Oral administration of cholera toxin B-insulin conjugates protects NOD mice from autoimmune diabetes by inducing CD4(+) regulatory T-cells

Restoration of peripheral tolerance to target autoantigens during autoimmun e diseases has met with several limitations because of the limited efficacy of this approach in an already immune host. To optimize the induction of t olerance, we have shown that feeding insulin conjugated to cholera toxin B- subunit (CTB), a potent mucosal adjuvant, reduced by 5,000 the amounts of a ntigen necessary for delaying diabetes onset in NOD mice. To analyze these protective mechanisms, we have performed cotransfer experiments using splen ocytes from young females fed once with 10 mu g of CTB-insulin, mixed with diabetogenic T-cells, and intravenously injected into irradiated syngeneic male recipients, We demonstrated that the delayed onset of diabetes relied on CD4(+) T-cells. We studied the cytokine production from plate-bound anti -CD3-stimulated cells. Higher interleukin (IL)-4 amounts were observed in b oth splenocytes and pancreatic lymph node (PLN) cell cultures from CTB-insu lin-fed mice as soon as 4 h after the Feeding. An increase in the levels of transforming growth factor-beta was seen after 24 h only in the mesenteric lymph nodes (MLN). In both of these organs, a reduction of gamma-interfero n (IFN-gamma) production occurred after CTB-insulin treatment, at 24 h in t he PLN and at 7 days in the MLN. Reverse transcription-polymerase chain rea ction analysis indicated an increase in the level of IL-4 and a reduction i n IFN-gamma transcripts in the PLN of mice treated orally with CTB-insulin and of the recipients of regulatory T-cells. Using different strains of con genic NOD mice at the Thy1 locus, me showed that protection was associated with the accumulation of T-cells from CTB-insulin-fed mice in the lymph nod es Tram draining sites containing functional islets, i.e., the PLN in norma l mice and the renal lymph nodes after a syngeneic islet graft under the ki dney capsule of streptozotocin-treated mice. Taken together, our results cl early indicate that oral administration of CTB-insulin conjugates in NOD mi ce produced a shift from a T-helper type 1 to a type 2 profile with the ind uction of antigen-specific regulatory CD4(+) T-cells in the vicinity of the mucosal barrier and close to the inflamed islets.