Christopher C. Norbury

Rapid degradation of a large fraction of newly synthesized proteins by proteasomes

MHC class I molecules function to present peptides eight to ten residues long to the immune system. These peptides originate primarily from a cytosolic pool of proteins through the actions of proteasomes, and are transported into the endoplasmic reticulum, where they assemble with nascent class I molecules. Most peptides are generated from proteins that are apparently metabolically stable. To explain this, we previously proposed that peptides arise from proteasomal degradation of defective ribosomal products (DRiPs). DRiPs are polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding. Here we show, first, that DRiPs constitute upwards of 30% of newly synthesized proteins as determined in a variety of cell types; second, that at least some DRiPs represent ubiquitinated proteins; and last, that ubiquitinated DRiPs are formed from human immunodeficiency virus Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides.

Diabetic Retinopathy Seeing Beyond Glucose-Induced Microvascular Disease

Diabetic retinopathy remains a frightening prospect to patients and frustrates physicians. Destruction of damaged retina by photocoagulation remains the primary treatment nearly 50 years after its introduction. The diabetes pandemic requires new approaches to understand the pathophysiology and improve the detection, prevention, and treatment of retinopathy. This perspective considers how the unique anatomy and physiology of the retina may predispose it to the metabolic stresses of diabetes. The roles of neural retinal alterations and impaired retinal insulin action in the pathogenesis of early retinopathy and the mechanisms of vision loss are emphasized. Potential means to overcome limitations of current animal models and diagnostic testing are also presented with the goal of accelerating therapies to manage retinopathy in the face of ongoing diabetes.

Class I MHC presentation of exogenous soluble antigen via macropinocytosis in bone marrow macrophages.

Extracellular proteins are not generally presented on class I MHC molecules in vitro, yet many studies show that a pathway exists in vivo for the presentation of extracellular material on class I molecules to prime CD8+ T cell responses. Here, we provide morphological evidence that proteins taken up by macropinocytosis can gain access to the cytosol and therefore into the conventional class I MHC pathway. Class I presentation of soluble ovalbumin by mouse bone marrow macrophages was dramatically enhanced by MCSF or phorbol ester and blocked by amiloride, which stimulate and inhibit membrane ruffling and macropinocytosis, respectively. Brefeldin A, gelonin, and a peptide aldehyde inhibitor of proteasomal processing each blocked presentation of macropinocytosed antigen, demonstrating that unusual access to the conventional class I MHC pathway was occurring. This novel cell type-specific endocytic pathway may facilitate presentation of exogenous material on class I MHC molecules, allowing induction of CD8+ T cell responses to soluble proteins, tumor cell fragments, and some pathogens.

Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo

The rational design of vaccines that elicit CD8+ T cell responses requires knowledge of the identity of the antigen-presenting cell (APC), the location and time of presentation and the nature of the antigen presented by the APC. Here we address these questions for an antigen encoded by a recombinant vaccinia virus. We found that, following local infection, vaccinia virus infected macrophages and dendritic cells in draining lymph nodes. However, only the dendritic cells presented antigen to naïve CD8+ T cells, as determined by direct visualization of sectioned nodes by confocal microscopy. Presentation occurred as rapidly as 6 h after inoculation and quickly declined in parallel with the number of infected cells present in the nodes. These data provide direct evidence that virus-infected APCs prime naïve CD8+ T cells in vivo.

Mechanisms of Exogenous Antigen Presentation by MHC Class I Molecules in Vitro and in Vivo: Implications for Generating CD8+ T Cell Responses to Infectious Agents, Tumors, Transplants, and Vaccines

Publisher Summary Most cell types in the body constitutively express class I molecules that present thousands of distinct peptides from a diverse array of housekeeping and differentiation-specific cellular proteins. Under normal circumstances, these peptides fail to activate T CD8+ either because of the absence of T CD8+ bearing a complementary T cell receptor (TCR) or the silencing of such autoreactive T CD8+ in the thymus or periphery. The MHC class I system evolves to identify cells bearing foreign peptides that in the natural world derive from infectious agents—primarily viruses and a number of medically important prokaryotic and eukaryotic organisms. The vast majority of peptides constitutively presented by class I molecules derive from proteins synthesized by the cells own ribosomes. Polypeptides that provide these peptides are referred to as “endogenous” antigens, whereas polypeptides derived from all other sources are termed “exogenous” antigens. The chapter concludes by posing questions, the answers of which seem most important for achieving a basic understanding of exogenous antigen presentation in vitro and in vivo and assumes that this knowledge could have important practical applications, particularly in the area of vaccine development.

Interleukin-10 Prevents Diet-Induced Insulin Resistance by Attenuating Macrophage and Cytokine Response in Skeletal Muscle

OBJECTIVE Insulin resistance is a major characteristic of type 2 diabetes and is causally associated with obesity. Inflammation plays an important role in obesity-associated insulin resistance, but the underlying mechanism remains unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine with lower circulating levels in obese subjects, and acute treatment with IL-10 prevents lipid-induced insulin resistance. We examined the role of IL-10 in glucose homeostasis using transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10). RESEARCH DESIGN AND METHODS MCK-IL10 and wild-type mice were fed a high-fat diet (HFD) for 3 weeks, and insulin sensitivity was determined using hyperinsulinemic-euglycemic clamps in conscious mice. Biochemical and molecular analyses were performed in muscle to assess glucose metabolism, insulin signaling, and inflammatory responses. RESULTS MCK-IL10 mice developed with no obvious anomaly and showed increased whole-body insulin sensitivity. After 3 weeks of HFD, MCK-IL10 mice developed comparable obesity to wild-type littermates but remained insulin sensitive in skeletal muscle. This was mostly due to significant increases in glucose metabolism, insulin receptor substrate-1, and Akt activity in muscle. HFD increased macrophage-specific CD68 and F4/80 levels in wild-type muscle that was associated with marked increases in tumor necrosis factor-α, IL-6, and C-C motif chemokine receptor-2 levels. In contrast, MCK-IL10 mice were protected from diet-induced inflammatory response in muscle. CONCLUSIONS These results demonstrate that IL-10 increases insulin sensitivity and protects skeletal muscle from obesity-associated macrophage infiltration, increases in inflammatory cytokines, and their deleterious effects on insulin signaling and glucose metabolism. Our findings provide novel insights into the role of anti-inflammatory cytokine in the treatment of type 2 diabetes.

Regulation of T cell development by the deubiquitinating enzyme CYLD.

T cell receptor signaling is essential for the generation and maturation of T lymphocyte precursors. Here we identify the deubiquitinating enzyme CYLD as a positive regulator of proximal T cell receptor signaling in thymocytes. CYLD physically interacted with active Lck and promoted recruitment of active Lck to its substrate, Zap70. CYLD also removed both Lys 48– and Lys 63–linked polyubiquitin chains from Lck. Because of a cell-autonomous defect in T cell development, CYLD-deficient mice had substantially fewer mature CD4+ and CD8+ single-positive thymocytes and peripheral T cells.

Drinking a lot is good for dendritic cells.

Macropinocytosis is the actin‐dependent formation of large vesicles, which allow the internalization of large quantities of fluid‐phase solute. In the majority of cells examined, an exogenous stimulus is required to induce the initiation of this endocytic pathway. However, dendritic cells are thought to constitutively macropinocytose large quantities of exogenous solute as part of their sentinel function. In this review we discuss the evidence that dendritic cells macropinocytose exogenous solute and subsequently present antigenic peptides derived from internalized material to T cells. In addition, we put these data into the context of immune surveillance in vivo.

Multiple Paths for Activation of Naive CD8+ T Cells: CD4-Independent Help

CD8+ CTLs play a pivotal role in immune responses against many viruses and tumors. Two models have been proposed. The “three-cell” model focuses on the role of CD4+ T cells, proposing that help is only provided to CTLs by CD4+ T cells that recognize Ag on the same APC. The sequential “two-cell” model proposes that CD4+ T cells can first interact with APCs, which in turn activate naive CTLs. Although these models provide a general framework for the role of CD4+ T cells in mediating help for CTLs, a number of issues are unresolved. We have investigated the induction of CTL responses using dendritic cells (DCs) to immunize mice against defined peptide Ags. We find that help is required for activation of naive CTLs when DCs are used as APCs, regardless of the origin or MHC class I restriction of the peptides we studied in this system. However, CD8+ T cells can provide self-help if they are present at a sufficiently high precursor frequency. The important variable is the total number of T cells responding, because class II-knockout DCs pulsed with two noncompeting peptides are effective in priming.

Multiple Antigen-Specific Processing Pathways for Activating Naive CD8+ T Cells In Vivo

Current knowledge of the processing of viral Ags into MHC class I-associated ligands is based almost completely on in vitro studies using nonprofessional APCs (pAPCs). This is two steps removed from real immune responses to pathogens and vaccines, in which pAPCs activate naive CD8+ T cells in vivo. Rational vaccine design requires answers to numerous questions surrounding the function of pAPCs in vivo, including their abilities to process and present peptides derived from endogenous and exogenous viral Ags. In the present study, we characterize the in vivo dependence of Ag presentation on the expression of TAP by testing the immunogenicity of model Ags synthesized by recombinant vaccinia viruses in TAP1−/− mice. We show that the efficiency of TAP-independent presentation in vitro correlates with TAP-independent activation of naive T cells in vivo and provide the first in vivo evidence for proteolytic processing of antigenic peptides in the secretory pathway. There was, however, a clear exception to this correlation; although the presentation of the minimal SIINFEKL determinant from chicken egg OVA in vitro was strictly TAP dependent, it was presented in a TAP-independent manner in vivo. In vivo presentation of the same peptide from a fusion protein retained its TAP dependence. These results show that determinant-specific processing pathways exist in vivo for the generation of antiviral T cell responses. We present additional findings that point to cross-priming as the likely mechanism for these protein-specific differences.