The A/WySnJ mouse provides a model for studying B-cell selection into the stable peripheral B-cell pool. The investigators have shown that the A/WySnJ strain has a single, autosomal co-dominant defect, Bcmd-1, resulting in a profound peripheral B-cell deficiency and poor IgG responses. The Bcmd-1 defect is not present in the related A/J sub-line, is intrinsic to B-cells, and shortens the life span of peripheral B-cells by causing premature death at every maturation step after surface IgM acquisition. Two hypotheses can explain these observations: Bcmd-1 may be a loss-of-function mutation that either disrupts a life span lengthening pathway or disrupts a molecule that dampens a life span shortening pathway. The investigators propose biological, biochemical and genetic studies to distinguish these hypotheses and investigate the mechanism of Bcmd-1 action.
In Specific Aim one, the two genes that control B-cell deficiency in interspecies crosses, Bcmd-1 and Bcmd-2, will be mapped by analysis of simple sequence length polymorphisms in the extreme progeny of the (A/WySnJXCAST/Ei) F2 interspecific cross. A congenic strain will be constructed and used to map the Bcmd-1 locus that is polymorphic between strains A/WySnJ and A/J.
In Specific Aim two, the investigators test whether the steps in transit to the long-lived pool affected by Bcmd-1 comprise an """"""""all -or-none"""""""" event versus continuous signals. BrdU labeling studies of F1 individuals will be employed, since continuous signals predict an intermediate life span for all B-cells in F1 mice, whereas an all-or none event predicts separate pools of long-versus short-lived B-cells.
In Specific Aim three, assays will be established for B-cell life span, apoptosis, Bcl gene family induction, and cell division; then pathways that affect these parameters will be systematically probed in immature B-cells. When a pathway is found to affect A/J but not A/WySnJ B-cells, biochemical markers will be analyzed to determine whether Bcmd-1 disrupts the pathway.
In Specific Aim 4, the investigators assess Bcmd-1's effect on B-cell traffic, repertoire formation, and the germinal center reaction, through mixed marrow chimeras, limiting dilution and fine specificity analysis, and cytofluorimetric immunohistochemical studies in chimeric mice.
