Mixed lymphocyte reactions do not predict severity of graft versus host disease (GVHD) in HLA-DR compatible, sibling bone marrow transplants.

Mixed lymphocyte reactions (MLRs) were measured in 25 HLA-A, B and DR compatible sibling bone marrow transplants. Only four of 25 MLRs were positive and in these the low reactivity was of doubtful clinical importance. There was no correlation between MLR and the subsequent development or severity of graft versus host disease (GVHD). A survey of bone marrow transplant units in the United Kingdom showed that most centres perform HLA-DR typing as well as an assessment of the MLR. Factors other than histocompatibility are important in the pathogenesis of GVHD and the data from this study suggest that conventional MLRs can be omitted in HLA-A, B and DR compatible sibling bone marrow transplants.     

Typing of human fetal organs for the histocompatibility antigens A, B and DR

In the transplantation of human fetal pancreatic explants into diabetic man, the importance of matching the histocompatibility antigens of donor and recipient to decrease the chances of rejection is unknown. Before this question can be answered human fetuses must be tissue typed. We have shown that lymphocytes harvested from fetal liver, thymus, bone marrow and spleen can be successfully HLA DR typed in 64% and A and B typed in 57% of 58 fetuses aged 15 wk or more. Typing should ideally be carried out on unseparated T and B cells. Best results were achieved if all four of the above organs were available and more than one million viable cells were able to be harvested for typing. Whilst the DR antigens could be typed from all tissues, the A and B antigens could be typed, with few exceptions only from thymus, spleen and bone marrow. The efficacy of matching the histocompatibility antigens of recipient and donor fetuses, especially the DR antigens can now be tested in the human diabetic being transplanted with pancreatic explants.     

“Bare lymphocytes” without immunodeficiency

“Bare lymphocytes”, which cannot be typed for the HLA-A,B, and C antigens were observed in two siblings, nine and six years of age. The elder child presented with aplastic anemia and was being considered for bone marrow transplantation. The younger child was healthy. The inability to phenotype both children for these three gene products persisted throughout the 21-month period of observation. The DR antigens were demonstrable which rendered it possible to deduce their A, B, and C genotype from the typing of the other four family members. Although alloantisera failed to detect the antigens on peripheral blood lymphocytes, monoclonal antibodies demonstrated reduced amounts of the HLA-A,B, and C antigens on the cells. The reduced level was confirmed following EBV transformation of the cells. After prolonged culture, HLA antigens immunoprecipitated from the cell extracts were normal in amount, molecular weight, and polypeptide composition. Southern blot analysis did not reveal gross genomic rearrangements. A regulatory defect leading to the expression of these Class I antigens is postulated.     

Evaluation of HLA-class II identity between unrelated individuals by serological typing, DNA-RFLP method, and mixed lymphocyte reaction

Seven groups, each consisting of two to nine unrelated HLA-A, -B, and -DR serologically identical individuals, were analyzed by DNA-restriction fragment length polymorphism (RFLP) and mixed lymphocyte reactions (MLR) in order to evaluate HLA-class II identity between unrelated individuals and to assess the importance of HLA-class II incompatibilities detected by DNA-RFLP in the allogeneic reactions. It is clear that DNA-RFLP represents a powerful typing method for HLA-DR, -DQ, and -DP since the combinations of the RFLP band patterns define all the serological specificities and most of the cellular specificities to give a highly accurate typing. This report shows that an HLA-DP incompatibility induces proliferation in primary mixed lymphocyte culture (MLC) between unrelated HLA-A, -B, -DR, -DQ, and -DW identical individuals, which may suggest the importance of this molecule as a transplantation antigen, especially for unrelated bone marrow transplantations. Still, an isolated HLA-DPw4/HLA-DP a disparity did not induce any proliferation in MLC. Moreover, our results show that DQw7 (w3)/DQw8 (w3) disparity associated with HLA-DR4 represents a nonfunctional incompatibility in MLR. The HLA-Dw subtypes of HLA-DR specificities can induce a high proliferative response in MLC. The HLA-Dw subtypes of HLA-DR specificities can induce a high proliferative response in MLC. Finally, DNA-RFLP typing represents a reliable method for the selection of histocompatible donor-recipient pairs and could potentially reduce many logistic problems and delays in live-donor transplantation, especially for unrelated bone marrow transplantation.     

A simple and rapid method for the detection of lymphocytotoxic antibodies using cell panels frozen on Terasaki plates

A 40 cell panel of lymphocytes selected for HLA-A,B and DR antigens, frozen in Terasaki microtest trays can be used routinely to identify the presence of specific HLA antibodies within two hours. Blind testing using well defined HLA typing sera showed that specificities could be identified to a high degree of significance using this method. The method has proved successful for screening for T cell, including HLA-A,B and B cell, including HLA-DR antibodies. The method is particularly useful for the routine tissue typing laboratory as the frozen panel can be used without the need for complicated and time-consuming cell washing procedures which have been the downfall of previously published methods.     

Non-polarized cell surface expression of HLA-A,B,C and HLA-DR antigens in Graves' thyroid follicle cells.

The intrathyroidal distribution and cell surface location of HLA-A,B,C and HLA-DR antigens was studied in polarized thyroid follicle cells from Graves' (n = 11) and normal (n = 3) thyroid tissue, using light and electron microscopy. Cryosections and isolated, open follicle segments were incubated with monoclonal antibodies against HLA-A,B,C and HLA-DR antigens and with patient sera containing autoantibodies against the microsomal antigen/thyroperoxidase. Immunoreactivity for HLA-A,B,C and HLA-DR on isolated thyroid follicle cells was frequently detected in Graves' disease, but absent in normal glands. There was a large variation in the immunolabelling between follicles as well as between different glands. Both HLA-A,B,C and HLA-DR immunoreactivity were detected on the apical and the basal surface of the follicle cells. Microsomal antigen/thyroperoxidase immunoreactivity was restricted to the apical cell surface. In contrast to normal tissue, HLA-DR positive cells with a dendritic or macrophage-like morphology were frequent in Graves' tissue. These cells adhered directly to the basal surface of isolated follicle segments. We conclude that HLA antigens are, unlike thyroid-specific plasma membrane constituents, expressed in a non-polarized manner at the surface of the follicular epithelium. These observations might have implications on the immune recognition of thyroidal autoantigens in Graves' disease.     

Antisera Recognising HLA-A, -B and DRW Antigens Raised in Rhesus Monkeys

Rhesus monkeys were immunized with partially purified HLA-A, -B, -C and DR antigens. The resulting sera were shown to have activity against species-specific determinants on both HLA-A, -B, -C chains and beta 2 microglobulin by the use of somatic cell hybrids. When this was removed by absorption, the sera showed activity against three of the four HLA-A and -B antigens in the immunogen when tested on a panel of peripheral blood lymphocytes and T cells. Antibodies recognizing HLA-DR antigens were detected by testing platelet absorbed sera on a panel of typed lymphoblastoid cell lines. After absorption to remove activity against species-specific determinants on the HLA-DR antigens, two cross reacting specificities were defined. One consisted of a determinant in common between HLA-DRw1, 2 and 6 and the other a putative determinant in common between HLA-DRw4, and 5. The nature and significance of these cross-reacting groups of HLA-DR antigens is discussed in the light of current HLA-DR serology and the nature of HLA antigens in general.     

Reproducibility of HLA-A, B, and DR typing using peripheral blood samples: results of retyping in the collaborative corneal transplantation studies. Collaborative Corneal Transplantation Studies Group (corrected)

Recipients enrolled in the Collaborative Corneal Transplantation Studies were retyped as part of a quality assurance program. Because the observed percentage of HLA-DR homozygosity on original typing was more than twice as high as expected from CCTS allele frequencies, the sample selected for retyping was heavily weighted with patients whose original typing identified fewer than two DR antigens. Retyping was performed in a different laboratory from the laboratory performing the original typing. For the 129 patients who were retyped, agreement between the original and retyping laboratories was 88% for HLA-A, 79% for HLA-B, and 55% for HLA-DR. When criteria was relaxed to consider only discrepancies involving readily identifiable antigens, the agreement improved to 95% for HLA-A, 91% for HLA-B, and 59% for HLA-DR. Identification of a second HLA-DR antigen on retyping when only one DR antigen had been identified on original typing was by far the most common form of disagreement. There were no significant differences in the amount of disagreement among the laboratories. Of special interest is that 50% of the discrepancies involved DR3, DR5, and/or DR6, which have structural similarities. Based on the results of the project, we recommend: (1) replicate testing for all DR typing; and (2) retyping using a second source of antiserum for all subjects having DR blanks.     

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.     

The MHC in human bone marrow allotransplantation

In this chapter, we have considered the theoretical and practical background of bone marrow transplantation. The immune response and its regulation by genes within the major histocompatibility complex, particularly of the I region of the mouse and of the HLA-D/DR region in man, is of central importance in both graft acceptance (rejection) and graft-versus-host disease. Methods which are available for typing alleles at the HLA-A, -C, -B, -DR and complotype (BF, C2, C4A, C4B) loci, have been considered in detail. The extent to which recombination affects specific alleles on haplotypes within families is discussed, as is the occurrence of linkage disequilibrium and extended haplotypes in populations of unrelated individuals. Because the HLA-DR and complotype region in man is thought to be critical for the success of bone marrow transplantation, methods for typing of HLA-D by both the HTC and PLT approaches have been examined. Although HLA-D/DR assignments are easily made in normal subjects, they are ambiguous in about 50 per cent of candidates for bone marrow transplantation, including, particularly, patients with aplastic anaemia, leukaemia, and severe combined immunodeficiency. In this setting, it is particularly important to obtain additional information by modification of HLA-D typing procedures and through complotype and GLO allele determinations in all family members. Finally, we can hope that there will be an increased possibility of using non-family donors through methods for removing cytotoxic T cells from donor marrow and through the identification, in the general population, of individuals who are genotypically similar or identical to the recipient. In this regard, the recognition that some 30 per cent of chromosome 6 in caucasians (50 per cent of individuals) bear extended haplotypes, which include a relatively fixed set of alleles particularly in the HLA-B, -DR, complotype and GLO regions, offers considerable promise.     

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.     

Typing of HLA class II and class I antigens using PHA-activated, IL-2-propagated T lymphocytes

We describe here a simple procedure, by which HLA class II antigens can be accurately and reliably identified in those patients where there is minimal or absent expression of HLA-DR,DQw antigens on B cells, or when the total number of leukocytes recovered from the patients do not permit reliable typing. Ficoll-Hypaque-separated peripheral blood mononuclear leukocytes, fresh or cryopreserved, were activated by PHA and then propagated in IL-2-containing medium until enough cells for typing were obtained (usually 7-14 days). At this stage, the cultured cells were shown to be primarily T cells (greater than 90% CD3+). Since the activated T cells propagate in the presence of IL-2, even a small number (10(4] of fresh or cryopreserved patients' cells suffice for this protocol. To date we have been able to successfully HLA-DR,DQw type 34/34 bone marrow transplantation candidates and 12/12 long-term dialysis patients, who were untypable using fresh cells. HLA-DR,DQw antigens on activated T cells from normal individuals were identical to those found on their uncultured B cells. In addition, class I antigens that were undetectable on the uncultured cells of one patient could be identified on activated T cells. The HLA antigens identified on the patients' activated T cells were confirmed by phenotypic analysis of cells from family members.     

Approaches to managing volunteer marrow donor registry HLA data. Algorithms for directing donor center-initiated HLA-DR typing of selected donors

The German bone marrow donor center (DKMS) hasrecruited over 732 500 donors during the first 9 years of its existence. Initially, donors were typed for HLA-A and B, and DR typing was only done on request for a patient-initiated search. In 1994, a project was started which led to the donor center-initiated DR typing (DCI-DRT) of >35,000 donors. These donors were selected by donor-specific criteria (age, sex, height and weight) and according to HLA-A and B phenotypes. The latter was done to avoid unnecessary DR typing of the most common A, B phenotypes With a follow up of >6 years, this strategy has led to a number of confirmatory typings (CT) (n=4588) and stem cell harvests (n=568), which is at least comparable to those ensuing after patient-initiated HLA-DR typing (126 000 DR typings, 8,213 CTs, 888 resulting in stem-cell donation). DCI-DRT seems to be a cost-effective strategy which may help to reduce search times and improve search outcome, and improve the overall efficiency of donor center operations     

Ontogeny of T lymphocyte differentiation in the human fetus: acquisition of phenotype and functions

Phenotype and functions of cells of the T lymphocyte lineage from fetal liver, thymus, spleen and bone marrow were investigated at various ages. T lymphocyte differentiation was shown to be initiated in the thymus after the 7th week of gestation. In this organ, a large number of cells with a phenotype comparable to that of children thymocytes and with a high proliferative response to phytomitogens was observed at the 14th week. The fetal liver and bone marrow never contained many T-cells and the liver was shown to be virtually devoid of any of these cells before the 13th week. Fetal spleen contained appreciable amounts of T-cells after the 13th week. Helper and suppressor activities of fetal thymocytes and splenocytes were acquired between the 12th and the 16th week, but they were never as complete nor as potent as those of adult lymphocytes. HLA antigens were detected in very low amount in lymphocytes from the various organs at the beginning of the second trimester and their expression was significantly enhanced by in vitro incubation with alpha-interferon (alpha-IFN), a procedure that permits easier HLA typing of fetal cells.     

Reverse-SSO hybridization provides an accurate and simple HLA-DR typing--a comparative study with HLA-DR serologic typing.

The HLA-DR molecule is a polymorphic membrane glycoprotein and one of the key molecules causing allograft rejection and graft-versus-host disease in organ transplantation. Serologic typing using DR specific alloantisera has long been used, but several problems have limited its application. The purpose of this study was to establish an efficient reverse-SSO typing system that detects DRB1 and DRB3/B4/B5 alleles on a single membrane. A DR typing membrane was prepared by immobilizing 21 dT-tailed sequence specific oligonucleotides (SSOs) on a nylon membrane and was used in a hybridization assay with digoxigenin-labeled PCR-amplified target DNA. The positive signals were detected on X-ray film using chemiluminescence. A comparison study with serology using DNAs from 105 unrelated individuals demonstrated that the reverse-SSO typing system was superior to serologic typing in terms of accuracy (100% vs 90.5%), simplicity, range of application, rapidity, and cost of the test. These data indicate that the reverse-SSO typing system can replace serology as a routine DR test, and will be useful in time-restricted solid organ transplantation and in selection of an unrelated marrow donor prior to bone marrow transplantation.     

HLA antigens and complotypes in insulin-dependent diabetes mellitus

One hundred and thirty-six Finnish patients with insulin-dependent (type I) diabetes mellitus were investigated for the HLA-A, B, D and DR antigens as well as the Bf and C4 allotypes. The statistically significant increase in the frequencies of HLA-A9, B8, B15, Dw3, Dw4, DR3, DR4, C4A0 and C4B3 was observed when compared with the healthy controls. About 79% of the patients had HLA-DR4, and 53% had HLA-DR3 antigens. A rare C4 allele C4B3 was found in 21% of the patients, whereas only in 2% among the controls (relative risk 16.35). The etiological fraction (EF) values indicated that HLA D/DR alleles were the best markers for IDDM, the observed EF for HLA-DR4 in diabetes was as high as 0.70. Examination of HLA, Bf and C4 phenotypes suggested that at least two supratypes "B15 BfS C4A3B3 D(R)4" and "B8 BfS C4A0B1 D(R)3" were markers for the susceptibility to type I diabetes, one third of our patients had either of these supratypes. The protective role of DR2 and Dw2 antigens was also confirmed: no HLA-Dw2 positive patients and only one with HLA-DR2 was found.     

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.     

Histocompatibility Determinants in Israeli Jewish Patients with Coeliac Disease: Population and Family Study

The association between HLA and coeliac disease (CD) was studied in the Jewish population of Israel. A total of 112 patients were typed for HLA-A,B,C antigens, including 67 patients whose families were typed in order to deduce the genotypes. Forty-seven patients were typed for HLA-DR antigens. The HLA-A,B,C data show a pattern of association, which is similar to that found in European CD patients: HLA-B8 is increased, although to a lower degree; a suggestive, insignificant increase for Aw30, B13 and Cw6 and a decrease of Bw35 were noted. The DR antigens DR3 and DR7 are associated with CD in the Jewish population. An excess of DR3/DR7 heterozygotes was noted. The data from family and population studies support a model in which two different HLA-DR associated genes are intereacting.     

Frequencies of HLA-A, HLA-B, HLA-DR, and HLA-DQ phenotypes in the United Arab Emirates population

The high degree of polymorphism of the human leukocyte antigen (HLA) system provides means for the study of diversity in different populations. The aim of this work is to study the HLA phenotype frequencies in the United Arab Emiratis in comparison with other geographically related Arabs, Iranians, and Asians, all living in the United Arab Emirates (UAE). Healthy blood donors and potential kidney or bone marrow donors were typed for HLA class I (n = 1880) and class II (n = 2022). Only one representative member of each family was included to avoid bias. UAE Emiratis, Arabs of Arabian Gulf Peninsula (AGP), Arabs of South Mediterranean (SMR), North African Arabs (NA), Iranians, and Asians. HLA typing was done by microlymphocytotoxicity method and/or low-resolution polymerase chain reaction-sequence-specific primer techniques. As an individual antigen, HLA-A2 had the highest frequency in all populations studied, however, the frequency of the broad antigen A19 surpassed A2 in all the groups except the AGP Arabs and Iranians. B5 was the predominant B antigen in all groups except the SMR and Asians. Amongst the class II broad antigens, DR2 was the most frequent antigen in UAE, AGP Arabs, Iranians, and Asians. The overall frequency of DQ1 was high in all groups except the SMR Arabs who had an almost equal distribution of DQ1 and DQ3. In conclusion, this study indicates that the most frequent antigens in the UAE population are HLA-A19, HLA-A2, HLA-B5, and HLA-DR2. It also sheds light on the similarities between the UAE Emiratis, AGP Arabs, Iranians, and Asians, specially the predominance of DR2 of the class II antigens.     

Genetically Appropriate Expression of HLA and DR (IA) Alloantigens on Human Melanoma Cell Lines

Studies of the expression of HLA and DR alloantigens on cultured human melanoma cells in comparison with those expressed on peripheral lymphocytes and B-cells derived from the same patients have shown that the HLA-A,B, and C locus antigens expressed on the cultured tumor cells were consistent with those expected from the typing of peripheral blood lymphocytes. One melanoma cell line failed to express all the HLA antigens expected from donor typing. All five of the lines tested also expressed DR antigens and in three instances these could be demonstrated to have genetically consistent allotypes. However, in preliminary studies stimulation of allogeneic lymphocytes by the DR positive melanoma cells could not be demonstrated.