Delfina M. Mazzuca

Diabetes Is Reversed in a Murine Model by Marginal Mass Syngeneic Islet Transplantation Using a Subcutaneous Cell Pouch Device

Background Islet transplantation is a successful &bgr;-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation. Methods Syngeneic mouse islets were transplanted subcutaneously within Sernova Corps Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance. Results Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels. Conclusions The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies.

A novel technique for the transplantation of pancreatic islets within a vascularized device into the greater omentum to achieve insulin independence

BACKGROUND The greater omentum with its vascularization and blood flow has been considered as a location for islet transplantation; however, there is a need to provide a controlled and protected site for the islets within the omentum that would be applicable to donor islets and future stem cell technologies. Here we describe the use of a novel device implanted within the omentum with a subcutaneous delivery port that offers an environment for donor islets. METHODS A prototype cell pouch device was wrapped in the greater omentum and an islet implantation port was exposed subcutaneously in diabetic Lewis rats. After tissue growth throughout the device, islet isografts were implanted and long-term glucose control was evaluated. RESULTS By using this technique, 7 of 10 diabetic rat recipients showed long-term normal blood glucose levels after minimal islet dose transplants. Histologic assessment revealed collagen formation and vascularization within the device. CONCLUSIONS The implanted device assessed using this technique provides a safe and efficacious environment for the support of pancreatic islets contained within a removable device as a cell therapy in a highly vascularized setting.

CD1d-independent activation of mouse and human iNKT cells by bacterial superantigens

Invariant NKT (iNKT) cells are infrequent but important immunomodulatory lymphocytes that exhibit CD1d‐restricted reactivity with glycolipid Ags. iNKT cells express a unique T‐cell receptor (TCR) composed of an invariant α‐chain, paired with a limited range of β‐chains. Superantigens (SAgs) are microbial toxins defined by their ability to activate conventional T cells in a TCR β‐chain variable domain (Vβ)‐specific manner. However, whether iNKT cells are directly activated by bacterial SAgs remains an open question. Herein, we explored the responsiveness of mouse and human iNKT cells to a panel of staphylococcal and streptococcal SAgs and examined the contribution of major histocompatibility complex (MHC) class II and CD1d to these responses. Bacterial SAgs that target mouse Vβ8, such as staphylococcal enterotoxin B (SEB), were able to activate mouse hybridoma and primary hepatic iNKT cells in the presence of mouse APCs expressing human leukocyte antigen (HLA)‐DR4. iNKT cell‐mediated cytokine secretion in SEB‐challenged HLA‐DR4‐transgenic mice was CD1d‐independent and accompanied by a high interferon‐γ:interleukin‐4 ratio consistent with an in vivo Th1 bias. Furthermore, iNKT cells from SEB‐injected HLA‐DR4‐transgenic mice, and iNKT cells from SEB‐treated human PBMCs, showed early activation by intracellular cytokine staining and CD69 expression. Unlike iNKT cell stimulation by α‐galactosylceramide, stimulation by SEB did not induce TCR downregulation of either mouse or human iNKT cells. We conclude that Vβ8‐targeting bacterial SAgs can activate iNKT cells by utilizing a novel pathway that requires MHC class II interactions, but not CD1d. Therefore, iNKT cells fulfill important effector functions in response to bacterial SAgs and may provide attractive targets in the management of SAg‐induced illnesses.

Bacterial Superantigens Promote Acute Nasopharyngeal Infection by Streptococcus pyogenes in a Human MHC Class II-Dependent Manner

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as ‘trademark’ virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC –II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms.

Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non‐polymorphic CD1d molecules. They are capable of rapidly releasing pro‐ and/or anti‐inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2‐polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline‐induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen‐experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4+CD25+FoxP3+ regulatory T cells. Interestingly, administering the Th1‐promoting iNKT cell glycolipid ligand α‐C‐galactosylceramide into HLA‐DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline‐induced arthritis. Therefore, our findings indicate a role for Th1‐mediated immunopathology in citrulline‐induced arthritis and provide the first evidence that iNKT cell manipulation by Th2‐skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA.

The islet size to oxygen consumption ratio reliably predicts reversal of diabetes posttransplant.

β-Cell replacement therapy by either whole-organ pancreas or islets of Langerhans transplantation can restore carbohydrate control to diabetic patients and reduces complications associated with the disease. One of the variables inherent in islet transplantation is the isolation of functional islets from donor pancreata. Islet isolations fail to consistently produce good-quality functional islets. A rapid pretransplant assay to determine posttransplant function of islets would be an invaluable tool. We have tested the novel hypothesis that modified oxygen consumption rates (OCR), standardized to DNA quantity (nmol/min-mg DNA), would serve as a pretransplant assessment of the metabolic potency of the islets postisolation. This study compares the ability of current in vitro assays to predict in vivo restoration of normoglycemia in a diabetic nude mouse posttransplantation of adult pig islets. There is known to be a diversity of islet sizes within each preparation. This parameter has not heretofore been effectively considered a critical factor in islet engraftment. Our results suggest a surprising finding that islet size influences the probability of restoring carbohydrate control. Based on this observation, we thus developed a novel predictor of islet graft function that combines the effects of both islet OCR and size. When OCR was divided by the islet index (size), a highly significant predictor of graft function was established (p = 0.0002, n = 75). Furthermore, when OCR/islet index values exceeded 70.0 nmol/min-mg DNA/islet index, an effective threshold of diabetes reversal was observed. This assay can be performed with as few as 1,000 islet equivalents (IEQ) and conducted in less than 60 min. Our data suggest that, using this novel method to assess islet cell function prior to transplantation, OCR/islet index thresholds provide a valuable tool in identifying which islet preparations are most likely to restore glycemic control posttransplant.

Differential Regulation of Simultaneous Antitumor and Alloreactive CD8 + T-Cell Responses in the Same Host by Rapamycin

Rapamycin is an immunosuppressive agent routinely used in organ transplantation but also paradoxically exerts antiviral and antitumor activities. Pathogen‐specific memory CD8+ T‐cell (TCD8) responses were recently found to be augmented by rapamycin. However, whether rapamycin influences the magnitude and quality of anticancer TCD8 responses is unknown. Importantly, how rapamycin may regulate simultaneous virus/tumor‐specific and alloreactive TCD8 in the same host remains unexplored. To answer these questions, we primed wild‐type mice with allogeneic cells concomitantly expressing simian virus 40 large tumor antigen (T Ag), a viral oncoprotein with well‐defined epitopes. Rapamycin selectively enhanced the cross‐priming of TCD8 specific for T Ags most immunodominant epitope called site IV but not TCD8 alloreactivity. Rapamycin‐treated mice also had a high percentage of splenic CD127highKLRG1low TCD8 and an increased frequency of site IV‐specific T cells long after the peak of their primary response. When site IV was presented as a cytosolic minigene encoded by a recombinant vaccinia virus, rapamycin failed to boost the site IV‐specific response. Therefore, the nature and presentation mode of antigen determine the susceptibility to the adjuvant effect of rapamycin. Our findings reveal the unexpected benefit of rapamycin treatment in recipients of allografts co‐expressing tumor/viral Ags.

Nasopharyngeal infection by Streptococcus pyogenes requires superantigen-responsive Vβ-specific T cells

Significance Superantigen toxins were defined over 25 years ago for their ability to activate T cells in a T-cell receptor β-chain variable domain-dependent manner. This “Vβ-specific” T-cell activation is the hallmark feature of the superantigen, and although these toxins can mediate dangerous human disease such as toxic shock syndrome, mechanisms that explain why bacteria produce superantigens have remained enigmatic. Herein, we provide evidence that Streptococcus pyogenes utilizes superantigens to target functional, Vβ-specific T cells to promote a state of colonization providing a mechanism that helps explain why bacteria produce toxins that specifically activate T cells of the adaptive immune system. This work also implicates the superantigen exotoxins as potential vaccine candidates against this globally important, human-specific pathogen. The globally prominent pathogen Streptococcus pyogenes secretes potent immunomodulatory proteins known as superantigens (SAgs), which engage lateral surfaces of major histocompatibility class II molecules and T-cell receptor (TCR) β-chain variable domains (Vβs). These interactions result in the activation of numerous Vβ-specific T cells, which is the defining activity of a SAg. Although streptococcal SAgs are known virulence factors in scarlet fever and toxic shock syndrome, mechanisms by how SAgs contribute to the life cycle of S. pyogenes remain poorly understood. Herein, we demonstrate that passive immunization against the Vβ8-targeting SAg streptococcal pyrogenic exotoxin A (SpeA), or active immunization with either wild-type or a nonfunctional SpeA mutant, protects mice from nasopharyngeal infection; however, only passive immunization, or vaccination with inactive SpeA, resulted in high-titer SpeA-specific antibodies in vivo. Mice vaccinated with wild-type SpeA rendered Vβ8+ T cells poorly responsive, which prevented infection. This phenotype was reproduced with staphylococcal enterotoxin B, a heterologous SAg that also targets Vβ8+ T cells, and rendered mice resistant to infection. Furthermore, antibody-mediated depletion of T cells prevented nasopharyngeal infection by S. pyogenes, but not by Streptococcus pneumoniae, a bacterium that does not produce SAgs. Remarkably, these observations suggest that S. pyogenes uses SAgs to manipulate Vβ-specific T cells to establish nasopharyngeal infection.

Rapid and Rigorous IL-17A Production by a Distinct Subpopulation of Effector Memory T Lymphocytes Constitutes a Novel Mechanism of Toxic Shock Syndrome Immunopathology

Toxic shock syndrome (TSS) is caused by staphylococcal and streptococcal superantigens (SAgs) that provoke a swift hyperinflammatory response typified by a cytokine storm. The precipitous decline in the host’s clinical status and the lack of targeted therapies for TSS emphasize the need to identify key players of the storm’s initial wave. Using a humanized mouse model of TSS and human cells, we herein demonstrate that SAgs elicit in vitro and in vivo IL-17A responses within hours. SAg-triggered human IL-17A production was characterized by remarkably high mRNA stability for this cytokine. A distinct subpopulation of CD4+ effector memory T (TEM) cells that secrete IL-17A, but not IFN-γ, was responsible for early IL-17A production. We found mouse “TEM-17” cells to be enriched within the intestinal epithelium and among lamina propria lymphocytes. Furthermore, interfering with IL-17A receptor signaling in human PBMCs attenuated the expression of numerous inflammatory mediators implicated in the TSS-associated cytokine storm. IL-17A receptor blockade also abrogated the secondary effect of SAg-stimulated PBMCs on human dermal fibroblasts as judged by C/EBP δ expression. Finally, the early IL-17A response to SAgs was pathogenic because in vivo neutralization of IL-17A in humanized mice ameliorated hepatic and intestinal damage and reduced mortality. Together, our findings identify CD4+ TEM cells as a key effector of TSS and reveal a novel role for IL-17A in TSS immunopathogenesis. Our work thus elucidates a pathogenic, as opposed to protective, role for IL-17A during Gram-positive bacterial infections. Accordingly, the IL-17–IL-17R axis may provide an attractive target for the management of SAg-mediated illnesses.

Synthesis, self-assembly, and immunological activity of α-galactose-functionalized dendron-lipid amphiphiles

Nanoassemblies presenting multivalent displays of biologically active carbohydrates are of significant interest for a wide array of biomedical applications ranging from drug delivery to immunotherapy. In this study, glycodendron-lipid hybrids were developed as a new and tunable class of dendritic amphiphiles. A modular synthesis was used to prepare dendron-lipid hybrids comprising distearylglycerol and 0 through 4th generation polyester dendrons with peripheral protected amines. Following deprotection of the amines, an isothiocyanate derivative of C-linked α-galactose (α-Gal) was conjugated to the dendron peripheries, affording amphiphiles with 1 to 16 α-Gal moieties. Self-assembly in water through a solvent exchange process resulted in vesicles for the 0 through 2nd generation systems and micelles for the 3rd and 4th generation systems. The critical aggregation concentrations decreased with increasing dendron generation, suggesting that the effects of increasing molar mass dominated over the effects of increasing the hydrophilic weight fraction. The binding of the assemblies to Griffonia simplicifolia Lectin I (GSL 1), a protein with specificity for α-Gal was studied by quantifying the binding of fluorescently labeled assemblies to GSL 1-coated beads. It was found that binding was enhanced for amphiphiles containing higher generation dendrons. Despite their substantial structural differences with the natural ligands for the CD1d receptor, the glycodendron-lipid hybrids were capable of stimulating invariant natural killer T (iNKT) cells, a class of innate-like T cells that recognize lipid and glycolipid antigens presented by CD1d and that are implicated in a wide range of diseases and conditions including but not limited to infectious diseases, diabetes and cancer.