A T-cell dormant state in the autoimmune process of nonobese diabetic mice treated with complete Freund's adjuvant.

Abstract

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% ...

Reversal of established autoimmune diabetes by restoration of endogenous β cell function

“…Three aspects of this treatment regimen appear to operate in parallel and in a synergistic manner: (a) Injection of CFA, and the consequent induction of TNF-α, results in the elimination of TNF-α-sensitive cells, which have been shown previously to transfer existing disease (28)(29)(30)(31); (b) the introduction of functional MHC class I peptide complexes expressed on the surface of either normal islet cells or normal lymphocytes results in partial but stable reselection of the T cell population of the NOD host, leading to an increase in the abundance of long-term memory T cells (6,12,23); and (c) suppression of hyperglycemia, although not obligatory, promotes the functional restoration of endogenous β cells or their precursors.…”

“…Adoptive transfer was performed as described (29). Recipient male NOD mice, 4-8 weeks of age, were irradiated (790 rads) with a 137 Cs source and injected intravenously within 2 hours of irradiation with donor splenocytes (2 × 10 7 viable cells) suspended in 0.25 ml of serum-free medium.…”

“…Successful therapy generated a subpopulation of nonpathologic but TNF-α-resistant T cells that exhibited an increased tendency to undergo cell death in culture (Figure 7a). Disease reversal, even 210 days after cessation of treatment, was thus associated with the persistent elimination of TNF-α-sensitive T cells, a population of cells with a demonstrated ability to play a role in disease (29,30). The permanent elimination of these formerly abundant TNF-α-sensitive lymphoid cells, presumably in response to TNF-α (and, perhaps, to other CFA-induced cytokines), was observed uniformly in successfully treated diabetic NOD mice.…”

“…The increased susceptibility to apoptosis of misselected T cells that result from improper education by MHC class I peptide complexes suggested that the production of TNF-α in vivo might promote the selective death of such poorly educated lineages (10,26,27). Furthermore, treatment of diabetic NOD mice or BB rats with CFA, an inducer of TNF-α production, both impairs the transfer of disease by T cells from these animals to naive hosts (28)(29)(30)(31) as well as prolongs the survival of syngeneic islet grafts in spontaneously diabetic hosts (2,32). We therefore hypothesized that CFA treatment might eliminate, at least temporarily, the autoreactive lymphoid cells of NOD mice by promoting their apoptosis, in part through the induction of TNF-α.…”

Thymic and Postthymic Regulation of Diabetogenic CD8 T Cell Development in TCR Transgenic Nonobese Diabetic (NOD) Mice

“…Transfer of diabetes by spleen cells was conducted by the method described previously (18). In brief, spleen cells (2 ϫ 10 7 ) from diabetic mice were injected i.v.…”