Investigations during the past year have focused on purification of the natural human cytokine leukoregulin for amino acid sequencing and on expression of the gene(s) coding for leukoregulin. Biologically active leukoregulin has been further purified by addition of reverse phase chromatography to the ion-exchange, isoelectric focusing and molecular sizing chromatographic procedure previously developed in the laboratory. The most homogeneous leukoregulin is obtained following phorbol myristate acetate stimulation of human peripheral blood leukocytes but is secreted at only 2.5% the rate of lectin-induced leukoregulin. Several recently established human B lymphocyte cell lines have been identified which constitutively secrete leukoregulin at a rate similar to phorbol myristate acetate-induced lymphocytes and which may be useful for obtaining sufficient cytokine for amino acid sequencing. Transfection of pools of 350-500 clones each from a cDNA library constructed from mRNA extracted from lectin-stimulated lymphocytes into COS7 cells demonstrates leukoregulin expression by 0.05% of the 500,000 cDNA clones examined. The quality of the cDNA library has been validated by showing that it contains genes for IL-6 and lymphotoxin. The transfection procedure has been validated by demonstrating IL-6 and lymphotoxin secretion following transfection of a cloned IL-6 or lymphotoxin gene into the COS7 cells. A subtracted cDNA library from the lectin stimulated lymphocytes using mRNA from paired non-stimulated lymphocytes has been prepared to enrich for the gene coding for leukoregulin. Diagnostic probing of the subtracted library shows that both IL-6 and lymphotoxin which were expressed by the non-stimulated and stimulated cells have been eliminated from the subtracted library. IL-9 which was present only in the stimulated cDNA library remains in the library after subtraction by the non-stimulated library. This indicates the quality of the subtracted library for cloning of inducible lymphocyte genes important in the ability of the host to recognize and eliminate cells during their transition to neoplasia.
