Evidence for limited B-lymphopoiesis in adult rabbits

Rabbits are born with a limited VDJ gene repertoire formed primarily by rearrangement of one V-H gene, V(H)1. The VDJ genes are undiversified at birth but become diversified by similar to 2 mo of age. To investigate more closely the time during which this diversity occurs, we determined the nucleotide sequences of VDJ genes from peripheral blood leukocytes taken from young rabbits ar various time points, and we examined the extent of the diversified of the VDJ genes. At 4 wk of age there were, on average, 3 nucleotide changes per V-H region, with similar to 75% of the genes showing some diversification. The number of nucleotide changes per VH region increased to 12 by 6-8 wk of age, and all but 1 of the 35 sequences analyzed were diversified. Because only a limited number of genes can be examined by nucleotide sequence analysis, we used an RNase protection assay to examine a large number of genes and we determined the level of undiversified V(H)1 mRNA in lymphoid organs of both young and adult rabbits. In young rabbits, we found a high level of undiversified VDJ genes, but the level was greatly reduced by 2 mo of age. By adulthood, essentially all VDJ genes of cells from appendix, peripheral blood, and bone marrow were diversified. Because we had expected B lymphopoiesis to be ongoing in the bone marrow of adult rabbits, we were surprised not to find undiversified VDJ genes from newly generated B cells. Therefore, we searched for evidence of ongoing B lymphopoiesis in bone marrow by isolating and examining circular DNA for the presence of VD and DJ recombination signal joints. We found highly reduced levels of recombination signal joints in bone marrow of adult rabbits relative to the levels found in bone marrow of newborn rabbits. These data indicate that limited VD and DJ gene rearrangements occur in bone marrow of adults rabbits, and we therefore suggest that B lymphopoiesis is limited in adults.