HLA antigens on platelet membranes. In vitro and in vivo studies

In order to determine whether HLA-A,B antigens of platelets are integral membrane constituents or rather represent adsorbed plasma proteins, their presence in plasma and their adsorbability onto platelet membranes was studied by in vitro and in vivo experiments. The amount of HLA antigens was quantitated by inhibition of lymphocytotoxicity (LCT) and by enzyme-linked immunosorbent assay (ELISA) using operationally monospecific polyclonal HLA antibodies or murine HLA-specific monoclonal antibodies, respectively. We found that in 11 out of 13 HLA-A2 and in 9 out of 10 HLA-B13 experiments, platelets from antigen-negative donors pretreated with plasma from the same number of antigen-positive donors inhibited LCT to the same extent as platelets from antigen-positive donors. Nevertheless, the in vitro adsorbed HLA antigens onto antigen-negative platelets were, unlike those on antigen-positive platelets or in plasma, not reactive with monoclonal antibodies as quantitated by ELISA. Similarly, infusion of HLA-A2-negative platelets from single donors into 3 HLA-A2-positive, thrombocytopenic patients with bone marrow failure led to a good platelet increment, but did not convert the HLA type of donor platelets, neither at 2 h nor at 18 h posttransfusion. On the basis of these results, we conclude that soluble HLA antigens can be taken up by human platelets from plasma in small amounts. However, the major portion of HLA antigens appears to be integral membrane constituents.     

Influence of HLA-A2 on the effectiveness of platelet transfusions in alloimmunized thrombocytopenic patients

Platelet transfusions from donors selectively mismatched for cross- reactive and certain non-cross-reactive HLA antigens were found to be more effective in HLA-A2 negative than in HLA-A2 positive, alloimmunized thrombocytopenic patients. The two groups of patients responded equally well to platelets matched for antigens of the HLA-A and B loci. Certain alloimmunized patients negative for HLA-A2 continued to respond satisfactorily to platelets selectively mismatched for non-cross-reactive HLA antigens as long as platelets containing HLA- A2 were avoided. The data indicate that platelet transfusion support can be provided within a broader range of donor-recipient HLA antigenic disparity to HLA-A2 negative alloimmunized patients than to those who are positive for this antigen.     

Quantitative analysis of platelet surface HLA by W6/32 anti-HLA monoclonal antibody

Class I molecules of human major histocompatibility complex (HLA) are the most important antigenic system in determining the survival of transfused platelets in alloimmunized patients. Platelets with reduced expression of a specific type of HLA antigen may escape specific anti-HLA antibody-mediated destruction. By using 125I-labeled Fab fragments of W6/32 anti-HLA monoclonal antibody and competitive protein binding assays, we measured the range of total HLA concentrations on platelets. In 12 individuals examined, the mean number of HLA-A, B, and C molecules per platelet was 81,587 +/- 20,016 (mean +/- SD); its range was between 54,782 to 116,185 molecules per platelet. After treatment with chloroquine, 79.9 +/- 7.0% (mean +/- SD, n = 6) of HLA antigens were removed from platelets as determined by binding of 125I-W6/32 Fab. A similar result was obtained when HLA antigens on chloroquine-treated platelets were evaluated with immunofluorescence flow cytometry. In contrast, chloroquine treatment did not remove integral membrane protein such as P1A1 antigens on platelets. The presence of HLA antigens in the chloroquine eluate of platelets could be demonstrated to contain HLA antigens similar in mol wts to intact class I molecules by an immunoblotting technique. These data suggest that 70% to 80% of platelet HLA antigens are adsorbed and that such HLA antigens are not proteolytic products of integral membrane class I molecules. The origin of the adsorbed platelet HLA-antigens remains to be determined.     

Isolation of a human major histocompatibility complex class I gene encoding a nonubiquitous molecule expressed on activated lymphocytes

The human major histocompatibility complex is a multigene family containing at least 20 class I genes. Included within this family are the loci encoding the highly polymorphic HLA-A, -B, and -C antigens present at the surface of most nucleated cells. The large number of genes detected with class I probes by Southern blot analysis and the existence of serological reagents defining nonubiquitous, non-HLA-A,B,C class I antigens suggest that products other than HLA-A,B,C antigens are encoded within the class I gene family. These products might be the human counterparts of the murine Qa and TL antigens. In order to identify non-HLA-A,B,C genes, we have developed a probe, JF11, located in noncoding regions flanking the HLA-A locus. This probe detects only a limited number of class I genes and does not detect HLA-A,B,C-associated restriction fragments on Southern blots. This probe was used to screen a human cosmid library. Some of the cosmids isolated with this probe were then transferred into mouse fibroblasts expressing human beta 2-microglobulin. One of the transfectants specifically reacts with one alloantiserum (HA2) that detects HLA class I molecules specific to HLA-A2-positive, phytohemagglutinin-activated T cells and not found on resting T or B cells. Data presented in this paper provide evidence for the isolation and expression of a class I gene encoding a nonubiquitous class I antigen that could be a human analogue of the murine Qa antigens.     

An evaluation of crossmatching, HLA, and ABO matching for platelet transfusions to refractory patients

Refractoriness occurs in many patients receiving multiple platelet transfusions. We used a sensitive ELISA assay to assess the utility of crossmatching HLA-A,B matched single donor platelets in 51 consecutive, typical refractory patients. Of the 222 transfusions evaluated at 1 to 4 hours posttransfusion, only 17 of 54 (31%) with positive crossmatches had corrected platelet count increments of greater than or equal to 7,500/microL. In contrast, 95 of 168 (57%) of those with negative crossmatches had such increments (P less than .001). Regardless of the results of the crossmatch, HLA-A,B, and ABO matching had independent influences on transfusion outcome. The median corrected 1- to 4-hour increment for crossmatch negative transfusions was 13,300/microL for A/BU grade matches, 9,700 for BX, and 7,800 for C. Increments were 10,000/microL for ABO-identical transfusions and 5,900 for transfusions of platelets ABO incompatible with the recipient's plasma antibodies. When the donor platelets were ABO compatible, but the donor plasma contained ABO antibodies to the recipient's platelets, the increment was intermediate (8,200/microL). The most important factor in predicting platelet survival was the crossmatch, followed by HLA-A,B and ABO, each having independent predictive value. These data demonstrate that the predictive value of a negative crossmatch may be considerably less than that reported in previous studies with stable, less ill patients. In typical refractory patients, there appear to be mechanisms of platelet destruction that are related to HLA-A,B and ABO but are not detected with current crossmatch methods. We hypothesize that soluble plasma HLA-A,B and ABO antigens contribute to the destruction of donor and sometimes recipient platelets by an immune complex or other "innocent bystander" mechanism. With our crossmatching technique, HLA-A,B and ABO match grades remain relevant to platelet transfusion therapy in some refractory patients.     

HLA antibody formation within the HLA-A1 crossreactive group in multitransfused platelet recipients

The formation of intragroup antibodies, HLA antibodies directed against antigens in the same crossreactive group (CREG) as those of the serum donor, may be an important cause of transfusion failures in patients receiving HLA-matched platelets. Twenty-two patients whose HLA types included at least one antigen in the HLA-A1 CREG were studied. Of the ten patients who formed HLA antibodies, six produced antibodies that reacted with one or more antigens in the HLA-A1 CREG. Five of 12 patients whose HLA types included HLA-A3 formed antibodies directed against HLA-A1-10-11 or HLA-A1-10. In contrast, only one of ten individuals whose phenotypes included HLA-A1, HLA-A11, or both produced anti-HLA-A3. Eleven incompatible retrospective crossmatches were observed in recipients of HLA-matched platelets attributable to intragroup antibodies. Patients receiving incompatible platelets had unsatisfactory post-transfusion platelet count increments. It is concluded that intragroup antibodies, such as those directed against antigens in the HLA-A1 CREG, are an important cause of platelet transfusion failures in patients requiring long-term platelet transfusion support. These antibodies can be identified by routinely screening recipient sera for HLA antibodies and performing retrospective crossmatches using the lymphocytotoxicity technique.     

Cell cycle and the differential expression of HLA-A,B and HLA-DR antigens on human B lymphoid cells.

Monoclonal antibodies specific to HLA antigens and the fluorescence-activated cell sorter were used to analyze the changes in the density of human histocompatibility antigens HLA-A,B and HLA-DR on the surface of synchronously growing WI-L2 cells (a human B cell line) progressing through the cell cycle. The WI-L2 cells were synchronized by density-dependent arrest in G1, and samples from G0, G1, late S and late G2 phases were used to determine the frequency distribution of cell volume, DNA content, and the relative amounts of cell surface HLA antigens; the observed density changes were calculated from these values. The HLA-A,B density remained nearly constant throughout the cell cycle, whereas the HLA-DR density increased sharply at the G2-M stage. These results suggest a cell cycle-dependent differential control of the expression of these two sets of histocompatibility antigens on B cells.     

Successful transfusion of platelets "mismatched" for HLA antigens to alloimmunized thrombocytopenic patients

A critical factor limiting the availability of histocompatible platelet transfusions for alloimmunized, thrombocytopenic patients is the large pool of HLA-typed donors needed to procure platelets perfectly matched for HLA antigens. We have, therefore, investigated the effectiveness of platelets obtained from donors having lesser degrees of histocompatibility. In 421 transfusions administered to 59 alloimmunized patients who were refractory to "random donor" platelets, it was found that platelets mismatched for 1 or 2 "cross-reactive" HLA antigens were in most instances as effective in increasing circulating platelet levels as perfectly matched platelets. A significant number of patients also responded to platelets from donors selectively mismatched for non-cross-reactive HLA antigens. The latter group had a significantly reduced frequency of the antigen HLA-A2 (13%) in comparison to the total patient population (49%). Use of donors whose HLA antigens are serologically cross-reactive with those of alloimmunized patients provides approximately 10 times as many prospective donors as does selection based on matching for HLA and simplifies the procurement of hemostatically effective platelets for such patients.     

Insulin binding to human B lymphoblasts is a function of HLA haplotype

A variety of genetic and biochemical evidence points to an association between major histocompatibility complex (MHC) haplotype and several types of cell surface receptors including epidermal growth factor and insulin receptors. We report evidence for such associations between human class I MHC antigens, HLA antigens, and specific insulin binding sites on human B lymphoblasts. We have measured insulin binding to cells of an HLA-heterozygous, Epstein-Barr virus-transformed B-cell line, LCL 721, and to derivative mutants from which all or part of the HLA complex had been deleted. The affinity, Ka, of insulin binding sites is approximately 10(8) M-1 in mutants expressing antigen HLA-B5 together with other HLA antigens and in mutants expressing only HLA-C. HLA-A1; HLA-A1,B8; HLA-A2,C; and HLA null mutants (not expressing any HLA antigens) bind insulin to sites with an affinity of approximately 10(9) M-1.     

Membrane expression and synthesis of p23,30 (HLA-DR antigen) by human peripheral blood monocytes

The synthesis and expression of protein complexes of 23,000 and 30,00 dalton (p23,30) HLA-DR antigen, and of 44,000 and 12,000 dalton (p44,12) HLA-A,B antigen and beta 2-microglobulin was demonstrated on human peripheral blood monocytes and in cultures of purified, adherent monocytes. In indirect immunofluorescence assays, a gradation in the intensity of staining with rabbit anti-p23,30 serum was present but clearly p23,30-negative, actively phagocytic monocytes were not found. In contrast the fluorescence intensity of staining with a serum to p44,12 (HLA-A,B antigens and beta 2-microglobulin) was constant on all macrophages. Macrophage HLA-DR antigen was shown in SDS gels of immunoprecipitates of 35S-methionine-labeled, detergent-solubilized membrane proteins to be composed of 29,000 and 34,000 dalton, noncovalently linked chains, which form was indistinguishable from that of B lymphoblastoid cell line HLA-DR antigens. The rate of synthesis of HLA-DR antigen was about 17% of that of synthesis of HLA-A,B antigens in adherent macrophage populations. The variable expression of p23,30 on peripheral blood monocytes was consistent with the view of the existence of subsets of such monocyte populations. These findings were compared to studies by others of the variable expression of Ia antigens on human, murine and guinea pig monocytes populations.     

Membrane proteins on human megakaryocytes and platelets identified by monoclonal antibodies

We describe five monoclonal antibodies that react with four discrete antigens present on human platelets. Antibodies B2.12 and B59.2 precipitate the glycoprotein IIb-IIIa complex from radiolabeled platelet membrane extracts and inhibit platelet aggregation induced by adenosine diphosphate (ADP), collagen, or epinephrine. The antigen recognized by the two antibodies is present on megakaryocytes but either absent entirely or expressed in small amounts on platelets from Glanzmann's thrombasthenic patients. The antigen recognized by antibody B37.3 is absent from thrombasthenic platelets. Antibody B1.12 reacts with an antigen shared by platelets and 20% of peripheral blood lymphocytes and is a potent inducer of platelet aggregation. Antibody B2.10 reacts specifically with platelets and megakaryocytes but does not affect platelet functions. Thus, these reagents are useful tools in diagnostic and functional studies of both normal and abnormal platelets.     

Identification of human genomic clones coding the major histocompatibility antigens HLA-a2 and HLA-B7 by DNA-mediated gene transfer.

We have screened a large number of isolated human genomic clones that hybridize to a cloned HLA cDNA probe for their ability to direct the synthesis of HLA-A, -B, and -C surface antigens on mouse L cells following DNA-mediated gene transfer. The surface expression of human histocompatibility antigens, monitored by indirect immunofluorescence and the fluorescence-activated cell sorter, was examined at 60 hr after transfection and on hypoxanthine/aminopterin/thymidine-resistant (HATR) populations derived from cotransfer with the herpes simplex virus thymidine kinase gene. Two unique genomic clones designated JY B3.2 and JY 158, isolated from the human lymphoblastoid cell line JY (homozygous HLA-A2, -B7), were shown to contain gene sequences capable of directing expression of an HLA-A, -B, -C determinant. By using allo-specific antibodies, the gene products of these clones were identified as HLA-A2 and HLA-B7, respectively. HATR clonal populations isolated from cotransfections with these genomic clones displayed varying levels of surface HLA expression that correlated with the number of intact donor HLA sequences present in the cells. In general, these levels of expression were stable during 3 months in culture. This system provides a powerful tool for the study of human surface antigen gene structure, expression, and function on a mouse cell background.     

Ankylosing spondylitis without B27: no evidence for gene conversion.

Isoelectric focusing gel electrophoresis was used to look for variant HLA molecules in five patients with HLA-B27 negative ankylosing spondylitis (AS). The isoelectric points of the HLA-A and B antigens from these patients and HLA paired controls were identical. This implies that the HLA-A and B antigens from the patients with AS and the controls are similar. Gene conversion of a nucleotide sequence from a B27 positive gene is thus unlikely to be the explanation for the existence of AS in patients who are HLA-B27 negative by alloantisera typing.     

The origin of ABH antigens on human platelets

ABH antigens are present on platelets from individuals of the corresponding red cell phenotype, but the extent to which these antigens are intrinsic or adsorbed remains undefined. To evaluate platelets for intrinsic H substance, an IgM mouse monoclonal antibody against type 2H chain (the intrinsic H structure found on erythrocytes) was labeled with 125I and incubated with platelets from donors of different ABO type. The antibody showed dose-response saturation curves, and binding to platelets paralleled that of the red cell ABO type, with O greater than B greater than A1 greater than A1B greater Oh cells, giving a single factor variance F of 190 (P less than .0005). Passive adsorption of A antigens by platelets has been previously reported. To verify this phenomenon for A and B antigens and to quantitate the elution of A and B antigens from platelets, the following assay system was used. Platelets from group A1 and B donors were incubated in plasma from group O donors, and platelets from group O donors were incubated in plasma from different ABO, Lewis, and presumed secretor-type donors. Human IgG anti-A or anti-B was added to the platelets. The amount of antibody bound was determined with a 125I- labeled mouse monoclonal anti-human IgG. When incubated for 96 hours in group O plasma, group A1 platelets showed a 45% to 50% decrease in binding of anti-A. There was no significant change in the level of type 2H antigen on these platelets during the same incubation period. Group O platelets incubated in A or B plasmas rapidly acquired the antigens, but if returned to their original plasma, 95% of this passively adsorbed antigen eluted off within 18 hours. The maximum uptake of A and B substances was influenced by the Lewis and secretor type of donor plasma. Our present study demonstrates that ABH antigens on platelets consist of type 2H chains, which are presumably intrinsic as when found on red cells, and of passively adsorbed ABH structures, which are presumably type 1H chains.     

Recognition by xenogeneic cytotoxic T lymphocytes of cells expressing HLA-A2 or HLA-B7 after DNA-mediated gene transfer.

Murine L cells expressing HLA-A2 or -B7 antigens were isolated after cotransformation of thymidine kinase-negative cells with the herpes simplex virus thymidine kinase gene and the genomic clones containing either the HLA-A2 or -B7 genes. Monoclonal antibody binding analyses demonstrated the stable cell surface expression of HLA antigens by these cells at levels of up to 40% of the amount expressed by the human B lymphoblastoid cell line, JY. The HLA-A2 and -B7 antigens expressed by the L cells retained all of the antibody-defined, heavy-chain-associated antigenic determinants but lacked those determinants associated with human beta 2-microglobulin. These HLA transformants were capable of functioning as targets for monoclonal cytotoxic T lymphocytes (CTL) that specifically recognize the HLA-B7 or -A2 antigens expressed by JY cells. However, the efficiency of lysis, relative to the JY cell line, was 50-99% for individual CTL. In addition, not all of these CTL were capable of lysing the appropriate transformants. Because the antigens appear by serological criteria to be structurally intact and expressed at high levels, these results suggest that the complementation of the HLA heavy chains with mouse, rather than human, beta 2-microglobulin may alter the antigenic determinants that are important for CTL recognition.     

HLA antigen structural gene mutants selected with an allospecific monoclonal antibody.

The HLA-A2 antigen-specific monoclonal antibody BB7.2 and complement were used to immunoselect mutants from an ethyl methanesulfonate-mutagenized human B lymphoid cell line, T5-1. Surviving colonies were screened by radioimmune binding with BB7.2 and with a monospecific HLA-A2 alloantiserum, Stewart, and HLA antigens of selected clones were immunoprecipitated and studied by isoelectric focusing. Several classes of mutants could be distinguished: mutants that expressed no HLA-A2 heavy chain; mutants that expressed an HLA-A2 heavy chain that was unable to associate with beta 2-microglobulin (beta 2m) and was not expressed at the cell surface; mutants with reduced HLA-A2 heavy chain-beta 2m association and cell surface expression of HLA-A2 dimer with or without heavy chain charge alterations; mutants with normal HLA-A2 heavy chain-beta 2m association and normal quantitative cell surface HLA-A2 expression but with HLA-A2 heavy chain charge alterations; and mutants with as yet incompletely defined lesions. Mutants with altered cell surface HLA antigens were not found in previous selections with alloantisera and should be useful for epitope mapping and structure-function studies of HLA molecules.     

Human leukocyte antigens in hypertrophic cardiomyopathy patients in South India

Hypertrophic cardiomyopathy is characterized by massive ventricular hypertrophy, reduced diastolic function, and excessive ventricular contraction. The human leukocyte antigens HLA-A, HLA-B, and HLA-DR were studied in 14 hypertrophic cardiomyopathy patients with left ventricular obstruction from South India. They were compared with 81 normal age- and sex-matched individuals from the same ethnic background. The human leucocyte antigens were identified using the standard serological assay with a longer incubation for DR antigens. The odds ratio, frequency, chi-squared value, p-value, etiological fraction, preventive fraction, and haplotype frequency estimates were calculated. The HLA-B51 and HLA-DR2 levels were significantly increased in hypertrophic cardiomyopathy patients compared to controls, whereas HLA-A19, HLA-B7, and HLA-DR4 were decreased when compared to the controls. It was noticed that haplotype B51-DR2-DQ3 was significantly associated with hypertrophic cardiomyopathy patients from South India. Hypertrophic cardiomyopathy may be associated with genes in the human leukocyte antigen region, and immunogenetic factors linked to human leukocyte antigens appear to play a major role in the pathogenesis.     

Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.

The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells.     

Variation of class I HLA antigen expression among platelet density cohorts: a possible index of platelet age?

Platelet alloantigens and other surface markers were studied in platelet cohorts of different mean density, using monoclonal and polyclonal probes. High density (HD) platelets expressed 12% more P1A1 molecules (46,942) than low density (LD) platelets (41,892). However, LD platelets carried 42% more HLA-A2 molecules (6,267 +/- 184) than HD platelets (4,406 +/- 232) (P less than .01) and 55% more class I HLA antigens (17,034 +/- 2,062 v 11,007 +/- 2,190) (P = .05). The platelet subpopulations did not differ in their content of glycoprotein (GP)IIb/IIIa complex or Baka antigen. The difference in expression of class I HLA antigens on HD and LD platelets is consistent with two possibilities: either class I HLA molecules are acquired from plasma or they are released into plasma as platelets age in circulation. Accordingly, class I HLA molecules may provide a useful marker of platelet age.     

Loss of HLA-A,B,C allele products and lymphocyte function-associated antigen 3 in colorectal neoplasia.

The expression of HLA-A,B,C antigens and lymphocyte function-associated antigen 3 in human colorectal adenomas and adenocarcinomas was studied by immunohistochemistry. None of 10 adenomas and only 1 of 30 carcinomas had lost expression of all HLA-A,B,C molecules. On the other hand, focal loss of an HLA-B product was seen in 2 of the adenomas, and complete losses of tumor cell HLA-A2 (in 7 of 13 cases), HLA-Bw4 (in 4 of 13 cases), and HLA-A3 (in 1 of 6 cases) were seen in the carcinomas. No complete losses of HLA-A1 (in 6 cases) or HLA-Bw6 (in 22 cases) occurred in the carcinomas. In addition, 1 of 20 adenocarcinomas totally lacked lymphocyte function-associated antigen 3. Because a loss of tumor cell HLA-A,B,C antigen or lymphocyte function-associated antigen 3 could be selected for through an advantage in escape from cytotoxic T-lymphocyte attack, our results suggest that immunoselection may be a more important mechanism in tumor progression than has previously been assumed.