Tracking of an Oral Salmonella-Based Vaccine for Type 1 Diabetes in Non-obese Diabetic Mice.

Type 1 diabetes (T1D) arises secondary to immune-driven destruction of pancreatic ß-cells and manifests as insulin-deficient hyperglycemia. We showed that oral vaccination with live attenuated Salmonella, which simultaneously delivers autoantigens and a TGFß expression vector to immune cells in the gut mucosa, provides protection against the progression of T1D in non-obese diabetic (NOD) mice. In this study we employed the Sleeping Beauty (SB) transposon system that is composed of a transposase and transposon encoding the td-Tomato to express red fluorescent protein (RFP) to permanently mark the cells that take up the Salmonella vaccine. After animal vaccination, the transposon labeled-dendritic cells (DCs) with red fluorescence appeared throughout the secondary lymphoid tissues. Furthermore, Sleeping Beauty containing tgfß1 gene (SB-tgfß1) co-expressed TGFß and RFP. The labeled DCs were detected predominantly in Peyer's patches (PP) and mesenteric lymph nodes (MLN) and expressed CD103 surface marker. CD103+ DCs induced tolerogenic effects and gut homing. TGFß significantly increased programmed death-ligand-1 (PDL-1 or CD274) expression in the DCs in the MLN and PP of treated mice. Also, TGFß increased cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) levels in CD4+ cells in MLN and PP. Interestingly, DCs increased in all lymphatic organs of mice vaccinated with oral live Salmonella-based vaccine expressing preproinsulin (PPI), in combination with TGFß, IL10, and subtherapeutic-doses of anti-CD3 mAb compared with vehicle-treated mice. These DCs are mostly tolerogenic in MLN and PP. Furthermore the DCs obtained from vaccine-treated but not vehicle-treated mice suppressed in vitro T cell proliferation. These data suggest that the MLN and the PP are a central hub for the beneficial anti-diabetic effects of an oral Salmonella-based vaccine prevention of diabetes in rodents.

Ubiquitous antigen-specific T regulatory type 1 cells variably suppress hepatic and extrahepatic autoimmunity.

Peptide MHC class II-based (pMHCII-based) nanomedicines trigger the formation of multicellular regulatory networks by reprogramming autoantigen-experienced CD4+ T cells into autoimmune disease-suppressing T regulatory type 1 (TR1) cells. We have shown that pMHCII-based nanomedicines displaying liver autoimmune disease-relevant yet ubiquitously expressed antigens can blunt various liver autoimmune disorders in a non-disease-specific manner without suppressing local or systemic immunity against infectious agents or cancer. Here, we show that such ubiquitous autoantigen-specific T cells are also awakened by extrahepatic tissue damage and that the corresponding TR1 progeny can suppress experimental autoimmune encephalomyelitis (EAE) and pancreatic ß cell autoreactivity. In mice having EAE, nanomedicines displaying either ubiquitous or CNS-specific epitopes triggered the formation and expansion of cognate TR1 cells and their recruitment to the CNS-draining lymph nodes, sparing their liver-draining counterparts. Surprisingly, in mice having both liver autoimmunity and EAE, liver inflammation sequestered these ubiquitous or even CNS-specific TR1 cells away from the CNS, abrogating their antiencephalitogenic activity. In these mice, only the ubiquitous antigen-specific TR1 cells suppressed liver autoimmunity. Thus, the scope of antigen spreading in autoimmune disorders is larger than previously anticipated, involving specificities expected to be silenced by mechanisms of tolerance; the regulatory activity, but not the retention of autoreactive TR1 cells, requires local autoantigen expression.

Reversal of New Onset Type 1 Diabetes by Oral Salmonella-Based Combination Therapy and Mediated by Regulatory T-Cells in NOD Mice.

Autoimmune diseases such as type 1 diabetes (T1D) involve the loss of regulatory mechanisms resulting in increased tissue-specific cytotoxicity. The result is destruction of pancreatic insulin-producing ß-cells and loss of glucose homeostasis. We are developing a novel oral vaccine using live attenuated Salmonella to deliver TGFß, IL10, and the diabetic autoantigen preproinsulin combined with low-doses of anti-CD3 mAb. Here we show that oral administration of Salmonella-based anti-CD3 mAb combined therapy reverses new-onset T1D in non-obese diabetic (NOD) mice. The therapeutic effect of the combined therapy was associated with induction of immune suppressive CD4+CD25+Foxp3+ Treg and CD4+CD49b+LAG3+ Tr1 cells. In adoptive transfer experiments, adding or depleting Treg or Tr1 cells indicated that both are important for preventing diabetes in combined therapy-treated mice, but that Tr1 cells may have a more central role. Furthermore, induced Tr1 cells were found to be antigen-specific responding to peptide stimulation by secreting tolerance inducing IL10. These preclinical data demonstrate a role for Treg and Tr1 cells in combined therapy-mediated induction of tolerance in NOD mice. These results also demonstrate the potential of oral Salmonella-based combined therapy in the treatment of early T1D.

Factors affecting Salmonella-based combination immunotherapy for prevention of type 1 diabetes in non-obese diabetic mice.

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFß, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFß or PPI+TGFß+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFß+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.

The Role of Dendritic Cell Maturation in the Induction of Insulin-Dependent Diabetes Mellitus.

Dendritic cells (DCs) are the dominant class of antigen-presenting cells in humans and are largely responsible for the initiation and guidance of innate and adaptive immune responses involved in maintenance of immunological homeostasis. Immature dendritic cells (iDCs) phagocytize pathogens and toxic proteins and in endosomal vesicles degrade them into small fragments for presentation on major histocompatibility complex (MHC) II receptor molecules to naïve cognate T cells (Th0). In addition to their role in stimulation of immunity, DCs are involved in the induction and maintenance of immune tolerance toward self-antigens. During activation, the iDCs become mature. Maturation begins when the DCs cease taking up antigens and begin to migrate from their location in peripheral tissues to adjacent lymph nodes or the spleen where during their continued maturation the DCs present stored antigens on surface MHCII receptor molecules to naive Th0 cells. During antigen presentation, the DCs upregulate the biosynthesis of costimulatory receptor molecules CD86, CD80, CD83, and CD40 on their plasma membrane. These activated DC receptor molecules bind cognate CD28 receptors presented on the Th0 cell membrane, which triggers DC secretion of IL-12 or IL-10 cytokines resulting in T cell differentiation into pro- or anti-inflammatory T cell subsets. Although basic concepts involved in the process of iDC activation and guidance of Th0 cell differentiation have been previously documented, they are poorly defined. In this review, we detail what is known about the process of DC maturation and its role in the induction of insulin-dependent diabetes mellitus autoimmunity.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.

The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity.

Indoleamine 2, 3-dioxygenase (IDO) is the first and rate limiting catabolic enzyme in the degradation pathway of the essential amino acid tryptophan. By cleaving the aromatic indole ring of tryptophan, IDO initiates the production of a variety of tryptophan degradation products called "kynurenines" that are known to exert important immuno-regulatory functions. Because tryptophan must be supplied in the diet, regulation of tryptophan catabolism may exert profound effects by activating or inhibiting metabolism and immune responses. Important for survival, the regulation of IDO biosynthesis and its activity in cells of the immune system can critically alter their responses to immunological insults, such as infection, autoimmunity and cancer. In this review, we assess how IDO-mediated catabolism of tryptophan can modulate the immune system to arrest inflammation, suppress immunity to cancer and inhibit allergy, autoimmunity and the rejection of transplanted tissues. Finally, we examine how vaccines may enhance immune suppression of autoimmunity through the upregulation of IDO biosynthesis in human dendritic cells.

Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic ß-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

The role of dendritic cells in tissue-specific autoimmunity.

In this review, we explore the role of dendritic cell subsets in the development of tissue-specific autoimmune diseases. From the increasing list of dendritic cell subclasses, it is becoming clear that we are only at the beginning of understanding the role of these antigen presenting cells in mediating autoimmunity. Emerging research areas for the study of dendritic cell involvement in the onset and inhibition of tissue-specific autoimmunity are presented. Further, we compare tissue specific to systemic autoimmunity to demonstrate how development of dendritic cell-based therapies may be broadly applicable to both classes of autoimmunity. Continued development of these research areas will lead us closer to clinical assessment of novel immunosuppressive therapy for the reversal and prevention of tissue-specific autoimmunity. Through description of dendritic cell functions in the modulation of tissue-specific autoimmunity, we hope to stimulate a greater appreciation and understanding of the role dendritic cells play in the development and treatment of autoimmunity.