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Odorant receptors (OR) are G-protein-coupled receptors that are predominantly expressed in the membrane of olfactory neurons. Members of the two OR gene clusters on the short arm of human chromosome 6 could be involved in major histocompatibility complex (MHC)-associated behavioral traits, such as olfaction-influenced mate selection and cryptic female choice. In this context, OR gene polymorphisms and haplotypes are likely to play an important role. Here we report an investigation of polymorphisms within 12 MHC-linked OR genes in 10 human cell lines. Eight of these OR loci belong to the telomeric, smaller OR gene cluster, whereas four are located centromeric, between the first cluster and the MHC. We also assessed part of this genomic region using sequence data from eight additional cell lines that had previously been sequenced. Thirteen novel OR variants were found through direct DNA sequencing and cloning, in addition to the detection of OR polymorphisms already known, and the number of OR cluster haplotypes could be increased to 21. Two loci belonging to the telomeric cluster (OR2B8P and OR1F12) were found to exhibit nonfunctional and potentially functional alleles and should therefore be considered as segregating pseudogenes. The results provide a detailed picture regarding polymorphisms and phenotypic variation in an ethnically diverse sample of major histocompatibility complex-linked OR clusters and identify a subregion of unusually pronounced genetic variability. We expand these data by analyzing linkage disequilibrium both within these OR clusters as well as between them and the HLA complex in 11 unrelated HapMap populations. The sequence data described in this paper have been submitted to the GenBank database under the accession numbers GU251059, GU251060, GU251061, GU251062, GU251063, GU251064, GU251065, GU251066, GU251067, GU251068, GU251069, GU251070, GU251071, and GU251072.

The hundreds of human olfactory receptor (OR) genes are organized into clusters occurring on nearly every chromosome. Although their sequences are not always closely related, they share stretches of considerable similarity, both at the amino acid and nucleotide levels. We demonstrate here that an HLA complex-linked OR sequence, FAT11, for which recently a number of alleles have been claimed within the Hutterites, contains sequences derived from two closely related, linked OR genes, hs6M1-12 and hs6M1-16. Instead of indicating a difference between alleles of a given locus, two of the polymorphisms described for FAT11 (at amino acids 48 and 220 of the deduced protein sequence, respectively) may in fact reflect distinct sequences of hs6M1-12 and a further, closely related HLA-linked OR locus, hs6M1-13P. As a consequence, recombination rates in Hutterites in the region telomeric of HLA-G may have to be reconsidered.

The formation of a trimeric complex, composed of heavy chain (HC), beta(2)-microglobulin (beta(2)m) and antigenic peptide, is generally believed to be a prerequisite for the expression of HLA class I molecules at the cell surface in vivo. Therefore, a possible role in immunological processes for HC/beta(2)m complexes devoid of peptide has not been seriously considered. Using a novel HLA-B*2705-transgenic rat model and monoclonal antibodies that distinguish between structurally different forms of HLA-B27 molecules, we demonstrate here that class I molecules which appear to lack antigenic peptides are expressed in abundance on a variety of cell types in lymphoid organs. These results imply a role for structurally diverse, possibly empty, MHC molecules in physiological T cell selection which has so far not been sufficiently appreciated.

Strong peptide dependency of HLA-B∗3501-specific alloreactive T-cell clones was observed in the recognition of cells bearing closely related B35 variants. The single amino acid exchange in the s-pleated sheet of B∗3503 completely abolished the responses of all clones, whereas an amino acid exchange in the α2 helix of the newest B35 member (B∗3508) only altered allorecognition of one T-cell clone, demonstrating the differential impact of these positions on peptide binding to B35 molecules. In contrast to T cells, amAb (TU165) recognizing the B35 specificity in a peptide-dependent manner bound to the B35 variants irrespective of their sequence heterogeneity. However, quantitative binding differences were detected with cells bearing the same B35 alleles. This is most likely due to variations in the amount of peptide(s) that associates with B35 and forms the ligand seen by this mAb. These results reveal how naturally occurring single amino acid substitutions have led to generation of functionally distinct molecules of another multimember HLA class I cluster.

Aim β 2-microglobulin ( β 2m) is a small, monomorphic protein non-covalently bound to the heavy chain (HC) in polymorphic major histocompatibility complex (MHC) class I molecules. Given the high evolutionary conservation of structural features of β 2m in various MHC complexes as shown by X-ray crystallography, β 2m is often considered as a mere scaffolding protein. We investigate here whether β 2m residues at the interface to the HC exhibit changes depending on HC polymorphisms and the peptides bound to the complex in solution. Methods We employ High-Cell-Density Fermentation (HCDF) to obtain deuterated β 2m and Nuclear Magnetic Resonance (NMR) spectroscopy to examine the β 2m-HC interface. Results Following complexation of β 2m, the HLA-B*27:09 HC, and a peptide, the NMR resonance assignments are used to examine the β 2m-HC interface. We then compare the resonances of β 2m in two minimally distinct subtypes, HLA-B*27:09 and HLA-B*27:05, that are differentially associated with the spondyloarthropathy Ankylosing Spondylitis. Each of these subtypes is complexed with three self-peptides (TIS, pVIPR, pGR) and a viral peptide (pLMP2) for which structural information is already available. The resonance of β 2m-Trp95 does not show any variation in chemical shift, thus serving as an ideal internal control. However, there are distinct resonance signals for β 2m-Trp60 in each of the complexes. Conclusions As these signals are not only distinguishable for a given HLA-B27 subtype, but are also in characteristic positions within the spectra for each of the four peptides employed here, this indicates the existence of an unexpected plasticity that enables β 2m to accommodate changes depending on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

Aim The expression of major histocompatibility complex (MHC, in humans HLA) antigens is widely regarded as a property of all nucleated cells. These molecules are expressed very strongly by cells of lymphatic tissues, while many cells in other tissues, (e.g. muscle cells, neurons) express only small amounts of these antigens. The functional consequences of this differential expression and the molecular control mechanisms are not well understood. Immunologically privileged tissues (e.g. brain, seminiferous tubules within the testis) are separated from circulating immune effector cells by blood–tissue barriers. These barriers can be surmounted only under pathological conditions. Therefore, since cells that bear the classical interaction partners of MHC-I such as T cell receptors are usually lacking, it would appear that MHC-I expression by cells within immunologically privileged sites serves no immunologically meaningful function. The purpose of the present study was to investigate the expression of structurally distinct forms of HLA class I molecules in testicular tissue. Methods Immunohistochemistry, Confocal laser scanning microscopy, Western blotting, in situ hybridization. Results Immunohistological analyses using monoclonal antibodies (mAb) revealed reactivity within seminiferous tubules of the testis exclusively with mAb directed against free HLA class I heavy chains (HC), while reagents against β 2-m and HC/ β 2-m complexes stained only cells in the interstitium. The former mAb reacted with sperm precursor cells, mainly preleptotene spermatocytes and pachytene spermatocytes I, but not spermatogonia or more mature germ cells including spermatozoa. Sertoli cells were also found to be unreactive. Locus-specific mAb such as HC10 or LA45 indicate tubular expression of highly polymorphic HC. Conclusions Our results suggest a function for polymorphic HLA class I HC, possibly in the context of an interaction with sperm-expressed guidance receptors within immunologically privileged sites of the testis.

The influence of the TAP complex on T-cell allorecognition of MHC class II molecules was examined using human B-cell lines that have mutations in the TAP 1 or 2 genes. The TAP mutations led to the loss of allorecognition for two of 28 anti- HLA-DR T-cell clones. Restoration of TAP expression by transfection of a TAP 2 cDNA clone led to recovery of the alloresponse for both clones. These results could be explained in two ways. First, TAP dependence could reflect specificity for a peptide derived from an MHC class I molecule that is less efficiently generated by the endocytic pathway in the TAP-deficient stimulator cells owing to reduction in surface class I expression. The proliferative responses of these clones to the TAP-deficient stimulator cells was not restored by rescue of cell-surface expression of class I molecules by low temperature culture or by the addition of class I-binding peptides. These data therefore favor the alternative explanation that class II loading by some peptides is TAP dependent. Circumstances that lead to the amplification of this minority pathway of endogenous presentation by class II MHC molecules may have the potential to interrupt self-tolerance.

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (± weak DC) (22 MoAbs); Dr + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tu109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tu109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tu109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tu109 ascites fluid, however, extra-reactivity on all HLA-Bw6+ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6+ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tu109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.

A complement-fixing murine monoclonal IgM antibody (TU 101) strictly directed against a supertypic determinant present on the HLA-A locus antigens A2 and A28 was defined from a fusion experiment employing T cell blasts as immunizing cells. The specificity of this antibody was established in the microcytoxicity assay on 91 normal Caucasian blood donors, as well as in the SpA-Ig assay on a panel of lympho- and hematopoietic cell lines. TU 101 segregates only with its defined HLA allotypes in families. This reagent may be of particular value as a probe for analyzing a molecular relationship of different antigenic determinants on the HLA-A2 and A28 specificities in comparison with two recently defined anti-HLA-A2/A28 monoclonal antibodies and may help to characterize structural variations of these HLA-molecules on a serological and immunochemical basis.

A murine monoclonal antibody (TU 110) prepared against blast cells of a patient with acute "undifferentiated" leukemia was tested in the microcytotoxicity assay on peripheral blood lymphocytes of 122 normal Caucasian donors. The TU 110 reactivity was found to show a correlation coefficient of 1.0 in population analysis for the presence of the HLA-B locus specificity B 13 as defined by alloantisera. Family segregation studies confirmed MHC linked inheritance of the TU 110 antigenic determinant strictly on HLA-B 13 positive haplotypes. As the first monoclonal reagent against the private specificity of this HLA-B locus antigen, TU 110 provides the possibility to study the structural relationships of sub- and supertypic determinants on this allotype and may help to correlate antigenic domains of HLA-B 13 with definable functional properties.