SUPPRESSION OF NORMAL AND LEUKEMIC HEMOPOIESIS

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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.