THE ROLE OF UROKINASE IN LUNG T LYMPHOCYTE RESPONSES

我们已经证明，uPA是有效的肺部防御体内新型隐球菌（C. neo）感染所必需的。宿主对新冠病毒的防御完全依赖于T细胞。在缺乏uPA的情况下，肺部CD4细胞的积累不能对感染做出反应，uPA阴性/阴性的动物死于播散性隐球菌病。我们发现，在缺乏uPA的情况下，T细胞损伤并不是C. neo特有的。在体内，uPA阴性/阴性小鼠对卡氏疟原虫的反应不能增加肺部CD4细胞，也不能清除感染。我们发现在缺乏uPA的情况下，T细胞的增殖反应严重受损。在体外，uPA负/负淋巴细胞不会发生抗原特异性增殖，也不会响应cd3交联或有丝分裂原产生的TCR介导信号而增殖。体内对t细胞特异性病原体的有效宿主防御在抗原、有丝分裂原或cd3交联诱导的体外T细胞增殖试验中，正常反应是由主要通过tcr -蛋白激酶级联介导的共同信号通路连接的，并与CD28和CD25传递的共刺激信号耦合。我们的假设是，在缺乏uPA的情况下，T淋巴细胞通过Jak/STAT的细胞内信号传导减少，导致细胞因子产生不足和细胞因子反应性降低。缺乏Jak/STAT信号导致T细胞活化和增殖受阻；T细胞对进一步的tcr介导的信号没有反应。1. 定位生化阻滞在T细胞信号级联中的作用。a)确定uPA的缺失是否会减弱TCR介导的信号传导。b)确定uPA的缺失是否会减少CD28和cd25介导的信号传导。c)确定uPA是否激活小鼠T淋巴细胞中的Jak/STAT信号。2. 确定异常T细胞信号传导的功能后果。a)通过活化抗原的表达、细胞因子的表达和对IL-12的反应来确定淋巴细胞活化对uPA的需求。b)确定uPA负/负淋巴细胞信号缺陷是否导致能量、细胞周期阻滞或细胞凋亡。3. 测定体外T细胞活化和增殖过程中uPA和uPAR的细胞表达和运输。确定重组uPA是否能逆转uPA阴性/阴性淋巴细胞的功能异常。

We have demonstrated that uPA is required for effective pulmonary defense against Cryptococcus neoformans (C. neo) infection in vivo. Host defense against C. neo is absolutely T cell dependent. In the absence of uPA, lung accumulation of CD4 cells fails to occur in response to infection, and the uPA minus/minus animals die from disseminated Cryptococcosis. We found that the T cell impairment in the absence of uPA is not specific to C. neo. In vivo, uPA minus/minus mice fail to increase pulmonary CD4 cells in response to P. carinii, and do not clear the infection. We have found that T cell proliferative responses are profoundly impaired in the absence of uPA. In vitro, uPA minus/minus lymphocytes do not undergo antigen-specific proliferation, nor do they proliferate in response to TCR- mediated signals generated by CD3-cross-linking, or by mitogen. Effective host defense to T-cell specific pathogens in vivo; and normal responses in vitro T cell proliferation assays induced by antigen, mitogen, or by CD3-cross-linking, are linked by a common signaling pathway mediated primarily through the TCR-protein kinase cascade, and coupled to co-stimulatory signals delivered by CD28 and CD25. It is our hypothesis that in the absence of uPA, T lymphocyte intracellular signaling via Jak/STAT is diminished leading to inadequate cytokine production and diminished cytokine responsiveness. The lack of Jak/STAT signaling results in blocked T cell activation and proliferation; and T cell unresponsiveness to further TCR-mediated signaling. 1. Localize the biochemical block(s) in T cell signaling cascades. a) Determine if the absence of uPA diminishes TCR- mediated signaling. b) Determine if the absence of uPA diminishes CD28- and CD25-mediated signaling. c) Determine whether uPA activates Jak/STAT signaling in murine T lymphocytes. 2. Determine the functional consequences of aberrant T cell signaling. a) Determine the requirement for uPA in lymphocyte activation as measured by expression of activation antigens, elaboration of cytokines, and responsiveness to IL-12. b) Determine whether the defects in uPA minus/minus lymphocyte signaling result in anergy, cell cycle arrest, or apoptosis. 3. Determine the cellular expression and trafficking of uPA and uPAR during T cell activation and proliferation in vitro. Determine if recombinant uPA can reverse the functional abnormalities in uPA minus/minus lymphocytes.