krn-7000 and Diabetes-Mellitus--Type-1

T cells reactive to lipids and restricted by major histocompatibility complex (MHC) class I-like molecules represent more than 15% of all lymphocytes in human blood. This heterogeneous population of innate cells includes the invariant natural killer T cells (iNK T), type II NK T cells, CD1a,b,c-restricted T cells and mucosal-associated invariant T (MAIT) cells. These populations are implicated in cancer, infection and autoimmunity. In this review, we focus on the role of these cells in autoimmunity. We summarize data obtained in humans and preclinical models of autoimmune diseases such as primary biliary cirrhosis, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, psoriasis and atherosclerosis. We also discuss the promise of NK T cell manipulations: restoration of function, specific activation, depletion and the relevance of these treatments to human autoimmune diseases.

Topics: Animals; Arthritis, Rheumatoid; Atherosclerosis; Autoimmunity; Clinical Trials, Phase I as Topic; Diabetes Mellitus, Type 1; Female; Galactosylceramides; Humans; Liver Cirrhosis, Biliary; Lupus Erythematosus, Systemic; Male; Mice; Multiple Sclerosis; Natural Killer T-Cells; Psoriasis

Natural killer T (NKT) cells are a conserved subpopulation of lymphocytes that recognize glycolipid antigens in a CD1d context. Upon activation through their semi-invariant T cell receptor, these cells rapidly release large amounts of immunomodulating Th1 and Th2 cytokines. NKT cells have therefore been implicated in immune responses controlling various diseases, including infection, cancer, transplantation, and autoimmunity. Stimulation of the immunoregulatory capacity of NKT cells by the prototypical antigen alpha-galactosylceramide results in amelioration of disease in several animal models. This review will focus on the current knowledge of human NKT cells and their role in autoimmune diseases. The features of these cells and their importance in regulation of autoimmunity suggest that NKT cell-based therapies might be an interesting approach for the treatment of autoimmune diseases.

Topics: Animals; Autoimmune Diseases; Autoimmunity; Diabetes Mellitus, Type 1; Encephalomyelitis, Autoimmune, Experimental; Galactosylceramides; Humans; Killer Cells, Natural; Lymphocyte Activation; Multiple Sclerosis; T-Lymphocyte Subsets; T-Lymphocytes; Thymus Gland

CD1d-restricted Natural Killer T cells (NKT cells), a novel lymphocyte lineage, are considered to play an intermediary role bridging innate and acquired immunity. This review discusses the characteristics of NKT cells and their biological significance in the immune system, and summarizes their in vivo functions observed in a number of pathological settings, including infectious diseases, cancer, autoimmunity, and transplantation. Further, we discuss recent data that have generated considerable interest in utilizing NKT cells as targets of new therapeutic interventions in various human diseases.

Topics: Animals; Antigens, CD1; Antigens, CD1d; Apoptosis; Autoimmunity; Cell Transplantation; Cytokines; Diabetes Mellitus, Type 1; Encephalomyelitis, Autoimmune, Experimental; Galactosylceramides; Glycolipids; Humans; Immune System; Immunity, Innate; Killer Cells, Natural; Multiple Sclerosis; Neoplasms

Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention.

Topics: Animals; Autoimmune Diseases; Cytokines; Diabetes Mellitus, Type 1; Drug Implants; Estradiol; Estrogen Replacement Therapy; Estrogens; Female; Galactosylceramides; Immune Tolerance; Killer Cells, Natural; Lymphocyte Activation; Lymphocyte Depletion; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Mutant Strains; Ovariectomy; Prediabetic State; T-Lymphocytes, Regulatory

Invariant natural killer T (iNKT) cells belong to the innate immune system and exercise a dual role as potent regulators of autoimmunity and participate in responses against different pathogens. They have been shown to prevent type 1 diabetes development and to promote antiviral responses. Many studies in the implication of environmental factors on the etiology of type 1 diabetes have suggested a link between enteroviral infections and the development of this disease. This study of the pancreatropic enterovirus Coxsackievirus B4 (CVB4) shows that although infection accelerated type 1 diabetes development in a subset of proinsulin 2-deficient NOD mice, the activation of iNKT cells by a specific agonist, α-galactosylceramide, at the time of infection inhibited the disease. Diabetes development was associated with the infiltration of pancreatic islets by inflammatory macrophages, producing high levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and activation of anti-islet T cells. On the contrary, macrophages infiltrating the islets after CVB4 infection and iNKT-cell stimulation expressed a number of suppressive enzymes, among which indoleamine 2,3-dioxygenase was sufficient to inhibit anti-islet T-cell response and to prevent diabetes. This study highlights the critical interaction between virus and the immune system in the acceleration or prevention of type 1 diabetes.

Topics: Animals; Coxsackievirus Infections; Diabetes Mellitus, Type 1; Female; Galactosylceramides; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-gamma; Interleukin-13; Islets of Langerhans; Macrophages; Mice; Mice, Inbred NOD; Mice, Transgenic; Natural Killer T-Cells

We have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist α-galactosylceramide C26:0 (α-GalCer) or its T helper type 2 (Th2)-biasing derivative α-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to α-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of α-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with α-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by α-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than α-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.

Topics: Animals; B7-1 Antigen; B7-H1 Antigen; Cell Movement; Cells, Cultured; Clonal Anergy; Dendritic Cells; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Galactosylceramides; Interferon-gamma; Interleukin-4; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Natural Killer T-Cells; Peptides; Th2 Cells; Time Factors

Type 1 diabetes is an autoimmune disorder resulting from lymphocyte-mediated destruction of insulin-producing β cells in pancreas. Natural killer T cells are regulatory immune components controlling autoreactivity and immune homeostasis. Although early studies supported that amelioration of autoimmune diabetes by natural killer T cells was associated with Th1/2 shift, other Th2-independent regulatory mechanisms were also suggested. Since natural killer T cells are critical for the generation of CD8(+) regulatory T cells controlling anterior chamber-associated immune deviation and CD8(+) regulatory T cells also participate in suppression of immune responses like experimental autoimmune encephalomyelitis, we investigate whether the similar suppressive effects are involved in α-galactosylceramide-induced immune tolerance in non-obese diabetic mice. We demonstrate that repeated exposure of α-galactosylceramide reveals a hyporesponsiveness of total or antigen-presenting cells-depleted splenocytes upon anti-CD3/28 antibodies stimulation. The dispensability of dendritic cells in the hyporesponsiveness is consistent with the comparable expression of costimulatory molecules on CD11c(+) subsets between α-galactosylceramide- and vehicle-treated mice. α-Galactosylceramide treatment not only affects the effector T cell subsets and their cytokine production but also increases the secretion of transforming growth factor-β by splenocytes, implying the suppressive regulation. The adoptive transfer experiments demonstrate the suppressive effect of T cells from α-galactosylceramide-treated non-obese diabetic mice when co-transferred with vehicle-treated littermates. Finally, it reveals that CD8(+) subset among antigen-presenting cells-depleted splenocytes tends to confer the suppression since the protective ability vanishes upon withdrawal of CD8(+) subset. These results suggest that repeated exposure of α-galactosylceramide ameliorates autoimmune diabetes in non-obese diabetic mice mediated by CD8(+) T cell-associated suppression.

Topics: Animals; Antigen-Presenting Cells; CD8-Positive T-Lymphocytes; Cytokines; Diabetes Mellitus, Type 1; Female; Galactosylceramides; Lymph Nodes; Male; Mice; Mice, Inbred NOD; Pancreas; Spleen

Because invariant natural killer T (iNK T) cells link innate and adaptive immunity, the structure-dependent design of iNK T cell agonists may have therapeutic value as vaccines for many indications, including autoimmune disease. Previously, we showed that treatment of non-obese diabetic (NOD) mice with the iNK T cell activating prototypic glycolipid α-galactosylceramide (α-GalCer) protects them from type 1 diabetes (T1D). However, α-GalCer is a strong agonist that can hyperactivate iNK T cells, elicit several side effects and has shown only limited success in clinical trials. Here, we used a structure-guided design approach to identify an iNK T cell agonist that optimally protects from T1D with minimal side effects. Analyses of the kinetics and function of a panel of synthetic α-GalCer fatty acyl chain derivatives (C8:0-C16:0) were performed in NOD mice. C16:0 elicited the highest protection from insulitis and T1D, which was associated with a higher frequency and survival of iNK T cells and enhanced activity of tolerogenic dendritic cells (DCs) in draining pancreatic lymph nodes (PLN), inability to transactivate NK cells and a more rapid kinetics of induction and recovery of iNK T cells from anergy. We conclude that the length and structure of the acyl chain of α-GalCer regulates the level of protection against T1D in mice, and propose that the extent of this protection depends on the relative capacity of the acyl chain to accommodate an endogenous spacer lipid of appropriate length and structure. Thus, our findings with the α-GalCer C16:0 derivative suggest strongly that it be considered as a lead glycolipid candidate in clinical trials of T1D.

Topics: Animals; Cytokines; Dendritic Cells; Diabetes Mellitus, Type 1; Drug Design; Female; Flow Cytometry; Galactosylceramides; Immunization; Injections, Intraperitoneal; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Molecular Targeted Therapy; Natural Killer T-Cells; Pancreas; Quantitative Structure-Activity Relationship

The ectoenzyme ADP-ribosyltransferase 2.2 (ART2.2) can apoptotically delete various T-cell subsets. Depending on the involved apoptotic T-cell subset, enhanced ART2.2 activity could result in immunosuppression or autoimmunity. Diminished activity of the CD38 ectoenzyme that normally represents a counter-regulatory competitor for the NAD substrate represents one mechanism enhancing ART2.2 activity. Hence, it would be desirable to develop an agent that efficiently blocks ART2.2 activity in vivo. While the llama derived recombinant s+16 single domain antibody overcame the difficulty of specifically targeting the ART2.2 catalytic site potential therapeutic use of this reagent is limited due to short in vivo persistence. Thus, we tested if a modified version of s+16 incorporating the murine IgG1 Fc tail (s+16Fc) mediated long-term efficient in vivo suppression of ART2.2. We reasoned an ideal model to test the s+16Fc reagent were NOD mice in which genetic ablation of CD38 results in an ART2.2 mediated reduction in already sub-normal numbers of immunoregulatory natural killer T-(NKT) cells to a level that no longer allows them when activated by the super-agonist alpha-galactosylceramide (alpha-GalCer) to elicit effects inhibiting autoimmune type 1 diabetes (T1D) development. Treatment with s+16Fc efficiently mediated long-term in vivo inhibition of ART2.2 activity in NOD.CD38(null) mice, restoring their iNKT cell numbers to levels that upon alpha-GalCer activation were capable of inhibiting T1D development.

Topics: ADP Ribose Transferases; ADP-ribosyl Cyclase 1; Animals; Diabetes Mellitus, Type 1; Female; Galactosylceramides; Immunoglobulin Heavy Chains; Immunosuppression Therapy; Lymphocyte Activation; Lymphocyte Depletion; Mice; Mice, Knockout; Mice, Transgenic; Natural Killer T-Cells; Protein Engineering; Recombinant Fusion Proteins; T-Lymphocytes, Regulatory

Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigen Presentation; Bystander Effect; Cytokines; Dendritic Cells; Diabetes Mellitus, Type 1; Drug Evaluation, Preclinical; Female; Galactosylceramides; Hypoglycemic Agents; Immunologic Factors; Interleukin-12; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Natural Killer T-Cells; Specific Pathogen-Free Organisms; Spleen; Structure-Activity Relationship; Th1 Cells; Th2 Cells

Stimulated by an agonistic ligand, alpha-galactosylceramide (alphaGalCer), invariant NKT (iNKT) cells are capable of both eliciting antitumor responses and suppressing autoimmunity, while they become anergic after an initial phase of activation. It is unknown how iNKT cells act as either activators or regulators in different settings of cellular immunity. We examined effects of alphaGalCer administration on autoimmune inflammation and characterized phenotypes and functional status of iNKT cells and dendritic cells in alphaGalCer-treated NOD mice. Although iNKT cells became and remained anergic after the initial exposure to their ligand, anergic iNKT cells induce noninflammatory DCs in response to alphaGalCer restimulation, whereas activated iNKT cells induce immunogenic maturation of DCs in a small time window after the priming. Induction of noninflammatory DCs results in the activation and expansion of islet-specific T cells with diminished proinflammatory cytokine production. The noninflammatory DCs function at inflammation sites in an Ag-specific fashion, and the persistence of noninflammatory DCs critically inhibits autoimmune pathogenesis in NOD mice. Anergic differentiation is a regulatory event that enables iNKT cells to transform from promoters to suppressors, down-regulating the ongoing inflammatory responses, similar to other regulatory T cells, through a ligand-dependent mechanism.

Topics: Animals; Autoimmune Diseases; CD8-Positive T-Lymphocytes; Cell Differentiation; Clonal Anergy; Dendritic Cells; Diabetes Mellitus, Type 1; Dose-Response Relationship, Immunologic; Galactosylceramides; Immunophenotyping; Inflammation; Islets of Langerhans; Killer Cells, Natural; Ligands; Lymphocyte Activation; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, Transgenic; Self Tolerance; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Natural Killer T (NKT) cells can effect both T cell development and peripheral immune responses through T(H)1/T(H)2 cytokines. Some humans with Type 1 Diabetes Mellitus (T1DM) have numerical and functional NKT deficiencies that contribute to disease severity. Correcting these deficiencies inhibits diabetes in the non-obese diabetic (NOD) T1DM model, which shares similar deficiencies. Here we show that antibodies to CD1d, when given during early thymic development, induce specific increases in surface TCR of developing NOD and C57BL/6 CD4(+)CD8(+) (DP) invariant NKT (iNKT) cells. However, the addition of anti-CD1d causes distinct strain-specific population changes in response to treatment. These changes include: (1) a dose-dependent increase in NOD iNKT(TCR)(+) cells and, conversely, (2) an inhibition of B6 iNKT(TCR)(+) cell production. The observed NOD iNKT expansions correlated with diabetes inhibition in an in vitro T1DM system, suggesting that intrathymic anti-CD1d treatment may correct NOD numerical iNKT deficiencies through developmental TCR enhancement.

Topics: Animals; Antibodies; Antigens, CD1; Antigens, CD1d; Diabetes Mellitus, Type 1; Galactosylceramides; Killer Cells, Natural; Mice; Mice, Inbred NOD; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Thymus Gland

CD1d-restricted natural killer T (NKT) cells and CD4+CD25+ regulatory T (Treg) cells are two thymus-derived subsets of regulatory T cells that play an important role in the maintenance of self-tolerance. Yet the functional changes of the two subsets of regulatory T cells in the development of diabetes in non-obese diabetic (NOD) mice remain unclear, and how NKT cells and CD4+CD25+ Treg cells cooperate functionally in the regulation of autoimmune diabetes is also uncertain. We provide evidence that in NOD mice, an animal model of human type 1 diabetes, the functions of both NKT cells and CD4+CD25+ Treg cells decrease in an age-dependent manner. We show that treatment with alpha-galactosylceramide increases the size of the CD4+CD25+ Treg cell compartment in NOD mice, and augments the expression of forkhead/winged helix transcription factor and the potency of CD4+CD25+ Treg cells to inhibit proliferation of CD4+CD25- T cells. Our data indicate that NKT cells and CD4+CD25+ Treg cells might cooperate in the prevention of autoimmune diabetes in NOD mice treated with alpha-galactosylceramide. Induced cooperation of NKT cells and CD4+CD25+ Treg cells could serve as a strategy to treat human autoimmune disease, such as type 1 diabetes.

Topics: Aging; Animals; Cell Communication; Cells, Cultured; Diabetes Mellitus, Type 1; Female; Galactosylceramides; Killer Cells, Natural; Mice; Mice, Inbred NOD; Obesity; T-Lymphocytes, Regulatory

Non-obese diabetic (NOD) mice develop diabetes mediated by pathogenic T-helper type 1 (Th1) cells. V alpha 14 Natural killer (NKT) cells are a unique lymphocyte subtype implicated in the regulation of autoimmunity and a good source of protective Th2 cytokines. We recently developed a Th2-skewing NKT cell ligand, OCH. OCH, a sphingosine truncated derivative of alpha-galactosylceramide (alpha-GC), stimulates NKT cells to selectively produce Th2 cytokines. Here we show that OCH prevented the development of diabetes and insulitis in NOD mice. The suppression of insulitis by OCH was more profound compared to alpha-GC. Infiltration of T cells, B cells and macrophages into islets is inhibited in OCH-treated NOD mice. OCH-mediated suppression of diabetes is associated with Th2 bias of anti-islet antigen response and increased IL-10 producing cells among islet-infiltrating leukocytes. Considering the non-polymorphic and well conserved features of the CD1d molecule in mice and humans, these findings not only support the proposed role of NKT cells in the regulation of self-tolerance but also highlight the potential use of OCH for therapeutic intervention in type I diabetes.

Topics: Animals; Autoantibodies; Autoantigens; Cytokines; Diabetes Mellitus, Type 1; Female; Galactosylceramides; Glutamate Decarboxylase; Glycolipids; Immunoglobulin G; Inflammation; Islets of Langerhans; Killer Cells, Natural; Ligands; Mice; Mice, Inbred NOD; Self Tolerance; Th2 Cells