Purification and Characterization of an Inhibitor of Thymidine Uptake From Culture Supernatants of Human Tonsil Lymphocytes

Lymphocytes from human tonsils were cultured in the absence of serum for 3 days. In the presence of the concentrated culture supernatant the proliferative response of PBL, to con A, as measured by the uptake of (\u273)H-tdr, was significantly reduced. The suppressor substance was referred to as SMAL (suppressor of mitogen activated lymphocytes). The estimated molecular weight of SMAL under nondenaturing conditions was 100,000-300,000. SMAL also suppressed the incorporation of (\u273)H-tdr by a variety of mouse and human tumor cell lines. The activity of SMAL was sensitive to pronase and heating at 100(DEGREES)C for 30 minutes but insensitive to RNase. Treatment with DNase, however, enhanced the activity of SMAL. SMAL activity was also destroyed by treatment with 5% TCA, 0.4 M HCl or 60% acetonitrile, but resistant to 6 M urea or dialysis against pH 2 buffer for 24 hours. SMAL activity was precipitated in 40-80% ammonium sulfate saturation. When applied to a phenyl-sepharose column no activity was recovered. SMAL was not produced by heat-killed tonsil lymphocytes or lymphocytes-treated with cycloheximide. Maximal production occurred in the first 24 hours of culture, and progressively less was produced in subsequent 24-hour intervals. Both T- and B lymphocyte-enriched culture supernatants contained SMAL. SMAL adhered strongly to DEAE-cellulose, but less than two-fold purification was achieved. Using QMA-Accell anion exchange medium, a 5-fold purification of SMAL with higher specific activity was obtained with HPLC. Activity of SMAL was recovered after native polyacrylamide gel electrophoresis by electroblotting to DEAE-cellulose paper followed by eluting the bound materials with salt. Two active components, one corresponding to a large and/or less negatively charged molecule and another corresponding to a small and/or highly acidic molecule, were recovered. HPLC-purified SMAL at relatively low doses inhibited the uptake and phosphorylation of (\u273)H-tdr, without significant effect on cell proliferation. The inhibition of (\u273)H-tdr uptake was favored over that of (\u273)H-udr or (\u273)H-adr, and this effect was reversible. At relatively high doses of HPLC-purified SMAL, the growth of mouse thymoma EL-4 and human T cell leukemia CEM-CM(,3) cell lines was inhibited