Mechanism of Chimeric Vaccine Mediated Immune Suppression of Human Dendritic Cells

Type 1 diabetes mellitus is a chronic inflammatory disease in which insulin producing β-cells of the pancreatic islets are killed by autoreactive cells of the immune system in response to a loss of tolerance. Dendritic cells (DC) interact predominantly with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this dissertation, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-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) shown to be directly linked to CTB-INS-induced DC activation and maturation. Treatment of vaccinated DCs with the pharmacological NF-κB inhibitors ACHP or DHMEQ inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in CTB-INS vaccine up-regulation of dendritic cell IDO1. Further, blocking the NF-κB-inducing kinase (NIK) phosphorylation of IKK-α dimers and translocation of p52/RelB complexes to the nucleus with anti-NIK siRNA, significantly inhibited IDO1 expression in human DCs. Chromatin immunoprecipitation (ChIP) analysis, showed that RelB-p52 dimers bound dendritic cell NF-κB consensus sequences in the IDO1 promoter region, demonstrating vaccine stimulation of NF-κB non-canonical pathway activation of IDO1 expression in human DCs in vivo. Addition of the Tumor Necrosis Factor Associated Factors (TRAF) TRAF6 and TRAF 2, 3 blocking peptides to vaccinated DCs dramatically reduced the level of IDO1 biosynthesis and stimulated DC suggesting the vaccine may function in the same signaling pathway as TNFR super-family for the up-regulation of IDO1 biosynthesis in vaccinated dendritic cells. Together, these data confirm CTB-INS vaccine activation of the non-canonical NF-κB signaling pathway TNFR receptor family up-regulation of dendritic cell IDO1 biosynthesis permitting vaccine inhibition of DC maturation leading to the suppression of type 1 diabetes development

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 β-cells and loss of glucose homeostasis. We are developing a novel oral vaccine using live attenuated Salmonella to deliver TGFβ, 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

First Round of DC+ and vaccine Sequest 3752 peptides

<p>Proteomic data generated in DCs treated by CTB-INS</p

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

Exploring the Potential of Plant-Based CTB-INS Oral Vaccines in Treating Type 1 Diabetes

The 19th century saw the development of vaccines, which were biological preparations designed to enhance immunity against specific diseases. Edible vaccines function by stimulating both systemic and mucosal immune responses against foreign pathogens, and they may potentially protect the host from autoimmunity. The mucosal surfaces provide a convenient and rapid route for delivering therapeutic small molecules. This is due to their large surface areas and easy administration. The effectiveness of mucosal immunization relies on the fact that mucous membranes represent the body’s largest immunogenic organ. Within this interface, there is a well-organized lymphatic structure known as MALT (mucosa-associated lymphoid tissue), which includes both T and B cells and encompasses the adaptive arms of the immune system. Oral vaccines specifically stimulate immune responses in the gut-associated lymphoid tissue (GALT), which consists of lymph nodes, Payer’s patches (where B cells make up about 75% of the population and T cells account for approximately 20%), and isolated lymphoid follicles within the gastrointestinal tract (GIT). However, a significant challenge in developing vaccines is the rapid degradation of antigens within the harsh environment of the digestive tract, which hampers effective protein delivery to the GIT. In light of recent proteomic analysis revealing strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in DCs inoculated with the Cholera toxin B-subunit-Insulin fusion protein vaccine (CTB-INS), we are interested in investigating the effects of transgene integration into a selected plant cell as an edible vaccine

Lipopolysaccharide-Induced Immunological Tolerance in Monocyte-Derived Dendritic Cells

Bacterial lipopolysaccharides (LPS), also referred to as endotoxins, are major outer surface membrane components present on almost all Gram-negative bacteria and are major determinants of sepsis-related clinical complications including septic shock. LPS acts as a strong stimulator of innate or natural immunity in a wide variety of eukaryotic species ranging from insects to humans including specific effects on the adaptive immune system. However, following immune stimulation, lipopolysaccharide can induce tolerance which is an essential immune-homeostatic response that prevents overactivation of the inflammatory response. The tolerance induced by LPS is a state of reduced immune responsiveness due to persistent and repeated challenges, resulting in decreased expression of pro-inflammatory modulators and up-regulation of antimicrobials and other mediators that promote a reduction of inflammation. The presence of environmental-derived LPS may play a key role in decreasing autoimmune diseases and gut tolerance to the plethora of ingested antigens. The use of LPS may be an important immune adjuvant as demonstrated by the promotion of IDO1 increase when present in the fusion protein complex of CTB-INS (a chimera of the cholera toxin B subunit linked to proinsulin) that inhibits human monocyte-derived DC (moDC) activation, which may act through an IDO1-dependent pathway. The resultant state of DC tolerance can be further enhanced by the presence of residual E. coli lipopolysaccharide (LPS) which is almost always present in partially purified CTB-INS preparations. The approach to using an adjuvant with an autoantigen in immunotherapy promises effective treatment for devastating tissue-specific autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes (T1D)

Lipopolysaccharide-Induced Immunological Tolerance in Monocyte-Derived Dendritic Cells

Bacterial lipopolysaccharides (LPS), also referred to as endotoxins, are major outer surface membrane components present on almost all Gram-negative bacteria and are major determinants of sepsis-related clinical complications including septic shock. LPS acts as a strong stimulator of innate or natural immunity in a wide variety of eukaryotic species ranging from insects to humans including specific effects on the adaptive immune system. However, following immune stimulation, lipopolysaccharide can induce tolerance which is an essential immune-homeostatic response that prevents overactivation of the inflammatory response. The tolerance induced by LPS is a state of reduced immune responsiveness due to persistent and repeated challenges, resulting in decreased expression of pro-inflammatory modulators and up-regulation of antimicrobials and other mediators that promote a reduction of inflammation. The presence of environmental-derived LPS may play a key role in decreasing autoimmune diseases and gut tolerance to the plethora of ingested antigens. The use of LPS may be an important immune adjuvant as demonstrated by the promotion of IDO1 increase when present in the fusion protein complex of CTB-INS (a chimera of the cholera toxin B subunit linked to proinsulin) that inhibits human monocyte-derived DC (moDC) activation, which may act through an IDO1-dependent pathway. The resultant state of DC tolerance can be further enhanced by the presence of residual E. coli lipopolysaccharide (LPS) which is almost always present in partially purified CTB-INS preparations. The approach to using an adjuvant with an autoantigen in immunotherapy promises effective treatment for devastating tissue-specific autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes (T1D)

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

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

CTB-INS fusion protein was expressed from the <i>E.co</i>li pRSET A expression vector and purified using Ni-NTA agarose with the indicated imidazole concentration in the wash and elution steps.

<p>Panel <b>(A)</b> is a plasmid map of the <i>E</i>. <i>coli</i> expression vector pRSET A (Invitrogen, Carlsbad, CA), carrying the CTB-INS fusion gene. Panel <b>(B)</b> show the SDS-PAGE. Proteins were visualized by Coomassie staining. Lane NI: non-induced <i>E</i>. <i>coli</i> (BL21) cell; I: induced E. <i>coli</i> cell; M: protein size marker; CL: cell lysate; FT: flow-through; W: wash; E: elution. Panel <b>(C)</b> Western blot detection of recombinant CTB-INS fusion protein identified with anti-CTB primary antibody. The arrow indicates the purified CTB-INS proteins. Panel <b>(D)</b> shows the predicted CTB-INS protein structure using its protein sequence generated by the RaptorX server.</p