Organization, sequence and expression of the HLA-B27 gene: a molecular approach to analyze HLA and disease associations.

Among the numerous autoimmune diseases associated with various HLA alleles, the one with the highest relative risk so far reported has been ankylosing spondylitis with HLA-B27. To examine this relationship more directly, we have cloned the gene encoding the HLA-B27 antigen and determined its complete DNA sequence. Comparison of the HLA-B27 sequence with that of the allelic HLA-B27 shows a high level of homology. Mutations are distributed evenly between exons and introns. Exon 1 and intron 1 are the most divergent ones, and the degree of divergence distinctly declines towards the 3' end. The HLA-B57 gene when transfected into murine L cells is expressed on the cell surface and reacts with a panel of monoclonal antibodies directed against monomorphic and polymorphic determinants associated with HLA-B27 antigen. The isolation of this gene allows for the first time a search for structural features which make the HLA-B27 antigen a high risk genetic factor for a group of rheumatoid disorders, in particular ankylosing spondylitis.

Vaccination of class I major histocompatibility complex (MHC)-restricted murine CD8+ cytotoxic T lymphocytes towards soluble antigens: immunostimulating-ovalbumin complexes enter the class I MHC-restricted antigen pathway and allow sensitization against the immunodominant peptide.

In vivo induction of anti-ovalbumin (OVA) cytotoxic T cell responses was brought about in an MHC class I-restricted fashion by immunizing H-2b mice with OVA in immunostimulating complexes (ISCOM). ISCOM formation with the hydrophilic soluble protein OVA was achieved upon unmasking hydrophobic protein domains by treatment at low pH values. The effector cells induced were MHC restricted, specific for the immunodominant peptide of OVA (258-276), and expressed the CD8+ CD4- phenotype. These results suggest that ISCOM-based vaccines may be useful to direct hydrophilic soluble antigens into the MHC class I presentation pathway in order to vaccinate CD8+ T lymphocytes.

Distinct recognition sites on histocompatibility antigens for antibodies and cytotoxic T lymphocytes.

Studies with monoclonal antibodies show that allodeterminants are concentrated on H-2 molecules in several distinct epitope regions. Different parts of H-2 molecules can also be recognized by alloreactive or H-2 restricted cytotoxic T lymphocytes, but these parts do not appear to be identical to those recognized by antibodies. These data show that an H-2 molecule can be subdivided into several specialized regions and that the B and T cell compartments of the immune system recognize different regions on H-2 molecules.

Alloreactive cytolytic T-cell clones preferentially recognize conformational determinants on histocompatibility antigens: analysis with genetically engineered hybrid antigens.

Hybrid genes were constructed for the localization of allodeterminants on murine class I antigens recognized by antibodies and cytolytic T lymphocytes. By using deletion subclones of the H-2Kd and H-2Kk genes, homologous regions were exchanged between the two alleles. The altered genes were introduced and expressed in mouse fibroblast and fibrosarcoma cells. Cells expressing hybrid antigens were analyzed with 29 monoclonal anti-H-2Kd and anti-H-2Kk antibodies and with 150 short-term alloreactive cytolytic T-cell clones. When only the first or only the second amino-terminal domain was exchanged, most T cells and 60% of the antibodies lost their reactivity to the H-2K antigen. No T-cell clone was directed against the third extracellular domain, whereas three antibodies could bind to this domain. This implies that nearly all determinants essential for a cytolytic T-cell response or for antibody binding lie on the two external domains and are conformational structures generated by the interaction of these two domains.

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles.

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allele-specific residue: arginine at position 81 located on the alpha 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central beta sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the alpha 1 helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and -Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the alpha 1 and alpha 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.

Expression and immunogenicity of HLA-B27 in high-transfection recipient P815: a new method to induce monoclonal antibodies directed against HLA-B27.

The immunization of a (BALB/c x C57BL/6) FI mouse with murine transfectants expressing the HLA-B27 antigen resulted in a panel of polymorphic monoclonal antibodies with specificity for HLA-B27 and some additional HLA-antigens. Specificity of the antibodies was defined by cytofluorometric analysis on a panel of lymphoblastoid cell lines (LCL) derived from HLA typed individuals. Three of these antibodies are cytotoxic, and one of them inhibits B27-specific T cell cytotoxicity. Our results indicate that HLA-class I transfectants could be used to generate polymorphic antibodies, and that these antibodies may be helpful for HLA typing and for definition of epitopes recognized by T cells.

Molecular biology of the HLA-B27 locus.

The molecular biology of the HLA-B27 locus is reviewed. The HLA-B27 gene itself does not differ between healthy individuals and ankylosing spondylitis patients. Several unique features of the HLA-B27 molecule have been identified and one epitope was proposed to cross-react with bacterial proteins.

Recognition of chlamydial antigen by HLA-B27-restricted cytotoxic T cells in HLA-B*2705 transgenic CBA (H-2k) mice.

OBJECTIVE: The association of reactive arthritis (ReA) with HLA-B27 and the presence of bacterial antigen in joints with ReA suggest that bacterial peptides might be presented by the HLA-B27 molecule and thus stimulate CD8 T cells. This study was performed to investigate the B27-restricted cytotoxic T lymphocyte (CTL) response to Chlamydia trachomatis, using the model of HLA-B27 transgenic mice. METHODS: CBA (H-2k) mice homozygous for HLA-B*2705 and human beta2-microglobulin expression were immunized with C trachomatis or with the chlamydial 57-kd heat-shock protein (hsp57) coupled to latex beads. Cytotoxicity of lymphocytes from in vivo-primed transgenic mice was tested against C trachomatis-infected targets. Blocking experiments were performed with monoclonal antibodies (MAb) against class I major histocompatibility complex molecules. RESULTS: A Chlamydia-specific lysis of both B27-transfected and nontransfected target cells was observed. This response could be inhibited by anti-B27 and anti-H2 MAb. CTL from mice immunized with hsp57 were not able to lyse Chlamydia-infected target cells, and Chlamydia-specific CTL could not destroy targets loaded with hsp57. CONCLUSION: These results suggest the existence of at least 2 CTL populations in this mouse model: one recognizing peptide of bacteria-infected cells restricted by HLA-B*2705 and the other recognizing peptide of bacteria-infected cells restricted by the murine H-2Kk molecule. It does not appear that hsp57 is a major target for the CD8 T cell response directed against Chlamydia. This animal model opens the way for identifying bacterial epitopes presented by HLA-B27, and might thus help to clarify the pathogenesis of B27-associated diseases.

Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases.

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.