lymphocytes from diabetic mice into male NOD mice transferred the diabetic state, but this transfer was markedly suppressed when the recipients were treated with CFA. In all three cases the spleen cells from the normoglycemic mice treated with CFA induced diabetes when transferred into NOD male mice. CFA, therefore, induces a state of T-cell dormancy, in which the islets are no longer subject to an immune attack.The autoimmune diabetic process of the nonobese diabetic (NOD) strain of mice is easily suppressed by a variety of inflammatory and/or immunological stimuli (1-7). These stimuli have ranged from infections with viruses or intracellular pathogenic bacteria to injection of complete Freund's adjuvant (CFA) (6, 7). Perhaps the effect of these different stimuli is to release the acute cytokines-such as interleukin-1 (IL-1), interleukin-6, or tumor necrosis factor (TNF) that, in some way, modulate the autoimmune state. Indeed, there are reports showing the ameliorating effect of repeated injections of TNF (8, 9) or IL-1 (9) on the diabetic state of NOD mice. Intrigued by these surprising findings, we now attempt to define the status of the T cells in CFA-treated NOD mice that do not exhibit diabetes.
MATERIALS AND METHODSMice. Male NOD mice were bred in our own colony under specific pathogen-free conditions or purchased from Taconic (Germantown, NY). Female NOD mice were bred in our own colony. After reaching 16 weeks of age female mice were tested weekly for diabetes by analysis of urine glucose with Chemstrips-UG (Boehringer Mannheim). Blood glucose levels of >300 mg/dl usually were confirmed on the day of sacrifice. Blood glucose levels were measured on a Beckman glucose analyzer or an Ames Glucometer II on blood collected in heparinized capillary tubes from the retroorbital area.CFA. CFA (Bacto adjuvant H37-RA from Difco) was prepared by emulsification with an equal volume of phosphate-buffered saline.Experimental Conditions. Three sets of experimental conditions were evaluated. In each the diabetic process was inhibited by CFA injections; subsequently, lymphocytes were harvested from the spleens and transferred i.v. into male nondiabetic NOD mice that had received 790 R (1 R = 0.258 mC/kg) from a '37Cs source (Gammacell 40, Atomic Energy, Ottawa)].(i) The first condition consisted of NOD female mice that received CFA at 7-9 weeks of age (50 .ld i.p. and 100 ILI s.c.on the base of the tail).(ii) The second condition consisted of diabetic NOD that received islet transplants from male, nondiabetic NOD mice, followed by CFA (either 200 .ul i.p. or 50 juI i.p. and 100 dul s.c.). The pancreatic islets of Langerhans were isolated by the method of Lacy and Kostianovsky (10) from 8-to 10-week-old male NOD mice. Briefly, islets were isolated from NOD pancreas by collagenase digestion and centrifugation over a discontinuous Ficoll gradient, cultured for 7 days at 240C or 370C in CMRL medium (GIBCO) adjusted to 150 mM glucose and supplemented with glutamine, penicillin at 100 pug/ml, streptomycin at 50,ug/ml, and 5% ...