Enhancing HIV-specific CTL by CD27/CD70 costimulation

描述（申请人提供）：HIV感染个体虽然产生大量抗原特异性CD8 T细胞，但无法控制病毒，最终发展为临床艾滋病。我们和其他人已经证明，新分离的hiv特异性CD8 T细胞功能受损。进一步的初步结果表明，来自同一感染个体的hiv特异性T细胞而不是cmv特异性T细胞表现出不成熟的表型特征。hiv特异性CD8 T细胞与CD8 T细胞对其他控制良好的慢性病毒（如巨细胞病毒）的反应之间一个显著的表型差异是不能下调TNF家族共刺激分子CD27。CD27下调通常发生在与其配体CD70相互作用后，这种相互作用似乎对T细胞的终末成熟和关键细胞毒性分子穿孔素的表达至关重要。因此，HIV感染的潜在缺陷可能是不能表达CD70，导致不能产生CD27触发的共刺激信号。为了验证这一假设，在Aim 1中，我们将研究外源性提供CD70是否能恢复或增强hiv特异性CD8 T细胞的增殖、细胞毒性和ifn - γ分泌。由于ebv转化的B淋巴母细胞样细胞系（BLCLs）大量表达CD70，我们将首先测试在自体BLCLs上阻断/去除CD70分子是否会降低它们刺激hiv特异性CTL的能力。我们还将在体外培养有或没有抗原刺激的细胞中以可溶性形式外源性或慢病毒递送CD70，并测试它们杀死hiv感染和肽脉冲靶标以及产生细胞因子的能力。

DESCRIPTION (provided by applicant): Although HIV infected individuals generate a large number of antigen-specific CD8 T cells, they are unable to control the virus and eventually develop clinical AIDS. We, and others have shown that freshly isolated HIV-specific CD8 T cells are functionally impaired. Further preliminary results suggest that HIV-specific T cells but not CMV-specific T cells from the same infected individual exhibit phenotypic features of immaturity. One striking phenotypic difference between HIV-specific CD8 T cells from CD8 T cells responding to other well-controlled chronic viruses such as CMV, is the failure to downmodulate the TNF family costimulatory molecule CD27. CD27 downmodulation normally occurs after interaction with its ligand CD70, and this interaction appears to be critical for the terminal maturation of T cells and expression of the key cytotoxic molecule perforin. Thus, the underlying defect in HIV infection may be a failure to express CD70, resulting in the failure to generate CD27 triggered costimulatory signals. To test this hypothesis, in Aim 1, we will investigate whether providing CD70 exogenously restores or enhances proliferation, cytotoxicity and IFN-gamma secretion of HIV-specific CD8 T cells. As EBV-transformed B lymphoblastoid cell lines (BLCLs) express CD70 abundantly, we will first test if blockade/abrogation of the molecule on autologous BLCLs diminishes their ability to stimulate HIV-specific CTL. We will also provide CD70 exogenously in soluble form or by lentiviral delivery during ex vivo culture of the cells with and without antigen stimulation, and test their ability to kill HIV-infected and peptide-pulsed targets and to produce cytokines. Because a small subpopulation of HIV-infected individuals, the long-term nonprogressors are able to control the virus in the absence of treatment, in the second specific aim, we will examine whether an intact CD70 costimulation pathway is responsible for maintenance of a functional virus-specific CD8 T cells in these subjects. We will block CD27/CD70 interactions during ex vivo stimulation using blocking antibody, CD27Ig chimera and RNAi and test if this treatment diminishes their responsiveness.