T cell recognition of the dominant I-A(k)-restricted hen egg lysozyme epitope: critical role for asparagine deamidation.

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL(48-62)) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-A(k) in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide-MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity.

The effects of deimination of myelin basic protein on structures formed by its interaction with phosphoinositide-containing lipid monolayers.

The recombinant 18.5-kDa charge isoform of murine myelin basic protein (rmMBP) is unmodified posttranslationally and was used to study the effects of deimination, i.e., the conversion of arginyl to citrullinyl residues, on the protein's interactions with itself and with lipids. The unmodified species rmMBP-Cit(0) (i.e., containing no citrullinyl residues) interacted with binary monolayers containing acidic (phosphatidylinositol) and nickel-chelating lipids to form paracrystalline arrays with 4.8-nm spacing. A sample of protein was deiminated to an average of 9 citrullinyl residues per molecule of protein, yielding rmMBP-Cit(9). Under both low- and high-salt conditions, this species formed better-ordered domains than rmMBP-Cit(0), viz., planar crystalline assemblies. Thus, deimination of MBP resulted in a significant alteration of its lipid-organizing and self-interaction properties that might be operative in myelin in vivo, especially in progression of the autoimmune disease multiple sclerosis. Comparisons of amino acid sequences indicated significant similarities of MBP with filaggrin, a protein that is deiminated in another autoimmune disease, rheumatoid arthritis, suggesting that comparable epitopes could be targeted in both pathologies. In contrast, binary lipid monolayers consisting of phosphatidylinositol-4-phosphate (or phosphatidylinositol-4,5-bisphosphate) and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by rmMBP, especially in its deiminated form. Sequence comparisons with other actin- and phosphoinositide-binding proteins (vinculin, ActA, MARCKS) suggested that the carboxyl-terminal segment of MBP could form an amphipathic alpha helix and was the phosphoinositide binding site.

Characterization of a recombinant murine 18.5-kDa myelin basic protein.

A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G(M1), and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3-4 nm along them.

Enhanced co-stimulatory ability of synovial fluid accessory cells in rheumatoid arthritis.

We have established in vitro assays that allow the examination of co-stimulatory function of rheumatoid arthritis (RA) antigen-presenting cells (APC). Synovial fluid (SF) and peripheral blood (PB) APC co-stimulatory ability was compared in the activation of peptide-specific human T-cell clones. T-cell receptor (TCR) stimulation by peptide or anti-CD3 antibody allowed the direct comparison of SF and PB APC co-stimulatory activity, separately from their ability to process antigen. SF APC from 15 RA patients consistently enhanced T-cell proliferation when compared to their PB counterparts. Moreover, increasing the numbers of PB APC present resulted in only a minor increase in T-cell proliferation, failing to achieve levels stimulated by SF APC. We propose that the enhanced co-stimulatory function of synovial APC may be a significant factor in the persistence of local immune responses in RA.

Amino-terminal trimming of peptides for presentation on major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC) class II molecules bind antigenic peptides for display to T lymphocytes. Although the enzymes involved remain to be identified, it is commonly believed that class II associated peptides are released from intact antigens through a series of proteolytic steps carried out inside antigen presenting cells. We have examined the effect of amino acid substitutions on proteolytic processing of the model antigen hen-egg lysozyme (HEL). Altered HEL molecules, engineered by site-directed mutagenesis of a HEL cDNA, were expressed as separate stable transfectants in a B cell lymphoma line. Each transfectant processed a different mutant HEL protein for presentation on MHC class II. We purified the resulting class II-associated peptides and analyzed them by mass spectrometry. Our results strongly support the hypothesis that antigen processing continues after peptide binding to the MHC class II molecule and are most consistent with a scenario in which long peptides first bind to MHC class II and are then trimmed by exopeptidase.

TCR-mediated adhesion of T cell hybridomas to planar bilayers containing purified MHC class II/peptide complexes and receptor shedding during detachment.

T cell recognition of foreign Ag/MHC class II complexes is sensitive down to approximately 100 complexes per cell or approximately 0.2 complexes/micron2. To better understand the physical basis of the recognition stage of Ag presentation, we examined adhesion of the lysozyme- specific T cell hybridoma, 3A9, to artificial bilayers containing covalent MHC class II/peptide complexes or adhesion molecules. Adhesion of 3A9 cells required a superphysiologic density of the MHC class II/peptide complex and was partly dependent on CD4; cells adhered but did not crawl. No adhesion was observed to bilayers containing MHC class II molecules without the lysozyme peptide. Activated 3A9 cells adhered and crawled on bilayers containing ICAM-1. The physical strength of contacts was tested with fluid shear. 3A9 cells adherent to bilayers containing MHC class II/peptide complexes shed their contact, which remained on the substrate and contained TCR. In contrast, 3A9 cells peeled from the ICAM-1 bilayer, and held firmly on LFA-1 bilayers; in a manner dependent on filamentous actin. When ICAM-1 and the MHC/peptide complexes were combined, the 3A9 cells adhered tightly and spread, but did not crawl, on the bilayers and TCR clustered at the center of the contact area. Physiologically, the TCR is unlikely to directly initiate adhesion. TCR clusters formed with the assistance of adhesion mechanisms may have to be shed to allow de-adhesion, and this may contribute to TCR down-regulation.

A negatively charged anchor residue promotes high affinity binding to the MHC class II molecule I-Ak.

An allele-specific peptide-binding motif for the murine MHC class II molecule I-Ak has proven elusive. Here we demonstrate that the I-Ak molecule preferentially binds peptides that contain negatively charged amino acids at the primary anchor position (Asp or Glu at P1), and that I-Ak can also bind peptides with polar residues at P1 (Cys, Ser, Asn, Gin, or Thr), although with lower affinity. This preference for a negatively charged anchor residue is so pronounced that polyalanine peptides containing a single Asp can bind to I-Ak. Eight naturally processed peptides were found to use an Asp, as demonstrated by a drop in the I-Ak binding affinity of these peptides after Ala substitution. The chemical identity of the amino acid in the anchor position was also important in determining the ability of peptide-I-Ak complexes to resist denaturation on SDS-polyacrylamide gels. The P1 binding pockets of HLA-DR and H-2E molecules are reported to be large and hydrophobic, and these class II molecules prefer to bind peptides with large aliphatic or aromatic side chains at P1. Our results suggest that the structure of the I-Ak P1 binding pocket is different. Based on sequence comparisons, we suggest that the P1 binding pockets of H-2A molecules may prove more polymorphic than the P1 binding pockets of H-2E molecules, and that this additional polymorphism will cause H-2A molecules to display larger intra-allelic differences in peptide binding specificities.

Complexes generated by the binding of free peptides to class II MHC molecules are antigenically diverse compared with those generated by intracellular processing.

We investigated the specificity of T cell hybridomas isolated from mice immunized with synthetic peptides identical in sequence with the dominant, naturally processed, I-Ak-restricted peptides of hen egg lysozyme (HEL). Surprisingly, the majority of hybridomas showed little or no recognition of intact HEL after processing by different APCs. This was not an artifact caused by the use of synthetic peptides since the peptide-specific hybridomas responded to a tryptic digest of HEL or to naturally processed HEL peptides extracted from I-Ak. Thus, the interaction of free peptides with class II MHC molecules can generate complexes that are antigenically dissimilar to those resulting from intracellular processing of intact Ag. This has important implications both for the interpretation of experimental studies that involve peptide immunization and for the efficacy of peptide vaccination as a strategy for intervention in human disease.

Role of antigen presenting cells in rheumatoid arthritis.

Histological evidence indicates that activated antigen presenting cells (APC) are present in large numbers within the synovial compartment in rheumatoid arthritis. Their potent function has been demonstrated in vitro using isolated synovial APC populations, although the full picture of which APC populations are involved is still emerging. The ability of these APC to activate T cells which traffic to the joint is likely to play an important role in the maintenance of the synovial inflammatory response.

Identification of a major I-Ek-restricted determinant of hen egg lysozyme: limitations of lymph node proliferation studies in defining immunodominance and crypticity.

We have chemically analyzed the peptides presented by I-Ek molecules after processing of hen egg lysozyme (HEL) by a murine B-lymphoma line or by splenocytes. In both cases, the identified peptides were derived from a single region of HEL, containing the core residues 85-96 with heterogeneous N and C termini. This was a surprising result because this determinant had previously been described as cryptic--i.e., not presented after processing of intact HEL. Examination of the specificities of T hybridomas isolated after immunization with either HEL or 84-96 peptide (p84-96) provided an explanation for this controversy. Whereas hybridomas induced by immunization with HEL responded equally well to HEL and p84-96, those induced by peptide immunization showed a marked preference for p84-96 over intact HEL. In other words, hybridomas isolated after p84-96 immunization responded poorly to forms of the 84-96 determinant produced by natural processing, leading to the possible erroneous interpretation that 84-96 is a hidden determinant. We conclude that (i) p84-96 is efficiently presented on I-Ek molecules after processing of HEL, (ii) the explanation for the weak lymph node response to this epitope after immunization with HEL lies at the level of the T cell, not the antigen-presenting cell, and (iii) crypticity cannot be defined on the basis of T-cell proliferation studies alone.

Cross-sectional analysis of the differences between patients with systemic lupus erythematosus in England, Brazil and Sweden.

This study is a cross-sectional analysis of the differences between SLE outpatients seen in Rheumatology departments at University centres in England, Brazil and Sweden, using a standard protocol. The demographic characteristics, extent and activity of disease of 209 patients with SLE were studied; 112 patients were seen in England, 33 in Brazil and 64 in Sweden. The median age of disease onset of Brazilian and English patients was 25 years and of Swedish patients 31.5 years. Disease activity was measured by the BILAG index. In most systems Brazilian patients experienced more activity than English patients and English patients more activity than Swedish patients. Non-Caucasians experienced more active disease than Caucasians. No sex or occupational differences were observed in disease activity. English patients were the most likely to have experienced photosensitivity, oral ulcers and haematological disorders, Brazilian patients renal disorders and Swedish patients discoid rashes. Brazilian patients were the most likely to be prescribed only one drug for treatment of SLE and to be taking steroids and the highest dose of steroids, in contrast to the European patients who were often prescribed steroids and an antimalarial agent or azathioprine. The results of this cross-sectional assessment of disease activity using a standardized instrument indicate that there are real differences in the extent and degree of activity of SLE in different national groups. This reflects a combination of genetic, environmental and social influences on disease expression and has implications for treatment and monitoring of SLE patients.

A longitudinal study of peripheral blood mononuclear cell proliferative responses to bacterial antigens in reactive arthritis.

Reactive arthritis (ReA) is a sterile inflammatory arthritis which usually occurs after an enteric or genitourinary infection. In recent years it has been recognized that synovial fluid mononuclear cells from an affected joint demonstrate marked proliferative responses if incubated with preparations of the organism triggering the arthritis; peripheral blood mononuclear cell (PBMC) responses are typically much smaller. One interpretation of this finding is that recognition of the triggering organism is enhanced within the joint compared to peripheral blood, but it could also be argued that the PBMC responses are actually depressed during acute arthritis. We have examined this possibility in a longitudinal study of PBMC proliferative responses in patients with ReA. In this study we have demonstrated that PBMC proliferative responses to the triggering organism were indeed depressed during acute ReA, and showed a significant increase after recovery from the arthritis. These findings also applied to PBMC recognition of the recall antigen PPD, and to the response to IL-2.

Synovial fluid antigen-presenting cell function in rheumatoid arthritis.

We have previously demonstrated enhanced synovial fluid (SF) antigen-presenting cell (APC) function in inflammatory arthritis patients selected on the basis of marked SF mononuclear cell (MNC) responsiveness to reactive arthritis-associated bacteria (Clin Exp Immunol 1990; 79:189-94). In this study we have assessed whether similarly enhanced synovial APC function is present in other inflammatory arthritis patients by using two assay systems to study 18 rheumatoid arthritis patients whose MNC responsiveness had not been determined in advance. We demonstrate that rheumatoid SF APC are much more potent than peripheral blood (PB) APC in stimulating the responses of autologous PB T cells to a range of recall antigens. In addition, SF APC are shown to be efficient stimulators of the antigen-specific responses of MHC-compatible, cloned T cells. Enhanced synovial APC function is thus likely to be a general feature of inflammatory arthritis and may play an important role in its pathogenesis.

Isolation of Yersinia-specific T cell clones from the synovial membrane and synovial fluid of a patient with reactive arthritis.

Synovial fluid (SF) mononuclear cells from patients with reactive arthritis (ReA) proliferate in vitro when challenged with ReA-associated bacteria, the maximal response being for the organism causing the triggering infection. We report the results of a study of the antigenic specificity of synovial T lymphocytes from an HLA-B27 positive ReA patient whose SF mononuclear cells responded preferentially to Yersinia antigens. This is the first report of the isolation of Yersinia-specific T cell clones from synovial membrane (obtained by closed-needle synovial biopsy). We present a detailed analysis of these clones, together with others obtained from the SF.

Synovial fluid antigen-presenting cells unmask peripheral blood T cell responses to bacterial antigens in inflammatory arthritis.

We and others have previously shown that synovial fluid (SF) mononuclear cells (MC) from patients with both reactive arthritis and other inflammatory arthritides proliferate in vitro in response to bacteria clinically associated with the triggering of reactive arthritis. In all cases, such SFMC responses are greater than the corresponding peripheral blood (PB) MC responses, often markedly so, and the mechanism for this is unclear. We have investigated this phenomenon by comparing the relative abilities of irradiated non-T cells derived from PB and SF to support autologous T cell responses to ReA-associated bacteria. Seven patients whose SFMC had been shown previously to respond to bacteria were studied. We demonstrate antigen-specific responses of PB T cells to bacteria in the presence of SF non-T cells which are in marked contrast to the minimal responses of either unfractionated PBMC or PB T cells reconstituted with PB non-T cells. We also show that PB, but not SF T cells respond strongly to autologous SF non-T cells in the absence of antigen or mitogen. These findings demonstrate that SF antigen-presenting cells (APC) are potent activators of PB T cells. We conclude that the contrasting responses of SFMC and PBMC to bacterial antigens may be accounted for at least in part by an enhanced ability of SF APC to support T cell proliferative responses.