Terry L. Delovitch

The Nonobese Diabetic Mouse as a Model of Autoimmune Diabetes: Immune Dysregulation Gets the NOD

Considerable evidence obtained during the past 15 years suggests a major role for T cell immune dysregulation in the initiation of IDDM in NOD mice. T cell anergy and deficient T cell–mediated suppression may mediate susceptibility to IDDM in NOD mice, and Th2 cell anergy may be responsible for a failure in immune regulation. The key to the onset of immune dysregulation and aberrant increase in the number of autoreactive T cells in the periphery may be that the activation threshold required for TCR-stimulation is markedly increased in T cells from NOD mice and humans with IDDM. This increase in the number of peripheral T cells may arise from the weak peptide-binding affinity of I-Ag7 molecules on NOD APCs and the resulting reduced capacity of APCs in the thymus of NOD mice to negatively select T cells with potential reactivity to islet autoantigens. While unresponsiveness to peptide/I-Ag7 complexes may preclude stimulation of autoreactive T cells to a sufficiently high threshold level to induce their deletion, the levels of activation reached by these T cells may suffice to render them anergic to subsequent TCR stimulation. Nonetheless, this anergic state resulting from a failure of central and peripheral tolerance mechanisms remains capable of maintaining the autoimmune phenotype of these T cells. These T cells may still retain the capacity to initiate and contribute to the development of autoimmune disease.Numerous fundamental questions related to IDDM remain to be explored in the NOD mouse model, and include the following. (1) Are I-Ag7 MHC class II molecules underexpressed on the cell surface of APCs, and if so, is this the result of their generally low peptide-binding affinity and inherent instability? Does this weak binding affinity for peptides mediate the positive selection and exit into the periphery of an increased number of islet autoantigen reactive T cells? (2) What are the critical autoantigens in IDDM, and is there a primary autoantigen that induces the onset of IDDM? (3) What are the mechanisms of induction of islet β cell death, and which pathways (e.g., Fas/FasL, TNF/TNF receptor, or perforin) are most relevant to β cell apoptosis? (4) What agents, internal or external (e.g., viral, bacterial, or diet), trigger the onset of inflammation and IDDM? (5) What is the antigenic specificity and mechanism of action of regulatory T cells that may mediate protection from IDDM? (6) Finally, which cytokines and chemokines are most active in the down-regulation of the autoimmune response, and what is their mechanism of action? These and other questions will direct our exploration of the mechanisms underlying IDDM and other autoimmune diseases.*To whom correspondence should be addressed (e-mail: del@rri.on.ca).

Activation of natural killer T cells by α -galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6–9). Thus, given that NKT cells respond to the α-galactosylceramide (α-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by α-GalCer might prevent the onset and/or recurrence of T1D. Here we show that α-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, α-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to α-GalCer. Protection from T1D by α-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet β cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility thatα-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.

JANUS-LIKE ROLE OF REGULATORY iNKT CELLS IN AUTOIMMUNE DISEASE AND TUMOUR IMMUNITY

Invariant CD1D-restricted natural killer T (iNKT) cells function during innate and adaptive immunity and regulate numerous immune responses, such as autoimmune disease, tumour surveillance, infectious disease and abortions. However, the molecular basis of their functions and the nature of disease-associated defects of iNKT cells are unclear and have been the subject of recent controversy. Here, we review recent findings that underscore the potential importance of interactions between iNKT cells and dendritic cells (DCs) that indicate that iNKT cells regulate DC activity to shape both pro-inflammatory and tolerogenic immune responses. The ability to modulate iNKT-cell activity in vivo using the ligand α-galactosylceramide and to treat patients with autoimmune disease or cancer is evaluated also.

Differential Expression of CC Chemokines and the CCR5 Receptor in the Pancreas Is Associated with Progression to Type I Diabetes

We investigated the biological role of CC chemokines in the Th1-mediated pathogenesis of spontaneous type I diabetes in nonobese diabetic (NOD) mice. Whereas an elevated ratio of macrophage inflammatory protein-1α (MIP-1α):MIP-1β in the pancreas correlated with destructive insulitis and progression to diabetes in NOD mice, a decreased intrapancreatic MIP-1α:MIP-1β ratio was observed in nonobese diabetes-resistant (NOR) mice. IL-4 treatment, which prevents diabetes in NOD mice by polarizing intraislet Th2 responses, decreased CCR5 expression in islets and potentiated a high ratio of MIP-1β and monocyte chemotactic protein-1 (MCP-1): MIP-1α in the pancreas. Furthermore, NOD.MIP-1α−/− mice exhibited reduced destructive insulitis and were protected from diabetes. Neutralization of MIP-1α with specific Abs following transfer of diabetogenic T cells delayed the onset of diabetes in NOD.Scid recipients. These studies illustrate that the temporal expression of certain CC chemokines, particularly MIP-1α, and the CCR5 chemokine receptor in the pancreas is associated with the development of insulitis and spontaneous type I diabetes.

Regulatory lymphocytes: Janus-like role of regulatory iNKT cells in autoimmune disease and tumour immunity

Invariant CD1D-restricted natural killer T (iNKT) cells function during innate and adaptive immunity and regulate numerous immune responses, such as autoimmune disease, tumour surveillance, infectious disease and abortions. However, the molecular basis of their functions and the nature of disease-associated defects of iNKT cells are unclear and have been the subject of recent controversy. Here, we review recent findings that underscore the potential importance of interactions between iNKT cells and dendritic cells (DCs) that indicate that iNKT cells regulate DC activity to shape both pro-inflammatory and tolerogenic immune responses. The ability to modulate iNKT-cell activity in vivo using the ligand α-galactosylceramide and to treat patients with autoimmune disease or cancer is evaluated also.

Evidence That a Peptide Spanning the B-C Junction of Proinsulin Is an Early Autoantigen Epitope in the Pathogenesis of Type 1 Diabetes

The expression of pro(insulin) in the thymus may lead to the negative selection of pro(insulin) autoreactive T cells and peripheral tolerance to this autoantigen in type 1 diabetes (T1D). We investigated whether proinsulin is expressed in the thymus of young nonobese diabetic (NOD) mice, whether T cells from naive NOD female mice at weaning are reactive to mouse proinsulin, and the role of proinsulin as a pathogenic autoantigen in T1D. Proinsulin II mRNA transcripts were detected in the thymus of 2-wk-old NOD mice at similar levels to other control strains. Despite this expression, proinsulin autoreactive T cells were detected in the periphery of 2- to 3-wk-old naive NOD mice. Peripheral T cells reactive to the insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, and islet cell Ag p69 autoantigens were also detected in these mice, indicating that NOD mice are not tolerant to any of these islet autoantigens at this young age. T cell reactivities to proinsulin and islet cell Ag p69 exceeded those to GAD67, and T cell reactivity to proinsulin in the spleen and pancreatic lymph nodes was directed mainly against a p24–33 epitope that spans the B chain/C peptide junction. Intraperitoneal immunization with proinsulin perinatally beginning at 18 days of age delayed the onset and reduced the incidence of T1D. However, s.c. immunization with proinsulin initiated at 5 wk of age accelerated diabetes in female NOD mice. Our findings support the notion that proinsulin p24–33 may be a primary autoantigen epitope in the pathogenesis of T1D in NOD mice.

Intravenous Transfusion of BCR-Activated B Cells Protects NOD Mice from Type 1 Diabetes in an IL-10-Dependent Manner

Although B cells play a pathogenic role in the initiation of type 1 diabetes (T1D) in NOD mice, it is not known whether activated B cells can maintain tolerance and transfer protection from T1D. In this study, we demonstrate that i.v. transfusion of BCR-stimulated NOD spleen B cells into NOD mice starting at 5–6 wk of age both delays onset and reduces the incidence of T1D, whereas treatment initiated at 9 wk of age only delays onset of T1D. This BCR-activated B cell-induced protection from T1D requires IL-10 production by B cells, as transfusion of activated B cells from NOD.IL-10−/− mice does not confer protection from T1D. Consistent with this result, severe insulitis was observed in the islets of NOD recipients of transfused NOD.IL-10−/− BCR-stimulated B cells but not in the islets of NOD recipients of transfused BCR-stimulated NOD B cells. The therapeutic effect of transfused activated NOD B cells correlates closely with the observed decreased islet inflammation, reduced IFN-γ production and increased production of IL-4 and IL-10 by splenocytes and CD4+ T cells from NOD recipients of BCR-stimulated NOD B cells relative to splenocytes and CD4+ T cells from PBS-treated control NOD mice. Our data demonstrate that transfused BCR-stimulated B cells can maintain long-term tolerance and protect NOD mice from T1D by an IL-10-dependent mechanism, and raise the possibility that i.v. transfusion of autologous IL-10-producing BCR-activated B cells may be used therapeutically to protect human subjects at risk for T1D.

Neonatal activation of CD28 signaling overcomes T cell anergy and prevents autoimmune diabetes by an IL-4-dependent mechanism.

Optimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.

Protection from Type 1 Diabetes by Invariant NK T Cells Requires the Activity of CD4+CD25+ Regulatory T Cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by α-galactosylceramide (α-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+CD25+ Treg, for this protection. We found that CD4+CD25+ T cells from NOD.CD1d−/− mice deficient in iNKT cell function similarly in vitro to CD4+CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon α-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+CD25+ Tregs from NOD mice pretreated with α-GalCer demonstrated that activated iNKT cells do not influence the ability of Tregs to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+CD25+ T cells, because splenocytes pretreated with α-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+CD25+ T cells before α-GalCer treatment were also not protected from T1D. Our data suggest that CD4+CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+CD25+ Tregs is required for iNKT cells to transfer protection from T1D.

Expression of ia antigens by murine keratinizing epithelial langerhans cells.

Immunofluorescent and immunoelectron-microscopic staining methods were utilized to investigate the localization of Ia antigens in murine keratinizing epithelia. Approximately 3–5% of epidermal cells were shown to be Ia positive. Only dendritic Langerhans cells in the interfollicular epidermis and outer root sheaths were found to express Ia antigens. These Ia determinants were shown to be controlled by both theI- A andI- EC subregions of theH-2 complex. The results were confirmed by identifying positively stained cells containing Langerhans cell granules at the ultrastructural level. No staining was noted on the surface of keratinocytes, melanocytes, or immigrant lymphocytes. The results presented are in close agreement with those previously reported for Ia-bearing Langerhans cells in human and guinea pig epidermis.