Development Of Diabetes In NOD Mice Research Articles

Dietary emulsifier consumption accelerates type 1 diabetes development in NOD mice

The rapidly increasing prevalence of type 1 diabetes (T1D) underscores the role of environmental (i.e. non-genetic) determinants of T1D development. Such factors include industrialized diets as well as the intestinal microbiota with which they interact. One component of industrialized diets that deleteriously impact gut microbiota is dietary emulsifiers, which perturb intestinal microbiota to encroach upon their host promoting chronic low-grade intestinal inflammation and metabolic syndrome. Hence, we investigated whether 2 dietary emulsifiers, carboxymethylcellulose (CMC) and polysorbate-80 (P80), might influence the development of T1D in NOD mice, which spontaneously develop this disorder. We observed that chronic emulsifier exposure accelerated T1D development in NOD mice, which was associated with increased insulin autoantibody levels. Such accelerated T1D development was accompanied by compositional and functional alterations of the intestinal microbiota as well as low-grade intestinal inflammation. Moreover, machine learning found that the severity of emulsifier-induced microbiota disruption had partial power to predict subsequent disease development, suggesting that complex interactions occur between the host, dietary factors, and the intestinal microbiota. Thus, perturbation of host–microbiota homeostasis by dietary emulsifiers may have contributed to the post-mid-20th-century increase in T1D.

1488-P: Reduced iPLA2b Expression in T-Lymphocytes Decreases Proinflammatory Eicosanoid Production and Cytokine Secretion Delaying Type 1 Diabetes Development in NOD Mice

Type 1 diabetes (T1D) is characterized by the destruction of insulin-producing pancreatic islet β-cells, but the causes of this process remain unclear. We find that Ca2+-independent phospholipase A2β (iPLA2β), which hydrolyzes membrane phospholipids at the sn-2 substituent to yield iPLA2β-derived lipids (iDLs), is induced under a diabetic milieu and mitigation of iPLA2β activity attenuates β-cell death. Several iDLs are pro-inflammatory and have been studied in different diseases but are not understood in diabetes. We investigated the role of immune-cell iDLs by utilizing T-cell adoptive transfer. Isolated CD4+/8+ T-cells from splenocytes prepared from donor female NOD and NOD. iPLA2β+/− (HET) mice, were transferred i.p. to NOD.scid recipient mice. We observed T1D onset for all T-cell recipient groups between 10-12 weeks of age. However, reduction of iPLA2β in CD4/8 T-cells delayed 50% incidence by 10 weeks in NOD4/HET8-recipient mice and HET4/NOD8 T-cell recipients compared to “matched” T-cell recipients. Furthermore, reduced insulitis and increased β-cell preservation was detected in HET4/NOD8-recipients compared to NOD4/8 recipients. The immune cell composition between our genotypes revealed no difference suggesting iDL signaling contributes to the observed incidence. Lipidomic analyses revealed a decrease in pro-inflammatory eicosanoids (PGs, DHETs and HETEs) in nondiabetic recipient mice, compared to diabetic counterparts. In addition, by targeting select eicosanoid signaling (PGE2 and DHET) utilizing chemical inhibitors (i.e., Grapiprant (EP4 receptor antagonist) and EC5026 (soluble epoxide hydrolase inhibitor)), we observed decreased IFN-γ production by Th1 helper cells in HET donors compared to NOD. These findings suggest that iPLA2β in T-cells is a critical contributor to the production of pro-inflammatory iDLs and could be a potential target in reducing T1D development. Disclosure T. White: None. S. Ramanadham: None. T.P. DiLorenzo: None. C.E. Chalfant: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK069455); National Institute of Allergy and Infectious Diseases (R21AI146743, R21AI169214); JDRF (2-SRA-2022-1210-S-B); University of Alabama at Birmingham (5T32GM8111-34)

74-OR: Adoptively Transferred T Lymphocytes with Reduced iPLA2b Expression Delays Type 1 Diabetes Development in NOD Mice

Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease characterized by the destruction of insulin-producing pancreatic islet β-cells. An understudied area is the role lipid signaling plays in this process. We find that the Ca2+-independent phospholipase A2β (iPLA2β) is induced under a diabetic milieu and mitigation of iPLA2β activity attenuates β-cell death. iPLA2β, a member of the PLA2 family, hydrolyzes the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid. When the fatty acid is arachidonic acid, it can be metabolized to eicosanoids, many of which are pro-inflammatory. To address our overarching hypothesis that iPLA2β-derived lipids (iDLs) contribute to T1D development, we investigated the role of immune cell-iDLs by utilizing a T-cell adoptive transfer. We isolated CD4+/8+ T-cells from splenocytes prepared from 11-week-old donor female NOD and NOD.iPLA2 β +/- (HET) . These were transferred i.p. to 4-week-old NOD.scid recipient mice. We find that NOD4/8-recipient mice develop T1D between 14-16 weeks of age. In contrast, T1D onset in HET4/8-recipient mice is delayed by 12 weeks and this was associated with better glucose tolerance, compared to NOD4/8 T-cell recipients. This finding is also supported by histology analysis of the pancreatic islet, which revealed less islet infiltration, more islet abundance, and the frequency of CD4 T cells were increased in the pancreas of HET4/8-recipient mice compared to NOD4/8 recipients. Further, lipidomics analyses revealed that HET donor T-cell production of proinflammatory lipids (i.e., PGE2 and DHETs) is decreased, relative to NOD T-cells. These findings suggest that pro-inflammatory iDLs generated by CD4 T-cells are critical contributors T1D development and can be targeted to counter T1D onset. Disclosure T.White: None. C.Chalfant: None. T.P.Dilorenzo: None. S.Ramanadham: None. D.Stephenson: None. Funding Training Program in Cell, Molecular, and Developmental Biology (5T32GM008111-34) NIH/NIDDK (R01DK110292) NIH/NIAID R21 (AI 146743) Cell, Developmental, & Integrative Biology (CDIB) , the University of Alabama at Birmingham

Chromogranin A Deficiency Confers Protection From Autoimmune Diabetes via Multiple Mechanisms.

Recognition of β-cell antigens by autoreactive T cells is a critical step in the initiation of autoimmune type1 diabetes. A complete protection from diabetes development in NOD mice harboring a point mutation in the insulin B-chain 9–23 epitope points to a dominant role of insulin in diabetogenesis. Generation of NOD mice lacking the chromogranin A protein (NOD.ChgA−/−) completely nullified the autoreactivity of the BDC2.5 T cell and conferred protection from diabetes onset. These results raised the issue concerning the dominant antigen that drives the autoimmune process. Here we revisited the NOD.ChgA−/− mice and found that their lack of diabetes development may not be solely explained by the absence of chromogranin A reactivity. NOD.ChgA−/− mice displayed reduced presentation of insulin peptides in the islets and periphery, which corresponded to impaired T-cell priming. Diabetes development in these mice was restored by antibody treatment targeting regulatory T cells or inhibiting transforming growth factor-β and programmed death-1 pathways. Therefore, the global deficiency of chromogranin A impairs recognition of the major diabetogenic antigen insulin, leading to broadly impaired autoimmune responses controlled by multiple regulatory mechanisms.

Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice.

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation of bone marrow–derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of interferon-γ–producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes.

Microencapsulated G3C Hybridoma Cell Graft Delays the Onset of Spontaneous Diabetes in NOD Mice by an Expansion of Gitr+ Treg Cells.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3+ Treg-cell expansion in the spleen of wild-type mice but not in Gitr-/- mice. G3C/cps also induced the expansion of nonconventional Cd4+Cd25-/lowFoxp3lowGitrint/high (GITR single-positive [sp]) Tregs. Both Cd4+Cd25+GitrhighFoxp3+ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Preproinsulin Designer Antigens Excluded from Endoplasmic Reticulum Suppressed Diabetes Development in NOD Mice by DNA Vaccination

DNA vaccines against autoimmune type 1 diabetes (T1D) contain a nonpredictable risk to induce autoreactive T cell responses rather than a protective immunity. Little is known if (and how) antigen expression and processing requirements favor the induction of autoreactive or protective immune responses by DNA immunization. Here, we analyzed whether structural properties of preproinsulin (ppins) variants and/or subcellular targeting of ppins designer antigens influence the priming of effector CD8+ T cell responses by DNA immunization. Primarily, we used H-2b RIP-B7.1 tg mice, expressing the co-stimulator molecule B7.1 in beta cells, to identify antigens that induce or fail to induce autoreactive ppins-specific (Kb/A12-21 and/or Kb/B22-29) CD8+ T cell responses. Female NOD mice, expressing the diabetes-susceptible H-2g7 haplotype, were used to test ppins variants for their potential to suppress spontaneous diabetes development. We showed that ppins antigens excluded from expression in the endoplasmic reticulum (ER) did not induce CD8+ T cells or autoimmune diabetes in RIP-B7.1 tg mice, but efficiently suppressed spontaneous diabetes development in NOD mice as well as ppins-induced CD8+ T cell-mediated autoimmune diabetes in PD-L1−/− mice. The induction of a ppins-specific therapeutic immunity in mice has practical implications for the design of immune therapies against T1D in individuals expressing different major histocompatibility complex (MHC) I and II molecules.

Low CD25 on autoreactive Tregs impairs tolerance via low dose IL-2 and antigen delivery

Dendritic cell (DC)-mediated T cell tolerance deficiencies contribute to the pathogenesis of autoimmune diseases such as type 1 diabetes. Delivering self-antigen to dendritic-cell inhibitory receptor-2 (DCIR2)+ DCs can delay but not completely block diabetes development in NOD mice. These DCIR2-targeting antibodies induce tolerance via deletion and anergy, but do not increase islet-specific Tregs. Because low-dose IL-2 (LD-IL-2) administration can preferentially expand Tregs, we tested whether delivering islet-antigen to tolerogenic DCIR2+ DCs along with LD-IL-2 would boost islet-specific Tregs and further block autoimmunity. But, surprisingly, adding LD-IL-2 did not increase efficacy of DC-targeted antigen to inhibit diabetes. Here we show the effects of LD-IL-2, with or without antigen delivery to DCIR2+ DCs, on both polyclonal and autoreactive Treg and conventional T cells (Tconv). As expected, LD-IL-2 increased total Tregs, but autoreactive Tregs required both antigen and IL-2 stimulation for optimal expansion. Also, islet-specific Tregs had lower CD25 expression and IL-2 sensitivity, while islet-specific Tconv had higher CD25 expression, compared to polyclonal populations. LD-IL-2 increased activation and expansion of Tconv, and was more pronounced for autoreactive cells after treatment with IL-2 + islet-antigen. Therefore, LD-IL-2 therapy, especially when combined with antigen stimulation, may not optimally activate and expand antigen-specific Tregs in chronic autoimmune settings.

CD137 Plays Both Pathogenic and Protective Roles in Type 1 Diabetes Development in NOD Mice.

We previously reported that CD137 (encoded by Tnfrsf9) deficiency suppressed type 1 diabetes (T1D) progression in NOD mice. We also demonstrated that soluble CD137 produced by regulatory T cells contributed to their autoimmune-suppressive function in this model. These results suggest that CD137 can either promote or suppress T1D development in NOD mice depending on where it is expressed. In this study, we show that NOD.Tnfrsf9-/- CD8 T cells had significantly reduced diabetogenic capacity, whereas absence of CD137 in non-T and non-B cells had a limited impact on T1D progression. In contrast, NOD.Tnfrsf9-/- CD4 T cells highly promoted T1D development. We further demonstrated that CD137 was important for the accumulation of β cell-autoreactive CD8 T cells but was dispensable for their activation in pancreatic lymph nodes. The frequency of islet-infiltrating CD8 T cells was reduced in NOD.Tnfrsf9-/- mice in part because of their decreased proliferation. Furthermore, CD137 deficiency did not suppress T1D development in NOD mice expressing the transgenic NY8.3 CD8 TCR. This suggests that increased precursor frequency of β cell-autoreactive CD8 T cells in NY8.3 mice obviated a role for CD137 in diabetogenesis. Finally, blocking CD137-CD137 ligand interaction significantly delayed T1D onset in NOD mice. Collectively, our results indicate that one important diabetogenic function of CD137 is to promote the expansion and accumulation of β cell-autoreactive CD8 T cells, and in the absence of CD137 or its interaction with CD137 ligand, T1D progression is suppressed.

Different immunological responses to early-life antibiotic exposure affecting autoimmune diabetes development in NOD mice

Environmental factors clearly influence the pathogenesis of Type 1 diabetes, an autoimmune disease. We have studied gut microbiota as important environmental agents that could affect the initiation or progression of type 1 diabetes especially in the prenatal period. We used neomycin, targeting mainly Gram negative or vancomycin, targeting mainly Gram positive bacteria, to treat pregnant NOD mothers and to study autoimmune diabetes development in their offspring. Neomycin-treated offspring were protected from diabetes, while vancomycin-treated offspring had accelerated diabetes development, and both antibiotics caused distinctly different shifts in gut microbiota composition compared with the offspring from untreated control mice. Our study demonstrated that neomycin treatment of pregnant mothers leads to generation of immune-tolerogenic antigen-presenting cells (APCs) in the offspring and these APCs had reduced specific autoantigen-presenting function both in vitro and in vivo. Moreover, the protection from diabetes mediated by tolerogenic APCs was vertically transmissible to the second generation. In contrast, more diabetogenic inflammatory T cells were found in the lymphoid organs of the offspring from the vancomycin-treated pregnant mothers. This change however was not transmitted to the second generation. Our results suggested that prenatal exposure to antibiotic influenced gut bacterial composition at the earliest time point in life and is critical for consequent education of the immune system. As different bacteria can induce different immune responses, understanding these differences and how to generate self-tolerogenic APCs could be important for developing new therapy for type 1 diabetes.

B-lymphocytes expressing an immunoglobulin specificity recognizing the natural pancreatic beta cell autoantigen, peripherin, are rendered proliferatively anergic but still accelerate type 1 diabetes development in NOD mice. (BA3P.107)

Abstract Type 1 diabetes (T1D) results from the destruction of insulin-producing pancreatic ß-cells by autoreactive CD4+ and CD8+ T-cells culminating in glucose dysregulation in both humans and the NOD mouse model. It is clear in the NOD mouse model and also likely humans that B-lymphocytes also play a key pathogenic role in T1D. A sizable proportion of B-lymphocytes isolated from pancreatic islets in NOD mice recognize the autoantigen, peripherin, a type II intermediate filament expressed widely in the peripheral and central nervous system. To further understand the role peripherin-reactive B lymphocytes play in the pathogenesis of T1D, we created NOD mice transgenically expressing non-secretory heavy and light chain transgenes encoding an immunoglobulin (Ig) recognizing peripherin derived from islet-associated B lymphocytes. These mice overwhelming produce B-lymphocytes expressing the transgenic peripherin-binding Ig molecule and develop T1D at a significantly accelerated rate compared with the standard NOD strain. Islet infiltration by lymphocytes also occurs earlier in these mice with the vast majority of islet-associated B-lymphocytes expressing the transgenic Ig. While these peripherin-reactive B lymphocytes are diabetogenic, they are also proliferatlively anergic and appear to require frequent replenishment from hematopoietic stem cells. These findings lend credence to the targeting of peripherin autoreactive B-lymphocytes as a possible T1D intervention.

WRNIP1 is a key regulator of antigen-specific Treg cells. (IRC4P.480)

Abstract It is well documented that, both Foxp3+ regulatory T cells (Treg) and antigen-expanded Foxp3lo/- Treg play critical roles in regulating immunity and autoimmunity, as well as in preventing autoimmune diseases. However, the molecular mechanisms and genes responsible for modulating function of these Treg still remain incompletely understood. WRNIP1 (Werner helicase-interacting protein 1) was identified as a protein that interacts with WRN, the Werner syndrome responsible gene product, and may participate in DNA synthesis, damage and repair. Here, we report an unexpected finding that WRNIP1 plays an important role in regulating the function of autoantigen-expanded Foxp3lo/- Treg. Down regulation of WRNIP1 in these Treg significantly increased their proliferation and reduced activation-induced cell death. More importantly, WRNIP1-downregulated Treg showed markedly enhanced suppressive function in vitro, partly due to an increased production of IL-10 and TGF-β. These Treg also significantly improved their ability in preventing diabetes development in NOD mice. To understand how WRNIP1 regulates Treg function, we found that elevated Foxo1 expression may contribute to the enhanced Treg function through promoting IL-10 and TGF-β production. These studies demonstrate that WRNIP1 negatively regulates Treg function and suggest previously undisclosed molecular pathways and targets for improving Treg-mediated immune tolerance to treat autoimmune diseases like type 1 diabetes.

Immune regulatory mechanisms, not alteration in Th1/Th2 skewing, are responsible for helminth-mediated protection against type 1 diabetes in NOD mice. (116.3)

Abstract Both Th2 immune responses and regulatory responses are increased during helminth infection and have the potential to be the mechanisms by which helminth infections protect against Th1-driven autoimmune diseases. We investigated these mechanisms by evaluating diabetes development in NOD mice infected with the filarial nematode Litomosoides sigmodontis in settings of depletion or absence of IL-4, regulatory T cells, IL-10, and TGFβ. Despite the absence of a Th2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4+CD25+FoxP3+ regulatory T cell frequencies and numbers, respectively, increased proliferation of CD4+FoxP3+ cells, and increased production of TGFβ. Depletion of FoxP3+ T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGFβ, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. This study demonstrates that helminth-mediated protection against autoimmunity is not dependent on the induction of a Th2 shift but does require TGFβ. These findings suggest it may be possible to develop helminth-derived therapies for autoimmunity that induce protective regulatory responses without upregulating potentially harmful proallergic Th2 responses.

The Role of Gr1+ Cells after Anti-CD20 Treatment in Type 1 Diabetes in Nonobese Diabetic Mice

Studies suggest that Gr1(+)CD11b(+) cells have immunoregulatory function, and these cells may play an important role in autoimmune diseases. In this study, we investigated the regulatory role of Gr1(+)CD11b(+) cells in protecting against type 1 diabetes in NOD mice. In this study, we showed that temporary B cell depletion induced the expansion of Gr1(+)CD11b(+) cells. Gr1(+)CD11b(+) cells not only directly suppress diabetogenic T cell function but also can induce regulatory T cell differentiation in a TGF-β-dependent manner. Furthermore, we found that Gr1(+)CD11b(+) cells could suppress diabetogenic CD4 and CD8 T cell function in an IL-10-, NO-, and cell contact-dependent manner. Interestingly, single anti-Gr1 mAb treatment can also induce a transient expansion of Gr1(+)CD11b(+) cells that delayed diabetes development in NOD mice. Our data suggest that Gr1(+)CD11b(+) cells contribute to the establishment of immune tolerance to pancreatic islet autoimmunity. Manipulation of Gr1(+)CD11b(+) cells could be considered as a novel immunotherapy for the prevention of type 1 diabetes.

Loss of Intra-Islet CD20 Expression May Complicate Efficacy of B-Cell–Directed Type 1 Diabetes Therapies

OBJECTIVEConsistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell–depleting agents, as well as attempts seeking diabetes prevention, are being considered.RESEARCH DESIGN AND METHODSAutoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset.RESULTSThe murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell–dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets.CONCLUSIONSThese findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention.

Characterization of antigen-specific CD4+CD25+ regulatory T-cells expanded with IL-2/IL-2mAb complexes in NOD mice (115.9)

Abstract Type-1 diabetes is an autoimmune disease caused by the destruction of insulin-producing pancreatic beta cells by auto-reactive T-cells. It results from a failure of the immune system to maintain tolerance to self-antigens. CD4+CD25+ regulatory T-cells represent one major mechanism by which auto-reactive T-cells are controlled in the periphery. Deficiency in this cell population number or function could tilt the regulatory cell-pathogenic cell balance and lead to type-1 diabetes development. In this study, we describe a new method to expand antigen-specific CD4+ CD25+ regulatory T-cells from autoimmune-prone non-obese diabetic mice. CD4+ CD25+ regulatory T-cells were expanded in vivo using a combination of IL-2, anti-IL-2 antibody, BDC 2.5 peptide, and rapamycin. The expanded CD4+ CD25+ regulatory T-cells expressed a classical phenotype, displayed suppressive functions in vivo, and preliminary data suggest that these cells can delay diabetes development in NOD mice. This new method of peripheral tolerance induction could be used in humans to promote tolerance to islet cell transplants without relying on immunosuppressive drugs.

IL-15 promotes regulatory T cell function and protects against diabetes development in NK-depleted NOD mice

IL-15, an anti-apoptotic cytokine, has been reported to promote the survival and function of NK cells and T cells, including regulatory T cells (Tregs). Here we examined the effect of repeated injections of IL-15 on the development of diabetes in NOD mice. Injection of recombinant murine IL-15, once a day for 2 weeks, neither inhibited nor accelerated diabetes development in untreated NOD mice. However, treatment with IL-15 significantly reduced the incidence and delayed the onset of diabetes in NOD mice that were depleted of NK cells, while NK cell depletion alone had no protection against the disease development. The protective effect in IL-15-treated, NK cell-depleted NOD mice was associated with an increase in immunosuppressive activity of CD4(+)CD25(+) Tregs. IL-15 also enhanced Foxp3 expression in CD4(+)CD25(+) cells in an in vitro culture system, and such an effect of IL-15 was abrogated by IL-15-activated NK cells. Inhibition of IL-15-induced Foxp3 expression by IL-15-activated NK cells likely resulted from their IFN-gamma production, as recombinant IFN-gamma, or the culture supernatant of IL-15-activated wild-type mouse NK cells but not of IL-15-activated IFN-gamma-deficient NK cells, mediated a similar inhibition. IFN-gamma also diminished the stimulatory effect of IL-15 on Treg function in vitro. These results indicate that IL-15 has the potential to promote Treg function and protect against diabetes development in NOD mice, but such an activity can be eliminated by simultaneous activation of NK cells in IL-15-treated mice.

CD4+CD25+ T-Cells Control Autoimmunity in the Absence of B-Cells

OBJECTIVETumor necrosis factor ligand family members B-cell–activating factor (BAFF) and a proliferation-inducing ligand (APRIL) can exert powerful effects on B-cell activation and development, type 1 T-helper cell (Th1) immune responses, and autoimmunity. We examined the effect of blocking BAFF and APRIL on the development of autoimmune diabetes.RESEARCH DESIGN AND METHODSFemale NOD mice were administered B-cell maturation antigen (BCMA)-Fc from 9 to 15 weeks of age. Diabetes incidence, islet pathology, and T- and B-cell populations were examined.RESULTSBCMA-Fc treatment reduced the severity of insulitis and prevented diabetes development in NOD mice. BCMA-Fc–treated mice showed reduced follicular, marginal-zone, and T2MZ B-cells. B-cell reduction was accompanied by decreased frequencies of pathogenic CD4+CD40+ T-cells and reduced Th1 cytokines IL-7, IL-15, and IL-17. Thus, T-cell activation was blunted with reduced B-cells. However, BCMA-Fc–treated mice still harbored detectable diabetogenic T-cells, suggesting that regulatory mechanisms contributed to diabetes prevention. Indeed, BCMA-Fc–treated mice accumulated increased CD4+CD25+ regulatory T-cells (Tregs) with age. CD4+CD25+ cells were essential for maintaining euglycemia because their depletion abrogated BCMA-Fc–mediated protection. BCMA-Fc did not directly affect Treg homeostasis given that CD4+CD25+Foxp3+ T-cells did not express TACI or BR3 receptors and that CD4+CD25+Foxp3+ T-cell frequencies were equivalent in wild-type, BAFF−/−, TACI−/−, BCMA−/−, and BR3−/− mice. Rather, B-cell depletion resulted in CD4+CD25+ T-cell–mediated protection from diabetes because anti-CD25 monoclonal antibody treatment precipitated diabetes in both diabetes-resistant NOD.μMT−/− and BCMA-Fc–treated mice.CONCLUSIONSBAFF/APRIL blockade prevents diabetes. BCMA-Fc reduces B-cells, subsequently blunting autoimmune activity and allowing endogenous regulatory mechanisms to preserve a prehyperglycemic state.

Type 1 Diabetes Development Requires Both CD4+ and CD8+ T cells and Can Be Reversed by Non-Depleting Antibodies Targeting Both T Cell Populations

Type 1 diabetes development in NOD mice appears to require both CD4(+) and CD8(+) T cells. However, there are some situations where it has been suggested that either CD4(+) or CD8(+) T cells are able to mediate diabetes in the absence of the other population. In the case of transgenic mice, this may reflect the numbers of antigen-specific T cells able to access the pancreas and recruit other cell types such as macrophages leading to a release of high concentrations of damaging cytokines. Previous studies examining the requirement for CD8(+) T cells have used antibodies specific for CD8alpha. It is known that CD8alpha is expressed not only on alphabeta T cells, but also on other cell types, including a DC population that may be critical for presenting islet antigen in the pancreatic draining lymph nodes. Therefore, we have re-examined the need for both CD4(+) and CD8(+) T cell populations in diabetes development in NOD mice using an antibody to CD8beta. Our studies indicate that by using highly purified populations of T cells and antibodies specific for CD8(+) T cells, there is indeed a need for both cell types. In accordance with some other reports, we found that CD4(+) T cells appeared to be able to access the pancreas more readily than CD8(+) T cells. Despite the ability of CD4(+) T cells to recruit CD11b class II positive cells, diabetes did not develop in the absence of CD8(+) T cells. These studies support the observation that CD8(+) T cells may be final effector cells. As both T cell populations are clearly implicated in diabetes development, we have used a combination of non-depleting antibodies to target both CD4-positive and CD8-positive cells and found that this antibody combination was able to reverse diabetes onset in NOD mice as effectively as anti-CD3 antibodies.

APC Activation Restores Functional CD4+CD25+ Regulatory T Cells in NOD Mice that Can Prevent Diabetes Development

BackgroundDefects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4+CD25+ regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4+CD25+ regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease.Methodology/Principal FindingsTo test these hypotheses, we used the well-documented ability of complete Freund's adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4+CD25+ regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4+CD25+ cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4+CD25+Foxp3+ cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4+CD25+ cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4+CD25+ cells in vitro compared to APC from untreated NOD mice.Conclusions/SignificanceThese data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC.