CD+CD25+ T Cells:Reservoir of Productive FIV Infection

描述(由申请人提供)：基本上所有接受HAART的患者中都存在正在进行的艾滋病毒复制的储存库。早期的研究发现，一种独特的CD4+CD25+T细胞是艾滋病毒生产性感染的潜在靶点。同样，来自FIV感染猫的CD4+CD25+T细胞在体外和体内都支持高效的FIV感染，而不是CD4+CD25-T细胞。这些CD4+CD25+细胞具有T调节细胞(Treg)的特征，即它们是无能的，并抑制T细胞对有丝分裂原的增殖反应。这些实验将定义调节Treg细胞的细胞因子和细胞内信号通路，并确定这些分子如何促进FIV复制。来自FIV+和FIV-CAT的PBMC和LN CD4+T细胞将通过带有特定抗体(例如CD25、B7.1、B7.2、CTLA4、MHCII和TGFbeta)的多色流式细胞术进行分析，以确定特定的CD4+细胞亚群。利用针对这些受体的mAb进行多参数高速细胞分选(例如，从αCD4到αCD25再到β7.1)将提供来自FIV+猫的CD4+CD25+和CD4+CD25-T细胞亚群，用于通过聚合酶链式反应和p24分析来确定潜伏性或生产性感染。纯化的FIV+和FIV-CAT的CD4+T细胞亚群也将通过TaqMan聚合酶链式反应和流式细胞仪染色分析细胞因子(如IL10、IL2、TGFbeta、IL4和IFNGamma)的表达。用AlphalL10和AlphaTGFbeta进行抗体阻断研究将确定它们在维持CD4+CD25+无能状态和FIV复制能力方面的作用。体内和体外FIV感染对CD4+CD25+Treg细胞功能和活性的影响将通过将病毒载量与a)无能(PI染色，3HTdR摄取)；b)对凋亡的敏感性(Anexin V，TUNEL)；以及c)对丝裂原或FIV肽刺激的CD4+CD25-Th细胞的抑制活性以及培养寿命(CFDA SE染色)相关联来确定。最后，将分析CD4+CD25+和CD4+CD25-细胞周期抑制因子p21cip1和p27kip1 cdk的表达，以及ATF和AP-1转录因子的表达，以期将这些蛋白的表达与CD4+CD25+T细胞无能和FIV复制相关联。调节这些蛋白质的表达将在调节无能的蛋白质和FIV复制之间建立因果关系。将对急性FIV感染的猫进行时间分析，以了解CD4+CD25+的激活情况，并建立FIV储存库。这些研究应该有助于深入了解维持Treg细胞无能状态的细胞因子和信号通路，并有助于确定促进FIV复制的分子环境，以及CD4+CD25+Treg细胞多快被激活，并在感染后建立FIV储存库。

DESCRIPTION (provided by applicant): A reservoir of on-going HIV replication persists in essentially all patients receiving HAART. Early studies identified a unique CD4+CD25+ T cell as a potential target of productive HIV infection. Similarly, CD4+CD25+ but not CD4+CD25- T cells from FIV-infected cats support a productive FIV infection in vitro and in vivo. These CD4+CD25+ cells have the characteristics of T regulatory (Treg) cells in that they are anergic and suppress T cell proliferative responses to mitogen. These experiments will define the cytokines and intracellular signaling pathways that regulate Treg cells and determine how these molecules may promote FIV replication. PBMC and LN CD4+ T cells from FIV+ and FIV- cats will be analyzed by multi-color flow cytometry with specific antibodies (e.g. CD25, B7.1, B7.2, CTLA4, MHCII and TGFbeta) to identify specific subsets of CD4+ cells. Multiparameter high speed cell sorting using mAb to these receptors (e.g. alphaCD4 to alphaCD25 to Beta7.1) will provide CD4+CD25+ and CD4+CD25- T cell subsets from FIV+ cats for determination of latent or productive infection by PCR and p24 analysis. Purified CD4+ T cell subsets from FIV+ and FIV- cats will also be analyzed for cytokine (e.g. IL10, IL2, TGFbeta, IL4 and IFNgamma) expression by TaqMan PCR and flow cytometry staining. Antibody blocking studies with alphalL10 and alphaTGFbeta will determine their role in maintaining the CD4+CD25+ anergic state and FIV replication capability. Effects of in vivo and in vitro FIV infection on CD4+CD25+ Treg cell function and viability will be established by correlating virus burden with a) anergy (PI staining, 3HTdR uptake); b) susceptibility to apoptosis (Anexin V, TUNEL); and c) suppressor activity against mitogen- or FIV peptide-stimulated CD4+CD25- Th cells; and longevity in culture (CFDA SE staining). Finally, CD4+CD25+ and CD4+CD25- will be analyzed for expression of p21 cip1 and p27 kip1 cdk inhibitors of G1 cell cycling and ATF and AP-1 transcription factor to correlate expression of these protein with CD4+CD25+ T cell anergy and FIV replication. Modulation of expression of these proteins will establish cause and effect relationships between proteins regulating anergy and FIV replication. Cats with acute FIV infection will be analyzed temporally for CD4+CD25+ activation and establishment of a FIV reservoir. These studies should yield insights into the cytokines and signaling pathways that maintain the anergic state of Treg cells and help define the molecular environment that promotes FIV replication and how soon CD4+CD25+ Treg cells are activated and establish a FIV reservoir after infection.