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

CD CD25 T 细胞：生产性 FIV 感染库

• 批准号: 7054124
• 负责人: Wayne A Tompkins
• 金额: $ 40.04万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7054124
• 关键词: AIDS therapy; AP1 protein; CD antigens; HIV infections; T lymphocyte; cAMP response element binding protein; cats; cell sorting; cytokine; disease /disorder model; enzyme inhibitors; enzyme linked immunosorbent assay; feline immunodeficiency virus; flow cytometry; gel mobility shift assay; helper T lymphocyte; immunodeficiency; immunoregulation; latent virus infection; mitogens; polymerase chain reaction; protein structure function; terminal nick end labeling; virulence; virus cytopathogenic effect; virus infection mechanism; virus replication

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.

描述（由申请人提供）：基本上所有接受 HAART 的患者体内都存在持续的 HIV 复制库。早期研究发现独特的 CD4+CD25+ T 细胞是有效 HIV 感染的潜在目标。同样，来自FIV感染猫的CD4+CD25+而非CD4+CD25-T细胞在体外和体内支持有效的FIV感染。这些 CD4+CD25+ 细胞具有 T 调节 (Treg) 细胞的特征，因为它们无反应性并抑制 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- 猫的 PBMC 和 LN CD4+ T 细胞将通过多色流式细胞术和特异性抗体（例如 CD25、B7.1、B7.2、CTLA4、MHCII 和 TGFbeta）进行分析，以鉴定 CD4+ 细胞的特定亚群。使用针对这些受体（例如 alphaCD4 至 alphaCD25 至 Beta7.1）的 mAb 进行多参数高速细胞分选将提供来自 FIV+ 猫的 CD4+CD25+ 和 CD4+CD25- T 细胞亚群，用于通过 PCR 和 p24 分析确定潜在或生产性感染。来自 FIV+ 和 FIV- 猫的纯化 CD4+ T 细胞亚群还将通过 TaqMan PCR 和流式细胞仪染色分析细胞因子（例如 IL10、IL2、TGFbeta、IL4 和 IFNgamma）表达。使用α1L10和αTGFβ的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) 无反应性（PI 染色、3HTdR 摄取）相关联来确定； b) 对细胞凋亡的敏感性（Anexin V、TUNEL）； c)针对丝裂原或FIV肽刺激的CD4+CD25-Th细胞的抑制活性；和培养寿命（CFDA SE 染色）。最后，将分析 CD4+CD25+ 和 CD4+CD25- 的 G1 细胞周期 p21 cip1 和 p27 kip1 cdk 抑制剂以及 ATF 和 AP-1 转录因子的表达，以将这些蛋白质的表达与 CD4+CD25+ T 细胞无反应性和 FIV 相关联。复制。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。将暂时分析患有急性 FIV 感染的猫的 CD4+CD25+ 激活和 FIV 储存库的建立。这些研究应该深入了解维持 Treg 细胞无反应状态的细胞因子和信号通路，并帮助定义促进 FIV 复制的分子环境，以及感染后 CD4+CD25+ Treg 细胞多久被激活并建立 FIV 储存库。