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From approximately 3,000 CML combinations, originally established in order to evaluate the qualitative and quantitative influence of the serologically defined HLA-A, B, and C antigens on cellular, complement independent cytolysis, 12 combinations were selected yielding reproducible positive cytolysis on allogenic target cells, although no HLA-antigenic sharing could be demonstrated between stimulator and target lymphocytes. These 12 CytoToxic Lymphocytes (CTL's) have been tested in parallell as "CML typing combinations" against lymphocytes from a random population sample of 100 unrelated Danes. Based on a pairwise analysis 11 of these CTL's could be classified into two groups of significantly correlated CTL's. These two groups do not define monospecific traits of allelic genetic origin as judged by a mutually positive correlation and a poor fit to Hardy-Weinberg equilibrium. The traits defined by these groups may be either partially identical or governed by closely linked loci. The same groups were identified and the same conclusions reached after exclusion of those individuals in the population sample where HLA-A, B, C, or D antigens may be targets for destruction. Thus, this study gives direct evidence that known HLA antigens are not sole target determinants in CML or that cytotoxic lymphocytes recognize HLA molecules in a different way than lymphocytotoxic antibodies. The studies underline the immunogenetic complexity of CML although this reaction is most probably governed by genes in the HLA region. It is suggested that cytotoxic lymphocytes may recognize "backbone structures" of the HLA molecules.

The relative influence on MIF release in MLC of different parts of the HLA region was studied in 1) MLCs between cells from HLA-A and B identical (probably HLA-D unidentical) unrelated persons and 2) MLCs between cells from HLA-D identical, HLA-A and B unidentical unrelated persons. It was found that the HLA-D part of the region has the major effect on the MIF production. Studies of MIF activity in supernatants from mitomycin treated cultures lent some evidence to the hypothesis that there is an extreme linkage disequilibrium between the HLA-D alleles and hypothetical separate MIF alleles. Examination of families with crossover between the HLA-A, B and HLA-D part of the region is required for confirmation of these findings.

Several normalization procedures have been applied to single MLC-typing experiments in order to neutralize experimental variation and hence to secure assignment of HLA-D phenotypes. The present paper concerns a statistical description and isolation of experimental and biological factors in repeated MLC-typing experiments. The statistical model used gives a meaningful description of the variables in MLC as judged by its application to MLC-typing data of HLA-identical siblings. Subsequent testing of the model in an HLA-A, -B,-C and -D-genotyped family material provides evidence that the level of typing response to selected homozygous typing cells may be influenced by factors coded for by the homologous HLA-D region. It is, however, concluded that such evidence must be accepted with caution due to the (for the present statistical purpose) suboptimal design for the HLA-D typing experiments and hence the relative insensitivity of the method. A general method for locating erroneous triplicates is devised.