Elizabeth D. Mellins

Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression

Summary:  In antigen‐presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen‐binding sites in pre‐endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii‐degrading proteases, the peptide exchange factor, human leukocyte antigen‐DM (HLA‐DM), and its modulator, HLA‐DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen‐binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post‐Golgi mechanisms; these factors include MHC class II‐associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.

Plasticity of T-cell phenotype and function: the T helper type 17 example

Mature T helper type 1 (Th1) and Th2 cells antagonize the development of the opposing subset to sustain lineage‐specific responses. However, the recent identification of a third distinct subset of helper T cells – the Th17 lineage – collapses the established Th1/Th2 dichotomy and raises intriguing questions about T‐cell fate. In this review, we discuss the Th17 subset in the context of the effector and regulatory T‐cell lineages. Initial studies suggested reciprocal developmental pathways between Th17/Th1 subsets and between Th17/regulatory T‐cell subsets, and identified multiple mechanisms by which Th1 and Th2 cells antagonize the generation of Th17 cells. However, recent observations reveal the susceptibility of differentiated Th17 cells to Th1 polarization and the enhancement of Th17 memory cells by the Th1 factors interferon‐γ and T‐bet. In addition, new data indicate late‐stage plasticity of a subpopulation of regulatory T cells, which can be selectively induced to adopt a Th17 phenotype. Elucidating the mechanisms that undermine cross‐lineage suppression and facilitate these phenotype shifts will not only clarify the flexibility of T‐cell differentiation, but may also shed insight into the pathogenesis of autoimmunity and cancer. Furthermore, understanding these phenomena will be critical for the design of immunotherapy that seeks to disrupt lineage‐specific T‐cell responses and may suggest ways to manipulate the balance between pathogenic and regulatory lymphocytes for the restoration of homeostasis.

T cell-mediated autoimmune disease due to low-affinity crossreactivity to common microbial peptides.

Environmental factors account for 75% of the risk of developing multiple sclerosis (MS). Numerous infections have been suspected as environmental disease triggers, but none of them has consistently been incriminated, and it is unclear how so many different infections may play a role. We show that a microbial peptide, common to several major classes of bacteria, can induce MS-like disease in humanized mice by crossreacting with a T cell receptor (TCR) that also recognizes a peptide from myelin basic protein, a candidate MS autoantigen. Structural analysis demonstrates this crossreactivity is due to structural mimicry of a binding hotspot shared by self and microbial antigens, rather than to degenerate TCR recognition. Biophysical studies reveal that the autoreactive TCR binding affinity is markedly lower for the microbial (mimicry) peptide than for the autoantigenic peptide. Thus, these data suggest a possible explanation for the difficulty in incriminating individual infections in the development of MS.

Susceptibility of Immature and Mature Langerhans Cell-Type Dendritic Cells to Infection and Immunomodulation by Human Cytomegalovirus

ABSTRACT Human cytomegalovirus (CMV) infection initiates in mucosal epithelia and disseminates via leukocytes throughout the body. Langerhans cells (LCs), the immature dendritic cells (DCs) that reside in epithelial tissues, are among the first cells to encounter virus and may play important roles in the immune response, as well as in pathogenesis as hosts for viral replication and as vehicles for dissemination. Here, we demonstrate that CD34+ progenitor cell-derived LC-type DCs exhibit a differentiation state-dependent susceptibility to CMV infection. In contrast to the small percentage (3 to 4%) of the immature LCs that supported infection, a high percentage (48 to 74%) of mature, LC-derived DCs were susceptible to infection with endotheliotropic strains (TB40/E or VHL/E) of CMV. These cells were much less susceptible to viral strains AD169varATCC, TownevarRIT3, and Toledo. When exposed to endotheliotropic strains, viral gene expression (IE1/IE2 and other viral gene products) and viral replication proceeded efficiently in LC-derived mature DCs (mDCs). Productive infection was associated with downmodulation of cell surface CD83, CD1a, CD80, CD86, ICAM-1, major histocompatibility complex (MHC) class I, and MHC class II on these cells. In addition, the T-cell proliferative response to allogeneic LC-derived mDCs was attenuated when CMV-infected cultures were used as stimulators. This investigation revealed important characteristics of the interaction between CMV and the LC lineage of DCs, suggesting that LC-derived mDCs are important to viral pathogenesis and immunity through their increased susceptibility to virus replication and virus-mediated immune escape.

Macrophage activation syndrome: advances towards understanding pathogenesis

Purpose of reviewMacrophage activation syndrome (MAS), a major cause of morbidity and mortality in pediatric rheumatology, is most strongly associated with systemic juvenile idiopathic arthritis (SJIA). There are no validated diagnostic criteria and early diagnosis is difficult. This review summarizes the progress in understanding of MAS pathophysiology that may help define specific diagnostic biomarkers. Recent findingsMAS is similar to the autosomal recessive disorders collectively known as familial hemophagocytic lymphohistiocytosis (FHLH), all associated with various genetic defects affecting the cytolytic pathway. Cytolytic function is profoundly depressed in SJIA with MAS as well. This immunologic abnormality distinguishes SJIA from other rheumatic diseases and is caused by both genetic and acquired factors. Phenotypic characterization of hemophagocytic macrophages has been another focus of research. These macrophages express CD163, a scavenger receptor that binds hemoglobin–haptoglobin complexes, and initiate pathways important for adaptation to oxidative stress induced by free iron. Expansion of these macrophages is seen in more than 30% of SJIA patients perhaps representing early stages of MAS. Recent gene expression studies linked expansion of these macrophages to distinct signatures. SummaryRecent advances in understanding of pathophysiologic conditions that favor expansion of hemophagocytic macrophages provide a source of new MAS biomarkers with applicability to clinical practice.

Modulation of Major Histocompatibility Class II Protein Expression by Varicella-Zoster Virus

ABSTRACT We sought to investigate the effects of varicella-zoster virus (VZV) infection on gamma interferon (IFN-γ)-stimulated expression of cell surface major histocompatibility complex (MHC) class II molecules on human fibroblasts. IFN-γ treatment induced cell surface MHC class II expression on 60 to 86% of uninfected cells, compared to 20 to 30% of cells which had been infected with VZV prior to the addition of IFN-γ. In contrast, cells that were treated with IFN-γ before VZV infection had profiles of MHC class II expression similar to those of uninfected cell populations. Neither IFN-γ treatment nor VZV infection affected the expression of transferrin receptor (CD71). In situ and Northern blot hybridization of MHC II (MHC class II DR-α) RNA expression in response to IFN-γ stimulation revealed that MHC class II DR-α mRNA accumulated in uninfected cells but not in cells infected with VZV. When skin biopsies of varicella lesions were analyzed by in situ hybridization, MHC class II transcripts were detected in areas around lesions but not in cells that were infected with VZV. VZV infection inhibited the expression of Stat 1α and Jak2 proteins but had little effect on Jak1. Analysis of regulatory events in the IFN-γ signaling pathway showed that VZV infection inhibited transcription of interferon regulatory factor 1 and the MHC class II transactivator. This is the first report that VZV encodes an immunomodulatory function which directly interferes with the IFN-γ signal transduction via the Jak/Stat pathway and enables the virus to inhibit IFN-γ induction of cell surface MHC class II expression. This inhibition of MHC class II expression on VZV-infected cells in vivo may transiently protect cells from CD4+ T-cell immune surveillance, facilitating local virus replication and transmission during the first few days of cutaneous lesion formation.

Determination of the HLA-DM Interaction Site on HLA-DR Molecules

HLA-DM removes CLIP and other loosely bound peptides from MHC class II molecules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Here, we describe mutations in class II that impair interactions with DM. Libraries of randomly mutagenized DR3 alpha and beta chains were screened for their ability to cause cell surface accumulation of CLIP/DR3 complexes in EBV-B cells. Seven mutations were associated with impaired peptide loading in vivo, as detected by SDS stability assays. In vitro, these mutant DR3 molecules were resistant to DM-catalyzed CLIP release and showed reduced binding to DM. All mutations localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange.

Interaction of HLA-DR with an Acidic Face of HLA-DM Disrupts Sequence-Dependent Interactions with Peptides

HLA-DM (DM) edits major histocompatibility complex class II (MHCII)-bound peptides in endocytic compartments and stabilizes empty MHCII molecules. Crystal structures of DM have revealed similarity to MHCII but not how DM and MHCII interact. We used mutagenesis to map a MHCII-interacting surface on DM. Mutations on this surface impair DM action on HLA-DR and -DP in cells and DM-dependent peptide loading in vitro. The orientation of DM and MHCII predicted by these studies guided design of soluble DM and DR molecules fused to leucine zippers via their beta chains, resulting in stable DM/DR complexes. Peptide release from the complexes was fast and only weakly sequence dependent, arguing that DM diminishes the selectivity of the MHCII groove. Analysis of soluble DM action on soluble DR/peptide complexes corroborates this conclusion.

Accessory molecules for MHC class II peptide loading

Accessory molecules, such as HLA-DM and invariant chain, modulate the ligands bound to MHC class II molecules in antigen-presenting cells. Recent investigations, including gene targeting experiments, have shed light on the functions of these molecules, their mechanisms of action, interactions with class II molecules, and the relationships with associated molecules such as tetraspanins and HLA-DO.

CD4+ T Cell Autoimmunity to Hypocretin/Orexin and Cross-Reactivity to a 2009 H1N1 Influenza A Epitope in Narcolepsy

Patients with narcolepsy carry CD4+ T cells that react to peptides from both the sleep-regulating neuropeptide hypocretin and a 2009 H1N1 influenza A protein. New Clues About Narcolepsy Most adults long for more sleep, but patients with narcolepsy would rather be free of their excessive sleepiness. Some things about narcolepsy are clear: It is caused by the loss of the peptide hypocretin from neurons that control wakefulness, and it has a remarkably strong association with a particular human leukocyte antigen (HLA) molecule (DQ0602), suggesting that there is an immune contribution to the disease. Other things are not so clear: We do not know what triggers the disease, and there is no way to prevent it. The neurons that are destroyed in narcolepsy contain the body’s only store of hypocretin, so an immune response against hypocretin could conceivably be responsible for the disease. Although no antibodies to hypocretin have been found in patients, now De la Herrán-Arita et al. have identified CD4+ T cells that react against several peptides derived from hypocretin when they are presented by HLA DQ0602. The two 13–amino acid peptides, corresponding to the N-terminal ends of the mature, secreted forms of hypocretin, triggered responses in T cells from 23 patients with narcolepsy but not in matched DQ0602-positive healthy control subjects. Similarly, in pairs of twins discordant for narcolepsy, the twin with disease carried cells that were activated by hypocretin peptides, whereas the healthy twin did not. Clues about environmental factors that might contribute to narcolepsy have come from epidemiology studies that associate Streptococcus, influenza, and other infections with the disease. In Scandinavia, a particular flu vaccine was associated with increased narcolepsy risk. To investigate these associations further, the authors searched for epitopes in proteins from the flu virus that might activate the same T cells that responded to the hypocretin peptides. Within the flu protein hemagglutinin, they found a small segment that had this effect, amino acids 275 to 287. When cells from patients were incubated with this peptide presented by DQ0602, there was an increase in the number of cells reactive to both the hemagglutinin peptide and the hypocretin peptides, suggesting cross-reactivity between these epitopes. Although more research is necessary to understand the effects of the hypocretin-reactive cells in patients, these results point to a possible molecular mimicry between epitopes on hypocretin and the influenza hemagglutinin protein. Narcolepsy, a disorder strongly associated with human leukocyte antigen (HLA)–DQA1*01:02/DQB1*06:02 (DQ0602), is characterized by excessive daytime sleepiness, cataplexy, and rapid eye movement sleep abnormalities. It is caused by the loss of ~70,000 posterior hypothalamic neurons that produce the wake-promoting neuropeptide hypocretin (HCRT) (orexin). We identified two DQ0602-binding HCRT epitopes, HCRT56–68 and HCRT87–99, that activated a subpopulation of CD4+ T cells in narcolepsy patients but not in DQ0602-positive healthy control subjects. Because of the established association of narcolepsy with the 2009 H1N1 influenza A strain (pH1N1), we administered a seasonal influenza vaccine (containing pH1N1) to patients with narcolepsy and found an increased frequency of circulating HCRT56–68– and HCRT87–99–reactive T cells. We also identified a hemagglutinin (HA) pHA1 epitope specific to the 2009 H1N1 strain, pHA1275–287, with homology to HCRT56–68 and HCRT87–99. In vitro stimulation of narcolepsy CD4+ T cells with pH1N1 proteins or pHA1275–287 increased the frequency of HCRT56–68– and HCRT87–99–reactive T cells. Our data indicate the presence of CD4+ T cells that are reactive to HCRT in narcolepsy patients and possible molecular mimicry between HCRT and a similar epitope in influenza pH1N1, pHA1275–287.