SINGLE CELL ANALYSIS OF T CELL RESPONSES TO ANTIGEN

T细胞介导的免疫应答是免疫系统行为的净结果。 T细胞区室中的单个细胞。TCR转基因小鼠的应用 已经使得能够鉴定抗原特异性细胞， 淋巴细胞群，用于测定细胞周期状态，活化 表型和活力。这些研究取得了重大进展，但 由于不能检查效应器功能， 增殖和细胞因子表达。最近我们 已经调整和改进了方法来跟踪增殖历史， 在群体流式细胞术内的单个细胞。我们的研究表明 TCR和CD 28介导共刺激作用协同增加 进入增殖池的细胞的频率， 有丝分裂的过程。令人惊讶的是，即使在最高水平的TCR 加上足以激活95%群体的共刺激信号， 如果T细胞不分裂但仍能存活的话，大约有40%。当被隔离时， 流式细胞术和再刺激，这些细胞表现出无反应性行为 （对再刺激难治，但可用IL-2挽救）。 这些研究提出了一系列有趣的问题，我们将 地址.为了做到这一点，我们已经调整了我们的系统，以与流式细胞仪一起使用。 细胞内细胞因子的检测和细胞表面的检测 转基因TCR。目标1将使用体外研究来定义参数 其决定进入增殖池的细胞的频率， 重点关注前体百分比、T细胞：APC比率、APC类型以及 共刺激和关闭信号的可用性。本文将分析 细胞周期状态和非增殖细胞的检查点。目标#2将 确定细胞因子分泌（定性或定量）是否 与增殖史有关研究将在体外进行， 使用体内过继转移技术。目标3将询问TCR和 CD 28信号在体外和体内影响细胞存活。这些研究 还将使用CD 28-T细胞系转基因小鼠， 可诱导的细胞存活基因bcl-x，使我们能够单独研究 CD 28刺激对细胞因子和bcl-x的诱导作用 表情这些研究应该提供重要的见解， 控制T细胞反应，并协助制定战略， 调节免疫系统并诱导耐受性。我们将与博士。 门罗的项目研究了B细胞反应过程中的类似参数。 博士格林的项目将描述无反应期间的生化事件， 我们将把他的发现应用到我们的系统中， 主要无反应。

T cell mediated immune responses are the net result of the behavior of individual cells in the T cell compartment. The use of TCR transgenic mice have enabled the identification of antigen-specific cells within a lymphocyte population, for determination of cell cycle status, activation phenotype, and viability. These studies have made important advances, but have been limited by the inability to examine effector functions such as proliferation and cytokine expression on a single cell basis. Recently, we have adapted and refined methods to track the proliferative history of individual cells within a population flow cytometry. Our studies indicate that TCR and CD28-mediated costimulation serve to cooperatively increase the frequency of cells which enter the proliferative pool, and the number of mitoses they undergo. Surprisingly, even with maximal levels of TCR plus co-stimulatory signals sufficient to activate 95% of the population, roughly 40% if T cells never divide yet remain viable. When isolated by flow cytometry and restimulated, these cells exhibit anergic behavior (refractory to restimulation but rescuable with IL-2). These studies raise a series of interesting questions which we will address. To do so we have adapted our system for use with flow cytometric detection of intracellular cytokines and cell surface detection of a transgenic TCR. Aim #1 will use in vitro studies to define the parameters which determine the frequency of cells which enter the proliferative pool, focusing on precursor percentage, T cell:APC ratio, APC type, and the availability of co-stimulatory and off signals. This aim will analyze the cell cycle status and checkpoint of non-proliferative cells. Aim #2 will determine if cytokine secretion (either qualitative or quantitative) is linked to proliferative history. Studies will be performed in vitro and using in vivo adoptive transfer techniques. Aim #3 will ask how TCR and CD28 signals influence cell survival in vitro and in vivo. These studies will also use mice transgenic (on the T cell lineage) for the CD28- inducible cell survival gene bcl-x to permit us to separately study the role(s) of the inductive effects of CD28 stimulation on cytokine and bcl-x expression. These studies should provide important insights into the control of T cell responses and assist in the development of strategies to modulate the immune system and induce tolerance. We will work with Dr. Monroe, whose project examines similar parameters during B cell responses. Dr. Greene's project will characterize biochemical events during anergy, and we will apply his findings to our system to examine the basis of primary non-responsiveness.