The characterization fo the B-cell repertoire specific for the 2,4-dinitrophenyl and 2,4,6-trinitrophenyl determinants in neonatal BALB/c mice.

Abstract

The process by which an individual's repertoire of unique antibody-forming cell precursor (B-cell)' specificities (clonotypes) is acquired has been the subject of extensive investigation and controversy over the past several years (1-3) . To help clarify the mechanism by which diversification of B-cell clonotypes might occur, this laboratory has conducted an investigation of the B-cell clonotypes available during the early stages of neonatal development, i .e ., at an important period in the initial development of the specificity repertoire .Previous reports from this laboratory have demonstrated that the adoptive transfer of neonatal spleen cells to an adult, carrier-primed environment maximizes the responsiveness of neonatal B cells (4-6) . The principle rationale for this approach-that neonatal B cells are indeed immunologically competent when provided with ancillary mechanisms by the adult host environment-has been verified by other investigators (7,8) . Previous analyses from this laboratory have also shown the similarity of neonatal and adult primary B cells, in terms of the kinetics and antigen dose dependence of the antibody response (4), and the relative ease of hapten inhibition of primary B-cell stimulation (6,9,10) . Recently, an analysis of the frequency of neonatal B-cell precursors specific for the haptenic determinants 2,4-dinitrophenyl (DNP) ; 2,4,6-trinitrophenyl (TNP) ; and fluorescein (FL) has demonstrated that a disparity exists in the rate of development of FL-specific precursor cells, as compared to DNP-or TNP-specific cells (5) . Thus, at an early stage in the generation of specificity, when the total number of available specificities is limited, significant disparities may exist due to potential "all or none" expressions .This report extends these observations by an analysis of the clonotypes available in neonatal BALB/c mice which are specific for the DNP and TNP haptenic *

In vitro tolerance induction of neonatal murine B cells.

“…The preparation ofLirnulus polyphernus hemocyanin (Hy), dinitrophenylated hemocyanin (DNP-Hy, i0 mol of DNP per 100,000 g of Hy), and trinitrophenylated hemocyanin (TNP-Hy, 10 tool of TNP per 100,000 g of Hy) have been previously described (29,31). Human gamma globulin (H-/G, Cohn fraction If, Nutritional Biochemicals Corporation, Cleveland, Ohio) was purified by the method of Chiller et al (33).…”

“…Thus, tolerance induction or immune induction of neonatal and adult B cells can be compared at the level of individual B cells. In addition, this technique is currently the only available method that permits measurable stimulation of neonatal B cells under defined conditions and allows the characterization of the isotype and clonotype (individual antigenic specificity) of the antibody product of each stimulated B cell (29)(30)(31)(32)). …”

“…20 ~l of culture fluids collected 10 or 13 days after stimulation were quantitatively assayed for antihapten antibody. The radioimmunoassays were performed by detecting immunoglobulin bound to 2,4-dinitrophenyl-lysine-bromoacetylcellulose immunoadsorbent (DNP-LYS-BAC), 2,4,6-trinitrophenyl-lysine-BAC (TNP-LYS-BAC), or dansyl-lysine-BAC (DANS-LYS-BAC) prepared by general procedures described previously (29)(30)(31). Bound immunoglobulins were quantified with ~25I-labeled purified rabbit antimouse Fab or goat antimouse IgM (p) or IgG1 (T1) antibody (39,40).…”

“…Previous studies from this laboratory have demonstrated that the splenic focus technique permits detectable stimulation of individual neonatal or adult clonal precursor cells (28)(29)(30)(31)(32). Since tolerogens can be added to fragment cultures and removed before stimulation, this system provides a unique opportunity to compare the interaction of tolerogen with individual neonatal and adult cells.…”

Ontogenic development of B-lymphocyte function and tolerance susceptibility in vivo and in an in vitro organ culture system.

“…This is in contrast to specificities directed against other haptens such as phosphorylcholine, which develop later in ontogeny (22). From these results Klinman and Press (21) predicted that the DNP clonotypes would be expressed as early as the 15th d of gestation in fetal spleen. Therefore, DNP appeared to be a suitable antigen for studies of B-cell function during fetal development.…”