Effect of Glycosidases on the Fate of Transfused Lymphocytes.

Small lymphocytes circulate through the body by a unique route; they selectively emerge from the bloodstream in lymphoid tissue and recirculate to the blood via lymphatics. Thus, after radioactively labeled thoracic duct lymphocytes (syngeneic or allogeneic) are transfused intravenously, a relatively large percentage of the radioactivity soon accumulates in all lymphoid tissue of the recipient (except the thymus), and by radioautography, labeled small lymphocytes can be seen concentrated in the white pulp of the spleen, Peyer's patches, and lymph nodes. Subsequently, labeled lymphocytes can be recovered from the thoracic duct of the recipient. 1-7 In the present investigation thoracic duct lymphocytes were incubated in vitro with glycosidases prepared from Clostridium perfringens and the effect of this treatment on their fate in recipients was studied. Materials and Methods.-Animals: Transfusions were performed between male OsborneMendel rats except where noted. These animals are kept as a closed (but not highly inbred) stock. Donor rats weighed 250-300 gm, and recipients 125-175 gm. Collection and handling of P32-labeled lymphocytes: A donor rat was injected subcutaneously with about 50 ;moles of H3P3204 (300 p&c) in two equal doses at 12-hr intervals. Twelve hours after the second injection, the thoracic duct was cannulated by usual techniques.8 1 The animal was then placed in a Bollman cage and allowed free access to commercial rat cake and 5% dextrose in saline. The lymph was collected for 8-12-hr periods in 5 ml of saline, containing 20 units of heparin per ml, and kept at 00. Clots, when present, were removed by passing the lymph through a thin layer of cotton wool. Lymphocytes were separated from the lymph by centrifugation at 40 for 5 min at 150 X g. The supernatant fluid was discarded, and the sedimented cells were gently resuspended in 4.0 ml cold isotonic saline; the suspension was divided into 0.5-ml aliquots, and kept at 00 until used. After appropriate treatment, each sample was injected rapidly into the tail vein of a recipient lightly anesthetized with ether. Recipients in each experiment received the same number of cells from a single 8-12-hr collection period, but from experiment to experiment this number varied from 10 to 75 X 101 cells. The injections were completed within an hour after the cells were separated from the lymph, at which time at least 90% of the p82 activity was associated with the lymphocytes, and about 95% of the cells were viable as determined by motility under phase contrast microscopy or by uptake of nigrosin dye.' Assay of radioactivity in organs of recipient rats: At various intervals after injection, the recipient rats were killed by cervical dislocation, and the spleens (and in some instances other organs) were removed. The organs were minced, spread on planchets, and dried on a hot plate. Radioactivity was then assayed with an end-window gas-flow counter. The dried samples weighed approximately 0.2 gm. Preparation of glycosidases: The Clostridial enzyme fraction used in this study is similar to the glycosidases used by Morgan and his co-workers' 11 to release sugars from blood group substances. In a typical preparation, 4 liters of thioglycollate media (Difco) were inoculated with a culture of Clostridium perfringens (ATCC 10873) and growth continued for 72 hr at 37°. Bacteria were removed by centrifugation, and to the supernatant solution was slowly added an equal volume of acetone while it was cooled to 100. The precipitate was collected by centrifugation, dissolved in 50 ml of water, and (NH4)2S04 was then added to 70% saturation. The protein precipitate was redissolved in 8 ml of distilled water, and the resulting solution dialyzed overnight against distilled water. After removal of insoluble material by centrifugation, the dialyzed prep-