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The HLA-D region of the Major Histocompatibility Complex has been subdivided since 1978 (Mawas et al. 1978) into two subregions separable by recombination: a telomeric subregion (closer to HLA-B), coding for the classical HLA-DR or Dw specificities (Mawas et al. 1980) as well as for the more recent MT series (Park et al. 1980); and a centromeric subregion (closer to GLO), coding for a new series of alleles provisionally named SB (for secondary B cell antigens) (Shaw et al. 1980, 1981a). Reagents allowing the identification of six independent alleles have been characterized in two laboratories (Charmot et al. 1980 and Shaw et al. 1980, 1981b) using the technology of primed lymphocytes typing (Sheehy et al. 1975; Mawas et al. 1975). The existence of this new locus is supported by the following arguments: population studies by Shaw demonstrating five traits distinct from DR behaving as alleles (Shaw et al. 1981b), analysis of two informative SB/DR recombinant families (Mawas et al. 1978; Mawas et al. 1980; Shaw et al. 1981a), and, finally, studies of mutants showing independent loss of DR expression without loss of SB expression (Kavathas et al. 1981). The present report summarizes the HLA-SB typing of 109 unrelated individuals from the South of France and segregation studies in 14 unrelated families; a first attempt to correlate local “SB” reagents with the NIH reference standards is presented.

Forty-six members of a family known to have Porphyria were studied. As the disease is often latent clinically, erythrocyte uroporphyrinogen I synthetase activity was determined to classify the subjects as being healthy or carriers. HLA--A, B, C, Bf, GLO antigens were determined. No linkage between acute intermittent Porphyria and the HLA system was noted in this family.

A monomorphic anti-Class I monoclonal antibody, ST01, found in our laboratory, was used to quantify Class I antigens on normal and leukemic cells, using a "CELISA" technique. Saturation graphs were used to compare the quantity of Class I antigens on normal PBL (25 cases) with that on the following types of leukemic cells: a) common acute lymphoblastic leukemias (cALL) (11 cases), b) mature B lymphocytic proliferations (16 cases), c) T hemopathies (9 cases), d) non-lymphoid leukemias (9 cases). In most cases the quantity of HLA Class I antigens was greatly reduced. No correlation was found between the quantitative expression of Class I antigens and the stage of maturation in each cell type, nor was any correlation found between the quantitative expression of Class I antigens on the leukemic cells and the proliferation of leukemic cells in the peripheral blood.

"Natural antibodies" directed against antigens of the Major Histocompatibility Complex have been observed in aged mice but very seldom in humans. It is likely that HLA natural antibodies are more common than previously supposed: our results suggest they may be found in 1% of normal blood donors. Most of these antibodies seem to be weak and can only be detected when B lymphocytes (BL) are used as target cells in the lymphocytotoxicity technique or when indirect immunofluorescence is used with peripheral blood lymphocytes (PBL). The behavior of these natural IgM antibodies was compared to that of weak immune IgG HLA antibodies detected in the same way. Absorption tests showed that "natural antibodies" were very specifically absorbed, whereas "immune antibodies" could also be absorbed by cells not carrying the specific antigen. Furthermore, about half of the "natural antibodies" detected to date carried the HLA-B8 specificity. Various hypotheses have been put forward to attempt to explain the appearance of these antibodies.

Class I allelic typing based on sequencing is reliable, immutable and easy to analyse when only one allele is amplified using a specific mono-allelic technique. A strategy has been developed to selectively amplify exons 2, 3 and 4 of each allele of the three class I loci, previously identified by generic typing, in order to sequence these alleles from their intronic parts in only one direction. This procedure is based mainly on the polymorphism of exon 1 and intron 1 of the HLA-A, -B and -C genes with allele group-specific forward primers and locus-specific reverse primers so as to perform mono-allelic amplification in a 'One Step' pre-sequence-based typing (pre-SBT) PCR. The 5' polymorphism found at each locus is nevertheless not sufficient to discriminate all allelic combinations. Hence exon 2 and exon 3 polymorphism had to be used in a 'Two Step' pre-SBT PCR method to selectively amplify the two alleles in the 1.8%, 7.6% and 0.9% of unresolved combinations found in our laboratory for, respectively, the HLA-A, -B and -C loci. Preparation and validation of 'ready-to-use' aliquots of primer-mixes, pre-SBT buffer and sets of Dye terminator reaction mixtures containing locus-specific intronic primers makes the procedure easy and efficient. The SBT method is the only allelic typing technique used in our laboratory (to date, 742 HLA-A*, 802 HLA-B* and 615 HLA-Cw* alleles have been sequenced) and our successful participation in the national and international quality controls of 4 years ago testifies to the accuracy of the results.

Thrombocytopenia in newborns is often due to maternal alloimmunization against platelet alloantigens of the foetus which have been inherited from the father and are absent in the mother. Our aim was to develop a "ready-to-use" typing kit based on polymerase chain reactions, using sequence-specific primers for rapid and simultaneous genotyping of the eight principal human platelet alloantigens. The typing technique uses two specific primer pairs for each bi-allelic system and a monomorphic primer pair as amplification control, with a single temperature cycle programme and identical PCR stringency conditions for all pairs of primers. This kit allows typing of blood samples of small volume within three hours after reception. Validation criteria are essential to check the reliability and specificity of the test, and DNA controls carrying the targeted HPA alleles must be obtained from typed individuals or created in vitro by PCR.

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified but the corresponding antigen on the cell surface was not detected. In the present report, we describe three members of a family in whom an HLA-A24 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was nevertheless faintly detectable by isoelectric focusing (IEF) and FACS analyses. Sequencing of the HLA-A*24 allele from the promoter region to the eighth exonic region revealed a point mutation in the acceptor site of the second intron as compared to the normal HLA-A*24 allele. This mutation could lead to incorrect processing of mRNA through a cryptic acceptor site located at the beginning of the third exon and hence to alternative splicing with a frame shift introducing an early stop codon into the fourth exon.

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified whereas the corresponding antigen was not detected on the cell surface. In the present report, we describe four members of a family in whom an HLA-A1 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was undetectable by isoelectric focusing (IEF). Sequencing of the HLA-A*01 allele from the promoter region to the eighth exonic region revealed insertion of a "C" nucleotide at the beginning of the fourth exon as compared to the common HLA-A*0101 allele. This mutation causes a frame shift, giving rise to an early stop codon in the fourth exon.

A second HLA-A*68 null allele, HLA-A*6818 N, was identified in our laboratory after discrepant results were obtained between class I serological and molecular typing in a male patient suffering from narcolepsy. HLA-A*6818 N displays a sequence identical to that of the HLA-A*6802 allele, except in exon 2 where 20 nucleotides inserted at codon position 48 are a repeat of the 20 preceding nucleotides. This duplication creates a shift of the reading frame, which leads to a premature non-sense codon at position 59 of the null allele.

A total of 84 individuals were DP typed in parallel with the restriction fragment length polymorphism (RFLP) analysis and the primed lymphocyte test (PLT). Whereas 80% of the cases gave concordant results, the other 20% showed discrepancies for one of the two alleles carried by the typed individuals. Oligotyping, PCR-RFLP and sequencing confirmed the results found by PLT. The 20% discordant results obtained with RFLP led us to conclude that RFLP typing cannot replace PLT typing. From a more general point of view, the RFLP analysis revealed the DP region to be more complex than expected since for each given PLT DP defined specificity, more than one RFLP DP haplotype could be determined. These were possibly induced by crossing-overs or gene conversion events.

Twenty nine healthy unrelated individuals were carefully selected and divided into three groups according to their HLA (Human Leukocyte Antigens) phenotypes. A sensitive and reproducible limiting dilution analysis (LDA) based bioassay using CTLL-20 cells for detection of human IL-2 was set up and used to assess the hierarchical impact of highly polymorphic HLA molecules on individual's alloreactivity. Our main interest was to evaluate the role of HLA-DP molecules in this process. By calculating frequencies of IL-2 producing helper T cell precursors (HTLp) and amounts of IL-2 produced in each experiment, we were able to confirm that HLA-DR molecules are the most potent alloantigens. In 29 different combinations where a single HLA-DP mismatch between stimulating and responding cells was evaluated, some were reasonably tolerant, while the other ones evoked moderate to relatively strong alloimmune responses. Finally, two groups with statistically significant difference in alloimmune responses to stimulating HLA-DP molecules carrying D,E,A,V or G,G,P,M amino acid sequences at positions 84,85,86 and 87 in the sixth variable region F of the molecule could be formed, according to HTLp frequencies and amounts of IL-2 detected. Data presented are of great importance for the selection of unrelated as well as related bone marrow donors for haematological patients.

This study of 110 seropositive rheumatoid arthritis (RA) patients confirms the significant association of susceptibility to RA with HLA-DR4 specificity (P less than 0.001). The DR1 frequency is elevated in the entire seropositive patient group, reaching marginal significance (P less than 0.025). The DR4-negative patients, however, have a much higher prevalence of DR1 (P less than 0.001). Surprisingly, the DRw6 specificity is significantly increased in the remaining DR4- and DR1-negative patients (P less than 0.01). These results demonstrate that RA is not associated with a single HLA-specificity, but to various degrees with DR4, DR1, and DRw6. These findings, and particularly the newly recognized association with DRw6, support the hypothesis that functionally equivalent shared epitopes or conformations on otherwise distinct MHC molecules may confer risk for developing RA.

SUMMARY Previous results obtained in our laboratory showed that novel class I antigens, closely related to HLA-A (TM antigen related to HLA-A9 and GO antigen related to HLA-A24), were expressed on activated HLA-A9 or HLA-A24 peripheral blood lymphocytes (PBL), whatever activation factor was used [mitogenic stimulation (PHA, PWM, Con A), EBV transformation or alloactivation], but not on resting T and B lymphocytes. These antigens were also expressed on HLA-A9 or HLA-A24 common acute lymphoblastic leukaemias (CALL) and some ‘immature’ chronic lymphocytic leukaemias (B-CLL), but not in hairy cell leukaemias (HCL), most of the B-CLL, acute myeloblastic leukaemias (AML) and acute myelomonoblastic leukaemias (AMoL). In order to investigate the regulation mechanisms of these novel class I antigens, PBL and different leukaemic cell types were treated in vitro with recombinant IL-2 (rIL-2) and α and γ-interferon (rIFN). Our results showed that without previous activation, but after culture with rIL-2, TM and GO antigens could be induced in HLA-A9 or HLA-A24 PBL and enhanced in HLA-A9 or HLA-A24 B-CLL cases, whereas no expression was found in HLA-A9 or HLA-A24 HCL, T-ALL, AML or AMoL. After culture with rINFs, the expression of TM and GO antigens could be induced on HLA-A9 PBL and all HLA-A9 leukaemic cell varieties. Our results support the hypothesis that the expression of class I antigens is induced at an early stage of the cell cycle.

A group of 80 kidney graft recipients was studied for HLA immunization after loss of graft function. Sera were screened (1) by the classical lymphocytotoxicity technique using PBMC, CLL cells and normal B cells isolated with magnetic beads, with and without DTT and before and after platelet adsorption to remove class I antibodies and (2) by ELISA techniques.

Class I allelic typing based on sequencing is reliable, immutable and easy to analyse when only one allele is amplified using a specific mono-allelic technique. A strategy has been developed to selectively amplify exons 2, 3 and 4 of each allele of the three class I loci, previously identified by generic typing, in order to sequence these alleles from their intronic parts in only one direction. This procedure is based mainly on the polymorphism of exon 1 and intron 1 of the HLA-A, -B and -C genes with allele group-specific forward primers and locus-specific reverse primers so as to perform mono-allelic amplification in a 'One Step' pre-sequence-based typing (pre-SBT) PCR. The 5' polymorphism found at each locus is nevertheless not sufficient to discriminate all allelic combinations. Hence exon 2 and exon 3 polymorphism had to be used in a 'Two Step' pre-SBT PCR method to selectively amplify the two alleles in the 1.8%, 7.6% and 0.9% of unresolved combinations found in our laboratory for, respectively, the HLA-A, -B and -C loci. Preparation and validation of 'ready-to-use' aliquots of primer-mixes, pre-SBT buffer and sets of Dye terminator reaction mixtures containing locus-specific intronic primers makes the procedure easy and efficient. The SBT method is the only allelic typing technique used in our laboratory (to date, 742 HLA-A*, 802 HLA-B* and 615 HLA-Cw* alleles have been sequenced) and our successful participation in the national and international quality controls of 4 years ago testifies to the accuracy of the results.

A second HLA-A*68 null allele, HLA-A*6818 N, was identified in our laboratory after discrepant results were obtained between class I serological and molecular typing in a male patient suffering from narcolepsy. HLA-A*6818 N displays a sequence identical to that of the HLA-A*6802 allele, except in exon 2 where 20 nucleotides inserted at codon position 48 are a repeat of the 20 preceding nucleotides. This duplication creates a shift of the reading frame, which leads to a premature non-sense codon at position 59 of the null allele.

The authors describe an A*68 allele present at the molecular level but not expressed at the cell surface. This non expression results from the deletion of one nucleotide in exon 1, which causes a shift of the reading frame leading to an early non-sense codon in the same exon.

The polymorphism of the class I (HLA-A, B, C) and class II (HLA-DR, DQ, DP) antigens was for a long time investigated using serological methods. Today molecular biology methods are available to define the numerous HLA alleles by genotyping [(82 HLA-A alleles, 174 HLA-B, 38 HLA-C, 166 HLA-DRB1, 27 HLA-DQB1, 71 HLA-DPB1) (nomenclature 1996)]. Many different molecular biology methods can be used to define these alleles (PCR- RFLP, PCR-SSOP, PCR-SSP, PCR-SBT), the choice of method depends on the number of genotypes achieved per day and the time required to obtain a result. The resolution degree of results can reach two levels: low resolution: provides results almost identical to those obtained by serological methods. Low resolution is sufficient to find HLA-identical siblings for bone marrow transplantation, to type organ donor-recipient pairs and for diagnosis in most HLA disease associations; high resolution: defines HLA allele subtypes. High resolution is essential to type bone marrow donor-recipient pairs when the donor is unrelated. Molecular biology methods will gradually replace serological methods in the future. The only restriction is that some alleles, defined at the genomic level, are not expressed at the cell surface and are thus not functional.

Two siblings with a peptide TAP deficiency were recently described. Despite poor cell surface expression of HLA class I molecules, these patients were not unusually susceptible to viral infections. The majority of the cell surface-expressed class I molecules were HLA-B products as assessed by cytofluorometry and biochemical analysis. Analysis of two peptides eluted from the class I molecules expressed by TAP-deficient EBV B lymphoblastoid cell lines indicated that both were derived from cytosolic proteins and presented by HLA-B molecules. Peripheral alphabeta CD8+ T cells were present and their TCR repertoire was polyclonal. Most of the alphabeta CD8+ T cell clones studied (21 of 22) were nonreactive against cells expressing normal levels of the same HLA alleles as those of the TAP-deficient patients. However, it was possible to isolate one cytotoxic CD8+ alphabeta T cell clone recognizing the EBV protein LMP2 presented by HLA-B molecules on TAP-deficient cells. These observations suggest that in the TAP-deficient patients, CD8+ alphabeta T cells could mature and be recruited in immune responses to mediate HLA class I-restricted cytotoxic defense against viral infections. They also strengthen the physiologic importance of a TAP-independent processing pathway of the LMP2 protein, which was previously shown to contain several other TAP-independent epitopes.

The specific immune mechanisms of corneal graft rejection are not completely understood. Recently, the technique of growing T-cell lines from rejected allografts using recombinant IL-2 has enabled the cells involved in allograft rejection to be recognized. In the present study, this method was applied for the extraction and propagation of T lymphocytes from rejected, normal, and diseased corneas. T-cell lines could successfully be grown from rejected and normal corneas, but not from corneas with keratoconus or pseudophakic bullous keratopathy. The phenotypic repertoire of the grown cells was studied by FACS scan analysis. Rejected corneas were invaded by a mixture of activated CD4+ and CD8+ T-cell lines, with one population being predominant. In normal corneas only activated CD8+ cells with cytotoxic function were cultured. No cells were obtained from diseased corneas. The in vitro function of cell lines was assessed by primed lymphocyte testing. The present study shows that the technique of propagating invading T-cell lines from organ grafts can be applied to human corneas, offering a new approach to understanding the immunological mechanisms occurring inside this immune tissue.

•Background: Endogenous posterior uveitis (PU) can be associated with systemic diseases, and certain forms have strong association with HLA antigens. Much less is known regarding intermediate uveitis (IU). The purpose of this study was to determine whether IU is associated with the HLA system and whether it can be associated with systemic symptoms. •Methods: In 179 consecutive patients consulting for uveitis, a detailed history was obtained and a physical examination performed. HLA typing for 71 HLAA, B, DR and DQ antigens, laboratory tests, and radiography of the chest, sinuses, and sacroiliac joints were systematically performed. •Results: Thirty-two patients (18%) had IU; 51 (28.5%) had PU and constituted our internal control group. Nine of the patients with IU (28%) had the HLA-A28 antigen, compared with 8.1 % of a healthy control population and 8.6% of the patients with PU (P < 0.001). An associated disease was found in four patients with IU (12.5%) (none was HLA-A28) and in 45% of the patients with PU (P < 0.01). Some 67% of HLA-A28 patients with IU had arthralgias affecting the knee(s), compared with 17% of non-HLA-A28 patients and 18% of patients with PU (P

HLA-DP allodeterminant elicit weak cellular and humoral immune responses. This study was undertaken to find out if the low immunogenicity of HLA-DP could be accounted for by a low level of expression on peripheral blood mononuclear cells. Double staining experiments and flow cytometry were performed in 28 healthy individuals, HLA-DP was found to be as expressed as DR and DQ on B lymphocytes. Furthermore, a DP+/DR- lymphocyte sub-population was identified and characterized as freshly activated T lymphocytes.

This communication reports 1. the frequency of the DR3 allele among 86 patients exhibiting various evolutive courses of Crohn's disease (CD), diagnosed in the same hospital in Alsace, France, hospitalized at least once between 1964 and 1986 and followed for at least 1 year, 2. the frequency of the DR3 allele among a control population in the same area: 126 volunteers donors of bone marrow. The definition of the evolutive course was similar to the one published by Binder et al.: slow course (including: intermittent: "occurrence of symptom-free period(s) of at least one month's duration excluding period where the patient was in steroid treatment"; and inactive: "completely free of bowel symptoms during the year") as opposed to continuous evolution: "without symptom-free periods". The DR3 allele determination was established by classical serologic analysis. 26 patients were found with the DR3 allele; 14 belonged to the slow course group, 12 to the continuous evolution group; 60 patients did not exhibit the DR3 allele: 50 in the slow course group, 10 in the continuous evolution group. chi 2 = 8.28; p = 0.004. Therefore the frequency of the DR3 allele was very significantly higher in the group with the continuous course (55%) than in the slow course group (22%). Similarly the frequency of the DR3 allele in the group with the continuous course (12/22: 55%) was significantly higher than in the group of control subjects (34/126: 28%) chi 2 = 6.64; p = 0.01.(ABSTRACT TRUNCATED AT 250 WORDS)

The biochemical structure of the HLA class II DP antigens is close to that of the remainder DR and DQ isotypes. Nevertheless, they may play a peculiar functional role. Their importance in bone marrow transplantation, where a complete HLA class II identity between graft donor and recipient is requested, is not yet known. The detection of a DP mismatch by using the mixed lymphocyte reaction is quite difficult. The role of such a mismatch in the incidence of graft versus host disease (GVHD) or graft rejection has to be defined. Studies on large series of recombinant families, using oligotyping, are necessary.

SUMMARY Using dithiothreitol (DTT) technique, IgM anti-HLA anti-idiotypic antibodies were detected in a multiparous multitransfused woman. These antibodies were able to inhibit the binding of specific IgG anti-HLA antibodies on their corresponding antigen. The recognized determinants were crossrcactive determinants since they were partially found on anti-HLA antibodies from unrelated individuals. By studying the patient's sera over a period of 2 years, no IgM-IgG switch was observed bui the presence of these antibodies was stable in time, despite the disappearance of the idiotypes (anti-HLA antibodies). However, when looking at the patient's earlier serum, it was shown that these IgM anti-idiotypic antibodies were absent from the first available serum. Thus, these anti-idiotypic antibodies seem to behave both like natural and immune antibodies. The incidence of such antibodies in pretransplant patients is discussed.

In a previous work we showed that the two functionally different specificities DPw4 and DPa could only be differentiated by RFLP analysis using two mutually exclusive fragments (respectively, Bg1 II 5.29 kb for DPw4 and 7.24 kb for DPa). The DP Workshop synthetic analysis localized these fragments in the DPA2 pseudogene region. Our results demonstrate, however, that they are located between the A1 and B1 genes; the Bg1 II restriction site responsible for the 5.29 kb fragment was localized between the first and second exon of the DPB1 gene and inside the 7.24 kb fragment. A single mutation point inside this restriction site is responsible for the absence of the 5.29 kb fragment, changing the specificity attributed by RFLP typing.

Previous results obtained in our laboratory showed that novel class I antigens, closely related to HLA-A (TM antigen related to HLA-A9 and GO antigen related to HLA-A24), were expressed on activated HLA-A9 or HLA-A24 peripheral blood lymphocytes (PBL), whatever activation factor was used [mitogenic stimulation (PHA, PWM, Con A), EBV transformation or alloactivation], but not on resting T and B lymphocytes. These antigens were also expressed on HLA-A9 or HLA-A24 common acute lymphoblastic leukaemias (cALL) and some 'immature' chronic lymphocytic leukaemias (B-CLL), but not in hairy cell leukaemias (HCL), most of the B-CLL, acute myeloblastic leukaemias (AML) and acute myelomonoblastic leukaemias (AMoL). In order to investigate the regulation mechanisms of these novel class I antigens, PBL and different leukaemic cell types were treated in vitro with recombinant IL-2 (rIL-2) and alpha and gamma-interferon (rIFN). Our results showed that without previous activation, but after culture with rIL-2, TM and GO antigens could be induced in HLA-A9 or HLA-A24 PBL and enhanced in HLA-A9 or HLA-A24 B-CLL cases, whereas no expression was found in HLA-A9 or HLA-A24 HCL, T-ALL, AML or AMoL. After culture with rINFs, the expression of TM and GO antigens could be induced on HLA-A9 PBL and all HLA-A9 leukaemic cell varieties. Our results support the hypothesis that the expression of class I antigens is induced at an early stage of the cell cycle.

Tests were carried out to absorb cold lymphocytotoxic antibodies on different types of cells. These included lymphocytes and neuraminidase-treated lymphocytes from healthy donors, thymocytes from children undergoing open heart surgery, fetal thymocytes, human brain and cultured lymphocytes. The results showed these antibodies to be absorbed on some types of cells and eluted from others. The best absorption was obtained with fetal thymocytes and brain tissue.

SUMMARY The factorial correspondence analysis, which is a descriptive statistical method, was used to study the preferential associations between locus HLA-A, B, C and Bf alleles. This method does not limit the number ofloci that can be studied and must therefore be considered as an interesting method of studying haplotypic associations within the HLA system.