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The ABO blood group system is known to act as a major transplantation barrier in primates. Different primate species share the presence of A and B antigens. The polymorphism of the macaque ABO blood group genes was analyzed by cloning and sequencing the exon 7 region. In the case of the rhesus macaque (Macaca mulatta) and cynomolgus monkey (Macaca fascicularis) we were able to identify ABO blood group gene segments which cluster into two lineages, namely: *A/*O1 and *B. In addition allelic variation was observed. The 2 amino acid replacements at positions 266 and 268, which are thought to be crucial for A or B transferase activity, could be confirmed for both macaque species. Comparison of primate sequences shows that A and B reactivity was generated independently from each other in the hominoids and Old World monkey lineages. Hence, the primate A and B blood group genes are subject to convergent evolution.

By the use of restriction fragment length polymorphism analysis 10 Taq I fragments could be identified for the MhcMamu-DQA1 region. A strong correlation exists between the occurrence of Mamu-DQA1/Taq I fragments and Mamu-DQA1 allelic sequence variation. Most restriction fragments correspond with a unique Mamu-DQA1 allele, with one exception being the Taq I 4.5 kb fragment that is associated with two Mamu-DQA1 alleles. The RFLP technique allowed the identification of 15 Mamu-DQB1/Taq I restriction fragments, whereas sequence analysis has permitted the characterization of at least 20 different Mamu-DQB1 alleles. In this communication two unpublished Mamu-DQB1 sequences are described. For Mamu-DQB1, on only four occasions was it possible to demonstrate a correlation between a certain fragment and an allelic sequence. These analyses, performed on material from truly homozygous animals, allowed us to define which combinations of Mamu-DQA1 and -DQB1 molecules form heterodimers at the cell surface. In addition, these studies are helpful in typing non-human primate species that are used in biomedical research.

MLC inhibition studies were performed with two human alloantisera: one specific for HLA-B7, the other for HLA-DRw7. The stimulator cell inhibitory effects of these sera were tested in primary and secondary MLC tests. Both sera inhibited in the primary MLC, whereas only the anti-DRw7 serum was capable of blocking the secondary MLC test. The difference in inhibiting properties of these sera was further analyzed in primary MLC tests using selected MLC combinations, Fc receptor negative cell populations and pepsin digests of the anti-B7 serum. Anti-DRw7 antibodies could inhibit by masking the stimulatory DR antigens. The inhibition of the anit-B7 antiserum was dependent on Fc, which suggested that anti HLA-B antibodies inhibited by some other mechanism. This inhibition could have been caused by antibody dependent cellular lympholysis of the stimulator cells or by the induction of suppressor cell activity.

A population of 94 unrelated rhesus monkeys was typed for MLC antigens using 38 homozygous typing cells which define RhLA-D specificities. A genetic analysis showed that the ten D specificities are alleles of a single locus, the gene frequencies of which are in Hardy-Weinberg equilibrium. The association between the cellularly defined D and the serologically defined DR antigens in the population was usually high, confirming the close relationship between D and DR antigens. Two new associations were found, i.e. between the D-locus antigens 9 and 10 and the serologically defined B-cell antigens 109 and 101, respectively. This observation confirms prior speculations that the latter two antigens may belong to the DR series.

Certain characteristics of 38 homozygous typing cell (TC's) of rhesus monkeys were determined. These TC's define ten RhLA-D locus specificities. Eight of them are associated with established RhLA-DR antigens. Two other groups of typing cells, D9 and D10, were previously considered to be associated with "blank" antigens of the DR series; they now appear to be associated with B-cell antigens which are also likely to be controlled by the DR locus. No influence of RhLA-A or B antigens of MLC reactivity was observed. It was shown, however, that products of at least one locus other than D/DR is responsible for MLC stimulation. Whether those MLC antigens are associated with serologically identifiable B-cell antigens which are not controlled by the DR locus, is not yet clear.

A large number of related chimpanzees were tested in mixed lymphocyte cultures against each other. Several similarities among the D locus products coded for by different ChLA haplotypes were observed. Six animals were found to be homozygous for D locus antigens and two of these animals carried the same D specificity. Hence, the available "typing cells" permitted the identification of five D locus antigens of the chimpanzee. So far, no linkage disequilibrium has been found between any of the D locus antigens and ChLA-A or -B locus antigens.

A human alloantiserum was found which selectively inhibits responding cells in mixed leukocyte culture reactions. Inhibition was achieved by pre-incubation of responder cells in the antiserum followed by washing. The serum showed dual specificity as an inhibiting agent. First, inhibition was restricted to HLA-B7 or -B40 positive stimulator cells, specificities against which the antiserum also had cytotoxic activity. Second, inhibition was almost exclusively associated with the presence of the phenotype HLA-A1, -B8 on the responder cells The HLA associated specificity for responder cells was unexpected since no alloantibody activity directed to responder alloantigens could be detected by conventional serological-methods. The antiserum donor had not been immunized with HLA-A1, -B8 antigens nor with known crossreactive antigens. Furthermore, the serum donor did not carry HLA-A1, -B8 antigens herself. The inhibiting substance in the antiserum had physicochemical properties of IgG and was specifically reactive with HLA-B7 positive platelets. Pepsin digest preparations were not inhibitory. Fc receptor positive responder cells were required for inhibition. Responder cells, preincubated with the antiserum, suppressed the response of cells not incubated with the antiserum. Three possible explanations of these results are discussed: specific binding of the Fc part of the antibody with Fc receptors of responder cells, specific activation of suppressor cells and cross-reactivity.

Eleven lymphocytotoxic pregnancy sera were tested for inhibition in the MLC test. Responder inhibition and stimulator inhibition were analyzed separately by using cells from the serum producer as stimulator and responder respectively. Only three sera showed inhibitionof responder function, whereas all sera showed inhibition of stimulator function. The specificity of the latter inhibition was at least in part attributable to antibodies directed to specificities other than B-cell specificities (presumed to be coded for in the HLA-D region). This was deduced from the fact that platelet eluates devoid of anti B-cell antibodies effectively inhibited in MLC.

One-way mixed lymphocyte culture (MLC) tests were performed among the members of five chimpanzee harems in all possible combinations: parents, parent-child, siblings and half-siblings. The technical aspects of MCL testing in chimpanzees appeared to be very similar to those observed for human and rhesus monkey MLC's. Two unexpected observations were made for which no satisfactory explanation can as yet be give: firstly, the occurrence of animals with consistently high autologous values and secondly, the existence of chimpanzees displaying low MLC responsive. The high autologous values occurred mostly in older imported animals (25% with high autologous values), while only one of the 45 laboratory born animals showed this phenomenon. Low responsiveness occurred in a few offspring belonging to a single harem only and is therefore likely to be genetically controlled but, as it appeared, not by genes linked to ChLA. Data suggesting the existence of an MHC-linked "major MLC" or D locus were confirmed and extended. A gene dose effect for D locus antigens was demonstratable, i.e., combinations of related animals differing for one showed lower MLC responses than combinations differing for two ChLA haplotypes. The number of D locus alleles was estimated to be 10.

The incidence of MLC-inhibiting antibodies was determined in 42 pregnancy sera. MLC's were carried out between the cells from the serum donor and her husband in the presence of nonimmune AB serum and the test serum. Fifty per cent of the sera reduced the MLC response to less than 40% of the control values. Only four sera had lymphocytotoxic activity. The inhibition was strong against the specific immunizor, less strong against random unrelated cells and weak against cells which were SD-identical with the serum donor. Absorptions with lymphocytes and platelets were carried out. Lymphocytes removed activity in three of the four sera tested. Platelets removed activity from one serum. It was concluded that both anti-LD and anti-SD antibodies were able to inhibit the MLR at the stimulator cell level.

Some technical aspects of MLC inhibition by sera obtained from multiparous women were studied. The variability of the MLC response was very high. The serum source used in the cultures was probably to a large extent responsible for this variability. Aspecific inhibition, which was observed with some test sera, could be removed by dialysis against PBS. To make the evaluation of inhibition by immune sera objective, a scoring system was introduced for the degree of inhibition. Test sera were usually added to the cultures. Alternatively, stimulator and responder cells were preincubated with the test serum. Preincubation of the stimulator cells did not show a difference in inhibition pattern when this was compared with serum addition. Preincubation of the responder cells showed a completely different inhibition pattern. The MLC inhibition test compared very well with an indirect immunofluorescence test and a cytotoxicity test using B-cell enriched cell suspensions.