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)     

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.     

HLA-A33 and -B44 and susceptibility to postherpetic neuralgia (PHN)

HLA class I and class II alleles of 32 Japanese patients with postherpetic neuralgia (PHN) and 136 healthy controls were analyzed by serological (class I) and DNA (class II) typing for any significance in the susceptibility to varicella-zoster virus (VZV). We recognized positive associations of the development of PHN with the HLA class I antigens HLA-A33 and -B44, and the HLA-A33-B44 haplotype. This haplotype is tightly linked to DRB1*1302 in a Japanese healthy population. However, no significant association between PHN and HLA class II alleles was observed with no linkage of the HLA haplotype HLA-A33-B44 to HLA-DRB1*1302 in the patients with PHN. These findings suggest that HLA class I gene may genetically control the immune response against VZV in the pathogenesis of PHN.     

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.     

Analysis of cytotoxic T cell precursor frequencies directed against individual HLA-A and -B alloantigens

We describe here a limiting dilution analysis to determine cytotoxic T lymphocyte precursor (CTLp) frequencies against individual HLA-A or -B antigens. This assay is reproducible and showed that the CTLp frequency of an individual remains stable with time. Significant variations in CTLp frequency against the same alloantigen were found in different individuals and even in monozygotic twins, showing that these differences were not (completely) genetically determined. Within an individual, a wide range of CTLp frequencies can be found against different allo-antigens. Serologically cross-reactivity seems not to interfere in this assay. This LDA is a practicable tool for a systematic analysis of CTLp response against selected individual HLA-A or -B antigens and can be used for the selection of HLA mismatched donors for transplantation patients.     

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.     

Protein micelles of human histocompatibility antigens (HLA-A and HLA-B)

Human histocompatibility antigens (HLA-A and -B) are membrane proteins which have large hydrophilic domains outside the cell membrane and a small hydrophobic portion in the lipid bilayer. In this paper we describe optimal conditions for preparing micelles of detergent-solubilized HLA-A2 and -B7 antigens. These homogeneous protein aggregates are water soluble and free of detergent and lipid. Hydrophobic interactions between the intramembraneous portions of the HLA antigens are the driving forces in the formation of these protein micelles. The papain-solubilized fragment of the HLA antigens is not included in the micelle. The average molecular weight of the HLA micelles is around 9 × 10⁵ daltons, which suggests sixteen HLA-A2 and/or HLA-B7 antigenic molecules per protein aggregate. Electron microscopic studies revealed that the most frequent size of the micelles is 12 mm and that HLA-micelles are similar but not identical to micelles from Sindbis Virus glycoproteins (E1 and E2) The HLA-A2 and -B7 micelles retained full antigenic activity as judged by precipitations with allo- and heteroantisera. Such micelles will no doubt be important tools in further studies of the role of histocompatibility antigens.     

Unbalanced regulation of the expression of HLA-A and -B antigens in metastatic melanoma cells compared to autologous PBMC: A quantitative analysis of fourteen patients.

The quantitative cell surface expression of the gene products of HLA-A and -B loci is genetically predetermined (following Mendelian inheritance) in a given individual; in addition, the regulation of their expression is tightly coordinated since the ratio of HLA-A and -B antigens expression is constant on different cell types and the expression of HLA-B antigens is lower than that of HLA-A antigens. In view of these considerations and of the potential relevance of the quantitative expression of the gene products of HLA-A and -B loci in antigen presentation and for T cell-based specific immunotherapy, levels of cell surface HLA-A (mAb A131) and -B (mAb YTH) antigens were investigated by flow cytometry on fourteen primary cultures of melanoma cells (analyzed between in vitro passages 5 to 10) derived from unrelated melanoma patients and compared to those obtained with autologous PBMC. All melanoma cells and PBMC investigated were stained by mAb A131 and YTH (samples were considered positive when the mean value of fluorescence intensity with specific mAb was at least double than negative control mAb). The mean values of mean fluorescence intensity obtained for HLA-A and HLA-B antigens were 275±247 and 35±30 on melanoma cells and 1520±490 and 780±340 on PBMC and were both significantly different in a paired test between melanoma cells and PBMC (HLA-A, P=2×10⁻⁶; HLA-B, P=1×10⁻⁶); thus, the expression of HLA-A and -B antigens was 5.5 and 22.1 times lower on melanoma cells than on autologous PBMC. Simple linear regression analysis showed a high correlation (r=0.9; P=1×10⁻⁵) between the mean values of fluorescence intensity observed for HLA-A and -B antigens in PBMC; in contrast, a low correlation (r=0.6; P=1×10⁻²) was found in melanoma cells. Therefore, we calculated the ratio between the mean values of mean fluorescence intensity obtained for HLA-A and -B antigens in melanoma cells and autologous PBMC. The ratio HLA-A vs HLA-B was 10.9±8.0 (range: 2.1 to 32.6) and 2.1±0.7 (range: 1.28 to 3.58) in melanoma cells and PBMC, respectively (p=1×10⁻³, paired t test). Results similar to those obtained with mAb YTH were also obtained with the anti-HLA-B antigens mAb Q6/64 and H2-89-1. Our data, altogether, strongly suggest the existance of an alteration involving the coordinated regulation of the expression of HLA-A and HLA-B loci in melanoma cells.     

Lymphokines,vol. 5, monoclonal T cells and their products: edited by M. Feldmann and M.H. Schreier. Academic Press,New York, 1982 (xviii + 496 pp., illus.) $55.00

HLA-A and -B antigens were detected on fresh and dried peripheral blood lymphocytes by an enzyme-linked immunosorbent assay. Intact cells fixed to plates with glutaraldehyde were used as antigen and anti-HLA alloantisera as a source of antibodies. Determination of HLA antigens by the ELISA technique was comparable with the complement-dependent cytotoxicity test. The relative stability of HLA antigens as shown in this report and the extensive polymorphism of the HLA system make the ELISA technique a promising tool for the analysis of HLA antigens on non-living cells including, for example, medicolegal investigation of blood stains.     

Hyperimmunized patients do not need to wait for an HLA identical donor

Hyperimmunized patients tend to accumulate on renal transplant waiting lists because their high level of sensitization leads to positive crossmatches with almost all potential organ donors. The origins of sensitization and the different efforts made to find cross-match negative donors for these patients are discussed. Special emphasis is given to a local strategy based on the determination of HLA-A and -B mismatches, against which the patient did not form allo-antibodies, the so-called acceptable mismatches. Kidney donor selection is based on compatibility with the patient's own HLA-antigens in combination with the acceptable HLA-A and -B antigens, and can be operated from a central organ-sharing office. The acceptable HLA mismatches are often identical with or include the non-inherited HLA class I antigens of the mother (non-inherited maternal antigens: NIMA).     

Detection of HLA antigens on blood platelets and lymphocytes by means of monoclonal antibodies in an ELISA technique

The expression of HLA-A and -B antigens on peripheral blood lymphocytes and blood platelets was measured using monoclonal antibodies in a semi-quantitative ELISA technique. Reactively of monoclonal anti-HLA-A2 and anti-HLA-B7, with lymphocytes as well as platelets, was in agreement with the presence of these antigens as detected by conventional HLA typing of lymphocytes. When the actual amount of HLA antigens was measured, a gene-dose effect was seen: cells from HLA-B7-homozygous individuals bound more monoclonal anti-HLA-B7 antibodies compared to their HLA-B7-heterozygous siblings. At the same time, cells of different donors showed only very small differences in binding of monoclonal antibody against an HLA-"backbone" determinant. Relative to total HLA-A, -B and -C expression, the amounts of HLA-A2 on lymphocytes and platelets were similar. On the other hand, the expression of HLA-B7 on platelets was diminished compared to that on peripheral blood lymphocytes.     

The role of the human major histocompatibility complex in cytotoxic T-cell responses to virus-infected cells

The human major histocompatibility complex (HLA) has been demonstrated to play two roles in the generation and expression of cytotoxic T-lymphocyte responses to virusinfected cells: (1) cytotoxic T cells can only recognize viral antigens in conjunction with antigens encoded by HLA-A and -B genes; and (2) HLA-linked genes may control the capacity to generate T-cell responses to a given virus or to virus in conjunction with particular self HLA-A and -B antigens. Analysis of T-cell responses generatedin vivo to Epstein-Barr virus suggests that human T cells may recognize virus in conjunction with antigens other than the class I HLA polymorphic specificities.     

Unexpected HLA haplotype sharing in dizygotic twin pairs discordant for rheumatoid arthritis.

Dizygotic twins are generally believed to be no more genetically similar than sibs born from separate pregnancies. In the present study, a panel of 93 dizygotic twin pairs discordant for rheumatoid arthritis were typed for HLA-A, -B, -Cw, and -DR antigens. HLA haplotype sharing identical by descent between the twins showed a trend towards increased sharing of both HLA haplotypes; this increased sharing was statistically significant when the female/female twin pairs were considered separately. In contrast, the pattern of HLA haplotype sharing in sib pairs (n = 128) was consistent with a 1:2:1 ratio of 2, 1, or 0 haplotypes shared. An analysis of 16 normal dizygotic twin pairs was consistent with these results raising the possibility that dizygotic twins in general are genetically more similar at the HLA complex than sibs born from separate pregnancies.     

中华(上海)骨髓库北方人群HLA多态性调查

调查中华(上海)骨髓库北方人群HLA多态性及其频率分布特征。用微量淋巴细胞毒试验检测11995名无关供者HLA-A、B抗原,并用PCR-SSP和反向PCR-SSOP技术对疑难标本进行复检。计算其中3 736名北方人群 HLA-A、B的抗原频率、基因频率和HLA-A、B位点单倍型频率、连锁不平衡参数。调查中共检出A位点抗原26种,B位点抗原54种,最常见HLA-A、B抗原包括A2,A11,A24,A30,A33,B13,B46,B51,B58,B60,B61,B62等。连锁不平衡参数大于0.0005的单倍型有40多种,最常见的单倍型有A2-B46,A30-B13,A33-B58等。结果显示中华(上海)骨髓库北方人群HLA多态性有自身特点,频率分布介于南、北汉族之间。     

Human mumps virus-specific cytotoxic T lymphocytes: quantitative analysis of HLA restriction

To obtain quantitative information about the use of HLA antigens as restriction element by antiviral cytotoxic T lymphocytes (CTL), we have analyzed precursors of human mumps virus-specific CTL by limiting dilution. CTL generated by restimulation of peripheral blood T lymphocytes with autologous mumps virus (MV)-infected stimulator cells were restricted by autologous HLA class I antigens, and derived from the T4-8+ population. They were specific for MV and did not lyse autologous target cells infected with other viruses. Frequencies of MV-specific CTL precursors ranged from 1/500 to 1/8000. HLA restriction was analyzed by split-well analysis of individual CTL colonies. CTL recognizing HLA-A or B antigens were unequally distributed: HLA-B7, -B13, and -B27 were found to function as predominant, in some cases as exclusive, restriction elements, whereas other antigens such as HLA-A24 were never or rarely used. In several combinations, there was no evidence for antigenic variants of HLA molecules as reason for the failure to be recognized. The proportion of CTL precursors recognizing HLA-A2 and -B8 seemed to be dependent on the presence or absence of "dominant" restriction elements. We conclude that CTL precursors recognizing certain virus-HLA combinations are preferentially expanded during an infection, but that low responsiveness to a given combination is not necessarily absolute.     

Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism.

The extensive polymorphism of the classic class I antigens has been well described. In contrast, the nonclassic HLA antigens are distinguished by their low polymorphism. We examine here the HLA polymorphism of the HLA-E locus by examining the DNA sequence of cDNA from nine ethnically diverse individuals. From this analysis, we show that there is no polymorphism in the regions including exon 1 and from exon 4 to exon 8, the 3' untranslated exon. In exons 2 and 3, there are two base substitutions, one of which is at a replacement site and the other silent. The replacement substitution changes an arginine to a glycine at position 107, defining two alleles at the HLA-E locus. Using the PCR on exon 3 from genomic DNA and hybridization with oligonucleotide probes, we have examined 90 HLA-typed individuals to determine the relative frequency of the two alleles in the population and their association with the classical antigens. This analysis showed that these two alleles were present at nearly equal frequencies in the population. Surprisingly, both alleles were found in an essentially random association with all but one HLA-A and -B haplotype. The single exception was to the A1-B8 haplotype, which appeared to be linked to only one of the two alleles. One implication of this random association is that these HLA-E alleles may have existed before most of the HLA-A and B polymorphism. Thus, selection has maintained the HLA-E locus essentially unaltered during a time when considerable polymorphism was being selected for at the HLA-A and -B loci. This finding may also have important consequences in an unrelated bone marrow transplant, where it is predicted that 37% of HLA-A and -B matched donors are mismatched at the HLA-E locus.     

基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的 探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案.方法 人胚胎干细胞系SYSU-I、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotide probe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型.应用PCR-STR技术检测两株细胞系的基因遗传标记.结果 获得两株hESC细胞系的HLA高分辨分型和STR基因型.结论 可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案.     

基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案。方法人胚胎干细胞系SYSU-1、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotideprobe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型。应用PCR-STR技术检测两株细胞系的基因遗传标记。结果获得两株hESC细胞系的HLA高分辨分型和STR基因型。结论可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案。