Tieying Hou

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.

Cathepsin G: roles in antigen presentation and beyond.

Contributions from multiple cathepsins within endosomal antigen processing compartments are necessary to process antigenic proteins into antigenic peptides. Cysteine and aspartyl cathepsins have been known to digest antigenic proteins. A role for the serine protease, cathepsin G (CatG), in this process has been described only recently, although CatG has long been known to be a granule-associated proteolytic enzyme of neutrophils. In line with a role for this enzyme in antigen presentation, CatG is found in endocytic compartments of a variety of antigen presenting cells. CatG is found in primary human monocytes, B cells, myeloid dendritic cells 1 (mDC1), mDC2, plasmacytoid DC (pDC), and murine microglia, but is not expressed in B cell lines or monocyte-derived DC. Purified CatG can be internalized into endocytic compartments in CatG non-expressing cells, widening the range of cells where this enzyme may play a role in antigen processing. Functional assays have implicated CatG as a critical enzyme in processing of several antigens and autoantigens. In this review, historical and recent data on CatG expression, distribution, function and involvement in disease will be summarized and discussed, with a focus on its role in antigen presentation and immune-related events.

On the perils of poor editing: regulation of peptide loading by HLA-DQ and H2-A molecules associated with celiac disease and type 1 diabetes

This review discusses mechanisms that link allelic variants of major histocompatibility complex (MHC) class II molecules (MHCII) to immune pathology. We focus on HLA (human leukocyte antigen)-DQ (DQ) alleles associated with celiac disease (CD) and type 1 diabetes (T1D) and the role of the murine DQ-like allele, H2-Ag7 (I-Ag7 or Ag7), in murine T1D. MHCII molecules bind peptides, and alleles vary in their peptide-binding specificity. Disease-associated alleles permit binding of disease-inducing peptides, such as gluten-derived, Glu-/Pro-rich gliadin peptides in CD and peptides from islet autoantigens, including insulin, in T1D. In addition, the CD-associated DQ2.5 and DQ8 alleles are unusual in their interactions with factors that regulate their peptide loading, invariant chain (Ii) and HLA-DM (DM). The same alleles, as well as other T1D DQ risk alleles (and Ag7), share nonpolar residues in place of Asp at β57 and prefer peptides that place acidic side chains in a pocket in the MHCII groove (P9). Antigen-presenting cells from T1D-susceptible mice and humans retain CLIP because of poor DM editing, although underlying mechanisms differ between species. We propose that these effects on peptide presentation make key contributions to CD and T1D pathogenesis.

An Insertion Mutant in DQA1*0501 Restores Susceptibility to HLA-DM: Implications for Disease Associations

HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules, and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on Ag presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. In this study, to further characterize DQ2–DM interaction, we introduced point mutations into DQ2 on the proposed DQ2–DM interface to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface, and the presentation of α-II-gliadin epitope (residues 62–70) to murine, DQ2-restricted T cell hybridomas. We found that the three α-chain mutations (α+53G, α+53R, or αY22F) decreased the intrinsic stability of peptide–class II complex. More interestingly, the α+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that α-II-gliadin 62–70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2–DM interaction and have implications for mechanisms underlying disease associations of DQ2.

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms

DM catalyses class II‐associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation‐sensitive anti‐class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse‐chase experiments indicates that DM increases the half‐life of affected alleles. Unexpectedly, the pulse‐chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM‐dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide‐receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM‐mediated increases in the abundance of low‐CLIP‐affinity alleles.

Retraction of the research article: "CD4⁺ T cell autoimmunity to hypocretin/orexin and cross-reactivity to a 2009 H1N1 influenza A epitope in narcolepsy".

Post date 24 September 2014 Our Letter “Comment on ‘CD4+ T cell autoimmunity to hypocretin/orexin and cross-reactivity to a 2009 H1N1 influenza A epitope in narcolepsy’ ” ([ 1 ][1]) discussed the article by De la Herran-Arita et al . ([ 2 ][2]). That article provided evidence for CD4+ T

I-Ag7 is subject to post-translational chaperoning by CLIP

Several MHC class II alleles linked with autoimmune diseases form unusually low-stability complexes with class II-associated invariant chain peptides (CLIP), leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. We recently demonstrated a novel post-endoplasmic reticulum (ER) chaperoning role of the CLIP peptides for the murine class II allele I-E(d). In the current study, we tested the generality of this CLIP chaperone function using a series of invariant chain (Ii) mutants designed to have varying CLIP affinity for I-A(g7). In cells expressing these Ii CLIP mutants, I-A(g7) abundance, turnover and antigen presentation are all subject to regulation by CLIP affinity, similar to I-E(d). However, I-A(g7) undergoes much greater quantitative changes than observed for I-E(d). In addition, we find that Ii with a CLIP region optimized for I-A(g7) binding may be preferentially assembled with I-A(g7) even in the presence of higher levels of wild-type Ii. This finding indicates that, although other regions of Ii interact with class II, CLIP binding to the groove is likely to be a dominant event in assembly of nascent class II molecules with Ii in the ER.

Masking of a cathepsin G cleavage site in vivo contributes to the proteolytic resistance of major histocompatibility complex class II molecules

The expression of major histocompatibility complex class II (MHC II) molecules is post‐translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II‐degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent‐solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)‐DR isolated from both HLA‐DM‐expressing and DM‐null cells. Even following CatG cleavage, peptide binding was retained by pre‐loaded, soluble recombinant HLA‐DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane‐proximal β2 domain. All allelic variants of HLA‐DR tested and murine I‐Ag7 class II molecules were susceptible, whereas murine I‐Ek and HLA‐DM were not, consistent with their altered sequence at the P1’ position of the CatG cleavage site. CatG effects were reduced on HLA‐DR molecules with DRB mutations in the region implicated in interaction with HLA‐DM. In contrast, addition of CatG to intact B‐lymphoblastoid cell lines (B‐LCLs) did not cause degradation of membrane‐bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen‐presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context‐dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis.

pH-susceptibility of HLA-DO tunes DO/DM ratios to regulate HLA-DM catalytic activity

The peptide-exchange catalyst, HLA-DM, and its inhibitor, HLA-DO control endosomal generation of peptide/class II major histocompatibility protein (MHC-II) complexes; these complexes traffic to the cell surface for inspection by CD4+ T cells. Some evidence suggests that pH influences DO regulation of DM function, but pH also affects the stability of polymorphic MHC-II proteins, spontaneous peptide loading, DM/MHC-II interactions and DM catalytic activity, imposing challenges on approaches to determine pH effects on DM-DO function and their mechanistic basis. Using optimized biochemical methods, we dissected pH-dependence of spontaneous and DM-DO-mediated class II peptide exchange and identified an MHC-II allele-independent relationship between pH, DO/DM ratio and efficient peptide exchange. We demonstrate that active, free DM is generated from DM-DO complexes at late endosomal/lysosomal pH due to irreversible, acid-promoted DO destruction rather than DO/DM molecular dissociation. Any soluble DM that remains in complex with DO stays inert. pH-exposure of DM-DO in cell lysates corroborates such a pH-regulated mechanism, suggesting acid-activated generation of functional DM in DO-expressing cells.

Serum amyloid A induces mitogenic signals in regulatory T cells via monocyte activation.

Serum amyloid A (SAA) has recently been identified by our group as a mitogen for regulatory T cells (Treg). However, the molecular mechanism by which SAA induces Treg proliferation is unknown. Here we provide evidence that IL-1β and IL-6 are directly involved in the SAA-mediated proliferation of Treg. By engaging its several cognate receptors, SAA induces IL-1β and IL-6 secretion by monocytes and drives them toward an HLA-DR(hi) HVEM(lo) phenotype resembling immature dendritic cells, which have been implicated in tolerance generation. This monocyte-derived cytokine milieu is required for Treg expansion, as inhibition of IL-1β and IL-6 abrogate the ability of SAA to induce Treg proliferation. Furthermore, both IL-1β and IL-6 are required for ERK1/2 and AKT signaling in proliferating Treg. Collectively, these results point to a novel mechanism, by which SAA initiates a monocyte-dependent process that drives mitogenic signals in Treg.