Use of monocytes in HLA-A,B, C and DR typings

A simple method of improved serologic typing of monocytes for HLA-A, B, C and DR specificities is described. The method employs monocytes recovered from frozen samples of peripheral blood mononuclear cells; it chiefly involves pretreatment of monocytes with 0.01% iodoacetamide (IAA) prior to typing. The advantage of this method lies principally in the lowering of the background nonspecific cytotoxicities and false positive readings upon IAA addition to the monocyte preparations. Using this method monocytes can be typed for HLA-A, B, C determinants. Although the addition of IAA results in substantial typing improvements, we found the assignment of A, B, C specificities difficult due to the presence of extra positive reactions when monocytes were compared to T lymphocyte typings. probably due to the presence of DR or monocyte specific antibodies in the routinely used HLA antisera. This method proved to be most useful in DR typings where mono cytes in the presence of IAA were compared with autologous B cells in the absence of IAA. The differences in typings due to a decrease in false positive cytotoxic readings were significantly in favor of using IAA treated monocytes in DR typings (P < 0.0001). The use of IAA in the course of B cell or T cell typings bad no adverse consequences on either A, B, C or DR typings, respectively. Our results indicate a potential usefulness for the use of IAA in typing monocytes HLA determinants in general and for the DR determinants in particular.     

Avoidance of Certain Systematic Pitfalls in the Detection of HLA–DR Antibodies Defining Fine Specificities

A selective screening program has been established to identify rapidly and effectively the fine specificity of HLA-DR antibodies in pregnancy anti-HLA sera. Following initial HLA-A, B, C screening sera with extra- or multispecific reactions were selected and specifically tested after platelet absorption on isolated B- and T-lymphocyte populations of the serum donor's husband. Identification of the HLA-DR type of the husband, as well as screening and typing on HLA, A, B and -C typed heterozygous panel B cells led to a more precise characterization of the major specificity of a detected anti-HLA-DR serum. Multispecific anti-DR sera were defined and rendered operationally monospecific by titration.
Some critical steps in a reliable assessment of HLA-DR typing reagents could be worked out. Weak HLA-A, B antibodies, B-cell auto- and Lewis antibodies may cause positive reactivity preferentially or even selectively on B lymphocytes. Of particular importance was the hidden presence of HLA-C specific antibodies, since they cannot be absorbed out by stored platelets. In addition they are not readily detectable through screening on typed panel cells. Because of the frequently very high linkage disequilibrium between HLA-B and HLA-C alleles it is difficult to select appropriately dissecting panel cells. The two points demonstrated above gain even more weight when isolated T and B cell populations are used for HLA-DR typing, because HLA-C antibodies preferentially kill B cells. In this fashion contaminating HLA-C antibodies are not only difficult to detect but can mimic the presence of HLA-DR antibodies.     

Hydatidiform mole and HLA. Methods for HLA-A,B,C determination

The current Danish project on genetic markers and hydatidiform moles involves the examination of the histocompatibility between patient, spouse and molar tissue. For this purpose 2 techniques have been developed for HLA-A,B determination of molar tissue: (a) a cytotoxic microtechnique for the HLA typing of cultured molar cells, and (b) a microabsorption method using selected HLA antisera for the examination of frozen molar tissue. To date molar cell cultures or tissue have been examined in 22 of the cases. The results of HLA determination were compared with chromosomal markers to elucidate the origin of the haploid chromosome complements. In the cases examined a general agreement between the HLA-A,B type and the origin of the chromosomes was observed. The microabsorption method used seems to be less sensitive than the cytotoxic technique.     

There are multiple regulators of expression throughout the MHC

We have previously reported the use of deletion mutants to investigate the location and function of new MHC genes. Several new examples of these will be reported using a cell panel of deletion mutants derived from a heterozygous parental cell (A2-B44-DR12-DQ3//A1-B8-DR13-DQ1) and selected for the loss of HLA-A2 following spontaneous mutation or treatment with x-rays or mutagens. The extent of the haplotypic loss of the mutants has been characterised by serology, by Block typing and by using multiple heterozygous CA repeat markers on 6p. The panel of deletion mutants has been used to screen serological reagents. It has been found that some antibodies suggest reduced levels of expression of HLA-B44 and B8 (AHS#6 6034, 6039) in a mutant following deletion in a region close to D6S248 but not including HLA-A. This has been confirmed by flowcytometry using a B8 MoAb. Following deletion of HLA-A2, the expression of HLA-B was apparently restored. Further AHS#6 serum 6051 was found to have undefined non HLA extra reactivity in addition to HLA A10 and B16. It reacted strongly with the original cell and all mutant panel cells except for two cells in which the deletion included a segment between D6S265 and D6S273 (includes HLA-B). With further deletions centromeric of D6S273, cytotoxicity was restored. These data imply that there are several unidentified up and down regulatory elements within the MHC which react in cis and trans combinations. Serological techniques and deletion mutant panels are informative for the localisation of HLA regulatory elements, new genes, cofactors and/or conformational elements affected by the deletions.     

HLA class I sequence-based typing using DNA recovered from frozen plasma

We describe a rapid, reliable and cost-effective method for intermediate-to-high-resolution sequence-based HLA class I typing using frozen plasma as a source of genomic DNA. The plasma samples investigated had a median age of 8.5 years. Total nucleic acids were isolated from matched frozen PBMC (~2.5 million) and plasma (500 μl) samples from a panel of 25 individuals using commercial silica-based kits. Extractions yielded median [IQR] nucleic acid concentrations of 85.7 [47.0-130.0]ng/μl and 2.2 [1.7-2.6]ng/μl from PBMC and plasma, respectively. Following extraction, ~1000 base pair regions spanning exons 2 and 3 of HLA-A, -B and -C were amplified independently via nested PCR using universal, locus-specific primers and sequenced directly. Chromatogram analysis was performed using commercial DNA sequence analysis software and allele interpretation was performed using a free web-based tool. HLA-A, -B and -C amplification rates were 100% and chromatograms were of uniformly high quality with clearly distinguishable mixed bases regardless of DNA source. Concordance between PBMC and plasma-derived HLA types was 100% at the allele and protein levels. At the nucleotide level, a single partially discordant base (resulting from a failure to call both peaks in a mixed base) was observed out of >46,975 bases sequenced (>99.9% concordance). This protocol has previously been used to perform HLA class I typing from a variety of genomic DNA sources including PBMC, whole blood, granulocyte pellets and serum, from specimens up to 30 years old. This method provides comparable specificity to conventional sequence-based approaches and could be applied in situations where cell samples are unavailable or DNA quantities are limiting.     

The utility of class I HLA antibodies screening in a Tunisian obstetric department in order to select good HLA typing reagents

In order to reduce the cost of the serologic class I HLA typing, we have established a screening program for HLA antibodies among obstetric patients. Class I HLA antibodies were identified by standard microlymphocytotoxicity test using a panel of 100 lymphocytes. In the study period, carried out between January 2000 and June 2002, 817 women were tested, most of them (62%) during the last trimester of pregnancy and in the other cases after delivery. These patients, aged between 18 and 45 years, were in the majority (88%) multiparous. From the total number of 817 maternal serum samples screened, 194 specimens (23,74%) tested positive for HLA antibodies. Thirty eight HLA specificities were characterised in 110 maternal sera of which 62 contained monospecific HLA antibodies. HLA-A2 and HLA-B51(5) were the most common specificities characterised in this study. HLA alloimmunization was present since the first pregnancy. There was no statistically significant linear correlation between HLA alloimmunization and the number of pregnancies. Fifty maternal serum samples (6,11%) could be used as HLA typing reagents. Of these 50 sera, 36 had monospecific HLA antibodies. These useful antisera covered a total of 30 specificities: 8 HLA-A and 22 HLA-B. The cost of self - screening for useful antisera as estimated at dollars 62/mL, the personnel was not considered. However, the lowest cost of commercial HLA typing sera is approximately dollars 400/mL. Our study showed the utility and the net economic advantage to use maternal sera as reagents in our new HLA laboratory with limited budget.     

Techniques used to define human MHC antigens: serology

The classical, routine test employed for definition of HLA antigens expressed in humans (tissue typing) is the complement-mediated cytotoxicity assay developed by Terasaki and McClelland in the early 1960s. In both healthy persons and patients, the assay target cells are usually lymphocytes obtained from peripheral blood, but when typing cadaver donors, splenic or lymph node lymphocytes can be used. HLA-A, B, Cw (class I) antigens are expressed on all nucleated cells while HLA-DR, DQ (class II) are restricted to B lymphocytes and immune activated cells. Tissue typing has been achieved using culture cells from amniocentesis and typing of cell lines is possible with small modifications to the standardised cytotoxicity assay. Usually, target cells are incubated under oil with typing antisera at 22 degrees C in a 60- or 72-well Terasaki tray. After 30 min rabbit serum is added as a source of complement. After a further 60 min incubation the test is stained. A positive reaction results in target cell death. There are local variations to this test. Automation of the assay is now commonplace, from reagent dispensing to automated reading of finished assay. The use of antibody-coated magnetisable microspheres has enabled separation of pure B lymphocyte samples for class II typing and has reduced incubation times through antigen modulation. It is possible to define antibodies to HLA antigens in the same assay using target cells with known HLA phenotypes.     

Two monoclonal antibodies detecting allotypic determinants of HLA-A

Three mouse hybridomas producing cytotoxic antibodies against HLA were established. By standard microcytotoxicity test against panels of normal controls, the antigen defined by MA-9 antibody (IgM) showed a good correlation with HLA-A9 alloantigen detected by conventional typing alloantisera (r = 1.0). Family studies also showed that MA-9 determinant segregated with HLA-A9. MA-10 antibody (IgM) reacted with all HLA-A10 positive lymphocyte donors and cross-reacted with two thirds of HLA-AW33 positive donors. M1-1 antibody (IgG2a) reacted with all the panel cells tested and immunoprecipitated a molecule of 43,000 daltons from Nonidet P-40 lysates of ³H-glucosamine-labelled cells. The results showed that MA-9 and MA-10 antibodies can be used as routine tissue typing reagents.     

Expression and immunogenicity of HLA-B27 in high-transfection recipient P815: a new method to induce monoclonal antibodies directed against HLA-B27

The immunization of a (BALB/c x C57BL/6) FI mouse with murine transfectants expressing the HLA-B27 antigen resulted in a panel of polymorphic monoclonal antibodies with specificity for HLA-B27 and some additional HLA-antigens. Specificity of the antibodies was defined by cytofluorometric analysis on a panel of lymphoblastoid cell lines (LCL) derived from HLA typed individuals. Three of these antibodies are cytotoxic, and one of them inhibits B27-specific T cell cytotoxicity. Our results indicate that HLA-class I transfectants could be used to generate polymorphic antibodies, and that these antibodies may be helpful for HLA typing and for definition of epitopes recognized by T cells.     

A mouse monoclonal antibody detecting the allospecificity HLA-A3

Balb/c mice were immunized with a human B-lymphoblastoid cell line typed HLA-A3, B7. The splenocytes of the immunized mice were fused with a murine myeloma. Supernatants of the cultures were screened against the immunizing cell line in fluorochromasia. Positive cultures were expanded and cloned. One of the clones, X 15.4, was expanded and brought to ascites in Balb/c mice. Monoclonality of the antibody X 15.4, which belongs to the class IgM and immunoprecipites a molecule of 44000 daltons, was demonstrated by isoelectric focusing. By complement dependent citotoxicity the ascites only reacted with the lymphocytes of all HLA-A3 individuals from a panel of 146 donors, showing no crossreactions. X 15.4 appears to be one of the very rare xenomonoclonal antibodies suitable for HLA typing.     

Biochemical HLA typing: a population study in 112 Caucasians

In order to define frequencies and associations of biochemically defined HLA-A and -B specificities and their variants and subtypes in a Caucasian population, biochemical HLA typing was performed in a panel of 112 Austrians. Already known rare variants of HLA-A2, A3, A30, Aw33 and B39 and the more frequent subtypes of HLA-B27, B35, B44 and, in addition, a so far unknown variant of HLA-B18 were present in the panel. Family segregation analyses of the biochemically defined HLA class I variants revealed that they could be found only in certain rare haplotypes, most of them in high linkage disequilibrium. The basic variant of HLA-A30, for example, obviously occurred only in the HLA-A30, B49, Cw7 haplotype, similar to the Aw33 acidic variant, which was exclusively found in the Aw33, Bw58, Cw3 haplotype. None of the biochemical variants was found in frequent common haplotypes.     

Quantitative and qualitative differences in the distribution of HLA class I antigenic determinants in the human thymic compartments

The topographical distribution of HLA class I antigens has been investigated by the immunoperoxidase technique on normal frozen thymic tissue sections with a panel of mono- and polymorphic monoclonal anti-HLA antibodies. HLA class I framework determinants detected by the monoclonal antibody W6/32. HL were present on 80–90% of cortical thymocytes, as well as on all cortical epithelial and medullary cells. However the staining intensity of cortical thymocytes with this reagent was about threefold weaker than that of the medullary thymocytes. Labeling patterns of selected monoclonal antibodies against matching HLA-A/B allospecificities revealed striking variations in the quantitative expression of certain HLA-A vs. HLA-B locus alloantigenic determinants on cortical thymocytes compared to a consistent staining on almost all medullary cells.     

Assessment of fidelity and utility of the whole-genome amplification for the clinical tests offered in a histocompatibility and immunogenetics laboratory

Increasing emphasis on the use of molecular tests in a histocompatibility and immunogenetics laboratory (HIL) poses a potential problem of lack of sufficient DNA to perform multiple genetic analyses. In this study, we report the feasibility, fidelity and utility of multiple displacement amplification (MDA) method to perform whole-genome amplification (WGA) to generate DNA specimens that can be analyzed by multiple molecular techniques and can be used for different clinical tests offered by an HIL. The MDA-generated DNA when compared with the native DNA showed 100% congruency in genotyping of 37 genes/loci using multiple downstream molecular techniques: sequence-based typing and sequence-specific primer-based typing for 5 human leukocyte antigen (HLA) class I and II genes (HLA-A, B, C, DRB1 and DQB1), luminex-based sequence-specific oligonucleotide (SSO) genotyping for a panel of 16 killer immunoglobulin-like receptor (KIR) genes and automated fragment size analysis for a panel of 15 short tandem repeat (STR) loci and amelogenin gene. For post-allogeneic hematopoietic cell transplantation (HCT) chimerism analysis, MDA-generated DNA appeared useful for enriching pre-transplant DNA but not for enriching post-transplant chimeric DNA. Overall, our results show that MDA-based WGA could generate DNA of high yield and fidelity that can be used for various clinical tests and research purposes.     

QUANTITATIVE EXPRESSION OF HLA CLASS I MOLECULES IN ACUTE NON-LYMPHOBLASTIC LEUKAEMIA CELLS

The present study concerns a panel of 33 acute non lymphoblastic leukaemia (ANLL) patients, previously typed for HLA-A,B serological specificities and including samples with a normal HLA-A,B phenotype (3,4 detected specificities) as well as samples with missing and extra specificities. Samples were analysed at the protein and/or RNA level in order to verify whether the observed typing anomalies were due to a modified quantitative expression of class I molecules. The number of HLA-A.B assigned specificities correlated significantly with the cell surface class I expression detected by indirect immunofluorescence using the monomorphic anti-class I MoAb W6/32 (Spearman rank correlation test, P < 0.01) and with the amount of class I Heavy Chain (HC, P < 0.05) and beta-2-microglobulin (β2m, P < 0.05) evaluated by Western blot in whole cell extracts. The RNA analysis suggested a HC-β2m coordinated down regulation at the mRNA level in a patient with no assigned HLA-A,B specificities. Another patient with no detectable HLA-A,B specificities showed a low expression selectively of the β2m protein. The results reported here demonstrate a heterogenous quantitative HLA class I expression in ANLL blasts, analogous to results reported for solid tumours.     

A microELISA assay for detection of anti-HLA activity of mouse monoclonal antibodies using an Astroscan 2100 automated plate reader.

A microenzyme-linked immunosorbent assay (microELISA) method has been developed using an Astroscan 2100 system automated plate reader which was initially designed for tissue typing by a two colour fluorescent microcytotoxicity assay. A 96-well plate ELISA used for screening mouse monoclonal antibodies raised against surface HLA antigens has been modified for use with the Astroscan plate reader and 72-well typing trays. The existing substrate 4-methylumbelliferyl-beta-D-galactoside (4MUG) has been replaced with fluorescein-di-beta-D-galactopyranoside (FDG), to provide a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes have also been reduced tenfold for use with Terasaki microtest plates. The assay now has the major advantage of requiring only 5 microliters of test supernatant allowing hybridomas to be screened earlier during a fusion and on a wider cell panel. The use of the large panel which includes B lymphoblastoid cell lines (B-LCL) and mouse L cell transfectants expressing HLA genes, reduces the length of time the hybridomas need to be kept in tissue culture before selection. Other advantages include the reduction in the number of target cells required, smaller volumes of reagents throughout the assay and the ability to screen cytotoxic as well as non-cytotoxic monoclonal antibodies. The sensitivity of this microELISA proved to be comparable with the original assay and so provides an efficient screening method for monoclonal antibodies.     

Resolution of HLA class I sequence-based typing ambiguities by group-specific sequencing primers

The increasing demand for allele-level human leukocyte antigen (HLA) typing has led the sequence-based typing (SBT) to become the preferred method. In turn, the steady increase in the number of HLA alleles driven by the adoption of SBT as the ultimate typing method leads to the ever increasing number of cis/trans ambiguities. Over the last few years, additional sequencing with the commercially available group-specific sequencing primers (GSSPs) has replaced sequence-specific primer-polymerase chain reaction and group-specific amplification as the means of resolving cis/trans ambiguities in many laboratories. Here we summarize our 3-year experience in designing and utilizing GSSPs for resolution of HLA class I ambiguities. The panel of GSSPs used in our laboratory includes 14 primers for HLA-A, 18 for HLA-B, and 13 primers for HLA-C. The panel resolves 99.9% of all ambiguities.     

Sera from Volunteers Immunized by Planned Blood Transfusions as a Source of Dr Cytotoxic Typing Reagents

Sera from volunteers immunized with planned blood transfusions were tested for anti DR cytotoxic antibodies with a panel of HLA typed cultured human lymphoid cells using a variety of serological techniques. The majority of sera contained DR cytotoxic antibodies. The specificity of DR antibodies in seven sera was determined by testing them with a panel of B peripheral lymphocytes typed with DR alloantisera submitted to the 7th International Histocompatibility Workshop. The temporal evolution of DR and HLA-A and B cytotoxic antibodies was determined in two subjects by testing serial bleedings with B lymphoid cells, coated with Fab₂ fragments from anti β₂μ and anti DR xenoantisera. Results indicated a parallel evolution of DR and HLA-A and B cytotoxic antibodies.     

Two cytotoxic human-human hybridoma antibodies to HLA: TrAH10 (anti A3.1) and TrAG2 (anti B7,Bw42)

Two cytotoxic human-human hybridoma IgM antibodies to HLA were generated by EBV transformation of PBMC from multiparous women and fusion of EBV transformed cells with the human fusion partners KR4 or KR12. Both mAbs required the sensitive immunomagnetic cytotoxicity method to display killing of freshly prepared PBMC. One mAb (TrAH10) was specific for HLA-A3. Strikingly, TrAH10 reacted much more strongly with lymphoblastoid cell lines of HLA-A3.1 than of the rare variant HLA-A3.2, previously detected by cytotoxic T cells. Thus, in the microcytotoxicity test, the titer of concentrated TrAH10 was approximately 2000 times higher for A3.1 as compared to A3.2, and a clear difference was also observed in radioimmunoassay. Since the two HLA-A3 variants differ by only two amino acids at positions 152 and 156 of the alpha 2-domain's alpha-helix, the epitopes defined by the mAb TrAH10 and HLA-A3.1 specific cytotoxic T cells must be closely related. The observations with TrAH10 suggest that the HLA polymorphism detected by human mAbs may turn out to be as extensive as the T-cell defined HLA polymorphism. The other mAb (TrAG2) bound B7 and Bw42 with equal strength, and in addition bound weakly to some cells that were Bw22 or B39. Magnetic polymerbeads coated with affinity purified human mAbs TrAH10 or TrAG2 formed rosettes with EBV transformed cells carrying relevant HLA antigens; however, rosette formation with freshly isolated PBMC was very weak and unsuitable as a typing assay.     

HLA-A2 as a target for cell-mediated lympholysis: Evidence from immunoselected HLA-A2 negative mutant cell lines

Cloned mutants of the human B lymphoblastoid cell line SB have been isolated using mutagenesis with ethyl methanesulfonate followed by negative selection with an anti-HLA-A2 serum and complement. Absorption analysis with ¹²⁵l Staphylococcus aureus protein A binding to antibody sensitized cells. HLA typing, and immune precipitation analysis showed the mutants to be serologically identical to the SB parent except for the loss of HLA-A2. When tested as target cells for cell-mediated lympholysis by cytotoxic T lymphocytes generated in the mixed lymphocyte response, the SB and mutant cell lines demonstrated comparable susceptibility when the putative targets were HLA antigens other than HLA-A2. However, when compared for susceptibility to lysis by cytotoxic T lymphocytes considered to be HLA-A2 specific, the SB parent was effectively killed whereas little or no killing of the HLA-A2 mutants was observed. The results provide a new line of evidence that HLA antigens recognized by antibody can also be the true molecular targets for cytotoxic T lymphocytes.     

Rapid and simultaneous HLA class I (-A, -B and -C loci) DNA typing using the microtitre plate-reverse hybridization assay (MRHA).

We have established a precise, rapid, simple and practical HLA class I DNA typing method using the microtitre plate-reverse hybridization assay (MRHA), which enables us to perform simultaneous DNA typing of the HLA-A, -B and -C loci using the same PCR parameters and hybridization conditions. PCR-amplified products for the HLA-A, -B and -C loci were hybridized, respectively, with sequence-specific oligonucleotide (SSO) probes, which were immobilized covalently onto a microtitre plate, in hybridization buffer containing formamide at 37 degrees C. After washing at room temperature, the bound PCR products were detected by peroxidase-conjugate streptavidine followed by colour development such as enzyme immunoassay (EIA). In addition to the simple thermoregulation for hybridization and postwashing, strong positive signals, low background and high reproducibility, this DNA typing method enabled simultaneous typing of the HLA-A, -B and -C loci using a single microtitre plate as in HLA serotyping. The assignment of the HLA genotype was easily achieved by automated colorimetric reading and computer software, based on the cut-off value (threshold) established for each probe. For routine HLA class I typing, it may be possible to replace serological typing with the HLA class I DNA typing system using our MRHA method.