T CELL HYBRIDOMA LYMPHOKINE CHEMOTACTIC FOR T LYMPHOCYTE

局部T淋巴细胞聚集被认为是肿瘤发生的重要因素 排斥反应，可能预示着局部自身免疫性疾病和排斥反应， 移植器官 T淋巴细胞聚集的机制 初始抗原刺激位点并不明确，但已知 抗原刺激的T细胞产生淋巴因子， 对其他T细胞的趋化性。 有人提出，几种不同的淋巴因子，特别是吸引 辅助/诱导或抑制/细胞毒性T细胞可在 不同的时间，可能是不同的T淋巴细胞亚群 在局部炎症反应的整个过程中。 进一步应 这些淋巴因子可能在决定淋巴细胞增殖中起重要作用。 淋巴细胞亚群在局部炎症反应中积聚 从而调节免疫反应。 一种T细胞杂交瘤FS730.11，其产生大量T细胞， 特异性淋巴细胞趋化因子（LCF），但范围有限 其他淋巴因子的研究已经被开发出来。 Con A刺激的上清液 FS730.11的培养物将用作来自单细胞的LCF来源 类型来定义是否辅助或抑制T细胞亚群响应 这个特殊的LCF。 辅助和抑制T细胞亚群将 通过将尼龙毛非粘附性小鼠脾细胞接种在大鼠上获得 单克隆抗Lyt 1或Lyt 2抗体包被的塑料组织培养物 板. 将通过流式细胞仪评估细胞亚群的纯度。 细胞计数法。 这些T细胞亚群响应于LCF的趋化性， 然后通过通过8微米孔径的迁移来评估 改良Blindwell Boyden室中的硝酸纤维素过滤器。 将半纯化的LCF注射到小鼠的腹膜中。 的 腹膜渗出液细胞将被定量，通过苏木精-伊红染色区分， 流式细胞术检测T淋巴细胞。 LCF的纯化将通过连续超滤开始， 高效液相色谱法（HPLC）。 将描述LCF的特征 通过对几种酶失活的敏感性，通过圆盘凝胶 电泳，通过等电聚焦和通过柱色谱法。 将在大鼠中产生纯化的小鼠LCF抗体。 内在的， 放射性标记的LCF将通过Con A刺激FS730.11产生， 组织培养和放射免疫测定（RIA）的LCF开发。 这个RIA 将促进未来的实验，以确定T细胞亚群 LCF的来源和LCF生产的动力学。 大鼠B细胞杂交瘤 分泌抗-LCF抗体，且来自B抗体 细胞杂交瘤将在未来的实验中使用，以研究 通过抑制体内LCF活性来抑制免疫应答。

Local T lymphocyte accumulation is thought to be important for tumor rejection and may presage local autoimmune disease and rejection of transplanted organs. The mechanisms of T lymphocyte accumultion at the site of initial antigen stimulation are not well defined, but it is known that antigen stimulated T cells produce a lymphokine(s) that is (are) chemotactic for other T cells. It is proposed that several different lymphokines that specifically attract either helper/inducer or suppressor/cytotoxic T cells may be released at different times and perhaps by different T lymphocyte subpopulations throughout the course of a local inflammatory response. Further, it is probable that these lymphokines play a significant role in determining the lymphocyte subpopulations that accumulate in a local inflammatory response and thereby, modulate the immune response. A T cell hybridoma, FS730.11, that produces large amounts of T cell specific lymphocyte chemotactic factor (LCF), but only a restricted range of other lymphokines has been developed. Con A stimulated supernate from cultures of FS730.11 will be used as a source of LCF from a single cell type to define whether helper or suppressor T cell subpopulations respond to this particular LCF. Helper and suppressor T cell subpopulations will be obtained by plating nylon wool non-adherent, mouse spleen cells on rat monoclonal anti-Lyt1 or Lyt2 antibody-coated plastic tissue culture plates. The purity of cell subpopulations will be assessed by flow cytrometry. Chemotaxis of these T cell subpopulations, in response to LCF, will then be assessed by migration through 8 micron pore size nitrocellulose filters in modified blindwell Boyden chambers. Semi-purified LCF will be injected into the peritoneum of mice. The peritoneal exudate cells will be quantified, differentiated by hematoxylin staining, and evaluated by flow cytometry for T lymphocyte. Purification of LCF will be initiated by sequential ultrafiltration and high performance liquid chromatography (HPLC). LCF will be characterized by sensitivity to inactivation with several enzymes, by disc gel electrophoresis, by isoelectric focusing and by column chromatography. Antibody to purified mouse LCF will be produced in rats. Intrinsically, radiolabelled LCF will be produced by Con A stimulation of FS730.11 in tissue culture, and a radioimmunoassay (RIA) for LCF developed. This RIA will facilitate future experiments to determine the T cell subpopulation(s) source of LCF and the kinetics of LCF production. A rat B cell hybridoma that secretes anti-LCF antibody will be produced, and antibody from that B cell hybridoma will be used in future experiments to study modulation of the immune response by inhibiting LCF activity in vivo.