The Basques were previously shown to present a high frequency of HLA-B18 and BfF1, which are known to be associated with insulin dependent diabetes mellitus (IDDM). During the VIII International Histocompatibility Workshop, we studied HLA-A, B, C, DR; Bf, C4 and GLO.I polymorphisms in 51 unrelated French Basque IDDM patients and in 50 controls. Haplotypes were established by family studies in all controls and some patients. Two haplotypes were frequently found in the controls: HLA-A1, Bw57, BfS, C4 F1S, DR7 and HLA-Aw30, Cw5, B18, Bf F1, C4Fs degree, DR3. The first one was not found in the patients. All the components of the second haplotype had increased frequencies possibly as a consequence of linkage disequilibrium with HLA-DR3: a highly significant association between IDDM and HLA-DR3 was observed (90.2% vs 24.0%, relative risk (RR) = 29.1, P less than 10(-11)). The HLA-DR4 frequency was slightly increased (37.3% vs 16.0%), and HLA-DR2 was not found. The silent allele C4s degree was particularly associated with early diagnosed IDDM (86.7% in patients with age at onset under 20 years vs 57.1% in other patients, P less than 0.02). The high relative risk for HLA-DR3/DR4 heterozygous vs that of individuals, possibly HLA-DR3 homozygous, supported the hypothesis that two HLA-DR linked genetic factors could be involved in the inheritance of IDDM susceptibility.

Since 1980, a DR blank specificity undefined by serology but known to segregate in families linked to DQw1 has been recognized. We describe a Caucasoid family. BON… where 4 among 8 children are homozygous for such a specificity; their cells can be used as homozygous typing cells and the specificity defined is provisionally called D‐BON. There is no known consanguinity in this family. The homozygous D‐BON cells carry DR molecules as shown by their reactivity with monomorphic anti‐DR monoclonal antibodies. In a random family material consisting of 210 haplotypes, the gene frequency of D‐BON is between 1 and 2%, which corresponds to about 40% of the DR blank frequency. The D‐BON specificity seems associated with DQw1 and also with B35 and C4B2.

Human trophoblast cells have developed various efficient regulatory mechanisms to prevent cell surface expression of the classical HLA-A, -B, and (but not always) -C class I molecules. This allows them to escape maternal alloimmune attack during pregnancy. However, recent results have demonstrated that such a lack of expression could be reversed in villous cytotrophoblast cells purified from term placenta by in vitro IFN-gamma treatment. In this context, we investigated whether both maternal and paternal HLA class Ia antigens were co-dominantly expressed in such trophoblast cells. Using polymerase chain reaction sequence-specific primers for HLA-A and HLA-C alleles, we detected transcripts of both paternal and maternal origins, showing that these genes were not affected by genomic imprinting, at least in term placenta. After in vitro IFN-gamma treatment, the polymorphic HLA-A and HLA-B antigens of both parental origins become detectable at the cell surface, as assessed by flow cytometry and/or complement-dependent microtoxicity test. Appearance of paternal antigens on trophoblast cells upon IFN-gamma induction raises the question of the in vivo biological consequences of this phenomena, in term of materno-fetal tolerance and in particular of a potential allogeneic cytotoxic immune response.

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Several strategies have been proposed for the screening of alloantisera towards HLA class II antigens. Most often the absorption of the sera with platelets is required in order to remove their anti‐HLA‐A, B, C activity. We have developed a simple micromethod for platelet absorption of sera: platelet absorption on the test tray (PATT); 0.5 μl of platelet suspension is incubated with 0.5 μ1 of the serum to be absorbed in the same tray which is subsequently used for the microlymphocytotox‐icity by the two‐colour fluorescence method. This technique is proved to be almost as efficient as classical absorption procedures: out of 44 anti‐HLA‐A, B sera the T‐sell activity is completely removed in 43 by a classical procedure as compared to 41 by PATT; only 8% discrepancies were found among 394 reactions. PATT was then used for anti‐B lymphocyte screening in 419 anti‐HLA‐A,B sera: 4% of the sera remained cytotoxic towards T and B lymphocytes while 35% reacted only with B cells, several of them being DR specific. PATT can also be useful for T/B lymphocyte differential cross‐matches before kidney transplantation. The method is now routinely used in our laboratory for anti‐HLA class II antibody screening.

Fourteen HLA‐A and 18 HLA‐B antigens were studied in three samples of Pyrenean populations: 198 unrelated individuals of a “Pays Basque” group; 212 non‐Basque individuals from a valley in Beam, l'Ouzom; and 73 non‐Basque individuals from the neighboring valley of Bareges. The results in the Basque and the non‐Basque people from l'Ouzom were comparable: the gene frequencies of HLA‐A29, Aw19.2, B17 were increased and the haplotypes HLA ‐ Aw 19.2, B18; A29, B12; A2, B5; Al, B17 were found frequently with a striking linkage disequilibrium; HLA‐B18 had an increased gene frequency in all these Pyrenean populations, while Bw35 was frequent in l'Ouzom and Bareges, but not among the Basques. The characteristics of Bareges were very different: the gene frequencies of HLA‐A2, All, B7 were increased while the frequency of HLA‐B5 was low; the most characteristic haplotypes were HLA‐A2, B12; A2, B18; All, Bw35; All, B27. It is interesting to note discrepancies between ethnic and HLA classification of the Basques and the non‐Basque population of l'Ouzom. The HLA characteristics are quite different in the Bareges sample, more closely resembling those of Northern Europe.

Production of monoclonal antibodies directed against polymorphic epitopes of HLA class II molecules using whole human cells as immunogen has often proved ineffective, because most of the antibodies produced are directed against non-MHC human cell surface molecules. One approach to overcome this problem is the use of transfected mouse L cells expressing a single HLA class II allele as immunogen. By immunizing C3H mice with DR103-transfected L cells, we obtained 3 mAb, OHA TM901, OHA TM902, and OHA TM903, that recognize different polymorphic epitopes of the HLA-DR molecule. The molecular specificities of the 3 mAb were determined on a large panel of B-lymphoblastoid cell lines (B-LCL), peripheral blood cells and HLA class II transfectants from the XIth International Histocompatibility Workshop. Interestingly, the 3 polymorphic mAb detect new HLA-DR epitopes shared by several specificities: OHA TM901 reacts with DR1 (DR101, DR103), DR9 (DR901) and DR10 (DR1001) molecules; OHA TM902 recognizes the same molecules but also DR8 (DR801, 802, 803); OHA TM903 reacts with all DR types except DR3 (DR301, 302), DR7 (DR701, 702) and DR52. Surprisingly, OHA TM901 reacts with DR9 transfectants and B-LCL but not with DR9 peripheral blood lymphocytes. Biochemical analyses indicate that the 3 mAb immunoprecipitate HLA-DR products and react in western blots with DR alpha/beta-dimer but not with free alpha- or beta-chains. This study shows that transfected L cells are very useful tools for the production and the fine characterization of mAb recognizing polymorphic epitopes of HLA class II molecules.

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