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

CD CD25 T 细胞：生产性 FIV 感染库

• 批准号: 6888542
• 负责人: Wayne A Tompkins
• 金额: $ 32.85万
• 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/6888542
• 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，正在进行的艾滋病毒复制的水库仍然存在。早期研究将独特的CD4+ CD25+ T细胞确定为生产性HIV感染的潜在靶标。同样，来自FIV感染的CAT的CD4+CD25+而不是CD4+CD25- T细胞支持体外和体内生产性FIV感染。这些CD4+ CD25+细胞具有T调节（Treg）细胞的特征，因为它们是厌食症并抑制T细胞对有丝分裂原反应的增殖反应。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细胞将通过具有特定抗体（例如CD25，B7.1，B7.2，CTLA4，MHCII和TGFBETA）的多色流式细胞仪分析，以识别CD4+细胞的特定亚群。使用MAB对这些受体的多参数高速细胞分选（例如，至beta7.1的alphACD4至alphACD25）将提供来自FIV+ CAT的CD4+ CD25+和CD4+ CD25- T细胞子集，以通过PCR和P24分析确定潜在或生产性感染的潜在或生产性感染。 Taqman PCR和流式细胞仪染色也将分析来自FIV+和FIV- CAT的纯化的CD4+ T细胞亚群（例如，IL10，IL2，TGFBETA，IL4和IFNGAMMA）表达的细胞因子（例如IL10，IL2，TGFBETA，IL4和IFNGAMMA）。使用Alphall10和Alphatgfbeta进行的抗体阻断研究将决定它们在维持CD4+ CD25+厌食状态和FIV复制能力方面的作用。体内和体外FIV感染对CD4+ CD25+ Treg细胞功能和生存能力的影响将通过将病毒负担与A）厌食（PI染色，3HTDR摄取）相关联建立； b）对凋亡的敏感性（Anexin V，Tunel）； c）针对有丝分裂原或FIV肽刺激的CD4+CD25- TH细胞的抑制活性；和文化的寿命（CFDA SE染色）。最后，将分析CD4+CD25+和CD4+CD25-的P21 CIP1和P27 KIP1 CDK抑制剂的表达，ATF和ATF和AP-1转录因子与CD4+CD25+T细胞的含量和fivergy和fiveration的表达相关。这些蛋白质表达的调节将建立调节厌食和FIV复制的蛋白质之间的因果关系。将对CD4+ CD25+激活和FIV储层的建立进行时间分析急性FIV感染的猫。这些研究应产生对维持Treg细胞厌氧状态的细胞因子和信号通路的见解，并有助于定义促进FIV复制的分子环境，以及在感染后激活CD4+ CD25+ Treg细胞多久就会激活并建立FIV储层。