Origin and development of a scientific collaboration and a long-lasting friendship

The aim of this contribution is twofold: to honour Ivan Lefkovits with a short recollection of our scientific collaboration in the years 1972-1985 and a summary of our joint contribution to studies of the mechanisms and functions of the immune system and to acknowledge our long-lasting friendship. Ivan's limiting dilution microculture method was adapted to rabbit peripheral blood lymphocytes (PBL). The antibody produced in the responding cultures was shown to be electrophoretically homogeneous and, in rabbits heterozygous at the b locus, to express either one or the other allele. Thus, the antibody released in single microcultures was indeed the product of single B-cell clones and allelic exclusion, once achieved, was maintained throughout clonal proliferation. In the response to streptococcal polysaccharides, analysis of the clonotypes triggered in vitro provided information on mechanisms of clonal dominance. A two-stage culture system was established, where rabbit PBL were precultured at low cell density with antigen before being partitioned in limiting dilution cultures. This method provided a new tool for studies of various cellular aspects of the immune response. Moreover, it allowed the application of the limiting dilution analysis to PBL from unprimed animals. Later, the method was extended with success to human PBL, leading to studies of regulatory aspects of immunity in this species.