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To determine the extent of kappa chain diversity in the preimmune repertoire early in development, kappa cDNA libraries were analyzed from 15-d old fetal omentum, 18-d-old fetal liver, and 3-wk old bone marrow. An anchored polymerase chain reaction approach was used to avoid bias for particular V kappa families. From the sequence analysis of 27 bone marrow clones, 10 different families and 20 unique V kappa genes were identified. In contrast, the V kappa expression in the fetus is highly restricted and clearly differs from the broader distribution see in 3-wk-old bone marrow. Although several V kappa families were represented in the fetal library including V kappa 9, V kappa 10, V kappa 4,5, V kappa 8, and V kappa 1, one or two members of individual families were observed repeatedly. The fetal liver and omentum libraries were found to be largely overlapping. Given the V kappa families/exons identified in the fetal sequences, the mechanism of kappa rearrangements in the early repertoire appears to occur predominantly by inversion. Importantly, the fetal repertoire was further restricted by dominant V kappa-J kappa combinations such as V kappa 4,5-J kappa 5, V kappa 9-J kappa 4, and V kappa 10-J kappa 1. Since in some cases independent rearrangements could be established, the results indicate a bias for particular V kappa-J kappa joins. The results also suggest that clonal expansion/selection in the fetal repertoire takes place after light chain rearrangement as opposed to at the pre-B cell level in the bone marrow. The restriction observed in kappa light chain expression together with known restrictions in gene usage and junctional diversity at the heavy chain level indicate a remarkably conserved fetal repertoire.

In a clonal analysis of the IgE response, it was found that a small proportion of primary or nonimmune B cells in spleen and mesenteric lymph nodes can be stimulated by antigen to produce IgE-secreting clones. In addition, there appears to be no substantial difference in the frequency of such cells between the classical low and high IgE responder strains. An analysis of immune, or memory, B cells revealed substantial increases in the frequency of B cells secreting IgE as compared with primary B cells, although the actual proportion of B cells secreting IgE remained relatively low. When the IgE-secreting clones derived from either primary or secondary B cells were reanalyzed for the presence of other isotypes, it was found that all clones secreting IgE were secreting at least one other isotype, with the majority secreting two or three other isotypes in addition to IgE. This demonstrates that there is no distinct subpopulation of B cells committed to IgE expression per se.

Neonatal and adult splenic cell suspensions were labeled with fluorescein isothiocynate-anti-Ig and fractionated into surface-immunoglobulin- (s-Ig) positive and s-Ig-negative subpopulations by the fluorescence-activated cell sorter. The subpopulations were then tested by splenic focus assay for both frequency and tolerance susceptibility of clonable 2,4,-dinitrophenol (DNP) precursors. It was shown that both adult, and neonatal, s-Ig-negative subsets contained clonable DNP-specific B-cell precursors. However, because these precursors result in fewer clones secreting IgG, they appeared to be less mature than the s-Ig-positive precursors. In the absence of helper T cells, it was found that exposure of s-Ig-negative lymphocytes to tolerogen during the process in which they were acquiring surface receptors resulted in nearly total abrogation of potential DNP clones. This finding provides compelling evidence for clonal abortion.

The maturation of B-lymphocyte function during fetal development was studied in vivo and in an in vitro organ culture system. The results indicated that the progenitors for 2,4-dinitrophenol (DNP)-specific B cells are present as early as 14 d of gestation in liver and possibly as early as 15 d in spleen. In addition, it was found that the organ culture system supports the development of B lymphocytes as measured by an increase in both the percentage of surface immunoglobulin-positive cells and the frequency of clonable DNP-specific B cells after culturing. The majority of anti-DNP-secreting clones resulting from the antigenic stimulation of fetal B cells produced only the IgM isotype, and the ability to secrete the IgG isotypes increased as a function of gestational age. Because fetal DNP precursors from spleens and livers that had been incubated in organ culture resulted in a greater proportion of clones secreting IgG compared with age-matched uncultured controls, it was concluded that the maturation with regard to the ability to secrete IgG can occur in vitro. In studies relating to the ontogenetic development of tolerance susceptibility, it was found that up to one-half of the DNP-specific B-cell precursors from livers and spleens less than 18 or 19 d of gestation were resistant to tolerogen treatment for 24 h as if in a pretolerant phase. However, if tolerogen were present for 3--5 d during organ culture there was near total elimination of potential DNP clones. This finding suggested that the 24-h induction period was insufficient for affecting the DNP-specific precursors in livers and spleens from the earlier gestational ages, and that a proportion of precursors could subsequently form DNP clones in the splenic focus assay after the removal of tolerogen.

The functional B cell repertoire in BALB/c mice was assessed at various stages in ontogeny. This was done by analyzing VH gene family expression using the sensitive technique of in situ hybridization. The B cell repertoire was probed with the mitogen, LPS, and the antigen DNP. DNP was chosen because B cells responsive to this hapten appear very early in ontogeny. The APCs that developed after stimulation with LPS or DNP were analyzed for VH gene expression by in situ hybridization of individual cells using radiolabeled VH gene family probes. The results indicated that VH gene expression in fetal B cells after stimulation was distinct from adult B cells in that there was a biased expression of D proximal families. The results indicated that this bias was associated with developmental age and not a given differentiation stage in the B cell lineage. In addition, stimulation of fetal B cells with DNP resulted in a large increase in expression of member(s) of VH 36-60, suggesting that the early appearance of DNP-responsive B cells is not strictly correlated with preferential rearrangement of D proximal families, VH 7183 and VH Q52. However, the results suggested that a large proportion of pre-B cells that preferentially rearrange D proximal families early in ontogeny become part of the functional developing repertoire.