Metabolic Time Course and Immunologic Concomitants of Adoptive Transfer of Type I Diabetes in the BB Rat zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP Jean-Francois Yale, Thomas A. Seemayer, and Claire Vigeant The metabolic and cellular immune changes during adoptive transfer of type I diabetes mellitus in the BB rat were examined. Concanavalin A (Sigma Chemical Co, St Louis) stimulation of acutely diabetic BB rat splenocytes increased the la-positive cells, but no other lymphocyte subset. Each spleen cell preparation was divided into two and injected into two separate recipients. Thirty-day-old diabetes-prone BB rats received these splenocytes intravenously (62 t 6 x IO* cells, n = 30) or buffer alone (controls, n = 14). Seventy-seven percent of the cell-injected rats became diabetic before 60 days of age, 15 f 1 days after injection. They were glucose intolerant two to three days before onset, with normal fasting glucose. All controls maintained normal glucose tolerance. The morphology revealed intense insulitis in all the rats that became diabetic, and its absence in all the controls. Eighty-three percent of the spleen cell preparations produced the same outcome in both recipients. The cell-injected rats had an increase in lymphocyte counts eight days after injection compared with the controls. The most affected subsets were the pan T cells (OX1 9+ 1 and helper T cells (W3/25+ 1. While the rats that ultimately became diabetic had a decrease of their lymphocyte subsets to control levels between eight and 14 days, the injected rats that maintained normal glucose tolerance maintained elevated T cells. We conclude that (1) adoptive transfer of diabetes occurs in the presence of an increase of the helper T (W3/25+) lymphocytes after spleen cell injections: (2) glucose intolerance precedes by two to three days fasting hyperglycemia; and (3) while the lymphocyte counts are increased in all recipients of splenocyte preparations, these counts decrease rapidly only in the rats that develop diabetes, possibly by entrapment of lymphocytes in the insulitis. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA @ 1988 by Grune & Stratton, Inc. S PONTANEOUS type I insulin-dependent diabetes mel- litus in the BB rat shows many analogies to the human condition at the clinical, metabolic, pathological and immu- nologic levels.‘v2In 30% to 90% of rats from diabetes-prone litters, there is the appearance at 60 to 140 days of age of hyperglycemia, glycosuria, ketonuria, and weight loss, lead- ing rapidly to death unless insulin therapy is instituted. An insulitis is present prior to and at the time of onset. The majority of the mononuclear cells identified in the insulitis are macrophages and activated T cells3 The syndrome is associated with the RTI” MHC haplotype, and a severe lymphopenia (40% to 50% of normal) is nearly always present,4-‘2 affecting particularly the T cells. The passive transfer of insulitis to athymic nude mice, with intensity related to dose of transferred spleen and blood lymphocytes, has been shown 13*14 as has been the transfer of insulitis to BB recipients.” While the passive transfer of insulitis to athymic nude mice may suggest that the BB immune insulitis is not MHC-restricted, the MHC restric- tion of the autoimmune diabetes remains controversial.‘6’8 Koevary et al demonstrated an earlier-than-expected appearance of the overt syndrome in diabetes-prone BB rats by transfer of spleen cells previously incubated with Concan- avalin A.19 The passive transfer of diabetes to immunosup- pressed non-diabetes-prone rats also has been achieved.20V2’ Passive transfer of diabetes has been used as evidence of the autoimmune nature of the insulin-dependent diabetes in the BB rat. The passive transfer of a process that leads to /3 cell destruction and diabetes can also be a powerful probe for the study of diabetes. The identity of the effector cells and the immune alterations required to prevent their P-cell destruc- tive process could be studied using this model. However, a better comprehension of the events occurring when using unseparated cells in the absence of immunotherapy is first required. As a first step, we therefore describe in this study the metabolic time course and the cellular immune changes Mefabolism, Vol37, No 11 (November), 1988: pp 1015-1020 associated with the adoptive transfer of diabetes in the BB rat using unseparated Concanavalin A-activated spleen cells from acutely diabetic BB rats. MATERIALS AND METHODS Animals Litters of young 25 to 30-day-old diabetes-prone BB rats and acutely diabetic BB rats (less than three days after onset) were obtained from Dr Pierre Thibert (Animal Resources Division, Health and Welfare Canada, Ottawa). The rats were kept in metabolic cages in laminar flow hoods in humidity- (70%) and temperature- (20°C) controlled rooms, with 12-hour light cycles. They were fed ad libitum (Purina rat chow, Ralston Purina Co, St Louis), except for periods of fasting prior to oral glucose tolerance tests. Spleen Cell Injections The diabetic rats (n = 18) were killed under ether anesthesia by exsanguination through cardiac puncture, and their plasma was stored. Under sterile conditions, the spleen was carefully dissected and flushed with RPM1 culture solution (RPM1 1640 containing 5% FCS, 100 U/mL penicillin, 5 mmol/L L-Glutamine, and 5 x IO-’ From the McGill Nutrition and Food Science Centre and Depart- ment of Pathology, McGill University, Montreal. Supported by grants from the Medical Research Council of Canada (MA 9275) and the Juvenile Diabetes Fowzdation (385336). Jean-Francois Yale is the recipient of a scholarship from Diabeies Canada. Presented in part at the 22nd Annual Meeting of the European Association for the Study of Diabetes. Diabetologia (1986) 29.6084. Address reprint requests to Jean-Franqois Yale, MD, McGill Nutrition and Food Science Centre. Royal Victoria Hospital, 687 Pine Ave W, Montreal, QC, Canada H3A I Al. Q I988 by Grune & Stratton, Inc. 0026-0495/88/371 l-0003%03.00/0 1015