Long-term antibody synthesis in vitro. VI. Anti-allotype sera as probes of clonal products in affinity maturation.
A new experimental system is described for measuring the allotypic product of rabbit B cells during long-lasting in vitro antibody responses. The immunoenzymatic assays described allow determination of several parameters mapping in different regions of the same molecule, which can be measured and combined to yield a multidimensional picture of the time-course dynamics of antibody synthesis. The rabbit immune system responding to Escherichia coli beta-D-galactosidase was sample and disassembled by (a) culturing lymph node microfragments and (b) sorting out from among all anti-enzyme antibodies only those activating a mutant enzyme, AMEF, which bore the b4 or b9 allotype. A considerable simplification of the response was achieved in the microcultures as documented by cultures of heterozygous cells which produced only one allotype and by the fact that each culture showed a distinctive pattern when antibody titre, association constant, heterogeneity index, L-chain type, and k-chain allotype were considered together. This array of patterns was not an artifact but the result of disassembling a representative sample of the rabbit immune system into small components, since the b4/b9 ratio obtained by averaging the results of all cultures from a heterozygous rabbit lymph node was the same as the serum ratio. Despite the Poisson distribution of the responder microcultures, none of them was monoclonal; i.e. no antibodies homogeneous by all parameters tested were observed, This finidng supports the notion that in normal lymphoid tissue in its native tridimensional arrangement, one T cell can trigger several B cells clustered in one antibody-forming unit. This natural arrangement would ensure the monospecificity of the cluster (dictated by the T cell) while allowing for variation in affinity (depending upon the array of B cells in the unit). Accordingly our findings would results from the fact that as the size of the microfragments was reduced, the cells diluted out first were T cells, but as long as one of them was present, several B-cell clones were triggered. The b4/b9 pattern of any given culture remained constant over several months, but the ratio kappa/lambda underwent changes. An increase in molecules with non kappa-chains (which could not be reacted with anti-kappa-chain allotype antisera) was usually associated with a parallel decrease in antibody affinity. This occurred by the end of the antibody cycle and might be related to the regulation of antibody synthesis by T-cell suppressor factors.
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D000915:Antibody Affinity; D000917:Antibody Formation; D002478:Cells, Cultured; D002999:Clone Cells; D004926:Escherichia coli; D005838:Genotype; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D007126:Immunoglobulin Allotypes / Q000032:analysis* / Q000235:genetics; D007145:Immunoglobulin kappa-Chains; D008198:Lymph Nodes / Q000276:immunology; D011817:Rabbits; D013997:Time Factors; D001616:beta-Galactosidase / Q000276:immunology
D000907:Antibodies, Bacterial; D007106:Immune Sera; D007126:Immunoglobulin Allotypes; D007145:Immunoglobulin kappa-Chains; C496591:kappa allotype b4, rabbits; D001616:beta-Galactosidase