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

我的实验室在艾滋病毒/艾滋病领域的中心任务是开发技术，使直接 在体内测试与HIV感染的免疫发病机制和治疗相关的假说。在那个时候 这个奖项开始时，我们开发了一种稳定的同位素-质谱学技术来测量T细胞 人类的动力学，但有几个限制和问题无法解决。超过了 在过去的3-4年里，我的实验室一直在系统地推进技术，使更复杂的， 艾滋病毒致病机制的细微差别模型可以通过实验进行测试。技术进步一直是极大的 令人满意。通过结合我们的重水(2H2O)标记方法，大大降低了对细胞和 在多参数细胞分类方面，一些问题已经或现在可以得到解决。 识别长寿(“中央记忆”)和短寿的表面表型标记的协议 (“效应记忆”)T细胞已经建立。到目前为止测试的标记(例如CCR7、CD28)已经揭示 只是营业额略有不同。T细胞周转率作为病毒致病性预后指标的应用 已在早期艾滋病毒感染中进行了探索，慢性免疫激活的动力学后果已被 建立在小鼠模型上。动力学测量也被用来剖析局部的贡献。 抗原(Ag)的淋巴增殖性反应(LN)中克隆性扩增与细胞募集的比较 目的是为了更好地刺激T细胞的募集，并找出能具体抑制T细胞募集的药物。最后，从辅助方面看 HIV感染的免疫病理机制，如LN纤维化，现在是可以同时测量的，通过重 水标签。 在该项目的下一阶段，我们建议在这些方法进步的基础上解决 致病问题的数量。首先，将追求剩余的两项技术进步(减少电池 10-100个细胞所需的数量；以及将人类摄入重水所需的时间减少到 1-3天)。我们将继续检测不同的表面标志物(如整合素-a4b7、CD27、IL-7受体、IL-15R9、 和IL-18R)作为中枢和效应器记忆T细胞的标志物。一旦鉴定出来，这些种类就会被提纯 麦库恩实验室将进一步确定亚群的分子和功能特征。另外， 由于技术上的原因，现在可以表征抗原特异性T细胞和其他稀有亚群的动力学 在几个背景下注意到的进展：一)对黄热病疫苗的反应(与R.艾哈迈德)；二)艾滋病毒-GAG 未经治疗的血清阳性患者与HIV阴性患者中抗CMV特异性T细胞的比较 受试者(与R.Sekaly)；iii)对乙肝和流感疫苗的反应和T调节的动力学 细胞(M.McCune)；以及(Iv)以极低数量将T细胞克隆转移到小鼠体内的动力学(M.Jenkins)。 还将讨论两个重要的临床问题，使用T细胞动力学的这些微妙指标： HIV感染者在有效的抗逆转录病毒治疗10-15年后存在残余免疫异常 (与年龄匹配的血清阴性者，与S.Deek和P.Hunt相比)？而员工的流动率是否具有表型特异性？ T细胞亚群可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、有效接受抗逆转录病毒治疗或具有病毒学耐药性的艾滋病毒感染者，以及 T细胞亚群动力学标记物将与其他免疫激活指标进行比较。最后，动能 技术将应用于淋巴组织：在小鼠中建立CD62L-CD44光明T细胞的排泄和死亡 (W.Paul)；以及SIV感染中肠淋巴组织中T细胞和胶原(纤维化形成)的动力学(S. Dandekar)。 总而言之，量子！本奖项第一阶段在测量技术方面取得的进展 现在，让关于艾滋病毒免疫致病机制的有趣和基本的问题被直接解决。

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.