Epitope map of the HLA-B7 CREG using affinity-purified human alloantibody probes.

Monoclonal antibodies (mAb) recognizing the B7 CREG have been used to construct an epitopic map of HLA-B7. Similar studies with human HLA alloantisera have been lacking due to the polyclonal nature of the alloantibodies (aAb). Detergent-solubilized HLA Class I antigens were purified and coupled to activated CH-Sepharose 4B. Sequential affinity isolation of aAb populations using a series of HLA antigen columns enabled us to produce a battery of aAb eluates against both the private B7, B13, B27, and B omega 60 determinants and the public B7-42, B7-60, B7-60-61, B7-27-13-60, B7-42-22-27, B7-8-42-60-41, and B omega 6 epitopes. The topographic relationship of the B7 family of determinants recognized by the Ab probes was derived using crosscompetition Ab blocking assays with quantitation by indirect immunofluorescence and FACS analysis. We have found that aAb and mAb of similar specificity crossblock; Ab of different specificity give complex patterns including both overlapping blocking between the alpha domains and Ab-induced conformational change of the molecule. From these investigations, we conclude that HLA Class I alloantigens bear both multiple, topographically distinct public epitopes and separate private determinants that can be distinguished using human aAb probes. At least four discrete epitopes are expressed by each molecule of the HLA-B7 CREG and can be ascribed to unique aa substitutions on the hydrophilic beta loops of the distal heavy chain domains and also on several exposed areas of the alpha helices. These findings are extremely similar to those of the HLA-A2 CREG and suggest that possibly all Class I molecules possess a comparable, complex degree of serologic polymorphism.     

Antigenicity of HLA-A2 and HLA-B7. Loss and gain of serologic determinants induced by site-specific mutagenesis at residues 62 to 80.

The contribution of the hypervariable region spanning amino acid residues 62 to 80 to the serologic determinants of HLA-A2 and HLA-B7 has been examined by site-directed mutagenesis. Three HLA-A2 mutants, having changes as in HLA-B7 at positions 62, 76, and at the complete 65-to-80 segment, respectively, were obtained and expressed on class I HLA-deficient human cells upon transfection. The reactivity of 19 monoclonal antibodies (mAbs) against both broad public and allospecific determinants on HLA-A2 and HLA-B7 was analyzed. The results indicate that: (1) the change at residue 62 abrogated recognition of the corresponding HLA-A2 mutant by mAb MA2.1 (anti-A2 + B17); (2) the change at residue 76 did not effect any of the determinants analyzed, although its side chain is easily accessible at the surface of the molecule; (3) the replacement of the whole 65-to-80 segment in HLA-A2 by that from HLA-B7 abrogated recognition by MA2.1 and by 108-2C5, a mAb recognizing a public determinant from the HLA-A locus. Such replacement led to gaining the determinants recognized by mAbs GS145.2 (anti-B7 + B27) and SFR8-B6 (anti-Bw6); and (4) the HLA-A2-reactive mAbs whose reactivity was known to be abrogated by changes in alpha 2 were unaffected by the changes introduced in alpha 1, underlining the frequent segregation of serologic determinants on class I antigens to single domains.     

A limited number of HLA epitopes are recognized from HLA class I-specific antibodies detected in the serum of sensitized patients

The goal of this study was to evaluate the epitope specificity of HLA class I-specific antibodies detected in the serum of sensitized patients awaiting retransplantation. The study group consisted of 22 sensitized from previous graft patients, who produced stable IgG HLA class I-specific antibodies. A total of 60 serum samples were screened and analyzed by two techniques in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. All recipients and donors were typed for class I HLA antigens by a standard lymphocytotoxicity technique. The epitope identification was based on class I HLA antigens sequencing, where the multiple immunogenic epitopes are differentially shared among various HLA antigens. The unique epitope configuration on one HLA antigen represents the private epitope of the specific HLA antigen while epitopes shared by more than one HLA antigen represent public determinants. In some HLA antigens (HLA-A1), more than one private epitope has been defined, while in others (HLA-B35, -B51), the private epitopes are not yet known. In a total of 36 antibody reactivity patterns, the majority of the definable IgG HLA class I-specific antibodies corresponded to the A-locus (75%), and only 25% had specificities against the B-locus antigens, although the number of incompatibilities concerning both loci were almost identical (29 for the HLA antigens of the A-locus and 26 for those of B-locus). All patients produced HLA class I-specific antibodies with specificities against the private epitopes of the immunogenic mismatched HLA antigen(s). In 6/21 cases (28.6%), HLA class I alloreactivity spreading to nongraft HLA antigens was detected and 9 public (shared) immunogenic alloepitopes were recognized. In conclusion, appling the epitope analysis of HLA class I-specific antibodies produced by sensitized from previous graft patients, we were able to define the immunogenic alloepitopes. We consider that the immunogenic alloepitopes, during transplantation course, are mainly private epitopes of mismatched HLA antigens and, in certain cases, shared epitopes between the donor alloantigens and other HLA antigens. This knowledge may offer the potential of transplanting sensitized patients through improved donor selection.     

Specificity and functional characteristics of anti-HLA-A mAbs LGIII-147.4.1 and LGIII-220.6.2

Only three anti-HLA-A monoclonal antibodies (mAbs) have been described in the literature. Two of them recognize determinants shared by only a few HLA-A allospecificities. The third one, mAb 3G11, recognizes a determinant shared by most HLA-A allospecificities. Being an IgM, the latter mAb is not likely to be a useful probe in immunohistochemical reactions and in functional assays. Therefore, in the present study we have characterized the specificity of the mAbs LGIII-147.4.1 and LGIII-220.6.2. The two mAbs that do not share idiotypic determinants recognize distinct but spatially close antigenic determinants expressed on most of the gene products of the HLA-A locus. Specifically, the determinant recognized by mAb LGIII-220.6.2 is expressed on HLA-A1, -A2, -A3, -A26, -A28, -A29, -A30, -A33, -A36, -A74 and -A80 allospecificities. The determinant recognized by mAb LGIII-147.4.1, which appears to be located on the amino-acid residues 79-83 of the heavy chain, is expressed on all HLA-A allospecificities but HLA-A23, -A24, -A25 and -A32. Because of its broad reactivity, the mAb LGIII-147.4.1 was characterized in a number of assays. It was found to be a useful probe to measure the HLA-A antigen level in serum, to assess the HLA-A restriction of cytotoxic T lymphocytes (CTL) and to monitor HLA-A antigen expression in normal and malignant lesions.     

HLA Alloantibodies and the Mechanism of the Antiglobulin-Augmented Lymphocytotoxicity Procedure

ABSTRACT: HLA Class I alloantigens express multiple epitopes which can be defined serologically using human HLA alloantibodies (aAb). We have shown that the vast majority of HLA antisera exhibit the CYNAP phenomenon (complement-dependent cytotoxicity (CDC) negative, adsorption positive) which can be identified by conversion to direct CDC positive reactivity with the addition of an antihuman immunoglobulin (Ig) light chain (AHG) reagent.

In this study, the immunochemical mechanisms responsible for the CYNAP phenomena and how AHG overrides CYNAP have been further characterized using affinity-purified HLA aAb, class-specific anti-IgH reagents and human C1q binding assays quantified by flow cytometry. We have found that CYNAP reactions are not the result of low affinity aAb or generally caused by non-complement fixing HLA aAb. Our experiments illustrate that only anti-human IgL AHG reagents can consistently augment CDC and override CYNAP; anti-IgH have not effective. Two noncompeting HLA aAb of different epitopic specificity or one aAb in conjunction with the AHG-augmenting reagent results in striking synergy with a 200 to 400% increase in binding of C1q.

We conclude from these and other experiments detailed in this article that an IgM aAb or either two adjacent, noncompeting IgG HLA aAb bound to spatially distinct epitopes on a single HLA molecule or a monospecific IgG HLA aAb in concert with the AHG binding to this HLA aAb, is required for efficient (bivalent) C1q binding and initiation of C-mediated lympholysis. In contrast, the CYNAP phenomenon usually occurs because monospecific HLA aAb directed against a single epitope cannot effect high affinity, bivalent interaction with C1q and activate complement that would ultimately lead to cytolysis.     

HLA class I association with progression to end-stage renal disease in patients from Zulia,Venezuela

The aim of this study was to determine HLA associations with progression to end-stage renal disease (ESRD) in the mixed Zulian population in Venezuela, regardless of other factors. A retrospective study to determine HLA Class I association was performed on 188 patients with ESRD due to different types of glomerulonephritis, and 202 healthy controls. Patients and control groups were serologically typed by Terasaki microlymphocytotoxicity technique using commercial Class I plates including 26 HLA-A and 48 HLA-B specificities. The antigens positively associated to the ESRD were: HLA-B38, B51, B53 and B62. Negatively associated antigens were: HLA-A9, B12, B17, B40 and B48. The haplotypes positively associated were: HLA-A2-B51, A2-B53, A23-B38 and A68-B38. The negatively associated haplotypes were: HLA-A2-B12, A2-B48, A9-B35 and A28-B40. The high Odds ratio observed and its statistical corroboration reflect the strength of the described association between HLA antigens and ESRD. Further molecular studies should clarify types and subtypes of the HLA class I alleles involved in the progression to ESRD.     

HLA antigens and Graves' disease in Black South Africans

This study concerns the frequencies with which 36 HLA-A, -B and -C antigens occurred in 84 Black Africans with Graves' disease and in 311 Black controls. In the hyperthyroid patients significant reductions were found in the frequencies of HLA-B7 ( P <0.001, relative risk (RR) 0.33), HLA-Bw42 ( P <0.001, RR 0.32) and the HLA-B7-Bw42 crossreactive group (CREG) ( P <0.0001, RR 0.27), and in the frequencies of the phenotypic combinations HLA-A1, B7 ( P <0.001) and Aw30, B7-Bw42 ( P <0.001). HLA-B8 was increased in frequency ( P <0.01, RR 2.84). In patients without circulating antithyroglobulin or antimitichondrial antibodies the frequencies of HLA-A2 and B17 were increased when compared to those with antibodies or to the controls. In patients with and without clinically evident infiltrative ophthalmopathy the frequencies of HLA antigens were similar. In 62 Caucasian patients with Graves' disease, no antigens or phenotypic combinations occurred with increased or decreased frequency when compared to 278 controls.
Analysis of the frequencies of 9 HLA antigens and phenotypic combinations common in Caucasians but rare in Blacks revealed that only two antigens (A2 and B8) occurred with increased frequency in Black patients, suggesting that a contribution of Caucasian genes to the Black thyrotoxic subjects was unlikely.
Similarly, only one common Black antigen (A28) of 8 common antigens and phenotypic combinations, occurred in Caucasian patients with a frequency similar to that of Black controls. Thus it is unlikely that Black genes contributed to the lack of a significant increase of HLA antigens in the Caucasian thyrotoxic patients. The possession of HLA-B7-Bw42 CREG or related genes may be a protection against Graves' disease in Black Africans.     

RG1, a new murine monoclonal antibody recognizing a "supertypic" determinant on HLA-A molecules

Abstract: Monomorphic and polymorphic anti-HLA monoclonal antibodies (mAb) are valuable reagents for assessment of the structural and functional importance of different class I determinants. We have generated a new mAb, RG1, reacting with an epitope variably expressed on normal and leukemic hematopoietic cells of different lineages. Immunoprecipitation of the RG1 antigen disclosed a bimolecular complex characteristic of class I proteins. The RG1 epitope was expressed on an HLA-A2 transfected cell line but not on cells transfected with HLA-E, -F or -G molecules. MAb reactivity with reference B-lymphoblastoid cell lines and HLA typing of RG1 reactive and unreactive cells demonstrated that the epitope was expressed in conjunction with defined HLA-A molecules. Cells expressing HLA-A2, -A24(9) and -A68(28) proteins were brightly stained with RG1 whereas mAb binding to HLA-A1, -A11 and a split of A3 molecules was significantly lower. In contrast, the RG1 epitope was apparently not expressed on HLA-A23(9), -A25(10), -A26(10), -A29(19), -A30(19), -A31(19), -A32(19), -A33(19) and some HLA-A3 molecules. Based on class I α sequence data, these results suggest that the RG1 epitope is localized to a region of the α2 helix accessible to the T cell receptor for antigen on cytotoxic T lymphocytes. Lys in position 144 and His in position 151 are apparently critical for RG1 binding.     

A new murine lymphocytotoxic monoclonal antibody recognizing HLA-A2, -A28 and -A9

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immuno-biology of HLA molecules. We immunized BALB/c mice with a HLA-A2 transfectant and screened for hybridomas which reacted with a HLA-A2 trans-fectant but not with a HLA-B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1–145 was established. 1–145 reacted with Epstein-Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA-A2, -A28, -A23 and -A24. The titer of 1–145 in culture supernatant against HLA-A2 and -A28 antigens was similar and the titer against HLA-A23 was lower. 1–145 reacted with cells expressing HLA-A24 but the titer against HLA-A24 antigens was even lower than that againt HLA-A23 antigens. The HLA-A24 antigens on the peripheral blood lymphocytes were not detected by 1–145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxic-ity assay in which 1–145 culture supernatant lysed all PBLs expressing HLA-A2.-A28 and -A23 but did not lyse PBLs expressing HLA-A24. Published deduced amino acid sequence data of HLA class I molecules indicate that Lys in position 127 may be critical for 1–145 binding.     

Distinct epitopes are recognized by cytolytic T lymphocyte clones on the same class I molecule: direct demonstration using DNA-transfected targets and long-term cytolytic T cell clones

By producing long-term, stable, cytolytic T lymphocyte clones and utilizing targets expressing only a single gene product derived from the stimulator mouse strain, we have been able to directly demonstrate that T cells recognize distinct epitopes expressed on a single H-2 molecule. These multiple determinants are distinguishable by inhibition patterns with monoclonal antibodies (mAb). When two T cell clones, P-2.14 and P-2.17, are tested on an L cell transfected with the Dp gene (lambda 12a), the T cells kill the transfected targets as well as blasts derived from Dp mouse strains. mAb 7-16.10 inhibits recognition and killing of Dp targets by both P-2.14 and P-2.17. This mAb recognizes a private specificity H-2.m22. Interestingly mAb 11-20.3 which also recognizes the H-2.m22 specificity inhibits clone P-2.14 but not P-2.17. The mAb 7-16.10, however, competes with 11-20.3 for binding to the surface of L cells expressing the Dp gene. Thus the two T cells must recognize an overlapping specificity. Other mAb which bind to the H-2Dp molecule are unable to inhibit either of these two cytolytic T cell clones. Paradoxically, any of the mAb when tested individually are sufficient to inhibit the polyclonal response derived from in vitro mixed lymphocyte culture. Therefore, by using targets expressing only a single H-2 molecule derived by DNA-mediated gene transfer and cytolytic T cell clones we have been able to directly demonstrate the presence of multiple epitopes on a single molecule and define their inhibition with mAb directed to that same molecule.     

Public epitopes and the antigenic structure of the HLA molecules

Simplified procedures for determining amino acid sequences in proteins and nucleotide sequences in DNA have rapidly expanded the number of MHC molecules for which primary amino acid structure is known. These molecules will be especially valuable as tools to study the structure-function relationships of globular proteins because of the extensive polymorphism of genes coding the MHC genes products. The general three-dimensional structure of class I MHC molecules was recently deduced, but the more subtle topographical microconformations are still undefined. Definition and topographical mapping of epitopes, defined by serological or cellular immune effector products, will be critical probes for these three-dimensional studies. Comparative studies of amino acid sequences among various MHC and molecules have revealed distinct regions of hypervariability in the alpha-1 and -2 domains of class I heavy chains and the alpha-1 and beta-1 domains of most class II molecules. Mutant MHC molecules that differ from each other by no more than one to three amino acids can have structural changes which may result in a loss of the private epitopes that defined the allelic gene product. On the basis of these studies, the private epitopes are thought to be determined by one or more of the hypervariable regions. Similar studies of the relationships between specific regions of the molecule and public epitopes are not fully explored. Because public epitopes are partially conserved structures, one might expect that their structure is not principally determined by hypervariable region. In fact, however, some public epitopes, such as A2/B17 and BW4/Bw6, do map to diversity regions. Epitope mapping as a means of identifying specific topographic sites and relating these sites to specific functional regions of the molecule will be difficult unless the epitopes themselves are better defined. Thus, the capacity to distinguish spatially distinct public epitopes from cross-reactive homologous private epitopes will be important if epitope-specific immunological probes are use to map specific regions of an MHC molecule. Many investigators are interested in the possibility that some components of HLA alloimmunization are regulated through idiotypic networks. Suciu-Foca and colleagues have provided preliminary evidence that epitope-specific HLA alloantibodies bear dominant idiotypic determinants. Antibodies to these determinants appear during pregnancy, following blood tranfusion, and following renal allograft transplantation, also, the antibodies have been correlated with renal allograft survival.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic predetermination of quantitative expression of HLA antigens in platelets and mononuclear leukocytes

In view of the potential functional importance of quantitative expression of HLA antigens, a series of studies were conducted to determine the relative quantities of specific HLA-A and -B antigens expressed in MNLs and platelets of HLA-phenotyped family members and unrelated individuals. An mAb that reacts with a well-defined monomorphic epitope in the α₃ domain of the heavy chains of HLA molecules was developed and used to quantify each HLA-A or -B antigen on western blots of IEF gels. The results of these studies demonstrated that the relative quantities of HLA-A and -B antigens in platelets and MNLs of an individual did not change over time. Further studies showed that the relative quantities of HLA-A and -B antigens for haplotypes shared among the first-degree relatives were always the same and followed Mendelian inheritance. In contrast, the relative quantities of HLA-A and -B antigens for a haplotype shared by unrelated individuals varied significantly. All these findings support the hypothesis that the quantitative expression of HLA antigens is genetically predetermined and may play important roles in determining disease susceptibility and severity. Human Immunology 38, 243–250 (1993)     

Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210^bcr-abl fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210^bcr-abl is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210^bcr-abl breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.     

HLA antigens in South India: II. Selected caste groups of Tamil Nadu

HLA-A, B antigen and haplotype frequencies were studied in four different caste groups of Tamil Nadu living in Madurai. A total number of 101 Nadars, 36 Kallars, 54 Iyers and 57 Telugu-speaking Naidus were studied. HLA A3 and B15 were significantly higher in Nadars; A10 & B8 in Kallars and Aw19, B12 & B35 in Iyers. HLA A-B haplotypes A10-B7, A28-B17 & A24-B- were characteristic of Nadars; A10-B8 & A1-B-, Kallars; Aw19-B12 & A1-B15, Iyers and A2-B-, Naidus. Negative linkage disequilibria for Aw19-B7, A28-B15 & A9-B51 were significant in Nadars; A1-B5, A1-B12 & Aw19-B- in Iyers and A2-B17 in Naidus. Heterogeneity chi-square based on antigen frequency and genetic distance also suggest the heterogeneous nature of the population of South India. Will these caste groups with such diverse haplotypic combinations differ from one another in their immune response and susceptibility to a given epidemic or infection?     

Detection of two distinct public specificities on HLA-A28 bearing lymphocytes

We have used the antiglobulin augmented cytotoxicity assay and platelet absorption-elution techniques to investigate crossreactivity among certain antigens coded by the HLA-A locus. These serologic and absorption studies demonstrated two supertypic determinants, distinct from private determinants, associated with HLA-A locus gene products. One determinant was present on HLA-A2, Aw23, Aw24, and A28 bearing lymphocytes; the other was found on HLA-A28, Aw33, Aw34, and A26 bearing lymphocytes. The second determinant was not associated with HLA-A25. Both supertypic determinants are associated with HLA-A28.     

Transfection of HLA genes using genomic DNA

Mouse L cells expressing HLA genes are potentially useful for producing and analyzing monoclonal antibodies (mAb) to HLA molecules. This paper describes the preparation of transfectants using uncloned human DNA and three methods to isolate the HLA-expressing transfectants. Transfectant libraries were made by contransfecting mouse thymidine kinase (tk)-deficient L cells with a calcium phosphate precipitate containing genomic DNA and tk plasmid DNA. Transfectants expressing HLA genes were isolated using these methods: immunomagnetism, replicate-plating combined with cellular enzyme-linked immunoassay, and sorting using a fluorescence-activated cell sorter. Two HLA-A2 transfectants were isolated using immunomagnetism, two HLA-A24 transfectants by replicate plating, and one HLA-Bw60 transfectant by FACS. However, no transfectants were isolated that stably expressed class II genes. The class I transfectants have been useful in characterizing several locally prepared mAbs which bind to monomorphic determinants on class I HLA molecules. Two of the transfectant lines, one expressing HLA-A2 (8001) and the other HLA-A24 (8008), have been included in the collection of lines distributed for use in the Eleventh International Histocompatibility Workshop.     

Evaluation of individual specificities of class I HLA on platelets by a newly developed monoclonal antibody

In order to quantify each specific HLA-A or-B antigen on platelets, a monoclonal antibody against HLA heavy chains was developed and designated as 2F2 monoclonal antibody. This monoclonal antibody reacted on Western blot with platelet HLA from each 10 individuals with different HLA phenotypes and precipitated all ³⁵S-methionine-labeled HLA-A and -B antigens from three different Epstein-Barr Virus-transformed lymphoblastoid cell lines. The results indicate that the 2F2 monoclonal antibody recognizes an epitope shared by different HLA-A and -B antigens. The quantitative variation of specific HLA antigens on platelets was then studied in nine different donors by isoelectric-focusing gel electrophoresis and immunoblot using the 2F2 monoclonal antibody. The results of our studies showed that the shared HLA antigens such as A2, B35, and B62, varied three- to fivefold among different individuals and individual HLA-A or -B antigen was not equally expressed on a person's platelets. The relative quantities of specific HLA-A and -B antigens on lymphocytes were also noted to be the same as those on platelets. The finding suggests that differential expression of HLA specificities may not be restricted to platelets but is a more general phenomenon including other nucleated cells.     

Human Coronavirus OC43 Interacts with Major Histocompatibility Complex Class I Molecules at the Cell Surface to Establish Infection

Human coronaviruses have been associated with common colds, diarrhea and enterocolitis, and have been implicated in multiple sclerosis. HLA class I molecules may play a critical role as receptor for OC43 because monoclonal antibody (mAb)W6/32 to HLA-A, -B and -C specificities completely blocks infectivity in human rhabdomyosarcoma (RD) cells. The role of HLA class 1 antigen as the virus receptor was examined using HLA-A3.1 stably transfected human plasma cells and untransfected HMY.C1R cells which do not express HLA-A and -B molecules. When the cells (5x10⁶) were infected at a multiplicity of one, the HLA.A3 transfected cells produced 10⁸ PFU of virus whereas no replication occurred in the HMY.C1R cells mAb W6/32 reduced the virus yield by 99.9% Cell membranes from HMY.C1R, HMY.A3 cells and chicken erythrocytes were biotinylated as live cells. Immunoprecipitation with polyclonal antiviral antibody to detect binding of biotinylated cell membranes to virus revealed that biotinylated HMY.A3 membranes co-precipitated with virus-antibody complexes when the immunoprecipitates were electrophoresed on SDS-PAGE gel, electroblotted and stained with Avidin-horseradish peroxidase. The results provide direct evidence that OC43 virus can recognize HLA class I as receptor on the cell surface.     

Selective monomorphic and polymorphic HLA class I antigenic determinant loss in surgically removed melanoma lesions

The analysis of human leukocyte antigen (HLA) class I allospecificity expression in malignant lesions has been hampered by the limited availability of HLA class I allospecificity-specific monoclonal antibodies (mAbs) which stain tissues in immunohistochemical (IHC) reactions. During the 12th International Histocompatibility Workshop, the HLA and cancer component made available a panel of mAbs capable of detecting monomorphic, locus- and allo-specific HLA class I antigenic determinants in surgically removed frozen tissue sections by IHC staining. In the present study, we have utilized this panel of mAbs to analyze the expression of HLA class I allospecificities in 33 primary and in 11 metastatic lesions surgically removed from HLA-typed patients with malignant melanoma, as this information contributes to determine the extent of HLA class I antigen abnormalities in melanoma lesions. HLA class I antigens were downregulated in six (18.2%) of the primary lesions and in six (54.5%) of the metastatic lesions. Selective loss of HLA-A and HLA-B antigens was detected in two (6.1%) and in one (3.0%), respectively, of the primary lesions, but in none of the metastases. HLA-A and HLA-B antigens were downregulated in three (9.1%) and four (36.4%) of the primary and metastatic lesions, respectively. Selective loss of one or more HLA class I allospecificities was found in 10 (33.0%) and two (18.0%) of the 33 primary and 11 metastatic melanoma lesions analyzed, respectively. HLA class I antigen abnormalities were present in 16 (48.5%) of the 33 primary lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, two lesions demonstrating selective abnormal reactivity with HLA-B locus-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A and HLA-B locus-specific mAbs, and seven lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). Furthermore, HLA class I antigen abnormalities were present in nine (81.8%) of the 11 metastatic lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A locus-specific mAb, and two lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). It cannot be ruled out that the frequency of HLA class I allospecificity abnormalities is higher, as the expression of several HLA class I allospecificities could not be investigated because of the lack of appropriate probes. The frequency of HLA class I antigen defects in primary lesions was significantly correlated with primary lesion thickness, an important prognostic marker in melanoma, arguing for a potential clinical significance of HLA class I antigen abnormalities in melanoma. In conclusion, the results of the present study (i) demonstrate that the frequency of HLA class I allospecificity abnormalities in primary melanoma lesions is markedly higher than that of total HLA class I antigen downregulation described in the literature; (ii) corroborate our previous findings that staining of melanoma lesions with mAb to monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss; and (iii) demonstrate for the first time selective loss of antigenic determinants expressed on HLA class I molecules in melanoma lesions. The latter finding indicates that at least two mAbs recognizing distinct antigenic determinants on the HLA molecule being investigated should be used for IHC staining of tissue sections in order to prove that lack of immunostaining reflects actual loss of the corresponding HLA molecule and not selective loss of antigenic determinants.