Testing in vivo of HIV-immunopathogenic hypotheses using new kinetic techniques

使用新的动力学技术在体内测试 HIV 免疫致病假说

• 批准号: 8242831
• 负责人: MARC Kopel HELLERSTEIN
• 金额: $ 45.12万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242831
• 关键词: Address; Age; Anti-Retroviral Agents; Antigens; Award; B-Lymphocytes; Behavior; CD28 gene; CD44 gene; CD8B1 gene; Cell Count; Cell Cycle Kinetics; Cell Separation; Cells; Cessation of life; Chronic; Clinical; Clonal Expansion; Collagen; Complex; Cytomegalovirus; Deuterium Oxide; Gagging; Gene Expression; Gut associated lymphoid tissue; HIV; HIV Infections; HIV-1; Hepatitis B; Human; Immune; Infection; Integrins; Interleukin-7; Kinetics; Label; Laboratories; Life; Long-Term Survivors; Longevity; Lymphoid Tissue; Macaca; Measurable; Measurement; Measures; Memory; Metric; Mission; Modeling; Mus; Pathogenesis; Pathogenicity; Patients; Persons; Phase; Principal Investigator; Protocols documentation; Regulatory T-Lymphocyte; Residual state; Resistance; SELL gene; SIV; Sorting - Cell Movement; Surface; T memory cell; T-Lymphocyte; Techniques; Technology; Testing; Time; Vaccination; Viral; Work; Yellow Fever; Yellow Fever Vaccine; fibrogenesis; immune activation; in vivo; influenza virus vaccine; interest; lymph nodes; mouse model; prognostic; programs; quantum; receptor; response; stable isotope

The central mission of my laboratory in the field of HIV/AIDs is to develop techniques that allow direct testing in vivo of hypotheses relating to the immunopathogenesis and therapy of HIV infection. At the time that this award started, we had developed a stable isotope-mass spectrometric technique for measuring T-cell dynamics in humans, but there were several limitations and questions that could not be addressed. Over the past 3-4 years, my laboratory has worked systematically to advance the technology so that more complex, nuanced models of HIV pathogenesis can be tested experimentally. Technical progress has been extremely satisfying. By combining our heavy water (2H2O) labeling approach, greatly reduced requirements for cells and multiparameter cell sorting, a number of questions have been or can now be addressed. A protocol for identifying surface phenotypic markers of long-lived ("central memory") and short-lived ("effector memory") T-cells has been established. The markers tested so far (e.g., CCR7, CD28) have revealed only modest differences in turnover. The utility of T-cell turnover as a prognostic metric of viral pathogenicity has been explored in early HIV-infection and the kinetic consequences of chronic immune activation have been established in a mouse model. Kinetic measurements were also used to dissect out the contributions from local clonal expansion vs. cell recruitment into lymph nodes (LN), in the lymphoproliferative response to antigen (Ag) stimulation and to identify agents that specifically inhibit T-cell recruitment. Finally, ancillary aspects of the immunopathogenesis of HIV-infection, such as LN fibrogenesis, are now measurable concurrently, by heavy water labeling. In the next phase of this project, we propose to build on these methodologic advances to address a number of pathogenic questions. First, two remaining technical advances will be pursued (reducing cell numbers required to 10-100 cells; and reducing the time required for heavy water administration in humans to 1-3 days). We will continue to test different surface markers (e.g., integrin-a4b7, CD27, IL-7 receptor, IL-15R9, and IL-18R) as markers of central vs effector memory T-cells. Once identified, these sort-purified subpopulations will be further characterized molecularly and functionally, with the McCune lab. Also, the kinetics of Ag-specific T-cells and other rare subpopulations can now be characterized, due to the technical advances noted, in several settings: i) the response to yellow fever vaccination (with R. Ahmed);ii) HIV-gag specific T-cell kinetics in untreated seropositive patients, compared to anti-CMV-specific T-cells in HIVnegative subjects (with R.Sekaly); iii) response to hepatitis B and influenza vaccine and kinetics of T-regulatory cells (M.McCune); and iv) Kinetics of clones of T-cells transferred into mice in very low numbers (M. Jenkins). Two important clinical questions will also be addressed, using these subtle metrics of T-cell dynamics: are there residual immune abnormalities in HIV-infected person after 10-15 years of effective ARV therapy (compared to age matched seronegatives, with S.Deeks and P. Hunt)? And can the turnover of phenotypicallyspecific T-cell subpopulations serve as a marker of immune activation or viral pathogenicity? For the latter, HIV-infected subjects who are untreated, effectively ARV treated, or virologically resistant will be studied, and T-cell subpopulation kinetic markers will be compared to other metrics of immune activation. Finally, the kinetic techniques will be applied to lymphoid tissue: establish egress vs death of CD62L-CD44 bright T-cells in mice (W. Paul); and kinetics of T-cells and collagen (fibrogenesis) in gut lymphoid tissue in SIV-infection (S. Dandekar). In summary, the quanta! advances in measurement technology achieved in the first phase of this award now allow interesting and fundamental questions about HIV-immunopathogenesis to be addressed directly.

我的实验室在HIV/AIDS领域的中心使命是开发技术， 在体内测试与HIV感染的免疫发病机制和治疗相关的假设。的时候 从这个奖项开始，我们已经开发了一种稳定的同位素质谱技术来测量T细胞 人类的动力学，但有几个限制和问题无法解决。来 在过去的3 - 4年里，我的实验室系统地致力于推进技术，使更复杂， HIV发病机理的细微差别模型可以通过实验进行测试。技术进步已经非常 令人满意。通过结合我们的重水（2H2O）标记方法，大大降低了对细胞的要求， 在多参数细胞分选中，许多问题已经或现在可以得到解决。 用于鉴定长寿命（"中央记忆"）和短寿命（"记忆"）的表面表型标记的方案 （"效应记忆"）T细胞已经建立。到目前为止测试的标记物（例如，CCR7，CD28）已经揭示了 只有轻微的营业额差异。T细胞更新作为病毒致病性预后指标的实用性 已经在早期HIV感染中进行了探索，慢性免疫激活的动力学后果已经被 在小鼠模型中建立。动力学测量也被用来剖析出的贡献，从本地 在对抗原（Ag）的淋巴增殖反应中，克隆扩增与淋巴结（LN）中的细胞募集 刺激和鉴定特异性抑制T细胞募集的试剂。最后， HIV感染免疫发病机制，如LN纤维形成，现在可通过重 水标签 在本项目的下一阶段，我们建议在这些方法学进展的基础上， 致病问题的数量。首先，将追求两个剩余的技术进步（减少电池 将所需的细胞数量减少到10 - 100个细胞;以及减少对人体施用重水所需的时间， 1-3天）。我们将继续测试不同的表面标记（例如，整联蛋白-a4b7，CD27，IL-7受体，IL-15R9， 和IL-18 R）作为中枢与效应记忆T细胞的标志物。一旦被识别，这些分类纯化的 亚群将进一步表征分子和功能，与McCune实验室。还 由于技术原因，现在可以表征银特异性T细胞和其他罕见亚群的动力学特征 注意到的进展，在几个设置：i）黄热病疫苗接种的反应（与R。（二）艾滋病毒-gag 未治疗的血清阳性患者中的特异性T细胞动力学，与HIV阴性患者中的抗CMV特异性T细胞相比 受试者（使用R. Sekaly）; iii）对B型肝炎和流感疫苗的应答以及T-调节性 细胞（M.McCune）;和iv）以极低数量转移到小鼠中的T细胞克隆的动力学（M. Jenkins）。 两个重要的临床问题也将得到解决，使用这些微妙的T细胞动力学指标： HIV感染者经过10 - 15年有效的抗逆转录病毒治疗后， （与S. Deeks和P. Hunt的年龄匹配血清阴性结果相比）？表型特异性的基因转换 T细胞亚群作为免疫激活或病毒致病性的标志？对于后者， 将研究未经治疗、接受有效ARV治疗或病毒学耐药的HIV感染受试者， 将T细胞亚群动力学标志物与免疫活化的其他指标进行比较。最后，动力学 技术将应用于淋巴组织：建立小鼠中CD62L-CD44亮T细胞的排出与死亡 （W. Paul）;以及SIV感染中肠道淋巴组织中T细胞和胶原蛋白（纤维形成）的动力学（S。 Dandekar）。 总之，量子!本奖项第一阶段取得的测量技术进步 现在可以直接解决有关HIV免疫发病机制的有趣和基本问题。