SUPPRESSION OF NORMAL AND LEUKEMIC HEMOPOIESIS

几种人白血病T细胞系向培养基中释放一种蛋白质， 通过凝胶过滤测定的表观分子量为88 KD， 对正常和白血病祖细胞的抑制活性以及对 白血病细胞系 滴定研究表明， 细胞对该因子（集落抑制淋巴因子; CIL）的敏感性 在不同的造血谱系或在不同的阶段， 分化 从Jurkat T细胞纯化CIL至均一性 线表示SDS-PAGE上的MW为45 KD，pI为5.3。 理化 研究表明，这种因子是一种抑制性淋巴因子， 与已经描述的各种因素不同， CIL对造血靶细胞谱的影响表明，它可能在造血干细胞中起作用。 在正常和白血病造血下调中的关键作用 骨髓 本研究的目的是：1）确定生物化学 2）克隆CIL的cDNA并在大肠杆菌中表达， 合适的载体，3）表征CIL-靶细胞相互作用， 检查HLA-DR抗原在这种相互作用中的作用，以及4）检查 CIL在正常和白血病造血中的作用。 在蛋白纯化得到证实后，CIL的生物活性将 在体外进行检查，确定氨基酸序列和特异性 产生抗血清。 CIL的分子克隆将通过 筛选大肠杆菌cDNA表达文库。使用抗血清和/或 寡核苷酸探针或检测转染的COS-1的上清液 细胞的生物活性。 将检查CIL-靶细胞相互作用 使用125 I-CIL结合试验，并直接检查HLA-DR的作用 使用抗HLA-DR抗体。 更间接的作用的可能性 将使用以下方法检查CIL-靶细胞相互作用中的HLA-DR 易感靶点的HLA-DR阴性变体。 最后，新鲜的细胞 从白血病患者获得的细胞将用于克隆形成测定， 体外测定纯化的CIL对白血病克隆原细胞的作用， 并使用抗血清测定白血病患者的CIL水平。

Several human leukemic T cell lines release into the medium a protein with an apparent molecular weight by gel filtration of 88 KD with potent suppressive activity on normal and leukemic progenitor cells and on leukemic cell lines. Titration studies indicate that the sensibility sensitivity of the cells to this factor (colony-inhabiting lymphokine; CIL) varies in different hemopoietic lineages or at different stages of differentiation. Purification to homogeneity of CIL from the Jurkat T cell line indicates a MW on SDS-PAGE of 45 KD and a pI of 5.3. Physicochemical studies indicate that this factor is a suppressor lymphokine that is different from a variety of factors already described, and the activity of CIL on a spectrum of hemopoietic target cells suggests that it may play a critical role in the down-regulation of hemopoiesis in normal and leukemic bone marrow. The aims of the proposed research ar: 1) to define the biochemical characteristics of CIL, 2) to clone the cDNA of CIL and express it in suitable vectors, 3) to characterize the CIL-target cell interaction and examine the role of HLA-DR antigens in that interaction, and 4) to examine the role of CIL in normal and leukemic hemopoiesis. After protein purification has been confirmed, the bioactivity of CIL will be examined in vitro, the amino acid sequence determined and specific antisera generated. Molecular cloning of CIL will be accomplished through screening of a cDNA expression library in E. coli using antisera and/or oligonucleotide probes or testing of supernatants of transfected COS-1 cells for bioactivity. The CIL-target cell interaction will be examined using a 125I-CIL binding assay, and the role of HLA-DR examined directly using anti-HLA-DR antibodies. The possibility of a more indirect role of HLA-DR in CIL-target cell interaction will be examined using HLA-DR-negative variants of susceptible targets. Finally, cells freshly obtained from leukemic patients will be used in clonogenic assays to determine the effect of purified CIL in vitro on leukemic clonogenic cells, and the level of CIL in leukemia patients determined using antisera.