SummaryCentral nervous system (CNS) immune privilege is an experimentally defined phenomenon. Tissues that are rapidly rejected by the immune system when grafted in sites, such as the skin, show prolonged survival when grafted into the CNS. Initially, CNS immune privilege was construed as CNS isolation from the immune system by the blood-brain barrier (BBB), the lack of draining lymphatics, and the apparent immunoincompetence of microglia, the resident CNS macrophage. CNS autoimmunity and neurodegeneration were presumed automatic consequences of immune cell encounter with CNS antigens. Recent data have dramatically altered this viewpoint by revealing that the CNS is neither isolated nor passive in its interactions with the immune system. Peripheral immune cells can cross the intact BBB, CNS neurons and glia actively regulate macrophage and lymphocyte responses, and microglia are immunocompetent but differ from other macrophage/dendritic cells in their ability to direct neuroprotective lymphocyte responses. This newer view of CNS immune privilege is opening the door for therapies designed to harness autoreactive lymphocyte responses and also implies (i) that CNS autoimmune diseases (i.e. multiple sclerosis) may result as much from neuronal and/or glial dysfunction as from immune system dysfunctions and (ii) that the severe neuronal and glial dysfunction associated with neurodegenerative disorders (i.e. Alzheimer's disease) likely alters CNSspecific regulation of lymphocyte responses affecting the utility of immune-based therapies (i.e. vaccines).

Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inf lammatory immune reactions against self-or non-self-antigens. Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease. We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals. We now report that feeding small amounts (2-20 g) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice. The protective effect could be transferred by T cells from CTBinsulin-treated animals and was associated with reduced lesions of insulitis. Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration. These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.

Homeostasis of T cell numbers in the periphery implies an ability of lymphocytes to sense cell numbers. Although the mechanisms are unknown, we find that the chemokine CCL21 (also known as TCA4, SLC, 6Ckine), a ligand for the chemokine receptor CCR7, can regulate homeostasis of CD4 (but not CD8) T cells. In the absence of CCR7 ligands, transferred CD4 T cells failed to expand in lymphopenic hosts, whereas in the presence of CCL21 overexpression, homeostatic CD4 T cell proliferation occurred even in nonlymphopenic recipients. Ag-specific CD4 T cells transferred into Ag-expressing mice proliferated and induced autoimmunity only in lymphopenic recipients. Pancreatic expression of CCL21 was sufficient to replace the requirement for lymphopenia in the progression of autoimmune disease. These results suggest that CD4 T cells use local concentrations of CCR7 ligands as an index of T cell steady state numbers and that homeostatic expansion of the T cell population may be a contributing factor in the development of autoimmune disease.

Restoration of peripheral tolerance to target autoantigens during autoimmune diseases has met with several limitations because of the limited efficacy of this approach in an already immune host. To optimize the induction of tolerance, we have shown that feeding insulin conjugated to cholera toxin B-subunit (CTB), a potent mucosal adjuvant, reduced by 5,000 the amounts of antigen necessary for delaying diabetes onset in NOD mice. To analyze these protective mechanisms, we have performed cotransfer experiments using splenocytes from young females fed once with 10 microg 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 both splenocytes and pancreatic lymph node (PLN) cell cultures from CTB-insulin-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-interferon (IFN-gamma) production occurred after CTB-insulin treatment, at 24 h in the PLN and at 7 days in the MLN. Reverse transcription-polymerase chain reaction analysis indicated an increase in the level of IL-4 and a reduction in 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 congenic NOD mice at the Thy1 locus, we showed that protection was associated with the accumulation of T-cells from CTB-insulin-fed mice in the lymph nodes from draining sites containing functional islets, i.e., the PLN in normal mice and the renal lymph nodes after a syngeneic islet graft under the kidney capsule of streptozotocin-treated mice. Taken together, our results clearly indicate that oral administration of CTB-insulin conjugates in NOD mice produced a shift from a T-helper type 1 to a type 2 profile with the induction of antigen-specific regulatory CD4+ T-cells in the vicinity of the mucosal barrier and close to the inflamed islets.