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The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.

The metabolic requirements of tolerance induction of immature B cells has been analyzed through the use of various putative inhibitors. The study utilizes the splenic fragment assay in which tolerance induction of individual B cells can be examined. Concentrations of inhibitors were determined which, if removed after the first 18 hr of culture, before antigenic stimulation, had no inhibitory effects. Thus, by adding tolerogen in the presence or absence of inhibitor during the first 18 hr of culture, the effect of that inhibitor on tolerance could be assessed. By using this protocol, the data indicate that several metabolic functions of the cell are necessary for tolerance induction to occur, including RNA biosynthesis, DNA biosynthesis, and a methyltransferase reaction, because drugs that interfere with these metabolic processes also prevent tolerance induction. Our previous studies indicated that protein biosynthesis and energy generation are also required. However, drugs that interact with the cytoskeletal structure of the cell and inhibit surface immunoglobulin capping do not interfere with tolerance induction. Moreover, colchicine, which inhibits cell division, does not inhibit B cell tolerance. Collectively, the results provide compelling evidence that the mechanism of immature B cell tolerance involves an active process requiring several metabolic activities of the cell.

The clonal deletion model proposed by Burnet and Lederberg and expanded by Nossal was one of the first theories concerning the nature of tolerance of self constituents. The model proposed that during the maturation of lymphocytes into immunocompetent cells, there is a sensitive differentiation stage whereby contact wth antigen results in specific inactivation of the cell. Experimental evidence indicates that neonatal or immature B lymphocytes are indeed different from adult lymphocytes in their extreme sensitivity to tolerance induction even at low antigen concentrations and with antigens that are normally immunogenic. The present study examines the mechanism of this tolerance phenomenon by determining whether or not tolerance of immature B cells is an active process and what specific interactions can induce this event. We used various putative inhibitors of tolerance induction in the splenic focus assay which examines the tolerance susceptibility of individual B cells. The results suggest that tolerance requires protein synthesis and that this process is initiated only after a minimum threshold affinity of binding occurs between antigen and cell-surface receptor with subsequent receptor interlinkage.

Antiserum to bovine serum albumin were used as a probe for native structure to study the process of refolding of denatured bovine serum albumin. Different antigenically active fragments that represent domains and subdomains of the molecule were isolated. Restricted populations of antibody to albumin were obtained by fractionating antisera to the native intact molecule on immunoadsorbents bearing these fragments. The restricted antibody populations were then used to probe the surface of the molecule during the refolding process. Some regions of the molecule refolded more rapidly than other regions. Isolated domains of albumin also refolded to native antigenic structure demonstrating that the entire polypeptide chain was not necessary for reformation of native structure. However, there does seem to be some interdomain influence on the rate of refolding of a particular domain within the intact protein.

The fetal VH gene repertoire was shown previously to be characterized by overrepresentation of D-proximal families, VH 7183 and VH Q52, compared with adult bone marrow B cells in which VH genes were expressed in a more stochastic fashion. To determine the underlying mechanisms of these findings, adult vs fetal progenitors were placed in the same supportive microenvironment and the resulting B lineage cells analyzed for VH gene family expression. The supportive microenvironment was provided by established adult bone marrow stromal cell layers. In this way the relative importance of environmental vs genetic influences could be determined. The fetal B cells and pre-B cells that developed on adult stromal cells maintained a fetal-like VH gene family repertoire with preference for D-proximal families VH 7183 and Q52. In contrast, adult cultured B cells maintained the adult-like repertoire with predominance of the largest family VH J558. Only after long-term incubation was there a change in the expression of particular VH gene families. These findings suggest that the D-proximal VH gene family preference observed early in ontogeny is associated more with the inherent genetic potential of B cell progenitors that predominate during fetal life and less with environmental influences.

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.

Mice infected with the parasite Mesocestoides corti undergo a polyclonal antibody response that results in a hypergammaglobulinemia restricted to the IgM and IgG1 isotypes. It was found that a similar restriction to IgM and IgG1 could be observed in an in vitro lymphocyte culture system providing that the source of helper T cells was from infected animals. In order to characterize the helper T cells responsible for the restriction, helper T cell clones were generated. Attempts to obtain isotype-restricting helper T cell clones by using the intact, nonviable organism were unsuccessful in that these T cell clones promoted multiple antibody class expression. However, two types of CD4+ (cluster designation) T cell clones were generated by cultivation on the live organism that appeared relevant to the observed restriction. These T cells did not function as conventional carrier-specific helper T cells. Instead, they were shown to regulate T-dependent responses to 2,4-dinitrophenyl-keyhole limpet hemocyanin by 2,4-dinitrophenyl-specific B cells and keyhole limpet hemocyanin-primed T cells derived from uninfected mice. The helper phenotype of one regulatory clone enhanced the IgG1 response, whereas the other phenotype inhibited the production of the other non-IgM isotypes tested. It is concluded that the activities of these two prototype regulatory T cell clones may predominate in infected animals resulting in the IgM, IgG1 dominance of the antibody response.