T regulatory cell suppression of CD8+ cell responses during FIV Infection

FIV 感染期间 T 调节细胞抑制 CD8 细胞反应

• 批准号: 7779473
• 负责人: Jonathan Fogle
• 金额: $ 11.21万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7779473
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Acute; Address; Animal Model; Animals; Antibodies; Antigens; Autologous; Binding; Blood; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell membrane; Cell physiology; Cell surface; Cells; Clinical; Clonal Anergy; Clonal Expansion; Coculture Techniques; Complement 3d Receptors; Cyclin-Dependent Kinase Inhibitor; Cyclin-Dependent Kinase Inhibitor Gene; Cyclin-Dependent Kinases; Data; Disease Progression; Effector Cell; Enzyme-Linked Immunosorbent Assay; Epitopes; Event; Failure; Family Felidae; Feline Immunodeficiency Virus; Felis catus; Flow Cytometry; Future; G1 Phase; Genes; Goals; HIV; HIV Antigens; HIV Infections; Helper-Inducer T-Lymphocyte; Human; IL2RA gene; Immune; Immune System Diseases; Immune response; Immunology; Immunosuppressive Agents; Incubated; Indium; Infection; Internal Medicine; Laboratories; Leishmania; Measures; Mediating; Membrane; Messenger RNA; Mus; Outcome; Patients; Peptides; Peripheral Blood Mononuclear Cell; Phenotype; Phosphorylation; Plasma; Plasmodium; Production; Proliferating; Regulatory T-Lymphocyte; Research; Research Personnel; Residencies; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Signal Pathway; Signal Transduction; Staging; Subfamily lentivirinae; Surface; T cell anergy; T-Lymphocyte; Testing; Therapeutic; Time; Transforming Growth Factor beta; Up-Regulation; Veterinary Medicine; Viral; Viral Antigens; Viral Load result; Virus; anergy; arm; design; experience; human subject; in vivo; pathogen; programs; receptor; research study; response

DESCRIPTION (provided by applicant): Candidate: Jonathan E. Fogle, DVM, has completed a residency in small animal internal medicine at NCSU and is board certified in Veterinary Internal Medicine. Dr. Fogle's short term goal is to obtain a doctoral degree from the Immunology program at NCSU. Dr. Fogle's long term goal is to continue independent lentiviral research that draws from both his clinical veterinary medicine and doctoral research experience. Proposal: A significant fraction of untreated HIV+ patients (progressors) are characterized by CD4+ and CD8+ T cell immune dysfunction. HIV-specific CD8+ effector cells are induced during the acute stage infection, but become non-responsive or "anergic" to antigenic stimulation shortly after induction. We propose that the CD8+ T cell immune dysfunction is the result of "clonal anergy" induced by membrane-bound TGF-beta (mTGF-beta) on virus activated CD4+CD25+ T reg cells that engage TGF-betaRII on the target cell. This hypothesis cannot be addressed in human subjects but rather requires a well-characterized experimental AIDS lentivirus animal model such as FIV. SPF cats will be infected with the NCSU1 isolate of FIV, plasma and PBMC/LN cells will be collected at various times post infection and quantitated for virus burden by ELISA and Real Time-PCR. PBMC/LN will be phenotyped for surface receptors by flow cytometry, and for Foxp3 mRNA by RT-PCR. Treg cells will be assessed for suppressor function by CD4+CD25+ cell depletion and by incubating FACS purified CD4+CD25+ cells with ConA and rFIVgag-stimulated CD8+ T cells and measuring IFNgamma by ELISpot. Ab-blocking experiments will be designed to test the hypothesis that Treg cells from FIV-infected cats anergize CD8+ T cells via mTGF-beta signaling. Purified CD4+CD25+ cells +/- anti-TGF-beta and purified CD8+ cells +/- anti-TGF-betaRII will be co-cultured and Treg suppressor function analyzed by measuring IFNgamma by ELISpot. Critical elements in the TGFbeta/ TGF-betaRII signaling pathway leading to suppression of CD8+ cell function will be analyzed, including Smad 2/3 phosphorylation, up-regulation of Foxp3 and Smad 7, and suppression of IFNgamma mRNA production. HIV is currently estimated to infect more than 37 million people world wide. FIV is a well established animal model of HIV/AIDS. The results of these experiments will have implications on future therapeutic strategies against HIV and FIV, and contribute to our current understanding of Treg cell function.

应聘者：乔纳森·E·福格尔，DVM，已完成NCSU小动物内科住院医师资格，并获得兽医内科委员会认证。福格尔博士的短期目标是在NCSU的免疫学项目中获得博士学位。福格尔博士的长期目标是继续利用他的临床兽医和博士研究经验进行独立的慢病毒研究。建议：很大一部分未经治疗的HIV+患者(进展期)的特征是CD4+和CD8+T细胞免疫功能障碍。HIV特异性CD8+效应细胞在感染的急性期被诱导，但在诱导后不久就对抗原刺激变得无反应或“无能”。我们认为CD8+T细胞免疫功能障碍是由膜结合型转化生长因子-β(mTGF-β)在病毒激活的CD4+CD25+T细胞上与转化生长因子-β受体结合所致的“克隆性无能”的结果。这一假设不能在人类受试者身上解决，而是需要一个具有良好特征的实验性艾滋病慢病毒动物模型，如FIV。将FIV NCSU1分离株感染SPF猫，在感染后不同时间采集血浆和PBMC/LN细胞，用ELISA法和Real-Time-PCR法测定病毒载量。用流式细胞术检测PBMC/LN细胞表面受体的表型，用RT-PCR检测Foxp3mRNA的表型。通过去除CD4+CD25+细胞，将FACS纯化的CD4+CD25+细胞与ConA和rFIVgag刺激的CD8+T细胞孵育，并用ELISpot检测IFNGamma，来评估Treg细胞的抑制功能。AB阻断实验将被设计来检验来自FIV感染猫的Treg细胞通过mTGF-β信号来激活CD8+T细胞的假设。将纯化的CD4+CD25+细胞+/-抗转化生长因子-β和纯化的CD8+细胞+/-抗转化生长因子-βRII与纯化的CD8+细胞+/-抗转化生长因子-βRII共培养，并通过ELISpot检测干扰素-γ来分析Treg抑制功能。将分析导致CD8+细胞功能抑制的TGFbeta/TGF-betaRII信号通路中的关键元件，包括Smad2/3磷酸化，上调Foxp3和Smad7，以及抑制IFNGamma mRNA的产生。据估计，目前全球有超过3700万人感染艾滋病毒。FIV是一种成熟的艾滋病毒/艾滋病动物模型。这些实验的结果将对未来对抗HIV和FIV的治疗策略产生影响，并有助于我们目前对Treg细胞功能的理解。