CD4+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。早期的研究确定了一种独特的CD 4 + CD 25 + T细胞作为生产性HIV感染的潜在靶点。类似地，来自FIV感染的猫的CD 4 + CD 25+而不是CD 4 + CD 25- T细胞支持体外和体内的生产性FIV感染。这些CD 4 + CD 25+细胞具有T调节（Treg）细胞的特征，因为它们是无反应性的并抑制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传导途径，并确定这些分子如何促进FIV复制。将使用特异性抗体（例如，CD 25、B7.1、B7.2、CTLA 4、MHCII和TGF β）通过多色流式细胞术分析来自FIV+和FIV-猫的PBMC和LN CD 4 + T细胞，以鉴定CD 4+细胞的特定亚群。使用针对这些受体（例如，α CD 4至α CD 25至β 7.1）的mAb进行的多参数高速细胞分选将提供来自FIV+猫的CD 4 + CD 25+和CD 4 + CD 25- T细胞亚群，用于通过PCR和p24分析确定潜伏性或生产性感染。还将通过TaqMan PCR和流式细胞术染色分析来自FIV+和FIV-猫的纯化的CD 4 + T细胞亚群的细胞因子（例如IL 10、IL 2、TGF β、IL 4和IFN γ）表达。用α 1 L10和α TGF β进行的抗体阻断研究将确定它们在维持CD 4 + CD 25+无反应性状态和FIV复制能力中的作用。将通过将病毒负荷与a）无反应性（PI染色，3 HTdR摄取）; B）细胞凋亡易感性（Anexin V，TUNEL）;和c）对促分裂原或FIV肽刺激的CD 4 + CD 25- Th细胞的抑制活性;和培养物中的寿命（CFDA SE染色）相关联，确定体内和体外FIV感染对CD 4 + CD 25 + Treg细胞功能和活力的影响。最后，将分析CD 4 + CD 25+和CD 4 + CD 25-的G1细胞周期的p21 cip 1和p27 kip 1 cdk抑制剂以及ATF和AP-1转录因子的表达，以将这些蛋白质的表达与CD 4 + CD 25 + T细胞无反应性和FIV复制相关联。这些蛋白质表达的调节将在调节无反应性和FIV复制的蛋白质之间建立因果关系。将对急性FIV感染猫的CD 4 + CD 25+活化和FIV储库的建立进行时间分析。这些研究将有助于深入了解维持Treg细胞无反应性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，以及感染后CD 4 + CD 25 + Treg细胞多久被激活并建立FIV储库。

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.