A. Termijtelen

Effect of One-HLA-DR-Antigen–Matched and Completely HLA-DR–Mismatched Blood Transfusions on Survival of Heart and Kidney Allografts

Blood transfusions can influence the survival of organ allografts favorably, in spite of the danger of sensitization. We investigated the influence of HLA compatibility between blood donors and transfusion recipients on the production of HLA antibodies and on graft survival. Among recipients of transfusions who shared one HLA-DR antigen with their respective donors, antibodies developed in 6 of 28 who had received one transfusion, in 2 of 16 who had received three transfusions, and in 4 of 24 who had undergone renal transplantation. Among recipients who were mismatched with their donors for both HLA-DR antigens, the rate of sensitization was significantly higher in all three of these groups (18 of 30, P = 0.02; 12 of 16, P = 0.0007; and 12 of 22, P = 0.001). The survival of kidney allografts among graft recipients who were given transfusions and shared one HLA-DR antigen with their blood donors (81 percent at five years) was significantly higher than among recipients who were given transfusions and were mismatched for both HLA-DR antigens (57 percent; P = 0.02) or among recipients who were not given transfusions (45 percent; P = 0.001). There was no difference in graft survival between patients who received transfusions mismatched for two HLA-DR antigens and those who were not given transfusions. We conclude that allograft survival can be improved by pretransplantation blood transfusion when the transfusion recipients share at least one HLA-DR antigen with their donors. In view of the increased rate of sensitization and the lack of improvement in graft survival, the transfusion of blood mismatched for two HLA-DR antigens appears to be contraindicated in candidates for transplantation.

Polymorphisms within the HLA-DRw6 haplotype

We have analyzed the HLA class II gene products from HLA-DRw6 homozygous cells. Epstein-Barr virus-transformed B-cell lines were internally labeled with [35S]-methionine. An NP-40 lysate of the cells was subjected to immunoprecipitation, first with a DRw52-like-specific monoclonal antibody and subsequently with a DR-specific framework antibody. The DR region-encoded gene products were analyzed by one-dimensional gel isoelectric focusing and two-dimensional gel electrophoresis. It is shown that DRw6 homozygous cell lines contain at least two nonallelic DRβ chains, one carrying a DRw52 determinant and one DRw52-negative population. Both chains appear to be polymorphic between the cellularly defined subtypes of DRw6. The determinant responsible for the differential mixed lymphocyte culture reactivity of Dw18 and Dw19 cells resides on the DRw52-positive population, whereas the Dw6-Dw9 differences are attributed to determinants on both populations of DR light chains. The Dw16-derived DRw52+ chain much resembles the Dw18 DRw52+ light chain whereas there is a clear-cut difference between these two subtypes in the DRw52− population. We conclude that, for DRw6 homozygous cells, the cellularly recognized D determinants are probably located on DR-encoded molecules, both DRw52+ and DRw52−, and that charge shift of these chains is at least partly responsible for differential recognition of these cells in mixed lymphocyte cultures.

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

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

Positive correlation between oligonucleotide typing and T-cell recognition of HLA-DP molecules

The identification of 19 different HLA-DPB1 sequences implicates the existence of more DP specificities than can be typed for with cellular methods. How many of the DPβ sequences can be specifically recognized by T cells, and which of the polymorphic regions can contribute to the specificity of allorecognition, is not known. In order to investigate the distribution and the immunological relevance of recently described DPB1 alleles, we have typed a panel of 98 randomly selected Dutch Caucasoid donors for the HLA-DPB1 locus by oligonucleotide typing. Comparison of the typing results with primed lymphocyte typing (PLT) defined DP specificities shows an extremely good correlation. Moreover, additional alleles could be defined by oligonucleotide typing reducing the number of DP blanks in the panel. By selecting the appropriate responder stimulator combinations we were able to show that distinctive PLT reagents against oligonucleotide defined specificities DPB1*0401, DPB1*0402, DPB1*0901, and DPB1*1301 can be generated. To investigate in more detail which part of the DP molecule is responsible for the specificity of T-cell recognition, T-cell clones were generated against HLA-DPw3. The clones were tested for the recognition of stimulators carrying DPB1 alleles which had been defined by oligonucleotide typing and sequence analyses and which differed in a variable degree from DPB1*0301. The recognition patterns demonstrated that differences of one amino acid in polymorphic regions situated either in the beta sheets or alpha helix of the hypothetical model of the HLA class II molecule can eliminate T-cell recognition. Furthermore, sequence analyses revealed a new DPB1 allele designated DPB1*Oos.

Leucine33-proline33 substitution in human platelet glycoprotein IIIa determines HLA-DRw52a (DW24) association of the immune response against HPA-1a (Zwa/PIA1) and HPA-1b (Zwb/PIA2)

Alloantibody formation against HPA-1a (Zwa/PIA1) has, to date, only been found in HLA-DRw52(a+) (Dw24) individuals. Alloimmunization against the product of the other HPA-1 allele, HPA-1b, is rare. We have been able to evaluate ten cases of HPA-1b alloimmunization in Europe in order to study whether there is an association between HLA phenotype and anti-HPA-1b antibody formation. HLA typing of these patients was performed with particular attention to the DRw52a specificity using specific T-cell clones. No association with DRw52a or any other known HLA phenotype was found. This finding implies that the amino acid substitution leucine33-proline33 in GPIIIa, responsible for HPA-1a/b, is of primary importance for the association of anti-HPA-1a antibody formation with DRw52a. These data show that the amino acid polymorphism affects the presentation of the immunogenic oligopeptides of HPA-1a and -1b in the HLA class-II groove.

Mapping SB in relation to HLA and GLO1 using cells from first-cousin marriage offspring

A newly defined leukocyte antigen system, the secondary B-cell (SB) system, was shown to be linked to HLA. The SB marker has been investigated in lymphocyte donors presumed to be genetically homozygous for HLA-A through HLA-D/DR by virtue of descent from a first-cousin marriage and of phenotypic homozygosity for these HLA markers. Of 19 donors, 3 were found to be heterozygous for SB. Studies of the families of these three donors could not distinguish with certainty whether the heterozygosity resulted from SB/DR recombination or from “pseudohomozygosity” for HLA-A through -D/DR by inheritance of two genetically unrelated but similar haplotypes. However, our data favored the occurrence of SB/DR recombination with a meiotic distance perhaps as large as 3.3 cMorgan. Recombinations were identified which mapped SB between HLA-B and GL01. These studies demonstrate the usefulness of cells from first-cousin marriage offspring in mapping a polymorphic genetic system.

Phenotyping of HLA-D-region products by negative and positive mixed lymphocyte reactions

Individuals of known HLA phenotype were used to examine the specificity of secondary proliferative (restimulation) responses in lymphocyte populations which had been primed previously in long-term, one-way mixed lymphocyte reactions (MLR). The objective was to identify the genetic region responsible for this secondary mitogenic effect. Restimulation responses between unrelated and related individuals from a family with a recombinant betweenHLA-B andD produced similar results. In both cases responses were grouped into zero, intermediate, and positive. Restimulation was by three types of cell: those with HLA-D types compatible with the responder; those with HLA-D types specific to the primary stimulator; and those with HLA-D types of a third-party cell. Compatible responses fell into the zero category and the intermediate category, third-party responses fell into the intermediate category, and specific responses fell into the positive category. Our conclusion was that HLA-D products evoked secondary mitogenic responses, while structures coded for by the HLA-A and B regions appeared to have no effect.

Oligonucleotide typing is a perfect tool to identify antigens stimulatory in the mixed lymphocyte culture

An important criterion for the selection of donors for bone marrow transplantation is the grade of matching for HLA between donor and recipient. For patients that lack an HLA-identical sibling, an extending pool of unrelated volunteers for bone marrow donation is available. From these donors the best matched candidate can be selected by serological typing, followed by a mixed lymphocyte culture (MLC). Oligonucleotide genotyping for HLA class II antigens is considered to be valuable for the prediction of MLC reactivity. We investigated whether this typing method, in combination with serological typing, would cover the recognition of all MLC stimulatory determinants. One hundred thirty-six combinations of HLA-A, -B, and -DR serologically identical individuals were tested in the MLC. Additional typing for HLA-DRB and HLA-DPB by oligonucleotide genotyping made it possible to evaluate the influence of these genes on MLC reactivity. Combinations that were matched for HLA-DRB gave significantly lower responses than those that were mismatched. Nevertheless, in the matched combinations responses were observed to 94% relative response index. These responses could all be attributed to HLA-DP, since all combinations that were identical by HLA-DPB genotyping were negative in the MLC. In conclusion, with the combined use of serology and oligonucleotide genotyping, responder-stimulator combinations can be selected that are identical for all MLC stimulatory determinants.

Cell membrane polymorphisms coded for in the HLA-D/DR region I. Relation between D and DR

The relation of HLA-D and -DR determinants was studied in Dutch Caucasoids. The recognition of subgroups of DR4, DR5, and DR7, and the specificities LB12 and LB13 are described. Phenotype and gene frequencies and a Hardy--Weinberg analysis of DR and local (LB) B-cell groups are given. Excellent correlation between D and DR typing was obtained when HTCs were studied by selected B-cell antisera. When the same sera were used to type a panel of D typed cells, the correlation was decreased (with the exception of DR3 and Dw3). In the case of discrepancies the DR specificity, but not the corresponding D specificity, always could be found and not the other way around. The data fit best the assumption that HLA-D and -DR are carried by the same molecule, although they might be different determinants on this molecule. A number of possible explanations for the observed discrepancies has been given.

B-Cell Antibodies, la-Like Determinants, and their Relation to MLC Determinants in Man

The HLA supergene is located in the 6th chromosome. Its position to the centromere and the position of a number of polymorphic isoenzymes has been elucidated. The HLA supergene codes not only for determinants present on all nucleated cells, but also for determinants present on B cells and absent from T cells and platelets. These determinants can be recognized by serology, and evidence is presented that some of them are coded for by a hither to unrecognized locus Ag, which is very closely linked to the MLC determinants of the D locus can be recognized with the help of the MLC test using unprimed cells, homozygous for the MLC determinants, so-called typing cells primed against one MLC determinant in the PLT test. So far, 8 MLC determinants have been recognized. Significant disease-association studies in different racial groups appear to be especially informative. They already indicate that the association found so far must rest on different mechanisms. Whether some of them could be caused by partial deficiency for one or more of the complement factors remains to be proven.