Linkage disequilibrium in HLA cannot be explained by selective recombination.

Some combinations of HLA-A, -B and -DR antigens occur more frequently than would be expected from their gene frequencies in the population. This phenomenon, referred to as Linkage Disequilibrium (LD) has been the origin of many speculations. One hypothesis to explain LD is that some haplotypes are protected from recombination. A second hypothesis is that these HLA antigens preferentially recombine after cross-over to create an LD haplotype. We tested these 2 hypotheses: from a pool of over 10,000 families typed in our department, we analyzed 126 families in which HLA-A:B or B:DR recombinant offspring was documented. To overcome a possible bias in our material, we used the non-recombined haplotypes from the same 126 families as a control group. Our results show that the number of cross-overs through LD haplotypes is not significantly lower then would be expected if recombination occurred randomly. Also the number of LD haplotypes created upon recombination was not significantly increased.

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing.

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogenous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.

HLA-DR beta chain residue 86 controls DR alpha beta dimer stability.

Major histocompatibility complex class II molecules exist in two forms, which can be distinguished on the basis of their stability in sodium dodecyl sulfate (SDS) as SDS-stable and SDS-unstable alpha beta dimers. The ratio of stable vs. unstable alpha beta dimers varies between murine H-2 alleles and isotypes, but the molecular basis for this observation is unknown. Here we show that for the human HLA-DRB1 and HLA-DRB3 gene products this ratio is controlled by the valine/glycine dimorphism at position 86. Haplotypes coding for DR beta chains with a valine at position 86 express higher numbers of stable dimers compared to similar haplotypes expressing DR beta chains with a glycine at that position. Reverse-phase high-performance liquid chromatography analysis of iodinated peptides, which were eluted from DR dimers with either a DRB1*1101 or a DRB1*1104 beta chain which differ only at position 86, indicated that these DR dimers contain (partially) distinct sets of peptides. The valine/glycine dimorphism is highly conserved, present in most HLA-DR alleles and influences peptide-binding. Analysis of the occurrence of the Val86 and the Gly86 gene products revealed that these are not equally present in the population. Depending on the DR specificity either the Val86 of Gly86 allelic variant is favored. Thus, the natural, highly conserved dimorphism at HLA-DR beta chain position 86 influences peptide selection. The dimorphism is therefore likely to influence antigen presentation and forms the molecular basis for the observed differences in stability of Val86- and Gly86-containing DR dimers in the presence of SDS.

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.

HLA-DR peptide-induced alloreactive T cell lines reveal an HLA-DR sequence that can be both "dominant" and "cryptic": evidence for allele-specific processing.

Previously, we reported on a T cell line, ThoU6, which we obtained through stimulation of DPw3+ cells with a synthetic "DR3 peptide" with a sequence identical to the third hypervariable region of the DRB1*0301 chain. This T cell line recognizes both the synthetic peptide presented by DPw3 as well as DR3+ DPw3+ stimulator cells. This implies that the synthetic DR3 peptide has a natural counterpart in DR3-positive cells. Here we describe the recognition pattern of another T cell line that was sensitized with the same synthetic DR3 peptide. This T cell line, BieU6, shows both HLA-DRw13/Dw18 (self)-restricted recognition of the synthetic DR3 peptide and allorecognition towards DR13/Dw19, a molecule which is highly homologous to Dw18, in the absence of synthetic peptide. These results suggest that the epitope formed by the Dw18 molecule plus the synthetic DR3 peptide and recognized by T cell line BieU6 mimics the Dw19 molecule. The potential role for a Dw19-specific peptide is discussed. The inability of T cell line BieU6 to recognize Dw18+ DR3+ cells indicates that, in this case, the synthetic DR3 peptide is "cryptic", i.e. does not have a natural counterpart that is effectively presented to T cells. Mapping of the shortest peptides recognized by T cell lines ThoU6 and BieU6 indicate that these sequences are fully overlapping. We, therefore, suggest that the antigen-presenting molecules, HLA-DPw3 and HLA-Dw18, differ in their accessibility for self peptides derived from the third hypervariable region of DR molecules. These observations may be explained by allele-specific processing.

T-cell recognition of class II products that result from the combined presence of two different HLA haplotypes.

To analyze DR2 haplotypes as recognized by alloreactive T cells, lymphocytes from a DR7; DQw2 homozygous donor were cocultured with irradiated lymphocytes that were DRw15, DR7; DQw6, DQw2 heterozygous. In this report, we focus on two HLA-DQ-specific T-cell clones obtained from this priming. These two clones (c3518 and c3523) responded to the positive control (original stimulator) and five of 66 panel donors. Three of these donors typed DRw15, DR7; DQw6, DQw2, as did the positive control. One stimulatory donor typed DRw15, DR7; DQw6, DQw9 and one stimulatory donor typed DRw14, DR7; DQw5, DQw2. Oligonucleotide typing revealed that recognition by the clones depended on the simultaneous presence of the DQB1*0602 gene on one haplotype and DRB1*0701 or DQA*0201 on the other. The hypothesis that c3518 and c3523 recognize an HLA class II product that results from the combination of two different HLA haplotypes was further confirmed in family studies. In three families, it was shown that the DRw15, DR7; DQw6, (DQw2 or DQw9)-positive individuals were recognized, whereas the cells carrying either DRw15; DQw6, DR7; DQw2, or DR7; DQw9 were nonstimulatory. Our results can be explained in two ways: (a) the T cells recognize a class II dimer that results from trans-complementation of DQA1*0101 and DQB1*0602, and (2) the T cells recognize a DR7-derived peptide that is presented by DQw6.

T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.

No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta-sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide-specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products.

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.

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 DP beta 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.

Early recurrent miscarriage: histology of conception products.

The histopathological appearance of conception products from 44 women with recurrent miscarriage was compared with those obtained from 105 women with sporadic miscarriage. Abnormal villi, suggesting fetal chromosomal abnormalities, were found in 62% of women with a recurrent miscarriage and in 58% of those with sporadic miscarriage. This difference is not statistically significant.

Evidence for tumor necrosis factor-alpha involvement in the optimal induction of class I allospecific cytotoxic T cells.

We have investigated the functional interaction between IL-2 and TNF on the generation of alloreactive CTL. The study was performed by using primary mixed cultures of lymphocytes from a MHC-recombinant sibling identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. Our data show that MHC class I disparity can trigger the induction of TNF receptor without promoting significant TNF production. Addition of exogenous TNF at the sensitizing phase of the primary mixed lymphocyte reaction did not result in CTL activation. However, when simultaneously added with IL-2, TNF could promote an optimal induction of cytotoxic T cell generation. The enhanced lytic ability of MHC class-I primed CTL by TNF was associated with a selective up-regulation of Tac Ag and subsequent amplification of cell proliferation. Furthermore, TNF was also found to induce a considerable increase in IL-2-induced intracellular benzyloxycarbonyl-L-lysine thiobenzylester-esterase activity by MHC class I-primed cells. TNF did not affect the expression of LFA1, CD2, CD4, and CD8, molecules that are associated with CTL-target interactions, on responder cells. These results extend our earlier observations on the role of class I MHC molecules that may function to transduce activation signals and suggest that TNF may be a potent mediator involved in the IL-2-induced acquisition of optimal lytic competence by precursor cytotoxic T cells.

The natural course of habitual abortion.

In recurrent spontaneous abortion an immunological fetal-maternal imbalance has been postulated and successes of immunotherapy have been described. A prospective study on the reproductive performance of untreated women with three abortions of unknown etiology has not been performed. The benefit of therapy can only be estimated if the natural course of the disease is known. The aim of this study was to investigate prospectively the abortion rate in a well-defined group of women with a history of habitual abortion of unknown etiology and to test the hypothesis that immunologic factors are involved. Fourty-four couples were taken into study. The overall pregnancy rate in the first subsequent pregnancy was 62%. Neither significant increased sharing of Human Leucocyte Antigens (HLA) nor an aberrant mixed lymphocyte reactivity in this group was found when compared to control random matings. The presence or absence of antipaternal antibodies did not correlate with the outcome of the subsequent pregnancy. It is concluded that the prognosis of untreated patients with habitual abortion is favourable and that no aberrant immunologic reactivity could be demonstrated by means of up-to-date diagnostic procedures.

Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells.

To understand the effect of human MHC class II polymorphism on antigen recognition, we analyzed the memory T cell response to three tetanus toxin epitopes defined by three short synthetic peptides (p2, p4 and p30). We found that p2 and p30 are universally immunogenic, since they are recognized by all primed donors, irrespective of their MHC haplotypes. The analysis of specific clones indicates that both peptides are very promiscuous in their capacity to bind to class II. p30 can be recognized in association with DRw11(5), 7, 9 and with DPw2 and DPw4, while p2 can be recognized in association with DR1, DRw15(2), DRw18 (3), DR4Dw4, DRw11(5), DRw13(w6), DR7, DRw8, DR9, DRw52a and DRw52b. On the contrary, the third peptide, p4, can be recognized by only half of the donors in association with only DRw52a and DRw52c. Analysis of truncated peptides shows that p30 contains three distinct epitopes, each recognized in association with different class II molecules. Therefore, the restriction specificity is already set at the level of the peptide-MHC complex and, in all cases, T cells discriminate p30 bound to different class II molecules. On the contrary, p2 contains only one epitope, which is recognized in association with all DR molecules. In this case we found two different restriction patterns. Some clones are monogamous, since they recognize the peptide in association with one DR allele, while others are promiscuous, since they recognize by peptide in association with several different DR molecules. Thus, in this case, the restriction specificity is also set at the level of the T cell receptor. We suggest that both the promiscuous binding of peptides and the promiscuous recognition by T cells are dependent on the particular structure of the DR molecules, having a monomorphic alpha chain associated with a polymorphic beta chain.

A novel T-cell-defined HLA-DR polymorphism not predicted from the linear amino acid sequence.

Recent investigations have shown that alloreactive T cells are capable of responding to structures defined by specific linear amino acid sequences on class II molecules. In the present study we show that also a polymorphism can be recognized that is not defined by such linear amino acid sequences. Two human T-cell clones, sensitized to DRw13 haplotypes, are described. The description of clone c50 serves to exemplify the first model. This DRB1-specific clone responds to stimulator cells that carry DR molecules, different in their DRB1 first and second hypervariable regions (HV1 and HV2) but identical in their HV3 regions (i.e., DRw13,Dw18; DRw13,Dw19; DR4,Dw10; and DRw11,LDVII). The second clone, c1443, behaves nonconventionally. It responds to DRw13,Dw18; DRw13,Dw19; and DR4,Dw4 stimulator cells, although no specific amino acid sequence is shared between these specificities. The latter pattern of reactivity suggests the existence of a novel polymorphism recognized by alloreactive T cells. This particular polymorphism may also be biologically significant.

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.

Correlation of structure with T cell responses of the three members of the HLA-DRw52 allelic series.

A third allele at the DRB3 locus, DRw52c, represents an intermediate sequence between DRw52a and DRw52b and may have arisen by a gene conversion-like event. The recognition of cells bearing these molecules by a number of alloreactive and antigen-specific DR-restricted T cell clones was analyzed. On the basis of a theoretical model of HLA class II structure, distinct amino acid clusters have been identified as motifs controlling TCR recognition. These are located both in the cleft and in the alpha-helical edge of the MHC class II recognition platform. Motifs shared between two alleles may restrict public T cell clones.

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.

Specific suppression of mixed lymphocyte reactions by alloactivated cells is correlated with cytotoxicity.

Mixed lymphocyte reaction (MLR)-activated lymphoblasts can suppress both proliferation and the induction of cell-mediated lysis (CML) when added to a subsequent MLR. Antigen specificity and the mechanism of MLR-induced suppression was investigated, with special emphasis on the exclusion of cell-mediated stimulator cell lysis as the cause of suppression. We studied the suppressive properties of nine MLR-activated cultures, selected because of their specific suppressive properties. Suppression of a given MLR was obtained when the stimulator cell carried at least one of the mismatched HLA antigens of the original stimulator. The mismatched antigen which activated suppression was an HLA class I antigen in 3 out of 9 cases, an HLA class II antigen in another 3 cases. In the remaining 3 cases suppression was observed when either an HLA class I or an HLA class II antigen was shared between the original stimulator and stimulator cell of the MLR. When analyzed on the same panel, a positive linear correlation between suppression of an MLR and CML activity against the stimulator cell was found. We also observed that both the original MLR (A*B) and the reserve MLR (B*A) could be suppressed by these anti-A suppressor cells. These results are compatible with the hypothesis that in the protocol studied suppression is mediated by lysis of the stimulator cells.

T cells sensitized to synthetic HLA-DR3 peptide give evidence of continuous presentation of denatured HLA-DR3 molecules by HLA-DP.

T cell clones raised against a synthetic peptide, identical to the third hypervariable region of the DR3 BI chain, were tested for secondary proliferative responses against a panel of PBLs. All seven DR3 DPw3+ stimulators could induce proliferation. DR3- DPw3+ PBLs were recognized when the synthetic peptide was added to the cultures. Inhibition studies with mAbs showed that in both cases the HLA-DP molecule is involved in the recognition of both types of stimulators. We conclude that the clones recognize the DR3 peptide presented by HLA-DPw3. This stimulus can be obtained in two different ways: (a) by addition of synthetic peptide to DPw3+ PBLs or (b) by using DR3 DPw3+ stimulator cells where DR3 peptides are present in the culture as a product of denaturation of the DR3 molecule. Because all DR3 DPw3+ PBLs tested could stimulate the line and clones, we assume that the presentation of the DR3 peptide by DP is a naturally and continuously occurring phenomenon.