Homozygosity in the major histocompatibility complex region influences natural killer cell activity in man

The effect of homozygosity at HLA loci on natural killer (NK) cell activity has been examined. Lymphocytes obtained from heterozygous and homozygous individuals were incubated with 51Cr-labeled, NK-sensitive K562 cells at different effector/target ratios, and lytic activity was determined. Homozygous cells, obtained from individuals who are known HLA homozygotes (homozygous typing cells) and from selected families, had low NK activity compared to those heterozygous donors. This low cytotoxic activity had no correlation with sex, but did correlate with homozygosity at the HLA-A, B and/or DR loci. A significantly lower number of cells, which bind to anti-Leu 7 antibody, was found in homozygous donors. However, this reduced number of Leu 7+ cells could only partially account for the decrease in NK activity. These studies suggest that in some individuals homozygosity at HLA may be linked to genes that control NK activity.     

Lymphocytes modulated by β interferon express new non-HLA-A,B,C class I antigens

Peripheral blood lymphocytes were cultured with recombinant β interferon (INF-lymph). Three days in culture and 5 × 10⁴ units/ml produced the best modulatory effect. Platelet absorbed alloantisera previously shown to contain non-HLA-A,B,C class I antibodies were tested with the INF-lymph. The serologic reactivity could be blocked by turkey sera to human B₂-microglobulin but not by turkey anti-Ia-like. Absorption studies with INF-lymph obtained from donors expressing different HLA antigens indicated that the serologic reactivity is not due to HLA. Fourteen sera appeared to cluster in three groups, only one of which was found to be associated with HLA (HLA-A1). Antigenic modulation of INF-lymph obtained from HLA-A1⁺ individuals with HLA-A1 typing alloantisera eliminated subsequent lysis mediated by HLA-A1 alloantibodies and complement but not lysis with platelet absorbed antibodies included in the cluster associated with HLA-A1. These findings suggest independence from HLA-A1 and coexpression of HLA-A1 and A1-associated class I antigens. Family studies indicated that the reactivities segregated with HLA thus mapping it to chromosome VI. Similar to what we have described with PHA, β interferon modulates lymphocytes and induces the expression of new class I differentiation antigens probably analogous to the murine Qa-T1a antigens.     

Subtypes of HLA-A1 defined on the basis of CTL precursor frequencies

Recently we have shown that limiting dilution analysis can be used to detect cytotoxic T-cell precursor frequencies directed against individual HLA class I antigens. Using the same protocol, we have been able to define two subtypes of HLA-A1, which are indistinguishable by conventional typing sera as well as by cell-mediated lympholysis. One-dimensional isoelectric focusing analysis of the variants did not show any overall charge differences. However, family studies indicated that these HLA-A1 subtypes are genetically determined and can be distinguished on the bases of T-cell precursor frequencies in HLA-A1-negative blood donors.     

Beta-2 microglobulin-free HLA class I heavy chain (FHC) A3 and/or A30 soluble products contribute only minimally to serum FHC expression.

No monoclonal antibodies (mAbs) are presently available to measure the total amount of beta2-microglobulin-free HLA class I heavy chain (FHC) in sera. The available ELISA-based double determinant immunoassay (DDIA), established to measure FHC, uses two mAbs (TP25.99 and HC-10) that recognize a monomorphic determinant expressed on all HLA-B/C FHC products and a determinant expressed only on some HLA-A FHC products. This restricted reactivity implies that, in addition to HLA-B/C, HLA-A FHC products are also detected in individuals bearing HLA A3 and/or A30 allotypes. The aim of this study was to establish whether such restriction results in the detection of low FHC levels in individuals lacking HLA A3 and/or A30 allospecificities. The FHC mean concentration (+/- SD) in 294 healthy blood/bone marrow donors (HBDs) was 0.24 (+/- 0.2) mg/l. The grouping of HBDs according to their HLA-A FHC product reactivity with one, both or no mAbs did not result in any statistically significant differences (Mann-Whitney test: P > 0.05) between their median FHC concentrations. Since the absence of differences in their FHC levels was not attributable to a difference in the percentage distribution of HLA allotypes associated with high or low HLA-B/C FHC expression, our results indicate that FHC HLA A3 and/or A30 products detected in DDIA by these two mAbs only minimally contribute to FHC serum expression and that the assay is not limited by the failure to detect HLA-A FHC products in A3- and/or A30- individuals.     

Population studies of HLA-linked SB antigens and their relative importance in primary MLC typing. Analysis of HLA-D homozygous typing cells and normal heterozygous populations

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propagated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium (p approximately equal to 0.20). Estimated gene frequencies in the German population appeared to be significantly different (p less than 0.002) from the North American Caucasian population: the principal differences were increased frequencies of the specificities SB1 and SB4, and decreased frequencies of blanks. Of HLA heterozygous donors 41% typed for two distinct SB specificities; 57% typed for one; and 2% were blank. In the HTC group, 20% typed for two specificities; 68% typed for one; and 12% were blank. Thus, a significant proportion of HLA-D homozygous test cells were, nonetheless, heterozygous for HLA-linked SB antigens. Performance of checkerboard mixed leukocyte cultures (MLCs) between 16 SB typed HLA-Dw3 HTCs, however, did not indicate that the observed mutual or one-way responses were influenced in any simple way by SB antigens; neither heterozygosity nor assumed homozygosity for SB antigens appeared to influence the frequency of MLC typing responses of HLA-Dw3-positive donors on these HTCs. These results add further confirmation of the genetic and functional independence of the SB gene product(s) and the HLA-D/DR gene product(s).     

An HLA class I specific monoclonal antibody that fails to bind to all HLA-A antigens

A monoclonal antibody, MHM.5, specific for HLA class I antigens, bound to lymphocytes of all donors tested and was thought to bind to a monomorphic determinant. When the antibody was used to precipitate 35S methionine labeled HLA class I molecules from lymphoid cells, which were then isoelectric focused, it was found that the HLA-A1,A2 and A3 antigens were not precipitated. Similarly, MHM.5, which is IgG1, failed to block complement mediated lysis by alloantisera specific for HLA-A1, 2 and 3, and most other HLA-A antigens. HLA-Aw24, A25, and A32, and all other HLA-B and C typing reactions tested were blocked. Thus the antibody binds to an epitope that is lacking on most A antigens, but present on Aw24, A25, A32 and all B and C locus antigens. Comparison of the published amino acid sequences of HLA-A2, A3, Aw24, A28, Cw3, B7, and B40 suggests some possible sites for this epitope.     

HLA-DR and HLA-A, B, C typing of human fetal tissue

In anticipation of clinical trials of fetal pancreas transplantation we have investigated the feasibility of performing HLA-DR and HLA-A, B, C typing on fetal lymphoid cells other than PBL. Using the standard NIH microcytotoxicity test modified for HLA-DR typing it was possible to demonstrate HLA-DR antigens on subpopulations of bone marrow cells and splenocytes but not on thymocytes or hepatocytes. In contrast, HLA-A, B, C antigens could be detected on all four tissues. Excellent HLA-DR typing, confirmed by maternal typing, was obtained for 19 fetuses (14 to 23 weeks old) using bone marrow cells isolated by two-fold purification on discontinuous Percoll buoyant density gradients. Similar purification of splenocytes resulted in weak reactions with anti-DR sera; however, adherent splenocytes recovered from nylon wool columns proved to be primarily DR-bearing and also provided excellent DR typing. As a corollary to these results, non-adhering splenocytes depleted of DR-bearing cells were ideal for HLA-A, B, C typing since spurious reactions due to DR antigens were greatly diminished, whereas strong specific reactions were obtained with anti-HLA-A, B, C sera. Despite weaker reactions with HLA-A, B, C antisera obtained for thymocytes, reliable HLA-A, B, C typing could be obtained when results from thymocytes were evaluated together with typing from bone marrow cells or splenocytes. The possible benefits of fetal HLA typing for fetal pancreas transplantation are discussed.     

HLA and IgA deficiency in blood donors

We have identified 67 IgA deficient healthy blood donors in our region by systematic screening of 24,782 blood samples. HLA typing results on 36 of these donors indicated a significant association with both HLA-A1 and HLA-B14 antigens. The frequency of A29 and B8 antigens was also increased. However, B8 association may be secondarily involved due to linkage disequilibrium (e.g., A1-B8-DR3). The frequency of HLA-A1 and HLA-B8 antigens was increased in the group of IgA deficient donors who developed anti-IgA antibodies (53.9%) compared to those who did not (26.1%). Although the sample size appears to be too small to show a statistical significance (chi 2 = 2.76, 0.05 less than p less than 0.1), this is the first report to imply a possible HLA association with anti-IgA antibody formation by IgA deficient healthy individuals.     

Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.     

Antigen-specific suppression of human antibody responses by allogeneic T cells. III. Role of the major histocompatibility complex

Specific antibody responses obtained in vitro from human blood mononuclear cells (PBM) were profoundly suppressed by allogeneic T cells. Experiments carried out with combinations of cells from HLA identical siblings, and HLA identical but unrelated donors, showed that suppression depended upon HLA incompatibility between responding PBM and allogeneic Ts. In order to map the specific HLA loci concerned, a series of experiments were undertaken using combinations of cells from a large number of HLA typed donors. Significant suppression was found to occur in every combination of HLA incompatible cells tested, including those with nonidentity at HLA-A, B, DR, A and DR, or B and DR, suggesting that suppression can be generated by nonidentity at class I or class II loci. With some HLA-A homozygous donors, however, a dominant role for class I (HLA-A) antigens was indicated by the finding of one-directional suppression in combinations where the HLA-A locus was seen as foreign by one partner only (A3,----A2,3; and A2----A2,26). Similar one-directional suppression was also seen with cells from a pair of siblings who were HLA identical except for a single A locus antigen arising from an HLA-A/B recombination (A3,----A3,1). These results indicate an important, but not exclusive role for class I MHC antigens in the activation of allogeneic Ts. The way in which this occurs is unknown, but one possibility is that it results from the activation of normal antigen-specific Ts by the interaction of their receptors for self-MHC with cross-reacting alloantigens.     

Characterization of the HLA-A polymorphism by locus-specific polymerase chain reaction amplification and oligonucleotide hybridization

This report describes a PCR-based typing protocol for the HLA-A polymorphism. Locus-specific primers selectively amplified HLA-A sequences from exon 1 to exon 3 in a single PCR that avoided co-amplification of other classical and nonclassical class I genes. The allelic variation in exons 2 and 3 of the HLA-A gene was examined with a set of 44 oligonucleotide probes. According to the recognized HLA-A sequences the protocol is potentially able to distinguish all known HLA-A alleles with unique nucleotide sequences in this gene region. The related HLA-A genotypes can also be identified in both homozygous and heterozygous individuals. Thus the protocol provides the highest resolution for HLA-A typing. The PCR-SSO typing technique is accurate, reliable, and particularly suitable for a large number of samples. The DNA typing results from 42 Tenth IHWS B-cell lines are compatible with the serologic and IEF definitions. Sixty-six unrelated donors from a northern Chinese population were also tested, with 16 HLA-A alleles detected. Four subtypes of HLA-A2 were found in this population. The distribution of HLA-A subtypes in the population indicated that 40% of donor-recipient pairs thought to be matched for HLA-A by serology would be mismatched. Two novel HLA-A alleles were identified by unusual oligonucleotide hybridization patterns.     

Anti-HLA-B7,B27,Bw42,Bw54,Bw55,Bw56, Bw67,Bw73 monoclonal antibodies: specificity, idiotypes, and application for a double determinant immunoassay

The monoclonal antibodies (MoAbs) KS3 and KS4 are secreted by hybridomas constructed with splenocytes from a BALB/c mouse sequentially immunized with the cultured lymphoid cells JKu and LG-2 which share only the HLA-B27 specificity. Serologic and immunochemical assays have shown that the two MoAbs recognize the same (or spatially close) determinant expressed by HLA-B7,B27,Bw42,Bw54,Bw55,Bw56,Bw67, and Bw73 alloantigens. This determinant is spatially close but distinct from those defined by the anti HLA-B27 monoclonal antibodies described in the literature. The syngeneic antiidiotypic MoAb T12-105 and T12-211 elicited with MoAb KS4 were shown to recognize idiotopes within the antigen combining site of MoAb KS3 and KS4. Neither idiotope was detected on the anti HLA class I and anti HLA class II monoclonal antibodies tested. The MoAb KS4 in combination with the anti human beta 2-microglobulin MoAb NAMB-1 was utilized to develop a double determinant immunoassay (DDIA). The latter represents a sensitive method to detect and quantitate HLA-B27 antigens in spent culture medium of lymphoid cell lines and in serum. Typing for HLA-B27 antigens with the DDIA of sera from HLA typed donors yielded results highly correlated with those of the conventional lymphocytotoxicity assay.     

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.     

Enzyme-linked immunosorbent assay for determination of HLA: gene dose effect

In a previous publication we demonstrated that polymorphic HLA antigens could be detected on fresh and dried peripheral blood lymphocytes using the enzyme-linked immunosorbent assay (ELISA) with HLA alloantisera (Bishara et al. 1983). In the present study we investigated whether the ELISA technique can be used in determination of gene-dose effect for antigens of the HLA-A and B loci. Lymphocytes from HLA-A1 and HLA-B14 homozygous individuals are shown to bind significantly more anti-HLA alloantibodies than their heterozygous siblings for the same HLA antigens. These results indicate that ELISA is a sensitive and reliable technique for the qualitative and quantitative assay of polymorphic HLA determinants.     

HLA-A2 class I antigens in couples with recurrent spontaneous abortions

Class I human leukocyte antigen (HLA) antigens, locus A and B, were typed in fertile and infertile couples in cases where one of the spouses carried the HLA-A2 antigen. HLA-class I typing data were obtained from 282 participants, 63 fertile couples and 78 infertile couples with recurrent spontaneous abortions (RSA). Locus A antigens were grouped into eight broad specificities (A1, A2, A3, A9, A10, A11, A19, A28) and locus B antigens were grouped, according to HLA epitopes, in two classes (BW4 and BW6). Although the number of cases is small, significant differences in the distribution of locus A antigens were found between HLA-A2-positive (A2+) women from fertile and infertile couples. HLA-A3, A11 and A28 cross-reacting antigens were absent in women from fertile couples and present in women from infertile couples. HLA-A19, which is associated with amino acid triplets of low immunogenicity, was significantly more often observed in A2+ fertile than in infertile women. An excess of the BW4 epitope was found in A2(-) husbands from infertile couples compared to fertile ones. The results of this study support the idea that in the presence of the HLA-A2 molecule the distribution of HLA-A and B loci antigens may be different in fertile couples compared to couples with recurrent spontaneous abortions. It can be suggested that the HLA-A2 molecule, in context with specific genotypes, may contribute to the overall maternal immune response in normal and disturbed pregnancy.     

Reanalysis of sequence-based HLA-A, -B and -Cw typings: how ambiguous is today's SBT typing tomorrow

The permanently increasing number of human leukocyte antigen (HLA)-alleles and the growing list of ambiguities require continuous updating of high-resolution HLA typing results. Two different kinds of ambiguities exist: the first, when two or more allele combinations have identical heterozygous sequences, and the second, when differences are located outside the analyzed region. The number of HLA-A, B and C alleles recognized in 1999 was almost tripled in 2006. Two hundred individuals, sequence-based typing (SBT) typed in the period from 1999 to 2002, were reanalyzed using the 2006 database. A final allele typing result of at least four digits was obtained for HLA-A, -B and -C by heterozygous sequencing of exons 2 and 3 and, if necessary, additional exons and/or allele-specific sequencing. Storage of the individual sequences in a specially developed database enabled reanalysis with all present and future HLA releases. In the 5-year period HLA-A, -B and -C typing results became ambiguous in 37%, 46% and 41% of the cases. Most were because of differences outside the analyzed region; ambiguities because of different allele combinations with identical heterozygous sequences were present in 7%, 8% and 13% of the HLA-A, -B and -C typings. These results indicate that within 5 years, approximately half of the HLA SBT typings become ambiguous.     

Study of HLA system in a Mataco population:

A search for antigens of the HLA system has been carried out in 53 Mataco Indians of Argentina living in a geographically isolated area in the northeast of the country. Samples were mostly collected from adults of both sexes who were not directly related.
Lymphocyte typing was performed using the microcytotoxicity technique of NIH. 118 sera specific for 15 antigens of the first HLA locus, 22 antigens of the second and 6 of the third were used.
The most frequently found alleles were HLA-A28, Aw31 and A2 for the first locus; B15 and B40 for the second; and Cw3 and Cw4 for the third.
In addition to previously published investigations on South American Indians, our typing work shows a remarkable homogeneous gene pool and a restricted range of polymorphism; therefore, a further set of haplotypes rendered us also restricted. The most frequent haplotypes that showed a significant statistical linkage desequilibrium were: A2-Cw4, A28-Bx, A2-Cw3, Aw31-Bw16, Aw24-Cw3, B15-Cw3, Bw16-Cw3 and A28-B5.
Some of these haplotypes have also been found in other indian populations.     

A new set of reagents and related software used for NGS based classical and non-classical HLA typing showing evidence for a greater HLA haplotype diversity

To evaluate the HLA typing performance of a new Long-Range PCR NGS set of reagents and its dedicated software, a panel of 41 reference homozygous cell lines from the International Histocompatibility Working Group (IHWG) and a panel of 376 volunteer bone marrow donors were analyzed for classical and non-classical HLA class I and class II genes. All results, except HLA-DPB1, were obtained without any ambiguities at the 3rd field level. Based on the high resolution performance of the reagents, a number of new alleles have been described not only for classical but also for non-classical HLA class I genes, leading to a more accurate haplotype definition. Linkage disequilibrium between HLA-A and HLA-G genes has been defined at 4th field level of resolution. Moreover, for the first time, HLA-DQA2 and DQB2 polymorphisms and their linkage disequilibrium with DQB1 were described.     

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-A, B, C and DR antigens in immunoglobulin A deficiency

HLA-A, B, C and DR typing was performed in 21 unrelated healthy blood donors with selective IgA deficiency (< 0.02 G/l of IgA). A significant increase in HLA A1 ( P < 0.05), HLA B8 ( P < 0.01) and HLA DR3 ( P < 0.001) was found. The frequency of HLA A28 was also increased ( P < 0.05). Furthermore, HLA A28 was present in all four donors lacking DR3.