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治疗的患者体内都存在持续的HIV复制库。早期的研究确定了一种独特的CD4+CD25+ T细胞作为生产性HIV感染的潜在靶标。同样，来自感染FIV的猫的CD4+CD25+ T细胞，而不是CD4+CD25- T细胞，在体外和体内都支持生产性FIV感染。这些CD4+CD25+细胞具有T调节性（Treg）细胞的特征，它们是无能的，抑制T细胞对有丝分裂原的增殖反应。这些实验将确定调节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-猫的PBMC和LN CD4+ T细胞将通过多色流式细胞术分析特异性抗体（例如CD25、B7.1、B7.2、CTLA4、MHCII和TGFbeta），以识别CD4+细胞的特异性亚群。使用mAb对这些受体（如alphaCD4到alphaCD25到Beta7.1）进行多参数高速细胞分选，将提供FIV+猫的CD4+CD25+和CD4+CD25- T细胞亚群，用于通过PCR和p24分析确定潜伏或生产感染。FIV+和FIV-猫的纯化CD4+ T细胞亚群也将通过TaqMan PCR和流式细胞术染色分析细胞因子（如IL10、IL2、tgf β、IL4和IFNgamma）的表达。用alphalL10和alphagfbeta进行抗体阻断研究将确定它们在维持CD4+CD25+无能状态和FIV复制能力中的作用。体内和体外FIV感染对CD4+CD25+ Treg细胞功能和活力的影响将通过将病毒负荷与a)能量（PI染色，3HTdR摄取）相关联来确定；b)细胞凋亡敏感性（Anexin V， TUNEL）；c)抑制分裂原或FIV肽刺激的CD4+CD25- Th细胞的活性；和寿命（CFDA SE染色）。最后，将分析CD4+CD25+和CD4+CD25- G1细胞周期的p21 cip1和p27 kip1 cdk抑制剂以及ATF和AP-1转录因子的表达，以将这些蛋白的表达与CD4+CD25+ T细胞能量和FIV复制联系起来。调节这些蛋白的表达将在调节能量的蛋白与FIV复制之间建立因果关系。急性FIV感染的猫将暂时分析CD4+CD25+激活和FIV库的建立。这些研究将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细胞在感染后被激活和建立FIV库的速度。

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.