Redundancy in antigen-presenting function of the HLA-DR and -DQ molecules in the multiple sclerosis-associated HLA-DR2 haplotype

The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction.     

Molecular analysis of thymopentin binding to HLA-DR molecules

Thymopentin (TP5) triggers an immune response by contacting with T cells; however the molecular basis of how TP5 achieves this process remains incompletely understood. According to the main idea of immunomodulation, we suppose that it would be necessary for TP5 to form complex with human class II major histocompatibility complex DR molecules (HLA-DR) before TP5 interacts with T cells. The uptake of TP5 by EBV-transformed B cells expressing HLA-DR molecules and the histogram of fluorescence intensities were observed by using fluorescent- labeled TP5, testifying the direct binding of TP5 to HLA-DR. The binding specificity was confirmed by the inhibition with unlabeled TP5, suggesting the recognition of TP5 by HLA-DR. To confirm the interaction between TP5 and HLA-DR, the complex formation was predicted by using various modeling strategies including six groups of trials with different parameters, alanine substitutions of TP5, and the mutants of HLA-DR. The results demonstrated that TP5 and its alanine substitutions assumed distinct conformations when they bound to HLA-DR. The observation further showed that there was flexibility in how the peptide bound within the binding cleft. Also, the molecular analysis supplemented a newly important discovery to the effect of Val anchor on TP5 binding HLA-DR, and revealed the important effects of Glu11 and Asn62 on the recognition of TP5. These results demonstrated the capability of TP5 to associate with HLA-DR in living antigen presenting cells (APC), thereby providing a new and promising strategy to understand the immunomodulation mechanism induced by TP5 and to design potential immunoregulatory polypeptides.     

Selecting peptides to optimize Th1 responses to an equine lentivirus using HLA-DR binding motifs and defined HIV-1 Th peptides

Three moderately to broadly recognized equine infectious anemia virus (EIAV) peptides that contained helper T-lymphocyte (Th) 1 epitopes were previously identified. Although lipopeptide immunization was only weakly immunostimulatory in a preliminary study, as measured by T-lymphocyte proliferation responses, it was of interest to define additional broadly recognized Th1 epitopes to include in future immunization trials. Using broadly cross-reactive and conserved Th epitopes known in the related human immunodeficiency virus-1 (HIV-1) and binding motifs defined in human leukocyte antigen DR molecules as guides, this work identified three new peptides containing Th1 epitopes recognized by 60–75% of EIAV infected horses. The observed similarity across species of major histocompatibility complex (MHC) class II binding motifs and the conservation of Th peptides between related viruses should allow easier targeting of Th epitope regions in less well characterized pathogens and/or in species whose MHC class II molecules are poorly defined.     

Conformational and structural characteristics of peptides binding to HLA-DR molecules.

A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.     

Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis

Disease-associated HLA-DR molecules, which may present autoantigens, constitute the greatest genetic risk factor for rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). The peptides presented by HLA-DR molecules in synovia have not previously been defined. Using tandem mass spectrometry, rigorous database searches, and manual spectral interpretation, we identified 1,427 HLA-DR-presented peptides (220-464 per patient) from the synovia of four patients, two diagnosed with RA and two diagnosed with LA. The peptides were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. A few epitopes were found only in RA or LA patients. However, two patients with different diseases who had the same HLA allele had the largest number of epitopes in common. In one RA patient, peptides were identified as originating from source proteins that have been reported to undergo citrullination under other circumstances, yet neither this post-translational modification nor anti-cyclic citrullinated peptide antibodies were detected. Instead, peptides with the post-translational modification of S-cysteinylation were identified. We conclude that a wide range of proteins enter the HLA-DR pathway of antigen-presenting cells in the patients' synovial tissue, and their HLA-DR genotype, not the disease type, appears to be the primary determinant of their HLA-DR-peptide repertoire. New insights into the naturally presented HLA-DR epitope repertoire in target tissues may allow the identification of pathogenic T cell epitopes, and this could lead to innovative therapeutic interventions.     

血清标记物在类风湿关节炎相关间质性肺疾病中的作用

类风湿关节炎相关间质性肺疾病(rheumatoid arthritis-associated interstitial lung disease, RA-ILD)是类风湿关节炎(rheumatoid arthritis, RA)最具破坏性的并发症,一旦发展成普通型间质性肺炎模式,患者的死亡率急剧上升,且缺乏有效的治疗手段和特异性的诊断方法。血清标记物,特别是MMPs、KL-6、SP-D、CCL18、OPN、WNT5A、Anti-CarP抗体、抗MAA抗体、抗PAD抗体等,可以早期识别RA-ILD的高危患者,预测亚型、评价疗效、监测预后,日益受到人们关注。本文就血清标记物与RAILD异常表达和肺纤维化发生、进展及预后的相关性作一综述,旨在为RA-ILD寻找可靠的血清标记物,为临床诊治工作提供参考。     

Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines

Staphylococcal enterotoxin A (SEA) is a potent polyclonal T cell activator. Its activating effect is entirely dependent upon its binding to accessory cells. Monocytes, B cells, and B lymphomas can bind SEA and support activation of T cells. We have earlier found that Raji cells are particularly efficient as accessory cells for SEA-induced T cell proliferation. In the present investigation we have used this cell line for the isolation and characterization of the membrane molecule to which SEA binds. Flow cytometric analysis of cells dually stained with SEA and anti-HLA-DR mAb showed that the amount of bound SEA was proportional to the HLA-DR expression. Electrophoresis of detergent extracts of Raji cells revealed one distinct SEA-binding band with a Mr of 60 to 65 kDa. This band had the same electrophoretic mobility as the MHC class II molecules. A mAb (G8) with the ability to block SEA binding to Raji cells was established. This mAb was shown to bind to the HLA-DR molecule. Both the G8 mAb and an anti-HLA-DR mAb 9-49 inhibited SEA binding to accessory cells and also inhibited SEA-induced, but not PHA-induced, T cell proliferation and production of IL-2. Immunoprecipitation with specific anti-HLA-DR and anti-HLA-DQ mAb demonstrated that SEA binds to the HLA-DR molecule but not to the HLA-DQ molecule. Binding SEA to Raji cells followed by cross-linking and detergent solubilization of cell membranes, electrophoresis, and Western blotting resulted in two SEA-containing bands corresponding to a Mr of 90 and 105 kDa, respectively. Both these bands also contained the HLA-DR molecule and their appearance could be blocked by preincubation of the Raji cells with the G8 mAb. Collectively the results show that the HLA-DR molecule is the main functional molecule for binding of SEA to accessory cells and that this binding of SEA to HLA-DR is a necessary requirement for SEA-induced T cell activation.     

Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Staphylococcal enterotoxin B and toxic shock syndrome toxin-1 bind to distinct sites on HLA-DR and HLA-DQ molecules

Staphylococcal enterotoxins (SE) activate human T cells in vitro. This requires the presence of Ia+ accessory cells but does not require processing of the toxin by the accessory cell. We and others have recently demonstrated direct binding of SE to human MHC class II molecules. In this study, we have compared in detail the ability of class II molecules to bind two SE, toxic shock syndrome toxin-1 (TSST-1) and SEB. Scatchard analysis of equilibrium binding data indicate that SEB binds to Ia+ human cell lines with a 10-fold lower affinity than TSST-1. Likewise, SEB precipitates HLA-DR alpha- and beta-chains from detergent lysates of Ia+ cells less efficiently than TSST-1. The binding of TSST-1 and SEB to transfected L cells expressing HLA-DR and HLA-DQ gene products was differentially inhibited by anti-HLA-DR mAb. There was virtually no cross-inhibition of TSST-1 and SEB in competitive binding assays. We conclude that TSST-1 and SEB bind to two MHC class II sites which can be distinguished by their relative accessibility to blocking by anti-class II mAb and heterologous toxin.     

One NY-ESO-1-derived epitope that promiscuously binds to multiple HLA-DR and HLA-DP4 molecules and stimulates autologous CD4+ T cells from patients with NY-ESO-1-expressing melanoma

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87-111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87-111 stimulates Th1-type and Th-2/Th0-type CD4(+) T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4(+) T cells and CD4(+) T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-gamma and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4(+) T cells recognizing peptide NY-ESO-1 87-111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87-111 in the large number of cancer patients with NY-ESO-1-expressing tumors.     

Peptide binding motifs and specificities for HLA-DQ molecules

 HLA-DQ molecules have been associated with susceptibility to a number of autoimmune and other diseases, possibly through the peptide repertoire that can be presented by different allelic products. It is thus of importance to understand which peptides can be bound by different HLA-DQ allelic products. Recently, a model for HLA-DQ has been described and used to derive peptide positional environments for HLA-DQ allelic products. By combining the peptide positional environments with known HLA-DQ peptide binding motifs, a set of predictions of likely anchor motifs for many of the products of HLA-DQ allelic variants are made and presented in a table referred to as a roadmap for HLA-DQ peptide binding specificities. Received: 3 March 1999 / Revised: 20 May 1999     

Binding of Streptococcus mutans to extracellular matrix molecules and fibrinogen

We have determined the ability of Streptococcus mutans cells to bind to extracellular matrix (ECM) molecules and fibrinogen. S. mutans cells were found to bind fibronectin, laminin, collagen type I, and fibrinogen. An isogenic S. mutans strain with a defect in the expression of the major surface protein of S. mutans, antigen I/II, possessed a reduced ability to bind fibronectin, collagen, and fibrinogen but not laminin, suggesting that antigen I/II contributes during pathological processes to the interaction of S. mutans cells with fibronectin, collagen type I, and fibrinogen.     

Binding of fibrinogen molecules to pig platelets and their membranes

Following addition of ADP, 125I-labelled fibrinogen binds specifically to pig platelets. This binding is completely inhibited by the unlabelled fibrinogen. Quantitative analysis indicates the presence of 12,400-25,000 molecules of fibrinogen which can be bound with an association constant of 5 . 10(8) M-1 to platelets. Fibrinogen receptors were found to be active in the isolated platelet membranes as well. Quantitative analysis of the saturable binding of fibrinogen to the platelet membranes showed that these receptors react with the same affinity with fibrinogen molecules. In contrast to the intact platelets, the platelet membranes can specifically bind fibrinogen in the absence of ADP. We conclude that a specific receptor for fibrinogen is exposed on the surface as a result of cell damage which is the first step of the platelet membrane isolation.     

Invariant chain influences post-translational processing of HLA-DR molecules

HLA class II MHC molecule alpha- and beta-chains are normally synthesized in the presence of a third molecule, the invariant chain (Ii). Although Ii is not required for surface expression of HLA class II molecules, the influence of Ii on post-translational processing and maturation HLA class II molecules has not been thoroughly studied. In the present study, BALB/c 3T3 cells were transfected with HLA-DR alpha- and beta-chains with or without co-transfection with human Ii. Although Ii had no effect on the surface expression of DR, Ii did have a profound effect on the post-translational processing of both the alpha- and beta-chains. In the absence of Ii, the major species of alpha- and beta-chains were of lower m.w. than when expressed in the presence of Ii. The differences in m.w. were shown to be caused by differences in glycosylation with the majority of alpha- and beta-chains remaining unprocessed and endo H sensitive in the absence of Ii. The small proportion of alpha-chains that were processed in the absence of Ii showed an altered m.w. and altered sensitivity to treatment with endo H relative to alpha-chains processed in the presence of Ii. Pulse/chase studies demonstrated that although the majority of the alpha- and beta-chains remained unprocessed in the absence of Ii, the small amount that was processed was done so at a rate similar to that observed for alpha- and beta-chains processed in the presence of Ii. These studies demonstrate that Ii influences the post-translational processing of human class II molecules by affecting the proportion of alpha- and beta-chains that are processed and by determining the degree of processing of oligosaccharides on mature alpha-chains.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils

Collagenase released from rheumatoid synivial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24°C and 37°C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibril can be degraded. Binding was not inhibited by the presence of α₂ macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding the substrate, and that activation by proteases such as plasmin intiates collagen breakdown.     

Peptide binding to soluble HLA-DR4 molecules produced by insect cells.

HLA-DR4Dw4 molecules were expressed in insect Sf9 cells. The transmembrane and cytoplasmic domains of the DR4 alpha- and beta-chains were replaced by the carboxy terminal sequence of decay accelerating factor, leading to a phosphatidyl inositol glycan membrane anchor. This structure contains a cleavage site for phosphatidyl inositol-specific phospholipase C, allowing efficient solubilization of the rDR4 molecules. We present evidence that infected insect cells express properly associated surface heterodimers and are able to present antigenic peptides to DR4Dw4-restricted T cell clones. Phosphatidyl inositol-specific phospholipase-cleaved recombinant molecules exhibited in vitro binding characteristics similar to DR4 molecules purified from lymphoblastoid cells. In terms of peptide specificity, pH optimum, kinetics, and affinity they were indistinguishable within the limits of our assay system. However, the peptide binding capacity of the recombinant molecules was higher than that of native DR4 molecules.     

Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules

In previous studies, the shared cancer-testis Ag, NY-ESO-1, was demonstrated to be recognized by both Abs and CD8+ T cells. Gene expression of NY-ESO-1 was detected in many tumor types, including melanoma, breast, and lung cancers, but was not found in normal tissues, with the exception of testis. In this study, we describe the identification of MHC class II-restricted T cell epitopes from NY-ESO-1. Candidate CD4+ T cell peptides were first identified using HLA-DR4 transgenic mice immunized with the NY-ESO-1 protein. NY-ESO-1-specific CD4+ T cells were then generated from PBMC of a patient with melanoma stimulated with the candidate peptides in vitro. These CD4+ T cells recognized NY-ESO-1 peptides or protein pulsed on HLA-DR4+ EBV B cells, and also recognized tumor cells expressing HLA-DR4 and NY-ESO-1. A 10-mer peptide (VLLKEFTVSG) was recognized by CD4+ T cells. These studies provide new opportunities for developing more effective vaccine strategies by using tumor-specific CD4+ T cells. This approach may be applicable to the identification of CD4+ T cell epitopes from many known tumor Ags recognized by CD8+ T cells.