Rapid cloning of HLA class I cDNAs by locus specific PCR

The human major histocompatibility complex (MHC) class I loci, human leukocyte antigen (HLA)-A, -B, -C, encode highly polymorphic molecules that mediate immune recognition of infectious pathogens and can initiate the rapid rejection of transplanted tissue. Cloning of HLA class I alleles is complicated by polymorphism as well as interlocus homology. Here, HLA class I cDNAs are amplified by PCR using one common primer with one of three locus specific primers whose 3' ends map to conserved, locus specific nucleotides. Using these primers, HLA-A, -B, and -C alleles were cloned from a number of cell lines and two different HLA-B alleles were cloned from a single, heterozygous cell line. The amplified products encode the entire extracellular portion of the class I molecules. An amplified HLA-A allele was cloned into an expression vector and the protein product was detected on the surface of a transfected cell. A premature termination codon was engineered into the HLA-A allele by site directed mutagenesis and the soluble protein product was detected in the culture medium of transfected cells. Therefore, these primers can be used to rapidly clone, alter, and express HLA class I molecules. This method may expedite the generation of reagents for testing the antigen specificity of antibodies, natural killer cells, or T cells.     

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.     

Expression of HLA class I antigens in transporter associated with antigen processing (TAP)-deficient mutant cell lines

Abstract: Cells lacking expression of the transporter associated with antigen processing (TAP) are deficient in surface HLA class I, yet express reduced levels of HLA-A2 antigen through TAP-independent processing pathways. We have analysed the expression of HLA-A, -B and -C antigens on the 721.174 and T2 TAP-deficient mutant cell lines using a panel of monoclonal antibodies specific for the HLA antigens encoded by the genotype of these cells. Our study has shown the constitutive expression of HLA-Cwl molecules on the cell surface of both T2 and 721.174 cells and has confirmed that HLA-A2 and HLA-B51 are expressed at low levels. Transfection of 721.174 cells with cDNAs encoding TAP1 and TAP2 proteins did not fully restore HLA class I antigen expression on these cells, which appeared to be mainly due to a deficiency in expression of the HLA-B51-associ-ated Bw4 epitope. This suggests that additional antigen-processing genes may be required for optimal generation of HLA-B-binding peptides. Our results indicate that TAP-independent pathways of antigen-processing provide peptides for functional expression of all three classical HLA class I molecules.     

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.     

Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry

Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-μm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%–19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.     

Identification of p53 peptides recognized by CD8(+) T lymphocytes from patients with bladder cancer

In many types of cancer, p53 frequently accumulates in tumor cells and anti-p53 antibodies can be detected. However, only four CD8(+) T-cell epitopes from p53 have been identified in humans so far. To further analyze the development of a T-cell response against p53, peptides having binding motifs specific for HLA-A1, -A2, -A3, -A24, -B7, -B35, -B44, and -B51 molecules have been defined. The HLA-binding capacity of those peptides was tested, and the stability of formed complexes was defined. Thirteen peptides that bound to HLA-A24 and -B44 molecules are presented. The positive peptides were then used to detect the anti-p53 response of CD8(+) T lymphocytes from patients with bladder cancer. Six peptides, presented by HLA-A2, -B51, or -A24, were able to stimulate T cells from two patients (among 16) with tumor cells that strongly accumulated p53. On the contrary, p53 peptides systematically failed to stimulate T cells from healthy donors or patients with low or undetectable levels of p53 in their tumor cells. These results have led to the identification of four new potential T CD8(+) epitopes from p53: 194-203 associating with HLA-B51 and 204-212, 211-218, and 235-243 associating with HLA-A24.     

Influence of age and HLA type on interferon-gamma (IFN-gamma) responses to a naturally occurring polymorphic epitope of Plasmodium falciparum liver stage antigen-1 (LSA-1)

Antigenic polymorphism and HLA restriction may limit the immunogenicity of a subunit vaccine against liver-stage Plasmodium falciparum. We examined 59 clinical isolates and five laboratory clones of P. falciparum for polymorphism in the N- and C-terminal regions of LSA-1, evaluated binding of the corresponding peptides to selected HLA class I alleles, and measured IFN-gamma responses in residents of a malaria-endemic area of Papua New Guinea where HLA-A*1101, -24, -B13, and -B40 are the most common class I alleles. LSA-1 polymorphism was limited to a single non-synonymous mutation encoding serine (S), proline (P), or threonine (T) at amino acid 85. Nine-mer 84-92 peptides with S, T, or P at the primary anchor position bound differentially to HLA-A11, -A2, and -B7. IFN-gamma ELISPOT responses increased with age in malaria-exposed subjects: 14-16% and 30-36% of 2-5- and 6-54-year-olds, respectively, had > or =10 IFN-gamma-secreting cells/106 peripheral blood mononuclear cells when stimulated with at least one peptide variant (P<0.05). IFN-gamma responses to all three peptides were also greater for older than younger individuals. No children < 3 years old had lymphocytes that responded to all three 84-92 peptides, whereas 45% of adults (mean age 48 years) had aggregated IFN-gamma responses. These data support the notion that age-related cumulative exposure to P. falciparum increases the frequency of IFN-gamma responses to polymorphic epitopes of liver-stage antigens such as LSA-1.     

Allotyping for HLA class I using plasma as antigen source

Immunoadsorption of soluble HLA class I antigens onto immunobeads, one-dimensional iso-electric focusing of these proteins and subsequent immunoblotting allows a biochemical identification of HLA class I allotypes. The distinct protein bands can be clearly attributed to particular HLA antigens and are comparable to those observed after detergent solubilization of membrane-bound HLA antigens. Segregation analysis showed that the biochemically detected pattern of soluble class I gene products followed Mendelian inheritance. However, antigens such as HLA-A1, -A2, -B8, and -B51 were not always clearly detectable, a phenomenon attributable to either different plasma concentrations of these HLA antigens or variable affinity of the monoclonal antibody used to capture class I antigens. These results show that in principle allotyping of HLA class I using plasma as the antigen source is feasible, but with the limitation that some antigens may not be easily detected in some individuals.     

MHC-I-restricted epitopes conserved among variola and other related orthopoxviruses are recognized by T cells 30 years after vaccination

It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic T lymphocyte epitopes. Eight epitopes were confirmed to stimulate IFN-γ release by T cells in smallpox-vaccinated subjects. The epitopes were restricted by five supertypes (HLA-A1, -A2, -A24 -A26 and -B44). Significant T cell responses were detected against 8 of 45 peptides with an HLA class I affinity of K _D less than or equal to 5 nM, whereas no T cell responses were detected against 60 peptides with an HLA affinity of K _D more than 5 nM. All epitopes were fully conserved in seven variola, vaccinia and cowpox strains. Knowledge of the long-term response to smallpox vaccination may lead to a better understanding of poxvirus immunity and may aid in the development of new improved vaccines and diagnostic tools. S. T. Tang and M. Wang contributed equally to this work.     

Detection in primary chronic myeloid leukaemia cells of p210BCR-ABL1 in complexes with adaptor proteins CBL, CRKL, and GRB2

Chronic myeloid leukemia (CML) arises as a consequence of the expression of a chimeric fusion protein, p210BCR-ABL1, which is localized to the cytoplasm and has constitutive protein tyrosine kinase activity. Extensive publications report that p210BCR-ABL1 complexed with multiple cytoplasmic proteins can modulate several cell signaling pathways. However, while altered signaling states can be demonstrated in primary CML material, most of the reported analytical work on complexed proteins has been done in cell lines expressing p210BCR-ABL1. This has been necessary because primary hemopoietic cell lysates contain a degradative activity which rapidly and permanently destroys p210BCR-ABL1, precluding accurate p210BCR-ABL1 quantification by Western blotting or investigation of coimmunoprecipitating proteins in primary cells. This degradative activity has proven intractable to inhibition by conventional protease inhibitors. We show here that the degradative activity in primary cells is associated with cell lysosomes and is most likely to be an acid-dependent hydrolase. By lysing primary hemopoietic cells at high pH, we have demonstrated substantial inhibition of the p210BCR-ABL1-degradative activity and now report, to the best of our knowledge, the first published demonstration by coimmunoprecipitation of the association between p210BCR-ABL1 and cytoplasmic effector proteins in primary CML material.     

Specificity and function of immunoglobulin superfamily NK cell inhibitory and stimulatory receptors

Summary: Human NK cells express clonally distributed receptors specific for HLA-A, -B and -C molecules. These receptors belong to the immunoglobulin superfamily and can be functionally distinguished as inhibitory or stimulatory Inhibitory receptors block NK-cell-mediated cytotoxicity upon binding to HLA class I ligands. This function is mediated by phosphorylation of cytoplasmic tyrosines, which recruit the protein tyrosine phosphatase SHP-1. Stimulatory receptors also bind HLA class I, lack cytoplasmic tyrosine-based motifs, and trigger NK cytotoxicity and proliferation. Both types of receptor are characterized by a limited diversity allowing for recognition of distinct class I supertypic epitopes. This limited diversity is counterbalanced by the expression of different combinations ill inhibitory and stimulatory receptors with self and/or non-self HLA class I specificities on distinct NK cell clones. This peculiar strategy allows NK cells to detect loss of MHC class I molecules on autologous transformed and virally infected cells with maximal sensitivity.     

The HLA-DR phenotype of the responder is predictive of humoral response against HLA class I antigens

Recent studies suggest that the immunogenicity of an human leukocyte antigen (HLA) incompatibility should be considered in the context of the HLA phenotype of the recipient. The HLA-DR phenotype of the responder is thought to be predictive for the strength of the alloimmune response. In order to analyze the humoral response against HLA class I antigens in the context of the HLA-DR phenotype of the responder, we selected all HLA-DR homozygous Dutch patients that were present on the Eurotransplant waiting list between 1967 and 2000 (n=1,317 patients). By logistic regression it was determined whether antibody production against a specific HLA class I antigen is associated with a particular HLA-DR antigen in the patient. Furthermore, it was analyzed whether a patient, expressing a particular HLA-DR antigen, preferentially produces antibodies against particular HLA class I antigens. The results demonstrate that patients, homozygous for a certain HLA-DR antigen, cannot be considered high or low responders when analyzing the antibody response in terms of panel reactive antibody (PRA) value. However, a correlation can be found between the HLA-DR phenotype of the patient and the specific antibody response against HLA class I antigens. For example, antibodies against HLA-A10, -A11, -A19, and -B35 are produced more frequently by HLA-DR6 positive individuals, whereas antibodies against HLA-A3, -B5, -B7, -B8, and -B12 are produced more frequently by HLA-DR4 positive individuals. These data confirm that the HLA-DR phenotype of the responder plays a determinative role in the immunogenicity of mismatched HLA antigens. The results indicate that selection of HLA class I mismatches of the donor in the context of the HLA-DR phenotype of the responder might reduce the incidence of humoral graft rejection and minimize the sensitization grade of retransplant candidates.     

Disulfide bridge disruption in the alpha2 domain of the HLA class I molecule leads to low expression of the corresponding antigen

Using sequence-based typing, we have identified a novel human leukocyte antigen (HLA)-A*30 allele, HLA-A*3014L, with a low expression pattern. The sequence of HLA-A*3014L is identical to that of HLA-A*3001 except for a G to C substitution in exon 3 at nucleotide position 563, resulting in an amino acid difference at position 164 (Cys to Ser). Due to the cysteine substitution, a disulfide bridge in the alpha2 domain of the HLA class I heavy chain cannot be formed. By using the standard microlymphocytotoxicity test, the HLA-A30 antigen cannot be detected. By flow cytometric analysis of the cell-surface expression at either 37 degrees C or 30 degrees C, a temperature-sensitive expression pattern of the HLA-A*3014L antigen was observed. Only by incubating the cells at 30 degrees C, which increases the stability of HLA class I heavy chains, was a weak but clearly detectable HLA-A*3014L expression found. The mRNA expression level of the HLA-A*3014L allele was not affected by the nucleotide substitution. The intrachain disulfide bond formation in the alpha2 domain is essential for the normal expression of the HLA molecules. Reduced protein expression is probably caused by incorrect HLA class I heavy chain folding and HLA class I complex assembly.     

Exploring myelin basic protein for HLA class I-binding sequences

In view of the increasing evidence of the involvement of CD8⁺ T cells in the pathogenesis of multiple sclerosis (MS), we have scanned the sequence of the myelin basic protein (MBP), using 162 overlapping nonapeptides, for HLA-class I binding sites. Peptide binding was measured using the recently reported HLA class I α-chain-refolding assay, and the following HLA allelic products were analyzed: HLA-A2 (*0201, *0204), B27 (*2705), B35, B51, and B62. A considerable number of binding peptides were distinguished for each of the allelic products tested. In addition, three interesting points emerged. The first was the identification of several binding peptides which did not contain the known anchor motifs. The second was the evidence that several peptides showed a promiscuous binding profile, being able to bind to different HLA class I molecules that were either allelic or non allelic. The third was that in several cases two consecutive peptides could bind to the same HLA molecule.     

Mapping and ranking of potential cytotoxic T epitopes in the p53 protein: effect of mutations and polymorphism on peptide binding to purified and refolded HLA molecules

In many cancer cells, the p53 gene displays point mutations that result in stabilization and accumulation of the p53 protein. Therefore, p53 peptides could be presented to the immune system by tumor cells; thus, p53 might be a suitable target antigen for developing an immunotherapy against tumors using cytotoxic T lymphocytes (CTL). To map candidate CTL epitopes, we synthesized 150 peptides of 8–11 residues that contained putative anchor motifs required for binding to common HLA class I molecules. They were tested for their capacity to promote the assembly of purified and refolded HLA-A1, A2, B7 and B8 molecules. The following wild-type p53 peptides were found to be reactive with the HLA molecules tested: 196–205 and 226–234 bound moderately to HLA-A1; 25–35, 65–73, 129–137, 187–197, 263–272 and 264–272 bound strongly, and 187–195 and 256–264 moderately to HLA-A2; 26–35, 63–73, 189–197, 249–257 and 321–330 bound strongly to HLA-B7; and 135–143, 210–218 and 375–383 bound weakly to HLA-B8. We also analyzed the effects of p53 mutations occurring naturally in tumors on peptide/HLA assembly. We found substitutions that enhanced, diminished or had no effect on the peptide binding to HLA molecules. Polymorphism at position 72 mainly affected peptide/HLA-B7 binding, the proline allele P72 giving a less-reactive peptide (63–73) than the arginine allele R72. We have ranked potential p53 epitopes according to their reactivity for purified HLA molecules, allowing the selection of appropriate peptides and HLA molecules to attempt CTL induction in vitro.     

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.