated by limiting dilution cloning. The GR-/- T cell clones exhibited CMV-specific cytolytic reactivity comparable to CMV-specific GRwt clones. However, in contrast to GRwt T cells, the GR-/- T cells were resistant to lymphotoxic effects of corticosteroids in vitro, consistent with disruption of the GR gene and lack of a functional GR protein. We are currently generating autologous GR-/- CMV-specific macaque CD8+ T cell clones from 2 macaques that can be used for adoptive transfer studies to evaluate the safety and efficacy of adoptively transferred CD8+ CMV-specific T cell clones with an edited GR gene.