Role of L-selectin in the development of autoimmune diabetes in non-obese diabetic mice

Autoimmune diabetes is characterized by an early mononuclear infiltration of pancreatic islets and later selective autoimmune destruction of insulin-producing beta cells. Lymphocyte homing receptors have been considered candidate targets to prevent autoimmune diabetes. L-selectin (CD62L) is an adhesion molecule highly expressed in naive T and B cells. It has been reported that blocking L-selectin in vivo with a specific antibody (Mel-14) partially impairs insulitis and diabetes in autoimmune diabetes-prone non-obese diabetic (NOD) mice. In the present study we aimed to elucidate whether genetic blockade of leukocyte homing into peripheral lymph nodes would prevent the development of diabetes. We backcrossed L-selectin-deficient mice onto the NOD genetic background. Surprisingly NOD/L-selectin-deficient mice exhibited unaltered islet mononuclear infiltration, timing of diabetes onset and cumulative incidence of spontaneous diabetes when compared to L-selectin-sufficient animals. CD4, CD8 T cells and B cells were present in islet infiltrates from 9-week-old L-selectin-sufficient and -deficient littermates. Moreover, total splenocytes from wild-type, heterozygous or NOD/L-selectin-deficient donor mice showed similar capability to adoptively transfer diabetes into NOD/SCID recipients. On the other hand, homing of activated, cloned insulin-specific autoaggressive CD8 T cells (TGNFC8 clone) is not affected in NOD/L-selectin-deficient recipients. We conclude that L-selectin plays a small role in the homing of autoreactive lymphocytes to regional (pancreatic) lymph nodes in NOD mice.     

The CXCR4/CXCL12 (SDF-1) signalling pathway protects non-obese diabetic mouse from autoimmune diabetes

Chemokines and their receptors are part of polarized T helper 1 (Th1)- and Th2-mediated immune responses which control trafficking of immunogenic cells to sites of inflammation. The chemokine stromal cell-derived factor-1 CXCL-12 (SDF-1) and its ligand the CXCR4 chemokine receptor are important regulatory elements. CXCR4 is expressed on the surface of CD4(+) T cells, dendritic cells and B lymphocytes. Levels of CXCR4 mRNA were increased in pancreatic lymph nodes (PLNs) of 4-week-old non-obese diabetic (NOD) mice in comparison to Balb/C mice. However, a significant reduction of CXCR4 was noticed at 12 weeks both at the mRNA and protein levels while expression increased in the inflamed islets. The percentage of SDF-1 attracted splenocytes in a transwell chemotaxis assay was significantly increased in NOD versus Balb/c mice. SDF-1 attracted T cells completely abolished the capacity of diabetogenic T cells to transfer diabetes in the recipients of an adoptive cell co-transfer. When T splenocytes from NOD females treated with AMD3100, a specific CXCR4 antagonist, were mixed with diabetogenic T cells during adoptive cell co-transfer experiments, prevalence of diabetes in the recipients rose from 33% to 75% (P < 0.001). This effect was associated with an increase of interferon (IFN)-gamma mRNA and a reduction of interleukin (IL)-4 mRNA levels both in PLNs and isolated islets. AMD3100 also reduced IL-4 and IL-10 production of plate-bound anti-CD3 and anti-CD28-stimulated splenocytes. Immunofluorescence studies indicated that AMD3100 reduced the number of CXCR4(+) and SDF-1 positive cells in the inflamed islets. We can conclude that the CXCL-12/CXCR4 pathway has protective effects against autoimmune diabetes.     

The transfer of autoimmune diabetes in NOD mice can be inhibited or accelerated by distinct cell populations present in normal splenocytes taken from young males

The NOD mouse is characterized by the development of spontaneous autoimmune diabetes which begins with a peri-islet lymphocyte infiltration of the pancreas around 6 weeks of age and progresses to overt diabetes in 50-60% of females from about 12 weeks. Although infiltration occurs around islets in males, the incidence of overt diabetes is much less (about 1%) and suggests that there may be more effective regulatory circuits in these animals. This possibility was examined by using splenocytes from young males to reconstitute irradiated male recipients 6 d before the transfer of diabetogenic spleen cells from spontaneously diabetic females. Those animals which were not reconstituted with male spleen cells developed diabetes 3-5 weeks later, whereas the majority of the reconstituted mice remained normoglycaemic. Characterization of the protective population demonstrated a role for CD4+ T cells. An additional observation was that splenocytes from young normal males also contained a population of non-T cells which could advance the diabetogenic transfer of disease by at least a week.     

Immunomodulation through inhibition of multiple adhesion molecules generates resistance to autoimmune diabetes in NOD mice

The effect of simultaneous blockade of adhesion molecules on the development of long-term resistance to type 1 diabetes was investigated in an adoptive transfer model in NOD mice. Splenocytes isolated from acutely diabetic NOD mice injected into NOD-scid mice caused diabetes at 43 +/- 5.0 days. Treatment with anti-alpha4-integrin monoclonal antibody (mAb) delayed the onset of insulitis and significantly delayed hyperglycemia to 66 +/- 5.8 days. Combination treatment with anti-alpha4-integrin and anti-LFA-1 mAbs delayed the onset of diabetes to >100 days (p<0.0001). Combination-treated mice were subjected to a second challenge with diabetogenic splenocytes after 85 days of normoglycemia. Without additional mAb treatment they developed hyperglycemia after significant delay (72 +/- 8.1 days post-reinoculation). Splenocytes from combination-treated mice transferred protection from diabetes to naive NOD-scid mice when co-transferred with diabetogenic splenocytes. The long-surviving mice showed periislet infiltration with CD62L+ cells, which were not seen in the insulitis developing in control animals. These findings suggest that adhesion molecule blockade does not prevent homing and may affect effector cell action through activation of immunoregulatory suppressor cells, leading to protection against development of diabetes.     

Effects of streptozotocin on autoimmune diabetes in NOD mice

Non-obese diabetic (NOD) mice develop autoimmunity that destroys their native beta cells causing diabetes. Their autoimmunity will also destroy syngeneic transplanted islets and transfer both autoimmunity and diabetes via spleen cells to non-diabetic mice. In this report, we studied the effects of streptozotocin (STZ) on the autoimmune diabetes in NOD mice. We transplanted NOD.SCID islets into three groups of NOD mice: (1) spontaneously diabetic NOD mice (NOD-sp.); (2) prediabetic NOD mice made diabetic by streptozotocin (NOD-stz); and (3) diabetic NOD mice also treated with streptozotocin (NOD-sp./stz). In the first group, the transplants were rejected within 3 weeks. In the second and third groups, the transplants survived indefinitely. Alloxan, a drug similar to streptozotocin, did not have the same effect as streptozotocin. The ability of streptozotocin to prevent diabetes in young NOD mice was reversed by anti-CD8 antibody treatment but not by anti-CD4 treatment. Streptozotocin also made spleen cells from diabetic NOD mice less effective transferring diabetes. These results indicate that streptozotocin treatment both prevents and reverses the islet destructive autoimmunity in NOD mice. We postulate that the effects of streptozotocin treatment may be mediated in part by regulatory T cells.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.     

In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha

Immunization with mycobacterial preparation such as Bacille Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. We found that the potential of splenocytes from BCG-immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-alpha, IFN-gamma and IL-4 by splenocytes, increased the expression of Fas(high) (Apo-1/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-gamma and TNF-alpha in BCG-immunotherapy was demonstrated by (i) reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anti-cytokine antibodies, (ii) inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNFR1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-alpha and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells. Our results suggest that BCG down-regulates destructive autoimmunity by TNF-alpha/IFN-gamma-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. These studies provide a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.     

Protection of IFN-gamma signaling-deficient NOD mice from diabetes by cyclophosphamide

Non-obese diabetic (NOD) mice that are genetically deficient in either IFN-gamma or beta chain of the IFN-gammaR develop diabetes with similar kinetics to wild-type NOD mice. In the current study, we demonstrated that treatment of IFN-gamma signaling-deficient NOD mice with cyclophosphamide (CY) not only fails to induce acute diabetes but also confers permanent protection from diabetes. Protection was mediated by the preferential generation of regulatory T cells (Treg cells) that are capable of suppressing the diabetogenic process, with no change in the total number and function of Treg cells. Moreover, CY treatment of IFN-gamma signaling-deficient NOD mice reversed the ongoing pathogenic process and eliminated cellular infiltrates of pancreatic islets. While these results have been derived using a genetically modified mouse model of diabetes, they indicate that knowledge of host genetic factors and environmental factors influencing the development of Type I diabetes mellitus may provide a rational approach to develop a means to reverse the development of Type I diabetes in human.     

A novel mechanism of regulatory T cell-mediated down-regulation of autoimmunity

We have established a novel CD4 and CD8 double-positive CD25+ T regulatory (Treg) clone, MT-5B, from lymph nodes of type 1 diabetes prone non-obese diabetic (NOD) mice immunized with CFA. CFA has previously been shown to prevent the onset of diabetes by inducing Treg cells. In vitro, clone MT-5B was anergic to a panel of antigen stimulations and exerted an immunosuppressive effect in antigen-non-specific and cell contact-independent manners. In vivo, clone MT-5B blocked the adoptive transfer of diabetes. Proteomics and immunoadsorption studies identified the suppressive proteins secreted by clone MT-5B as granzyme B (GrB) and perforin (PFN). GrB-mediated immune suppression was PFN dependent. Removal of GrB or PFN from the culture supernatant (SN) of MT-5B cells or pre-incubation of MT-5B cells with ethyleneglycol-bis(aminoethylether)-tetraacetic acid which blocks PFN activity reduced the immunosuppressive effect in vitro. Pre-incubation of diabetogenic splenocytes from NOD mice with MT-5B SN impaired their ability to transfer disease by inducing T cell apoptosis, and removal of GrB from MT-5B SN by immunoadsorption decreased the effector function of MT-5B SN on diabetogenic splenocytes. Immunization of NOD mice with CFA increased the expression of GrB+ CD4 T cells, indicating that these cells are present in vivo. In conclusion, we describe a novel mechanism of cell contact-independent immune suppression in which Treg cells maintain immune homeostasis by secreting GrB/PFN.     

Treatment with sulfatide or its precursor, galactosylceramide, prevents diabetes in NOD mice.

Sulfatide (3'sulfogalactosylceramide) is a glycosphingolipid present within the nervous system and in the islets of Langerhans. Anti-sulfatide antibodies have been observed in both pre-diabetic and newly diagnosed type 1 diabetic patients. The aim of this study was to test in vivo, the therapeutic effect of sulfatide on the development of diabetes in the NOD mouse. In four separate experiments diabetogenic splenocytes from newly diabetic NOD mice were injected iv into 7-8 week old irradiated (700R) female NOD mice (4-10 million cells/mouse). Each experiment consisted of four treatment groups to which the mice were randomly divided: 1) sulfatide; 2) galactosylceramide (the precursor to sulfatide without sulfate); 3) GM1, a glycosphingolipid negatively charged as sulfatide but with a different sugar composition; and 4) phosphate buffered saline (PBS). The mice received 100 microg glycosphingolipid iv on the day of cell transfer and 1-3 times thereafter at four day intervals, and were screened for diabetes three times a week the next 52 days. Among all the 35 sulfatide-treated mice 54% became diabetic compared to 93 % of 43 PBS-treated animals (p < 0.00001). Correspondingly, galactosylceramide reduced diabetes incidence to 52% (25 mice, p < 0.00001). On the other hand, 86% of GM1-treated mice (n=28) became diabetic indicating that no effect was obtained by this glycosphingolipid. In two experiments in which less spleen cells were transferred (4-5 mill.) and glycosphingolipids were given 4 times, 35% of the sulfatide-treated animals (n = 17) developed diabetes compared to 85% of PBS-treated mice (n = 20, p < 0.001). A robust proliferative response to sulfatide, but none to GM1, was observed when spleen cells were rechallenged with glycosphingolipid in vitro. Thus, like insulin and GAD, sulfatide is able to prevent diabetes in NOD mice.     

Interleukin 2 receptor targeted fusion toxin (DAB486-IL-2) treatment blocks diabetogenic autoimmunity in non-obese diabetic mice.

Insulin-dependent diabetes mellitus (IDDM) is strikingly similar in the non-obese diabetic (NOD) mouse and humans. In IDDM, the systematic autoimmune destruction of insulin-producing beta cells within the pancreas is dependent on autoreactive T cells. This autoimmune process can be accelerated by transferring spleen cells from diabetic donors into irradiated syngeneic NOD mice. In a previous study we established that interleukin 2 receptor (IL 2R)-bearing cells propagated from pre-diabetic NOD mice promote IDDM. Therefore, we reasoned that specific elimination of IL 2R+ T cells should abort the diabetogenic process. T cell expressing IL 2R can be selectively destroyed with a diphtheria toxin-related IL 2 fusion protein (DAB486-IL-2). We set DAB486-IL-2 the challenging task of preventing fulminant IDDM accelerated by the adoptive transfer of diabetic spleen cells. Eight weeks after the adoptive transfer only 10% and 20% of NOD mice treated with 10 and 5 micrograms/day of DAB486-IL-2, respectively, became diabetic while 100% control mice (vehicle buffer) became diabetic within 5 weeks. A dose of 1 microgram/day of DAB486-IL-2 had no protective effect. Although the protection conferred by DAB486-IL-2 is not permanent, it is maintained for at least 4 weeks following cessation of treatment. Furthermore, even though these NOD mice do eventually become diabetic, the tempo of expression and severity of diabetes, as assessed by the level of hyperglycemia, is dramatically reduced. Although histologic examination of pancreas revealed minimal degree of mononuclear infiltrate within the islets in both groups, the vehicle control mice had fewer islets per section indicating many islets had already been destroyed. In addition, spleen cells from diabetic NOD mice which were pre-treated with DAB486-IL-2 (10 micrograms/day) for 1 week lost their ability to transfer disease. Taken together, these studies strongly support the concept that IL 2R-bearing T cells are essential for the induction of IDDM and suggest that DAB486-IL-2 would be a promising therapeutic approach in the treatment of human IDDM.     

alpha4 integrins and L-selectin differently orchestrate T-cell activity during diabetes prevention following oral administration of CTB-insulin

Oral administration of insulin conjugated to the B chain of cholera toxin (CTB-insulin) in non-obese diabetic (NOD) mice results in diabetes prevention. We investigated the respective contributions of L-selectin (CD62L) and alpha4-integrin pathways during CTB-driven tolerance. Purified CD62L+CD4+ cells from CTB-insulin fed mice significantly reduced the capacity of diabetogenic T cells to transfer diabetes in syngeneic recipients. In vivo antibody blockade of fed animals during adoptive co-transfer experiments indicated that both CD62L and alpha4-integrins pathways were necessary to develop a protective response after oral tolerance induction. In contrast, when antibodies were given to recipient mice, only CD62L was critical for the protection. In vitro stimulated CD62L+CD4+ cells from the spleen of fed animals secreted lower amounts of IL-4 and IL-10 but comparable levels of TGFbeta than CD62L-cells. A reduced IFN-gamma production between the two cell subsets was specifically observed in CTB-insulin fed mice. Furthermore, antibody treatments induced changes in T-cell migration to the spleen, mesenteric and pancreatic lymph nodes. The protective effect was also associated with migration of regulatory T cells into pancreatic islets. Taken together, our results suggest that L-selectin and alpha4-integrin have distinct but complementary roles in the generation and function of regulatory CD4+ T cells following CTB-insulin administration.     

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non‐obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well‐characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus‐like particles produced by co‐expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes‐resistant mice developed anti‐Env autoantibodies that increase in titer as disease progresses. A lentiviral‐based RNA interference knockdown of Gag revealed that Gag contributes to the MV‐induced T‐cell response, whose diabetogenic function can be demonstrated via cell‐transfer into immune‐deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet‐derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes‐resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti‐retroviral responses to trigger autoimmunity.     

Expression of transgene encoded TGF-beta in islets prevents autoimmune diabetes in NOD mice by a local mechanism

To analyse the effects of TGF-beta in insulin dependent diabetes mellitus (IDDM), we have developed non-obese diabetic (NOD) transgenic mice expressing TGF-beta under the control of the rat insulin II promoter. Pancreata of TGF-beta transgenic mice were roughly one twentieth of the size of pancreata of wild-type NOD mice and showed small clusters of micro-islets rather than normal adult islets. However, these islets produced sufficient levels of insulin to maintain normal glucose levels and mice were protected from the diabetes, which developed in their negative littermates. A massive fibrosis was seen in the transgenic pancreata that was accompanied with infiltration of mononuclear cells that decreased with age. Interestingly, these mice showed normal anti-islet immune response in their spleens and remained susceptible to adoptive transfer of IDDM by mature cloned CD8 effector cells. TUNEL assays revealed increased apoptosis of invading cells when compared to non-transgenic NOD mice. Taken together, these results suggest that TGF-beta protects islets by a local event.     

CCR2 and CCR5 chemokine receptors differentially influence the development of autoimmune diabetes in the NOD mouse

The infiltration of monocytes represents an important early event in the development of autoimmune diabetes in NOD mice. Given that chemokines are key regulators of leukocyte trafficking, we examined the requirement for the chemokine receptors β(CC)-chemokine receptor-5 (CCR5) and β(CC)-chemokine receptor-2 (CCR2), which recruit monocytes, in disease development in the NOD mouse. Whereas the onset of diabetes was significantly delayed in CCR2-/-NOD mice (25% at 30 weeks) compared to NOD mice (50% at 28 weeks), the pathogenesis of diabetes was accelerated in CCR5-/-NOD mice (75% at 23 weeks). The rapid development of diabetes in CCR5-/-NOD mice was associated with aggressive destructive insulitis and was accompanied by altered leukocyte migration into islets. In contrast, CCR2-/- NOD mice exhibited delayed inflammatory cell recruitment. Nevertheless, total diabetogenic splenocytes from CCR2-/-NOD and CCR5-/-NOD showed similar capability to adoptively transfer diabetes into NOD.scid recipients. Importantly, our data suggest that targeting of CCR2 may lead to therapies against Type 1 diabetes.     

Short administration of polyclonal anti-T cell antibody (ALS) in NOD mice with extensive insulitis prevents subsequent development of autoimmune diabetes

Treatment of overtly diabetic NOD mice with antilymphocyte serum (ALS), a polyclonal anti-T cell antibody, leads to cure of diabetes. Here, we investigated whether ALS-treatment of NOD mice after development of extensive insulitis prevents onset of diabetes. Female NOD mice were treated with two doses of ALS at 14, 19 or 23 weeks of age. No further treatment was given. In untreated female NOD mice, diabetes developed starting at 13 weeks and reached 68% by 37 weeks. ALS-treatment at 14, 19 or 23 weeks when histology showed progressive insulitis completely prevented onset of overt diabetes in 9/12, 11/12 or 12/12 mice, respectively. Intraperitoneal glucose tolerance tests in 43 week-old ALS-treated, diabetes-free mice showed a normal pattern. Co-adoptive transfer of lymphoid cells prepared from ALS-treated diabetes-free mice together with splenocytes from overtly diabetic NOD mice resulted in marked delay in diabetes onset in NOD.SCID mice, suggesting the presence of autoimmune regulatory cells in ALS-treated mice. Autoimmune regulatory cells were CD4(+)CD25(+), but not CD4(+)CD25(-), T cells. Thus, treatment of euglycemic individuals who already show signs of autoimmune diabetes with a short course of polyclonal anti-T cell antibody may effectively prevent onset of type 1 diabetes mellitus.     

Islet-infiltrating T cell clones from non-obese diabetic mice that promote or prevent accelerated onset diabetes

In humans and non-obese diabetic mice (NOD), insulin-dependent diabetes mellitus (IDDM) results from a spontaneous T cell-dependent autoimmune destruction of the insulin-producing pancreatic beta cells. Previous data suggest that a delicate balance between autoaggressive T cells and suppressor-type immune phenomena determine whether expression of autoimmunity is limited to insulitis or progresses to IDDM. To resolve the cellular basis of this intricate network of pathogenic CD4+ and CD8+ T cells and the role of T cells in suppressive immune phenomena. T cell clones were propagated directly from islets of NOD mice at the onset of insulitis. Insofar as insulitis, but not IDDM, is universal in NOD mice, we have screened for the in vivo effects of the islet-infiltrating T cell clones upon expression of IDDM, not insulitis. A CD4+ T cell clone, IS-3S7D, proliferates in response to islet antigen(s) and its transfer into prediabetic NOD mice promotes the rapid onset of IDDM. An interleukin 2 (IL 2)-dependent noncytolytic, V beta 11+ CD8+. T cell clones IS-2.15, prevents an accelerated onset diabetes in two distinct models. The present study, which documents the presence of CD4+ diabetogenic T cell clones and CD8+ T cell clones that dampen autoimmunity, gives tangible evidence that opposing autoimmune processes may determine whether an autoimmune-prone host develops frank disease.     

Protection of non-obese diabetic mice from autoimmune diabetes by Escherichia coli heat-labile enterotoxin B subunit

Autoimmune diabetes in the non-obese diabetic (NOD) mouse is associated with development of inflammation around the islets at around 4-5 weeks of age, which may be prolonged until frank diabetes begins to occur around 12 weeks of age. Although many interventions can halt disease progression if administration coincides with the beginning of the anti-beta cell response, very few are able to prevent diabetes development once insulitis is established. Here we describe a strategy which blocks cellular infiltration of islets and prevents diabetes. Intranasal treatment with the B-subunit of Escherichia coli heat labile enterotoxin (EtxB), a protein that binds GM1 ganglioside (as well as GD1b, asialo-GM1 and lactosylceramide with lower affinities), protected NOD mice from developing diabetes in a receptor-binding dependent manner. Protection was associated with a significant reduction in the number of macrophages, CD4(+) T cells, B cells, major histocompatibility complex class II(+) cells infiltrating the islets. Despite this, treated mice showed increased number of interleukin-10(+) cells in the pancreas, and a decrease in both T helper 1 (Th1) and Th2 cytokine production in the pancreatic lymph node. Disease protection was also transferred with CD4(+) splenocytes from treated mice. Taken together, these results demonstrated that EtxB is a potent immune modulator capable of blocking diabetes.     

Salmonella typhimurium infection halts development of type 1 diabetes in NOD mice

Infectious disease has been proposed as an environmental modifier of autoimmunity in both human populations and the NOD mouse. We found that infection of NOD mice with attenuated, but not killed, Salmonella typhimurium can reduce the incidence of type 1 diabetes (T1D), even if infection occurs after the development of a peri-islet pancreatic infiltrate. Functional diabetogenic effector T cells are still present, as demonstrated by the initiation of diabetes in NOD-scid recipients of transferred splenocytes. High levels of IFN-gamma are secreted by splenocytes of infected mice, but there is no evidence of involvement of IL-10 in the protective effect of the infection. Finally, prolonged changes in cell subsets are observed in infected mice involving invariant Valpha14Jalpha281 NuKappaTau and dendritic cells. These data reinforce the idea that prevention of T1D in the NOD mouse cannot be reduced to the simple Th1/Th2 paradigm and that different infections may involve different protective mechanisms.