SUPPRESSION OF NORMAL AND LEUKEMIC HEMOPOIESIS

几个人类白血病T细胞株向培养液中释放一种含有 凝胶过滤后的表观分子量为88kD，具有较强的 对正常和白血病祖细胞的抑制活性及其他 白血病细胞系。滴定研究表明，敏感性 细胞对该因子的敏感性(集落寄生淋巴因子；CIL) 在不同的造血谱系或在不同的阶段 差异化。Jurkat T细胞CIL的纯化及其同源性 LINE在SDS-PAGE上显示相对分子质量为45kD，等电点为5.3。物理化学 研究表明，这种因子是一种抑制淋巴因子， 不同于已经描述的各种因素，以及 CIL在一系列造血靶细胞上的表达表明，它可能在 正常和白血病患者造血功能下调中的关键作用 骨髓。 本研究的目的是：1)定义生物化学 CIL的特性：2)克隆CIL基因并在原核表达系统中表达 合适的载体，3)表征CIL-靶细胞相互作用和 检查人类白细胞抗原-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 在蛋白质纯化得到确认后，CIL的生物活性将 在体外进行检测，确定氨基酸序列并具有特异性 产生了抗血清。CIL的分子克隆将通过以下途径完成 用抗血清和/或抗血清筛选大肠杆菌中的cDNA表达文库 转染COS-1的寡核苷酸探针或上清液检测 用于生物活性的细胞。我们将研究CIL与靶细胞的相互作用 用125I-CIL结合实验，直接检测了人类白细胞抗原-DR的作用 使用抗人类白细胞抗原-DR抗体。更间接地发挥作用的可能性 CIL-靶细胞相互作用中的HL-DR将用 易感靶点的HLA-DR阴性变异体。最后，新鲜的细胞 从白血病患者身上获得的信息将用于克隆分析 体外测定纯化的CIL对白血病克隆形成细胞的影响 用抗血清检测白血病患者的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.