EFFECTOR/MEMORY CD8 T CELL FUNCTIONS IN HIV INFECTION

在HIV感染中，体外刺激离子可以很容易地引起病毒特异性细胞毒性I细胞（kC I L）反应。尽管如此，在大多数未经治疗的受试者中，病毒的产生仍保持在高水平。因此，需要对体内功能反应进行关键评估，以了解它们与宿主耐药性和疾病进展的相关性。使用高度敏感的MHC/肽四聚体染色检测病毒特异性CD8 T细胞，我们发现在体内慢性感染受试者中hiv特异性CTL功能缺陷。然而，四聚体染色只允许研究一个参与免疫反应的表位范围。使用一种新的检测方法来测量hiv感染的原发CD4 T细胞靶细胞攻击后的功能，我们能够检测到一些功能细胞。进一步的初步数据表明，虽然病毒！被感染的细胞预计会呈递所有的病毒抗原，功能性反应仅针对少数的t表位。在目标1中，我们将验证一个假设，即功能性表位在体内是免疫显性的，而非功能性表位代表不积极参与免疫反应的亚显性表位。我们将描述CD8 T细胞对HIV感染的原发CD4 T细胞产生ifn - γ的表型和表位识别，并将它们与使用肽库和MHC/肽四聚体鉴定的HIV表位的总体进行比较。在目标2中，我们将分析导致非功能性hiv特异性CD8 T细胞积累的机制。已知nef是一种病毒蛋白，可下调关键抗原递呈分子I类MHC。为了研究nef的作用，我们将使用nef+或nef-病毒感染CD4 T细胞，并使用这些刺激细胞来比较CD8 T细胞的反应。HIV的另一个突出特征是它能够迅速变异以逃避免疫反应。由于抗原在突变后不再呈现，原来的免疫显性表位可能变得无关紧要，因此成为亚显性。为了测试这种可能性，我们将通过分析患者自身（自体）病毒感染的CD4 T细胞靶细胞的T细胞受体库，比较免疫磁捕获的产生ifn - γ的T细胞对实验室采用的hiviib病毒的反应与患者自身（自体）病毒感染的T细胞靶细胞的克隆组成。我们还将测试响应HIVIIIB病毒的功能细胞是否能识别自身病毒，反之，响应自身病毒的细胞是否能识别HIVIIIB病毒感染的靶标。

In HIV infection, virus-specific cytotoxic I cell kC I L) response can be readily elicited by stimul ion in vitro. Despite this, viral production continues at a high level in most untreated subjects. Thus, a critical evaluation of the functional response in vivo is required to understand their relevance to host resistance and disease progression. Using the highly sensitive MHC/peptide tetramer staining to detect viral-specific CD8 T cells, we have found that HIV-specific CTL are functionally defective in chronically infected subjects in vivo. However, tetramer staining allows the study of only one of a gamut of epitopes involved in the immune response. Using a novel assay which measures function after challenge with HIV-infected primary CD4 T cell targets, we were able to detect some functional cells. Further preliminary data suggest that although virus! infected cells are expected to present all viral antigens, the functional response is directed only against few t epitopes. In aim 1, we will test the hypothesis that the functional epitopes are immunodominant in vivo and the non-functional epitopes represent subdominant epitopes that are not actively involved in the immune response. We will characterize the phenotype and epitope recognition of CD8 T cells that produce IFN-gamma in response to HIV-infected primary CD4 T cells and compare them to the total body of HIV epitopes identified using peptide pools and a panel of MHC/peptide tetramers. In aim 2, we will analyze the mechanisms that lead to an accumulation of non-functional HIV-specific CD8 T cells. One of the viral proteins, nef is known to down regulate the key antigen presenting molecule, class I MHC. To study the role of nef, we will use nef+ or nef- virus to infect CD4 T cells and use these stimulator cells to compare the CD8 T cell response. Another prominent feature of HIV is its ability to mutate rapidly to evade immune response. Because the antigen is no longer presented after mutation, the original immunodominant epitope may become irrelevant, and hence subdominant. To test this possibility, we will compare the clonal composition of immunomagnetically-captured IFN-gamma producing T cells responding to the lab adopted HIVIIIB virus vs patients' own (autologous) virus-infected CD4 T cell targets by analyzing their T cell receptor repertoire. We will also test if the functional cells responding to HIVIIIB virus can recognize autologous virus and conversely, whether cells responding to autologous virus can recognize HIVIIIB virus infected targets.