EFFECTOR/MEMORY CD8 T CELL FUNCTIONS IN HIV INFECTION

在HIV感染中，病毒特异性的细胞毒性反应可以通过体外刺激容易地引发。尽管如此，在大多数未经治疗的受试者中，病毒的产生仍处于高水平。因此，需要对体内功能反应进行严格评价，以了解其与宿主抗性和疾病进展的相关性。利用高灵敏度的MHC/肽四聚体染色检测病毒特异性CD 8 T细胞，我们发现HIV特异性CTL在体内慢性感染受试者中存在功能缺陷。然而，四聚体染色仅允许研究参与免疫应答的一系列表位中的一个。使用一种新的测定方法，该方法在用HIV感染的原代CD 4 T细胞靶细胞攻击后测量功能，我们能够检测到一些功能性细胞。进一步的初步数据表明，虽然病毒！预期受感染的细胞呈递所有病毒抗原，功能性应答仅针对少数T表位。在目标1中，我们将检验以下假设：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应答的亚显性表位。我们将表征产生IFN-γ的CD 8 T细胞的表型和表位识别，以响应HIV感染的原代CD 4 T细胞，并将其与使用肽库和一组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CD 8 T细胞积累的机制。已知病毒蛋白之一nef下调关键抗原呈递分子I类MHC。为了研究nef的作用，我们将使用nef+或nef-病毒感染CD 4 T细胞，并使用这些刺激细胞来比较CD 8 T细胞应答。艾滋病毒的另一个突出特点是它能够迅速变异以逃避免疫反应。因为抗原在突变后不再呈递，原始免疫显性表位可能变得不相关，因此是亚显性的。为了测试这种可能性，我们将通过分析其T细胞受体库来比较免疫磁性捕获的产生IFN-γ的T细胞的克隆组成，所述T细胞响应于实验室采用的HIVIIIB病毒与患者自身（自体）病毒感染的CD 4 T细胞靶标。我们还将测试响应于HIVIIIB病毒的功能细胞是否可以识别自体病毒，反之，响应于自体病毒的细胞是否可以识别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.