Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones.

The human T cell response to the myelin basic protein (MBP) has been studied with respect to T cell receptor (TCR) usage, HLA class II restriction elements, and epitope specificity using a total of 215 long-term MBP-specific T cell lines (TCL) isolated from the peripheral blood of 13 patients with multiple sclerosis (MS) and 10 healthy donors. In most donors, the anti-MBP response was exceedingly heterogeneous. Using a panel of overlapping synthetic peptides spanning the entire length of human MBP, at least 26 epitopes recognized by human TCL could be distinguished. The MBP domain most commonly recognized was sequence 80-105 (31% of MS TCL, and 24% of control TCL). Sequence 29-48 was recognized more frequently by control-derived TCL (24%) than by TCL from MS patients (5%). The MBP epitopes were recognized in the context of DRB1 *0101, DRB5*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*1402, and DRB3*0102, as demonstrated using a panel of DR gene-transfected L cells. The TCR gene usage was also heterogeneous. V beta 5.2, a peptide of which is currently being used in a clinical trial for treatment of MS patients, was expressed by only one of our TCL. However, within this complex pattern of MBP-specific T cell responses, a minority of MS patients were found to exhibit a more restricted response with respect to their TCL epitope specificity. In these patients 75-87% of the TCL responded to a single, patient-specific cluster of immunodominant T cell epitopes located within a small (20-amino acid) domain of MBP. These nested clusters of immunodominant epitopes were noted within the amino acids 80-105, 108-131, and 131-153. The T cell response to the immunodominant epitopes was not monoclonal, but heterogeneous, with respect to fine specificity, TCR usage, and even HLA restriction. In one patient (H.K.), this restricted epitope profile remained stable for > 2 yr. The TCR beta chain sequences of TCL specific for the immunodominant region of HK are consistent with an oligoclonal response against the epitopes of this region (80-105). Further, two pairs of identical sequences were established from TCL generated from this patient at different times (June 1990 and June 1991), suggesting that some TCL specific for the immunodominant region persisted in the peripheral repertoire. The possible role of persistent immunodominant epitope clusters in the pathogenesis of MS remains to be established.

Targeting fibroblast growth factor-inducible-14 signaling protects from chronic relapsing experimental autoimmune encephalomyelitis.

The TNF-related weak inducer of apoptosis (TWEAK) is a TNF family member mediating proinflammatory effects by its receptor fibroblast growth factor-inducible-14 (Fn14). We studied the role of TWEAK/Fn14 in experimental autoimmune encephalomyelitis (EAE) by protein vaccination with TWEAK and Fn14 and recombinant TWEAK-DNA, respectively. TWEAK-DNA vaccination worsened the clinical course of EAE and increased central nervous system (CNS) inflammation. TWEAK increased the secretion of CCL2 [monocyte chemotactic protein-1 (MCP-1)] by CNS endothelial cells and astrocytes in vitro, suggesting CCL2 as a critical mediator of TWEAKs proinflammatory effects. Vaccination with the extracellular domain of TWEAK or with Fn14 resulted in the induction of specific inhibitory antibodies and an amelioration of EAE signs in two different models in rats and mice. Spinal cord inflammatory infiltrates were significantly diminished. Purified IgG from TWEAK- or Fn14-vaccinated rats prevented TWEAK-induced production of CCL2 by endothelial cells. Blocking Fn14 signaling represents a novel approach with potential for the treatment of CNS autoimmunity.

The ClpB protein from Campylobacter jejuni: molecular characterization of the encoding gene and antigenicity of the recombinant protein.

The ClpB heat-shock protein is necessary for the survival of Escherichia coli cells upon sudden increase of temperature. Using a PCR-based genomic walking method, the nucleotide sequence of a clpB homolog from Campylobacter jejuni was determined. The clpB gene encodes a protein of 857 amino acid (aa) residues, with a predicted molecular mass of 95.3kDa. Alignment of the deduced aa sequence with other known bacterial ClpB proteins revealed overall identity from 47% (E. coli) to 61% (Helicobacter pylori). Within the clpB promoter region, as indicated by primer extension analysis, we identified a sequence identical to the E. coli sigma70 consensus promoter. Northern blot analysis confirmed that clpB is heat-inducible in C. jejuni. The ClpB protein, fused to a 6xHis tag, was synthesized in E. coli and purified by metal-affinity and size exclusion chromatography. In ELISA studies, IgA levels reactive to recombinant ClpB were significantly higher in sera of patients with prior C. jejuni infections than in sera obtained from healthy control persons.

Human T lymphocytes distinguish bovine from human P2 peripheral myelin protein: implications for immunological studies on inflammatory demyelinating neuropathies.

In patients with inflammatory demyelinating neuropathy, which is possibly mediated by autoreactive, myelin-specific T lymphocytes, most studies focusing on immune responses to the major neuritogenic myelin protein P2 have been performed with bovine P2. However, the primary structure of bovine P2 differs from the human protein by nine amino acid residues that may profoundly influence the antigen recognition by T lymphocytes. We purified bovine and human P2 from peripheral nervous tissue and established a total of 19 T cell lines (TCL) reactive with bovine P2 from blood of two patients with acute Guillain-Barré syndrome (n = 5 TCL) and from six healthy individuals. Only four of these TCL, all raised from the blood of the GBS patients, transiently cross-recognized human P2 protein. Our results suggest that the use of human autoantigen may be crucial for the characterization of T cellular immune responses against P2 protein both in patients with inflammatory demyelinating neuropathy and in healthy controls.

T-cell receptor V beta-element expression in peripheral nerves of Lewis rats suffering from experimental autoimmune neuritis.

In experimental autoimmune neuritis (EAN), peripheral nerves are infiltrated by T-lymphocytes and macrophages. By RT-PCR and sequence analysis we characterized TCR V beta-element usage in sciatic nerve tissue of Lewis rats suffering from EAN induced by immunization with peripheral myelin antigens. Several TCR V beta-chain sequences were detected, which did not show homology to sequences of P2-reactive T cells published so far. In EAN induced with peripheral nerve myelin, but not with P2-protein or P2 peptide aa 53-78, TCR V beta 8.2 sequences identical to sequences of encephalitogenic myelin basic protein (MBP) reactive T-cells were identified. These results provide further evidence for a contribution of MBP-directed T-cell reactivity to the pathogenesis of myelin induced EAN and may have implications for the pathogenesis of human demyelinating neuropathies.

T cell antigenic and neuritogenic activity of recombinant human peripheral myelin P2 protein.

The major neuritogenic protein of peripheral nerve myelin is the P2 protein. Human P2 is a candidate autoantigen in inflammatory demyelinating diseases of the peripheral nervous system. Since human P2 is not readily available, we produced full-length recombinant human P2 protein (rhP2) in Escherichia coli. RhP2 was recognized by neuritogenic rat T cell lines and induced experimental autoimmune neuritis in Lewis rats. Production of rhP2 allowed the generation of human T cell lines reactive to the autologous protein. Studies of human T cell autoreactivity as well as efforts to use hP2 as a tolerogen will be facilitated by the large-scale expression of rhP2.

Cloning and expression of the Campylobacter jejuni lon gene detected by RNA arbitrarily primed PCR.

Fingerprinting of RNA by arbitrarily primed PCR was used to identify a heat-inducible gene in Campylobacter jejuni. Comparing RNA fingerprints from C. jejuni cells before and after 20 min of heat shock at 48 degrees C, a differentially amplified PCR product was identified which displayed a high degree of homology to bacterial lon genes. By screening C. jejuni genomic libraries, the entire lon gene was cloned and sequenced. It encodes a protein of 791 amino acids with a calculated molecular mass of 90.2 kDa. Alignment of the Lon amino acid sequence with that of other bacterial species revealed an overall identity of up to 56.6% (Helicobacter pylori). Northern and RNA dot blot experiments confirmed heat induction of the C. jejuni lon gene, revealing a maximum 6-8-fold increase in the level of specific mRNA.

Structural analysis of the rat T-cell receptor Tcra V4 gene family.

The rat Tcra V gene locus is only poorly characterized, although rats are widely used in a variety of T-cell-mediated experimental animal models. Recently, we described the first monoclonal antibody, G99, directed against a rat Tcra V4 segment. We examined cDNA transcripts of G99-positively sorted T cells and show that the monoclonal antibody G99 most likely recognizes at least two members of the Tcra V4 family. Moreover, we analyzed the genomic repertoire of this VA family and report 15 novel Tcra V4 DNA sequences. Based on sequence and Southern blot analysis, the Tcra V4 family could be divided into four subgroups, which were also detected in mice. These findings corroborate previous findings of a similar genetic organization of the Tcra V loci in both species.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.

Preferential TCR V usage in rat repertoire selection: V alpha 8 imparts both positive thymic selection by and alloreactivity to RT1f.

Using a panel of newly developed mAb to two rat TCR V alpha and four TCR V beta segments, TCR V usage in CD4 and CD8 T cells of eight RT1 congenic strains sharing the LEW background was analyzed by flow cytometry. While no striking effects on V beta 8.2 and 8.5 usage were observed, a 3- to 4-fold over-representation of V beta 10 in the CD4 as compared with the CD8 subset in all strains suggested a preference of V beta 10 for MHC class II products. The degree of 'overselection' was mapped to the RT1.B/D region. In addition, an allele-specific overselection of V alpha 4+ CD4 T cells was mapped to RT1.B/Du and of V beta 16+ CD8 T cells to RT1.Au. Finally, a dramatic overselection of V alpha 8+ CD8 T cells by RT1f (14% in RT1f versus 1-2% in other haplotypes) provides the most striking case yet for an intrinsic affinity of a TCR V segment for an MHC product. V alpha 8+ CD8 T cells are not only overselected by RT1f in the thymus, but also during the alloreactive response of peripheral CD8 T cells to RT1f. The implications of these findings for the contribution of TCR V segments to TCR-MHC interactions in repertoire selection and alloreactivity are discussed.

Bacterial expression of a soluble T-cell receptor alpha chain.

A bacterial expression system was used to express the complete extracellular domain of a rat T-cell receptor alpha-chain, including variable-, joining and constant regions. By introduction of a His-tail at the C-terminus, a simple and rapid purification protocol utilizing Ni(+)-chelating chromatography was applied, with renaturation of the protein while bound to the matrix. This method produced large amounts of soluble protein, which provide a source for T-cell receptor analysis and further studies on T cell function.

Cloning and expression of the dnaK gene of Campylobacter jejuni and antigenicity of heat shock protein 70.

Campylobacter jejuni is a leading cause of infectious diarrhea throughout the world. In addition, there is growing evidence that Guillain-Barré syndrome, an inflammatory demyelinating disease of the peripheral nervous system, is frequently preceded by C. jejuni infection. In the present study, the hrcA-grpE-dnaK gene cluster of C. jejuni was cloned and sequenced. The dnaK gene consists of an open reading frame of 1,869 bp and encodes a protein with a high degree of homology to other bacterial 70-kDa heat shock proteins (HSPs). The overall percentages of identity to the HSP70 proteins of Helicobacter pylori, Borrelia burgdorferi, Chlamydia trachomatis, and Bacillus subtilis were calculated to be 78.1, 60.5, 57.2, and 53. 8%, respectively. Regions similar to the Escherichia coli sigma70 promoter consensus sequence and to a cis-acting regulatory element (CIRCE) are located upstream of the hrcA gene. Following heat shock, a rapid increase of dnaK mRNA was detectable, which reached its maximum after 20 to 30 min. A 6-His-tagged recombinant DnaK protein (rCjDnaK-His) was generated in E. coli, after cloning of the dnaK coding region into pET-22b(+), and purified by affinity and gel filtration chromatography. Antibody responses to rCjDnaK-His were significantly elevated, compared to those of healthy individuals, in about one-third of the serum specimens obtained from C. jejuni enteritis patients.

Alleles of highly homologous rat T cell receptor beta-chain variable segments 8.2 and 8.4: strain-specific expression, reactivity to superantigens, and binding of the mAb R78.

This study addresses the molecular basis of a Tcrb-V polymorphism in the reactivity to the superantigens staphylococcus enterotoxin B (SEB) and the mtv-7 sag (MIs1a) of T cells recognized by the mAb R78, which reacts with the T cell receptor beta-chain variable segment 8.2 (Tcrb-V8.2) of Lewis (LEW) rats. Tcrb-V8.2-like sequences were isolated from liver DNA of the responder strain LEW (I) and the nonresponder strain DA (a) and alleles of the Tcrb-V8.2 and the highly homologous Tcrb-V8.4 were identified. Their expression was analyzed by RNase protection studies and cDNA clones were characterized. A comparison of thymocytes, activated R78+ cells, Con A-stimulated and SEB-stimulated cells allows the following conclusions: the newly identified Lewis allele of Tcrb-V8.4 (Trcb-V8.4I) is nonfunctional due to a frame shift induced by deletion of one nucleotide. The R78 epitope is expressed by Tcrb-V8.2I and Tcrb-V8.4a but not by Tcrb-V8.2a. The implication of this finding for mapping of the R78 epitope and the study of V region usage in experimental autoimmune encephalitis are discussed. Finally, the expression of both Tcrb-V8.2 alleles but not of Tcrb-V8.4a in SEB-stimulated cells defines a polymorphism of the CDR2 and/or CDR4 as the molecular basis of the differential superantigen reactivity.

Diversity of T cell receptor alpha and beta chain genes expressed by human T cells specific for similar myelin basic protein peptide/major histocompatibility complexes.

T cell receptor (TcR) alpha and beta nucleotide sequences involved in the human autoreactivity to myelin basic protein (MBP) were studied by screening cDNA libraries derived from 11 independent T lymphocyte clones (TCC) established from multiple sclerosis patients and healthy donors. The TCC with defined MBP peptide specificity and HLA-DR restriction expressed multiple TcR. Even TCC recognizing the same human MBP peptide [amino acids (aa) 139-153] in identical or very similar HLA-DR context expressed diverse TcR. Two TCC which recognized peptide aa 139-153 equally well in the context of both HLA-DR2a and -DR1 molecules used distinct TcR alpha but identical beta chains. The knowledge of TcR beta and TcR alpha chain sequences of human MBP-specific T cells will allow studies correlating structure and function of TcR and their targets in MBP autoreactivity. This may have an impact on the development of immunotherapies in multiple sclerosis.

Antigen therapy eliminates T cell inflammation by apoptosis: effective treatment of experimental autoimmune neuritis with recombinant myelin protein P2.

Exposure of T cells to their specific antigen normally results in proliferation, but in the presence of high and repeatedly administered doses of antigen, T cells may undergo apoptosis. Here we demonstrate that i.v. administration of as little as 100 microg of recombinant P2 protein twice daily completely prevents experimental autoimmune neuritis induced by adoptive transfer of neuritogenic P2-specific T cells or by immunization with the neuritogenic P2-peptide-spanning amino acids 53-78. Antigen treatment started after disease onset markedly ameliorated experimental autoimmune neuritis. The mechanism of action may be through programmed T cell death; a profound increase of the rate of apoptosis was seen in inflammatory foci of peripheral nerves and in the spleen. There was no cytokine switch by our Th1 cells after exposure to their specific antigen, but increased secretion of interferon gamma and tumor necrosis factor alpha was demonstrated. High antigen dose therapy using recombinant, pathogen-free protein may prove useful for the treatment of autoimmune inflammatory disorders of the nervous system.

Rapid method based on reversed-phase high-performance liquid chromatography for purification of human myelin basic protein and its thrombic and endoproteinase Lys-C peptides.

Reversed-phase high-performance liquid chromatography was applied to isolate myelin basic protein from human brain, followed by separation of proteolytic peptides thereof on the same chromatographic system. Brain tissue was delipidated under conditions that keep copurifying proteases inactive. The crude brain protein fraction was applied directly to a C4 column. The homogeneous protein obtained in this way was digested with thrombin and endoproteinase Lys-C in order to produce short defined myelin basic protein peptides. The purified peptides were used to determine the antigen fine specificity of myelin basic protein recognizing T lymphocyte lines isolated from multiple sclerosis patients.

Restricted expression of CD2 among subsets of sheep thymocytes and T lymphocytes.

A monoclonal antibody (mAb) generated against sheep T-cell blasts, called I/35 A, blocks sheep autologous E rosetting and competes with purified T11 target structure (TS), the sheep form of LFA3, for binding sites on the sheep T-cell surface. Immunoprecipitation from lysates of surface iodinated sheep T cells identifies the cell surface molecule recognized by mAb I/35 A as a single chain polypeptide migrating as a diffuse band of MW 55,000. From its binding properties and the biochemical nature of the target antigen, we conclude that mAb I/35 A is directed at sheep CD2. This finding makes sheep the first animal model in which the CD2-LFA3 (T11TS) system is defined by mAbs to both receptor and ligand. When analysed by two-colour flow cytometry and by immunohistochemistry, the cellular expression of CD2 in sheep differs significantly to that reported in humans. In peripheral blood, CD2 is found exclusively on CD4+8- and CD4-8+ T cells, while the third, CD4-8- (predominantly SBU-T19+) subset of sheep T cells (around 20% in peripheral blood) is CD2-. In thymus, only low to moderate levels of CD2 expression occurs on 80% of cells. Among these, medullary 'single positive' thymocytes express the highest level of CD2, whereas the CD4-8- 'double negative' population (which in contrast to peripheral CD4-8- T cells contains only very few SBU-T19+ cells) consists of CD2- and weakly positive cells. In peripheral lymphoid organs, CD2+ lymphocytes occur in the T-cell regions of spleen, lymph nodes and jejunal Peyer's patches (JPP). Tissue macrophages found in B-cell follicles of lymph nodes and JPP are also CD2+. The implications of these findings are discussed in terms of the role CD2 plays in the proliferation of immature thymocytes and of the possible importance of CD2/LFA3 interactions in lymphocyte recirculation.

Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques.

BACKGROUND: A number of enzymatic techniques have recently been developed to detect DNA fragmentation in apoptosis at the cellular level. However, since DNA fragmentation also occurs in cellular necrosis, we studied to which extent the use of DNA polymerase (nick translation) or terminal transferase (tailing) allows the differentiation between internucleosomal DNA degradation, typical for apoptosis, and the more random DNA destruction in necrosis. EXPERIMENTAL DESIGN: We compared these techniques on in vitro and in vivo models for apoptotic or necrotic cell death. Apoptosis of thymocytes in vitro was induced by gamma-irradiation, necrosis by the cytotoxic action of antibody and complement. Cell death in vivo was examined on paraffin-embedded tissue material from animals with autoimmune encephalomyelitis that served as a model for apoptosis, or in kainic acid-induced nerve cell degeneration as a model for necrosis. RESULTS: DNA fragmentation was visualized by the incorporation of labeled nucleotides into the nuclei of affected cells utilizing tailing or nick translation techniques. In the early stages of cell degeneration in vitro, cells undergoing apoptosis were preferentially labeled by tailing, whereas necrotic cells were identified by nick translation. Similarly, early stages of necrosis in vivo were preferentially detected by nick translation, whereas tailing was slightly more sensitive for the detection of apoptosis. Results obtained with these enzymatic techniques were in accord with the assessment of cell death by morphologic criteria. Both techniques could be applied in tissue samples even after prolonged fixation in paraformaldehyde if the sections were pretreated with proteinase K digestion. CONCLUSIONS: Our studies show that both in situ nick translation and in situ tailing are useful in detecting DNA fragmentation in cell suspensions and tissue sections. These techniques may help to define the molecular mechanisms leading to cell death in experimental conditions and eventually in human tissue.

Usage of Vbeta3.3 T-cell receptor by myelin basic protein-specific encephalitogenic T-cell lines in the Lewis rat.

T-cell receptor (TCR) beta-chain usage in experimental autoimmune encephalomyelitis (EAE) seems to be much more heterogeneous than previously assumed even for a single autoantigen in an inbred animal strain. Owing to the lack of suitable antibodies, this has been demonstrated only at the RNA level so far. To characterize further the TCR elements used in the Lewis rat for the recognition of the main encephalitogenic region of myelin basic protein (MBP), BALB/c mice were immunized with T-cell hybridomas expressing non-Vbeta8.2 TCR specific for guinea pig MBP peptide aa 68-88. Two B-cell hybridomas (clones C-A11 and F-D6) producing TCR Vbeta3.3-specific monoclonal antibodies were selected. Specificity was demonstrated by RT-PCR and cloning of PCR products obtained from sorted T-cell lines and naive T cells. MBP-specific Vbeta3.3 T cells used the L-S motif in the VDJ region, were associated with Valpha2 or Valpha8 chains, and specifically recognized MBP peptide aa 68-88. Vbeta3.3 TCR-positive T cells were detected in all of a panel of six MBP-specific T-cell lines, although to a lesser degree than Vbeta8.2 TCR-positive T cells. After intravenous injection of sorted Vbeta3.3 T cells, animals developed EAE, and Vbeta3.3-positive cells were found by immunocytochemical analyses in the spinal cord. Furthermore, treatment of EAE induced by immunization with MBP was more effective when a combination of anti-Vbeta3.3 and anti-Vbeta8.2 mAbs was used. These results confirm the functional role of TCR Vbeta3.3 and thus underscore the heterogeneity of TCR usage in MBP-associated autoimmunity.

Heterogeneity of T-cell receptor usage in experimental autoimmune neuritis in the Lewis rat.

In experimental autoimmune neuritis (EAN), T-cell receptor (TCR) variable (V)-region gene usage by neuritogenic T cells has been reported to be clonally restricted at the RNA level. This study was designed to verify TCR usage by neuritogenic T cells at the protein level. We generated two monoclonal antibodies (mAbs) 7H4 and 8G8 specific for a Vbeta4/Valpha11 associated idiotype expressed by the majority of neuritogenic cells of P2-specific T-cell lines. The remaining neuritogenic P2-specific T cells either exhibited a dominant usage of the TCR Vbeta13 chain recognized by the recently generated mAbs 17D5 and 18B1 or showed diverse Vbeta usage. Treatment of adoptive-transfer (AT)-EAN or of EAN actively induced with the neuritogenic P2 peptide by mAbs 7H4 and 8G8 led to a partial, but significant, reduction of clinical disease. Treatment with Vbeta13-specific mAb 17D5 had no clear effect on active EAN. Our data show that at least three different TCR are used by P2-specific pathogenic T cells in EAN, an animal model for human inflammatory neuropathies.