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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 巨细胞病毒（CMV）的再活化是异基因造血干细胞和实体器官移植的免疫抑制受者由于CD 8 + T细胞免疫缺陷而发病和死亡的主要原因。使用抗原特异性T细胞克隆的连续免疫疗法已被证明可以安全地纠正允许病毒感染进展的T细胞的定量或定性缺陷。然而，T细胞疗法未能恢复接受免疫抑制性皮质类固醇治疗移植物抗宿主病或移植物排斥的患者亚组中的保护性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，用于选择性地干扰糖皮质激素受体（GR）信号转导，使用锌指核酸内切酶（ZFN）靶向删除成熟T细胞中的GR基因。我们已经开发了一种用于CMV特异性T细胞克隆过继转移的猕猴模型，并采用该模型研究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。我们鉴定了外周血中具有可检测的CMV特异性CD 8 + T细胞应答的免疫活性猕猴，并使用复制缺陷型Ad 5/35载体递送靶向猕猴T细胞中GR基因的ZFN。然后通过有限稀释克隆分离CMV特异性GR-/-CD 8 + 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特异性细胞溶解反应性。然而，与GRwt T细胞相反，GR-/- T细胞在体外对皮质类固醇的光致变色作用具有抗性，这与GR基因的破坏和功能性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特异性猕猴CD 8 + T细胞克隆，其可用于过继转移研究，以评估具有编辑的GR基因的过继转移的CD 8 + 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.