The natural history of lymphocyte subsets infiltrating the pancreas of NOD mice

Summary A longitudinal study of lymphocytic infiltration in the endocrine pancreas of non-obese diabetic mice was performed to investigate the role of different lymphocyte subsets in the pathogenesis of diabetes. The incidence of insulitis and the percentage of mononuclear cell subsets in the pancreas were evaluated in non-obese diabetic mice of various ages (5, 9, 13, 17, 22, 29 and 36 weeks). Cryostat sections of pancreas were stained with heamatoxilin-eosin or with different monoclonal antibodies against total T lymphocytes, helper T lymphocytes, cytotoxic/suppressor T lymphocytes, activated interleukin 2 receptor positive lymphocytes and B lymphocytes. A monoclonal antibody against Class-II antigens was also used. Positive cells were revealed by the immunoperoxidase technique. Insulitis was found in 5 weeks old mice but to a lesser extent than in adult animals. No significant variation between infiltrating cell subsets was found in different age groups. T lymphocytes ranged between 20.4% and 28.1%, B lymphocytes between 28.8% and 30.8% and Class-II positive cells between 22.8% and 32.2%. Interleukin 2 receptor positive cells ranged between 5.5% and 8.5% as detected with AMT-13 monoclonal antibody which recognise the interleukin 2 binding site. A higher percentage of activated cells was observed using another monoclonal antibody (7D4) directed against a different epitope of the interleukin 2 receptor, suggesting the presence of activated lymphocytes with interleukin 2 receptors saturated by interleukin 2. No insulin-containing cells were found to express Class-II molecules as demonstrated by a double immunofluorescence technique. Most infiltrating mononuclear cells were found to be positive for Class-II and L3T4 antigens or to be Class-II positive and express surface immunoglobulins. We conclude that pancreatic infiltration is an early expression of autoimmune phenomena occurring in these mice and that monocytes and B-lymphocytes predominate in the infiltrate. The role of interleukin 2 and interleukin 2 receptor positive lymphocytes in inducing and maintaining the immune response towards B cells is discussed.     

Expression of class II major histocompatibility complex antigens on pancreatic B cells in the NOD mouse

Summary To elucidate the role of class II major histocompatibility complex antigen expression on pancreatic B cells in the development of diabetes in the non-obese diabetic (NOD) mouse, indirect immunofluorescence was employed for I-A staining on Bouin-fixed pancreas sections of NOD mice (I-A of which was reported as d), B10.GD (I-A, d), BALB/c (I-A, d) and C3H/He (I-A, k). I-A positive islets were observed in all NOD mice examined. Positive reaction was detected in islets both with and without lymphocytic infiltrations. Double staining with anti-insulin, glucagon, somatostatin or pan creatic polypeptide antibodies revealed that I-A positive cells corresponded with insulin cells, while other types of pancreatic islet cells were virtually negative for I-A. Weaker staining was seen in islets of B10.GD and, to a lesser extent, in those of BALB/c mice. C3H/He mouse islet cells showed no I-A expression. These results demonstrated the expression of I-A antigens on pancreatic B cells in the NOD mouse.     

Transplantation analysis of B cell destruction in (NOD x CBA)F1 mouse bone marrow chimeras

Summary F1 hybrids produced by outcross of non-obese diabetic (NOD) mice with diabetes resistant strains are also diabetes resistant. This resistance is abrogated if F1 hybrids are lethally irradiated and then haematopoietically reconstituted with NOD bone marrow. This model was employed to determine whether T lymphocyte recognition and elimination of pancreatic B cells in NOD mice is restricted by the MHC haplotype of the target B cell. Diabetes resistant (NOD/Lt x CBA/J)F1 hybrids were lethally irradiated and reconstituted with NOD/Lt bone marrow. Following haematopoietic reconstitution, donor matched NOD/Lt or CBA/J pancreatic islet and anterior pituitary grafts were grafted under a renal capsule to determine whether effector cells derived from NOD/Lt marrow progenitors would reject islet grafts in a MHC restricted fashion. The H-2^k haplotype expressed by CBA/J mice differs from all known loci of the unique H-2 haplotype of NOD; therefore, if NOD/Lt T lymphocytes eliminate pancreatic B cells in a MHC restricted fashion, NOD islet grafts would be eliminated in these chimeras while CBA islet grafts would be retained. Overt diabetes developed in 80% of the female and 40% of the male F1 hybrids following reconstitution with NOD/Lt marrow, while no hybrids reconstituted with CBA/J marrow became diabetic through a year of age. The retention of CBA/J skin and pituitary grafts in NOD/Lt marrow reconstituted F1 hybrids confirmed that the F1 thymic environment imparted tolerance to CBA/J alloantigens. Nonetheless, responses to a T cell dependent model antigen were restricted to the unique MHC haplotype of NOD. This was associated in the hyperglycaemic chimeras with rejection (8–21 days post-implantation) of both CBA/J and NOD/Lt islet grafts. This indicated generation of islet specific effectors that were not restricted by the MHC haplotype of the target B cell. Islet specific effectors were less active in those chimeras remaining normoglycaemic following NOD/Lt marrow reconstitution, since both NOD/Lt and CBA/J islet grafts remained intact 21 days post-implantation. Islet and pituitary grafts of each genotype remained free of leucocytic involvement in CBA/J marrow reconstituted F1 hybrids. The generation of B cell specific, but not genotype specific, effectors elicited from NOD/Lt marrow was age-dependent. Following priming with porcine insulin in vivo, T lymphocytes from 8-weekold NOD male mice failed to respond to porcine insulin in vitro, while primed T lymphocytes isolated from 16-weekold mice proliferated vigorously. This suggested that as pancreatic insulitis progressed in aging NOD/Lt mice, T lymphocytes capable of responding to B cells in a tissue specific, but not genotype specific fashion, were activated. Thus, we propose that immunological effectors derived from NOD/Lt, but not CBA/J marrow, are capable of eliminating islet grafts in non-MHC restricted fashion. However, this population could be activated as a consequence of B cell erosion initated in the pancreas by another class of effectors which may be restricted by the MHC haplotype of the target B cell.     

Genetic engineering of insulin expression in nonislet cells: implications for beta-cell replacement therapy for insulin-dependent diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) occurs as a consequence of autoimmune destruction of the insulin-producing pancreatic β-cells. Although progress has been made in the field of islet transplantation, an appealing alternative strategy for β-cell replacement therapy for IDDM is to target insulin expression to non-islet cells. We have recently generated transgenic nonobese diabetic (NOD) mice in which insulin gene expression was targeted to proopiomelanocortin (POMC)-expressing cells of the intermediate lobe (IL) of the pituitary. We have shown that POMC-expressing IL pituitary cells secreted large amounts of mature insulin, similar to islet β-cells. However, in contrast to the insulin-producing islet β-cells, the insulin-producing IL pituitary cells were not attacked by the immune system. Remarkably, transplantation of small amounts of the transgenic IL tissues into diabetic NOD mice resulted in the restoration of near-normogylcemia and the complete reversal of diabetic symptoms. In separate experiments, IL allografts showed enhanced viability and were highly vascularized, compared with similarly transplanted islet allografts. These features are highly advantageous in the transplantation setting and demonstrate the considerable potential of these non-islet cell types for insulin-gene delivery in IDDM. Received: 20 December 1996 / Accepted in revised form: 24 December 1996     

Glucagon-like-peptide-1 (7–36) amide improves glucose sensitivity in beta-cells of NOD mice

The effect of the insulinotropic gut hormone glucagon-like-peptide-1 (GLP-1) was studied on the residual insulin capacity of prediabetic nonobese diabetic (NOD) mice, a model of insulin-dependent diabetes mellitus (type 1). This was done using isolated pancreas perfusion and dynamic islet perifusion. Prediabetes was defined by insulitis and fasting normoglycemia. Insultis occurred in 100% of NOD mice beyond the age of 12 weeks. K values in the intravenous glucose tolerance test were reduced in 20-week-old NOD mice compared with agematched non-diabetes-prone NOR (nonobese resistant) mice (2.4±1.1 vs 3.8±1.5% min^–1,P<0.05). Prediabetic NOD pancreases were characterized by a complete loss of the glucose-induced first-phase insulin release. In perifused NOD islets GLP-1, at concentrations already effective in normal islets, left the insulin release unaltered. However, a significant rise of glucose-dependent insulin secretion occurred for GLP-1 concentrations>0.1 nM. This was obtained with both techniques, dynamic islet perifusion and isolated pancreas perfusion, indicating a direct effect of GLP-1 on the beta-cell. Analysis of glucose-insulin dose-response curves revealed a marked improvement of glucose sensitivity of the NOD endocrine pancreas in the presence of GLP-1 (half-maximal insulin output without GLP-1 15.2 mM and with GLP-1 9.4 mM,P<0.002). We conclude that GLP-1 can successfully reverse the glucose sensing defect of islets affected by insulitis.     

Expression of interferon inducible protein-10 in pancreas of mice

AIM: To investigate the expression of interferon inducible protein-10 (IP-10) in pancreas of mice and to discuss its possible role in the pathogenesis of type 1 diabetes. METHODS: Non-obese diabetic (NOD) mice were used as experiment group and BALB/c mice as non-diabetic prone model. Immunohistochemistry method was used to evaluate the expression of IP-10 in the pancreas of NOD mice and BALB/c mice. Immunoelectron microscope was used to show the location of IP-10 in pancreatic islet (3 cells. RESULTS: Pancreatic islets were positively stained in all the NOD mice. Insulitis could be found in mice at the age of 4 wk. The weakly positive results were found in control group with no insulitis. Immunoelectron microscopy further demonstrated that IP-10 was produced by pancreatic (3 cells and stored in cytoplasm of the cells. CONCLUSION: IP-10 can be largely produced in pancreatic islets of NOD mice at the age of 2 wk when there is no significant insulitis, and may play an important part in the pathogenesis of type 1 diabetes by attracting immune cells to infiltrate the pancreatic islets.     

Expression of interferon inducible protein-10 in pancreas of mice

AIM: To investigate the expression of interferon inducible protein-10 (IP-10) in pancreas of mice and to discuss its possible role in the pathogenesis of type 1 diabetes.METHODS: Non-obese diabetic (NOD) mice were used as experiment group and BALB/c mice as non-diabetic prone model. Immunohistochemistry method was used to evaluate the expression of IP-10 in the pancreas of NOD mice and BALB/c mice. Immunoelectron microscope was used to show the location of IP-10 in pancreatic islet β cells.RESULTS: Pancreatic islets were positively stained in all the NOD mice. Insulitis could be found in mice at the age of 4 wk. The weakly positive results were found in control group with no insulitis. Immunoelectron microscopy further demonstrated that IP-10 was produced by pancreatic β cells and stored in cytoplasm of the cells.CONCLUSION: IP-10 can be largely produced in pancreatic islets of NOD mice at the age of 2 wk when there is no significant insulitis, and may play an important part in the pathogenesis of type 1 diabetes by attracting immune cells to infiltrate the pancreatic islets.     

IL-21 signaling is critical for the development of type I diabetes in the NOD mouse

IL-21 is a pleiotropic type I cytokine that shares the common cytokine receptor γ chain and plays important roles for normal Ig production, terminal B cell differentiation to plasma cells, and Th17 differentiation. IL-21 is elevated in several autoimmune diseases, and blocking its action has attenuated disease in MRL/lpr mice and in collagen-induced arthritis. The diabetes-associated Idd3 locus is at the Il2/Il21 locus, and elevated IL-21 was observed in the nonobese diabetic (NOD) mouse and suggested to contribute to diabetes by augmenting T cell homeostatic proliferation. To determine the role of IL-21 in diabetes, Il21r-knockout (KO) mice were backcrossed to NOD mice. These mice were devoid of lymphocytic infiltration into the pancreas, and only 1 of 20 animals had an elevated glucose compared with 60% of NOD mice on a wild-type (WT) background. Although TCR and Treg-related responses were normal, these mice had reduced Th17 cells and significantly higher levels of mRNAs encoding members of the Reg (regenerating) gene family whose transgenic expression protects against diabetes. Our studies establish a critical role for IL-21 in the development of type I diabetes in the NOD mouse, with obvious potential implications for type I diabetes in humans.     

Resistance of T-cells to apoptosis in autoimmune diabetic (NOD) mice is increased early in life and is associated with dysregulated expression of Bcl-x

Summary Activated lymphocytes of autoimmune non-obese diabetic (NOD) mice exhibit an increased resistance to programmed cell death (PCD) following withdrawal of interleukin-2 (IL-2). In the present study, we found that resistance of NOD T lymphocytes to PCD was increased as early as 1 week of age, hence several weeks before the invasion of the pancreas by inflammatory cells, which is compatible with a role of the NOD apoptotic phenotype in the autoimmune susceptibility of this strain. In the thymus, mature single positive but not double positive or double negative thymocytes were more resistant to PCD in NOD compared to B6 mice. Moreover, in both NOD and B6 mice, CD4+ T cells were more resistant to PCD induced by IL-2 deprivation than CD8+ cells. As a result, NOD CD4+ T cells were remarkably resistant to cell death induced in this manner. In relation with this increased resistance to apoptosis, expression of the anti-apoptotic Bcl-x protein was upregulated in activated T cells of NOD mice, most notably after 24 h of IL-2 deprivation. These results should help us to understand the relationship of the NOD apoptotic phenotype to the emergence of the NOD mouse autoimmune disease. [Diabetologia (1998) 41: 178–184]     

Prevention of recurrent but not spontaneous autoimmune diabetes by transplanted NOD islets adenovirally transduced with immunomodulating molecules

Pancreatic islet transplantation has the potential to maintain normoglycemia in patients with established type 1 diabetes, thereby obviating the need for frequent insulin injections. Our previous study showed that recombinant IL-12p40-producing islets prevented the recurrence of NOD diabetes. First, to see which immunomodulating molecule-secreting islet grafts can most powerfully prevent diabetes development in NOD mice without immunosuppressant, NOD islets were transfected with one of the following adenoviral vectors: Ad.IL-12p40, Ad.TGF-β, Ad.CTLA4-Ig, or Ad.TNF-α after which they were transplanted under the renal capsule of acutely diabetic NOD mice. The immunomodulating molecules produced by these adenovirus-transfected islets in vitro were 74 ± 19 ng, 50 ± 4 ng, 821 ± 31 ng, and 77 ± 18 ng/100 islets, respectively. Transplantation of IL-12p40, TNF-α, and CTLA4-Ig but not TGF-β-secreting islets displayed enhanced survival and delayed diabetes recurrence in recent-onset diabetic recipients. IL-12p40-producing islet grafts most powerfully prevented recurrent diabetes in NOD mice. In addition, local production of TNF-α and CTLA4-Ig significantly prolonged islet graft survival. In second series of experiment, these manipulated islets were transplanted under the renal capsule of 10-week-old NOD recipients and were also transplanted subcutaneously into 2-week-old NOD recipients. Transplantation of these islets into 2- or 10-week-old pre-diabetic mice failed to protect them from developing diabetes; in fact, transplantation of Ad.TNF-α-transfected islets into 2-week-old mice actually accelerated diabetes onset. Taken together, this approach was ineffectual as a prophylactic protocol. In conclusion, this study showed comparisons of the immunomodulating effects of 4 different adenoviral vectors in the same transplantation model and local production of IL-12p40, TNF-α and CTLA4-Ig significantly prevented recurrent diabetes in NOD mice.     

Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations

Interleukin (IL)-7 is a cytokine essential for T lymphocyte development and homeostasis. However, little is known about the roles of IL-7 receptor α-chain (IL-7Rα) in late stages of T-cell development. To address this question, we established IL-7Rα–floxed mice and crossed them with CD4-Cre transgenic mice. Resultant IL-7R conditional knockout (IL-7RcKO) mice exhibited marked reduction in CD8 single positive (SP) T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells in thymus. The proportion and proliferation of both mature CD4SP and CD8SP thymocytes were decreased without affecting Runx expression. In addition, expression of the glucocorticoid-induced TNF receptor was reduced in CD4SP and CD8SP thymocytes, and expression of CD5 was decreased in CD8SP thymocytes. IL-7RcKO mice also showed impaired Treg and NKT cell proliferation and inhibition of NKT cell maturation. Bcl-2 expression was reduced in CD4SP and CD8SP thymocytes but not in Tregs and NKT cells, and introduction of a Bcl-2 transgene rescued frequency and CD5 expression of CD8SP thymocytes. Furthermore, IL-7RcKO mice exhibited greatly increased numbers of B cells and, to a lesser extent, γδ T and dendritic cells in thymus. Overall, this study demonstrates that IL-7Rα differentially controls development and maturation of thymocyte subpopulations in late developmental stages and suggests that IL-7R expression on αβ T cells suppresses development of other cell lineages in thymus.     

Antidiabetogenic MHC class II promotes the differentiation of MHC-promiscuous autoreactive T cells into FOXP3+ regulatory T cells

Polymorphisms in MHC class II molecules, in particular around β-chain position-57 (β57), afford susceptibility/resistance to multiple autoimmune diseases, including type 1 diabetes, through obscure mechanisms. Here, we show that the antidiabetogenic MHC class II molecule I-A^b affords diabetes resistance by promoting the differentiation of MHC-promiscuous autoreactive CD4⁺ T cells into disease-suppressing natural regulatory T cells, in a β56–67-regulated manner. We compared the tolerogenic and antidiabetogenic properties of CD11c promoter-driven transgenes encoding I-A^b or a form of I-A^b carrying residues 56–67 of I-Aβ^g7 (I-A^b-g7) in wild-type nonobese diabetic (NOD) mice, as well as NOD mice coexpressing a diabetogenic and I-A^g7–restricted, but MHC-promiscuous T-cell receptor (4.1). Both I-A transgenes protected NOD and 4.1-NOD mice from diabetes. However, whereas I-A^b induced 4.1-CD4⁺ thymocyte deletion and 4.1-CD4⁺Foxp3⁺ regulatory T-cell development, I-A^b-g7 promoted 4.1-CD4⁺Foxp3⁺ Treg development without inducing clonal deletion. Furthermore, non–T-cell receptor transgenic NOD.CD11cP-I-A^b and NOD.CD11cP-IA^b-g7 mice both exported regulatory T cells with superior antidiabetogenic properties than wild-type NOD mice. We propose that I-A^b, and possibly other protective MHC class II molecules, afford disease resistance by engaging a naturally occurring constellation of MHC-promiscuous autoreactive T-cell clonotypes, promoting their deviation into autoregulatory T cells.     

Evidence that glucagon stimulates insulin secretion through its own receptor in rats

Summary Since glucagon-like peptide-1 (7–36) amide (7–37) (GLP-1) has been found to be a potent insulinotropic hormone, it has been postulated that glucagon stimulates insulin secretion from islet beta cells through the GLP-1 receptor. We therefore examined the effects of a GLP-1 receptor antagonist, exendin (9–39) amide, on glucagon- or GLP-1-stimulated insulin release from isolated perfused rat pancreas. When infusion of 100 nmol/l exendin (9–39) amide was started 5 min before that of 1 nmol/l glucagon, the stimulation of insulin release by glucagon was similar to that found in the control situation (preinfusion with vehicle alone). By contrast, when 0.3 nmol/l GLP-1 was used in the same experimental setting, exendin (9–39) amide clearly inhibited insulin release. These results indicate that glucagon stimulates insulin release mainly through glucagon receptors but not GLP-1 receptors on islet beta cells.Abbreviations GLP-1 Glucagon-like peptide-1 - BSA bovine serum albumin - IRI immunoreactive insulin     

CD8 cytotoxic T-cell clone rapidly transfers autoimmune diabetes in very young NOD and MHC class I-compatible scid mice

Summary A CD8 T-cell clone (YNK1.3) generated from acutely diabetic NOD mouse islets, showed proliferation and cytotoxicity when challenged with NOD and BALB/c islet cells and NOD-derived insulinoma cells. When 1–2 × 10⁷ YNK1.3 cells were administered to 7–10-day-old NOD mice, the cells transferred overt diabetes very rapidly in each of the 16 recipients within 4 days of cell transfer. However, of 14 recipients receiving YNK 1.3 cells above 14 days of age none became diabetic. Fluorescent dye-labelled YNK1.3 cells extensively accumulated in the islets by 36 h after transfer in 7-day-old NOD recipients, while no significant insulitis was seen in 21-day-old recipients. Over half of NOD-scid recipients (5/9) rapidly became diabetic within 5 days after transfer of 1–2 × 10⁷ YNK1.3 cells at 7 days of age, whereas only one of 12 recipients over 14 days of age became diabetic. Furthermore, YNK1.3 cells also transferred diabetes to H-2K^d-matched very young BALB/c-scid and CB17-scid mice, but not to C57BL/6-scid mice. Thus, optimally activated islet-specific CD8 T-cell clones are able to rapidly transfer diabetes to NOD and MHC class I compatible scid mice when a large enough number is administered at 7 days of age. Administration of monoclonal antibodies against adhesion molecules involved in the trafficking of lymphocytes from the circulation into the inflammatory tissues, could not prevent the cellular infiltration of YNK1.3 cells into the islets in 7-day-old NOD recipients. The results indicate that islet cells in the mouse around 7 days of age are generally susceptible to cytotoxic CD8 T cells, suggesting, therefore, that CD8 T cells may play an important role in the initiation of autoimmune diabetes in NOD mice. [Diabetologia (1997) 40: 1044–1052] Received: 18 February 1997 and in revised form: 29 April 1997     

High copy number I-Ab transgenes induce production of IgE through an interluekin 4-dependent mechanism.

To better understand the role of class II major histocompatibility complex molecules in both normal and autoimmune responses, we have produced a series of I-Ab transgenic mice. One of these transgenic constructs, designated NOD.PD, has the sequence of the NOD beta chain (Abeta(g7)) except at positions 56 and 57, where Pro-Asp replaces His-Ser. Several NOD.PD transgenic lines have been produced. One line of these mice carried a very high number of copies (>50) of the NOD.PD transgene. As has been described in other mice carrying high copy numbers of I-Ab transgenes, B-cell development was abnormal. The steady state numbers of mature B cells (IgM+/IgD(hi)) in the periphery were greatly reduced in transgenic mice compared to nontransgenic littermates. Surprisingly, rather than being accompanied by a generalized hypogammaglobulinemia, this B-cell deficiency was accompanied by elevated concentrations of IgG1 and IgE in the serum. Conversely, the levels of IgG2a were reduced in transgenic mice compared to nontransgenic littermates. Because this isotype pattern was characteristic of interleukin (IL)-4-induced class-switching, we then investigated the role of IL-4 in causing the observed phenotype. We crossed the high copy number transgenic mice with an IL-4-deficient strain of mice. As expected, the elevated levels of IgE in high copy number transgenic mice were eliminated when the IL-4 gene was inactivated. However, the reduction in the number of B cells was not ameliorated. These data indicate that the primary defect caused by the transgene was to reduce the number of B cells in these mice. This reduction was accompanied by a secondary increase in IL-4 production, which drove the remaining B cells toward the production of IgGl and IgE.     

Insulin as a target antigen in autoimmune diabetes: a natural repertoire as the source of antibody response

A solid-phase immunoenzymatic technique with B1- or B29-biotinylated insulin coupled to avidincoated wells was used to characterize serum anti-insulin antibodies and to locate insulin antibody-producing B lymphocytes in different organs of mice. Low natural serum anti-insulin IgM and IgG antibodies were found in ten different healthy inbred strains of mice. Prediabetic nonobese diabetic (NOD) mice had significantly higher measurements than BALB/c mice (P<0.05). Anti-insulin IgM antibody-producing B lymphocytes were found in bone marrow and spleen of NOD mice and healthy strains of mice, but not in peripheral lymph nodes, thymus, blood or pancreas. B29-fixed insulin was more frequently recognized than B1-fixed insulin. There was no relationship to the MHC or to other immune markers. IgG insulin antibody-producing cells were not detected. IgG insulin antibody-producing cells appeared in the draining lymph node and in the blood 10 days after immunization with insulin. IgM insulin-recognizing cells in the spleen were reduced in number during the same period (P<0.05–0.01 for BALB/c, DBA2, B10.D2 and NOD), suggesting migration of these cells. This was tested by in vivo staining of spleens with the red-fluorescent membrane linker PKH-26 on day 7 after immunization. Cells from immunized lymph nodes were FACS-sorted on day 10. Insulin antibody-producing B lymphocytes with red-fluorescence were found, indicating a splenic origin. Examination of IgG subclasses showed preferential production of complement-fixing IgG2b in sera and lymph node cells of immunized NOD mice (P<0.05 vs BALB/c). We conclude that a natural repertoire of insulin recognition exists in bone marrow and spleen of mice. Hydrophobic epitopes around B1 (B29-fixed insulin) are more frequently recognized than hydrophilic epitopes around B29 (B1-fixed insulin), indicating a genetically fixed pattern of autorecognition. Insulin-recognizing cells from the spleen function as the source of insulin antibody response. Preferential occurrence of complement-fixing IgG2b in NOD mice could contribute to autoimmune-mediated -cell damage.     

A defective Il15 allele underlies the deficiency in natural killer cell activity in nonobese diabetic mice

The nonobese diabetic (NOD) mouse strain has a genetic deficiency in natural killer (NK) cells. This defect underlies this strain''s utility in several experimental settings; in particular, it promotes engraftment of human tissue in NOD hosts during the generation of “humanized” mouse models. We have mapped the major NK-cell defect in the NOD vs. C57BL/6 (B6) strain to an inadequately expressed Il15 allele. Treatment of NOD mice with a reagent that specifically enhances interleukin (IL)-15 bioavailability normalized NK-cell numbers and activity in the absence of nonspecific stimulation. These findings raise the possibility of exploiting reagents that impact the IL-15 receptor pathway to facilitate construction of humanized mouse models on non-NOD genetic backgrounds.     

Blockade of both B7-H4 and CTLA-4 co-signaling pathways enhances mouse islet allograft survival

Costimulation blockade is an effective way to prevent allograft rejection. In this study, we tested the efficacy of two negative co-signaling molecules in protecting islet allograft function. We used local expression of B7-H4 by adenoviral transduction of islets (Ad-B7-H4) and systemic administration of CTLA-4.Ig to investigate the outcomes of allograft survival. Five groups of streptozotocin-induced diabetic C57BL/6 mice received 400 islets each from BALB/c donors. The groups consisted of control (G1); CTLA-4.Ig (G2); Ad-LacZ (G3); Ad-B7-H4 (G4); and Ad-B7-H4 and CTLA-4.Ig combined (G5). G1 and G3 developed graft failure on average of two weeks. G2, G4 and G5 survived for 43.8 ± 34.8, 54.7 ± 31.2 and 77.8 ± 21.5 d, respectively. Activated T and B cells in the lymph nodes were significantly controlled by CTLA-4.Ig treatment. Significantly reduced infiltrates were also detected in the allografts of G2 compared with G1. By contrast, B7-H4 significantly inhibited Th1-associated IFN-gamma secretion in the early stage and increased Foxp3⁺ T cells in the long-term surviving allografts. Our study suggests that CTLA-4 and B7-H4 inhibit alloimmune responses through distinct mechanisms, and that combination therapy which activates two negative co-signaling pathways can further enhance islet allograft survival.     

Interferon-alpha initiates type 1 diabetes in nonobese diabetic mice

With the goal of identifying changes in gene expression in CD4⁺ T cells during the development of diabetes in the nonobese diabetic (NOD) mouse, we used DNA microarrays to analyze gene expression in CD4⁺ T cells from the pancreatic draining lymph nodes of NOD/BDC 2.5 T cell receptor transgenic and WT NOD mice at different ages. At 4 and 6 weeks of age, we found up-regulation of a number of genes that are known to be induced by IFN-α. IFN-α levels and IFN-α–producing plasmacytoid dendritic cells were increased in the PLNs of 3- to 4-week-old NOD mice. Moreover, blockade of IFN-α receptor 1 in NOD mice by a neutralizing antibody at 2–3 weeks of age significantly delayed the onset and decreased the incidence of type 1 diabetes, increased the relative number of immature dendritic cells in the PLNs, and enhanced the ability of spleen CD4⁺ T cells to produce IL-4 and IL-10. These findings demonstrate that IFN-α in the PLNs is an essential initiator in the pathogenesis of type 1 diabetes in NOD mice.