Genetic and temporal control of neonatal antibody expression.

Two hybridoma cell lines were established with B cells derived from neonatal BALB/c spleen cells. The anti-dinitrophenyl (DNP) antibodies derived from these lines were characterized with respect to their isotype, affinity, and isoelectric point. Antiidiotypic reagents were prepared that permit an analysis of the representation of antibodies sharing idiotype with these two hybridomas in the developing and mature B cell pool of BALB/c mice (Igha) and other murine strains. One of the two antibodies, TF2-36, was found to be indistinguishable from 14% of anti-DNP monoclonal antibodies derived in fragment culture from spleen cells of 1-4-d-old BALB/c donors. B cells expressing this idiotype were found to represent approximately 2% of the anti-DNP-specific repertoire after the 1st wk of neonatal development and into adulthood. The second hybridoma antibody, TF2-76, was found to be expressed at very low levels during the first several days of neonatal development; however, B cells expressing this idiotype increased in frequency during the 2nd wk of neonatal development representing 7% of all DNP-responsive B cells 12-13 d after birth. The proportion of B cells expressing this idiotype also decreased to approximately 2% in adults. The relatively late appearance of B cells bearing this idiotype was confirmed by their susceptibility to tolerance induction after the 1st wk of neonatal development. Both the early neonatal clonotype, TF2-36, and the late neonatal antibody clonotype, TF2-76, were found to be expressed in a similar fashion in F1 mice constructed between Igha and Ighb parentals, but both were expressed at very low levels during the development of Ighb mice. Thus, the control of the magnitude of expression of these neonatal clonotypes appears to be associated with the Igh locus.

Regulated progression of B lymphocyte differentiation from cultured fetal liver.

Lymphoid fetal liver cultures (LFLC) are long-term, nontransformed cultures of early B lymphoid lineage cells which appear developmentally blocked at the pre-B stage in vitro. When injected into severe combined immunodeficient (SCID) mice, cells from LFLC could reconstitute splenic B lymphocytes and serum IgM. T lymphocyte reconstitution was not observed and serum IgG levels were very low. IgG3 was the predominant gamma subisotype in the serum of the LFLC-reconstituted mice, indicating impaired class switching in these B lymphocytes. When thymocytes were coinjected with LFLC, the B lymphocytes were able to class switch fully and respond to T-dependent antigens. These serological responses were heterogeneous. This experimental system allows separation of three B lymphocyte developmental stages: early differentiation in vitro, progression to IgM secretion in vivo, and late differentiation dependent upon mature T lymphocytes in vivo. The unique advantage of this system is the ability to regulate the B lymphocyte developmental pathway in a defined, stepwise manner.

Long-term cultures of murine fetal liver retain very early B lymphoid phenotype.

Long-term cultures of murine fetal liver have been successfully established using a modification of our in vitro bone marrow culture system (14, 15). Fetal liver cells from midgestation BALB/c embryos were plated onto BAB-14 bone marrow stromal cell-adherent layers. After a 3-5 wk period, cell growth began to increase and these cells were expanded in number on fresh feeder layers. The cultured fetal liver cells were lymphoid in morphology, 5-20% cytoplasmic Ig-positive, but less than 1% surface Ig-positive. Southern blot analysis of the cultured fetal liver cells, as well as cultured bone marrow-derived B cells, demonstrated a population with germline Ig heavy chain loci, possibly representing very early B cell precursors. Abelson murine leukemia virus (A-MuLV) clonal transformants of such cultured fetal liver cells had a phenotypic distribution similar to that seen with fresh fetal liver transformants but distinct from those obtained with the transformation of either cultured or fresh bone marrow. All A-MuLV transformants isolated had rearrangements at the mu heavy chain locus of both chromosomes, irrespective of Ig production. In addition, most mu heavy chain producers had at least one rearranged kappa gene locus. These long-term fetal liver cultures provide large numbers of cells for studying events early in the B lymphocyte lineage. The cultured fetal liver cells retained phenotypic traits similar to fresh fetal liver B cells and distinctive from bone marrow cells cultured under similar conditions.

A murine model for B-cell lymphomagenesis in immunocompromised hosts: c-myc-rearranged B-cell lines with a premalignant phenotype.

Activation of c-myc or bcl-2 protooncogene is a common event in B-cell lymphomagenesis. Alone, each is insufficient to produce lymphoma, prompting the search for the additional steps required to complete the malignant phenotype. Among the existing systems of murine or human B-cell neoplasia, no commonly occurring complementary oncogenic activation has been found. This study introduces a new series of murine B-cell lines with a phenotype suggesting that such additional events might not involve intrinsic growth control, but instead host immune mechanisms which normally suppress tumorigenicity of premalignant B-cells. Four murine B-cell lines were isolated from the long-term culture of normal lymphoid tissue bearing a premalignant phenotype. (a) Their phenotype resembled naturally occurring lymphoid tumors of immunocompromised hosts with regard to c-myc activation, aberrant or absent immunoglobulin expression, preferential rearrangement of the lambda light chain locus, and a distinctive pattern of tissue invasion and tumor histology. (b) Their tumorigenicity was strictly dependent on host permissiveness correlated with immunodeficient status: C.B-17-scid greater than BALB/c-nu/nu greater than normal BALB/c much greater than other H-2d strains (NZB x NZW F1, NZB, DBA/2). (c) Host passage selected for malignant variants distinguished by a 10(4)-fold increase in tumorigenicity (as judged by limiting cell dose) and by novel tumorigenicity in nonpermissive syngeneic hosts. These features are analogous to properties of human lymphomas arising in immunocompromised states and, to our knowledge, unique among previously reported murine B-cell lines.

Tyzzer's disease in an 11-day-old foal.

An 11-day-old pony became depressed, anorectic, and pyrexic 2 days after the topsoil of its paddock had been turned over. Rapid progression to colic and head pressing occurred, despite intensive therapy for Tyzzer's disease, and the foal died within 7 h of the appearance of central nervous system signs.

In vitro development of B lymphocytes from long-term cultured precursor cells.

An in vitro system for the study of B-lymphocyte differentiation from precursor elements has been established. Two separate sets of culture conditions are utilized to control B-lymphocyte precursor growth versus differentiation. The first, modeled after the bone marrow culture system of Dexter and colleagues [Dexter, T. M. & Testa, N. G. (1976) in Methods in Cell Biology, ed. Prescott, D. M. (Academic, New York), Vol. 14, pp. 387-395], allows precursor replication whereas the second set of culture conditions is permissive for differentiation to more mature members of the B-lymphocyte lineage. The shift from one set of conditions to the other allows us to follow the kinetics of this differentiation from precursor cells to pre-B and B lymphocytes over a 5-week period. The resulting population of B-lineage cells synthesizes heterogeneous immunoglobulin molecules as analyzed by two-dimensional gel electrophoresis and has a heterogeneous array of immunoglobulin gene rearrangements, suggesting an origin from very early uncommitted precursors. This in vitro system will allow the study of genes that regulate B-lymphocyte differentiation and will aid in the isolation of the cellular intermediates in the B-cell developmental pathway.

VH repertoire in progeny of long term lymphoid-cultured cells used to reconstitute immunodeficient mice.

VH gene utilization in the progeny of long term lymphoid-cultured cells used for reconstitution of severe combined immunodeficient mice under varying conditions was determined. Hybridomas made from the spleens of these animals were evaluated for clonality and donor origin and a panel of 146 independent hybridomas were subsequently examined for VH expression. Hybridomas derived from the spleens of SCID mice reconstituted with fresh cells, used as a control, utilized VH families in proportion to their numerical representation in the genome. However, hybridomas from the spleens of mice reconstituted with long term cultured cells utilized a predominance of the two VH gene families most proximal to JH, characteristic of cells early in B lymphocyte development. Coinjection of thymocytes with cultured fetal liver cells, to provide good levels of T lymphocytes, did not alter this pattern of VH utilization. Irradiation (3 Gy) of the mice before cultured cell injection, which leads to more complete reconstitution of the B cell compartment, was effective in removing this bias in the VH repertoire. Hybridomas derived from these mice expressed their VH genes more in proportion to family size, characteristic of cells later in B lymphocyte development. In this manner, long term lymphoid-cultured cells can be used to study the transitions that occur in VH repertoire expression which appear to be mediated by either B lymphocyte developmental microenvironment or population size.

Defining the immune mechanism with monoclonal antibodies.

It has long been realized that only the study of homogeneous antibodies or cell populations could enable a definitive understanding of much of the immune mechanism. Hybridoma technology has greatly facilitated such approaches. Hybridoma antibodies have been used to delineate both B cell and T cell subpopulations. T cell studies per se have been accomplished by the use of T cell hybridoma cell lines producing a variety of factors. Anti-idiotypes against B cell hybridoma antibodies have been used to characterize T cell receptors and factors. B cell studies have been facilitated by hybridomas that have made available the immunoglobulin of pre-B cells or defective B cell lines. Hybridoma antibodies have also been used to dissect closely related antibody families and the potential for responsiveness against a variety of antigenic determinants. Finally, hybridomas have provided a primary source of material for protein and DNA sequence analysis. In our laboratories hybridoma antibodies derived against the murine H-2 locus have demonstrated the ability of B cell antibodies to discriminate amongst H2 mutants--a capacity previously attributed only to T cell specificities. Hybridoma antibodies have also been generated by fusions with antigen stimulated neonatal B cells to provide homogeneous antibodies reflective of the earliest developmental immunoglobulin readout. Such probes should increase our understanding of the processes involved in the generation of both the T and B cell repertoires

Evidence for a novel exon in the coding region of the adenomatous polyposis coli (APC) gene.

Germline mutations of the tumor suppressor gene APC cause familial adenomatous polyposis. Somatic APC alterations are involved in several sporadic neoplasms, including colorectal, duodenal, gastric, and esophageal carcinoma. The APC mRNA is encoded by 15 exons. Additional transcripts have been reported, due to alternative splicing of coding as well as noncoding regions. Two mRNA isoforms occur due to a deletion of exon 7 or a partial deletion of exon 9. We have identified a novel exon, flanked by APC exons 10 and 11, which is expressed as an alternatively transcribed product of the gene. Further, we have shown that the novel exon consists of a heptad repeat motif and is conserved across species.

Human-human B cell hybridomas from in vitro stimulated lymphocytes of patients with common variable immunodeficiency.

Human-human B cell hybridomas have been established from the peripheral blood lymphocytes of patients with common variable immunodeficiency (CVI) by fusion with an HGPRT-negative B lymphoblastoid cell line. IgM-secreting hybridomas were successfully obtained from CVI lymphocytes after stimulation for 5 days in vitro with a combination of PWM and Staphylococcus aureus strain Cowan I. Fusion of peripheral blood lymphocytes that were stimulated for 5 days in vitro with a single mitogen resulted in no viable hybrids from a total of 600 X 10(6) CVI lymphocytes. The combination of PWM and Cowan I did not induce appreciable Ig secretion from the CVI lymphocytes during the 5-day course, although it did so in normal lymphocytes. After the 5-day stimulation with this mitogen combination, however, a large percentage of the original number of peripheral blood cells were recovered, and these had a fusion frequency of approximately 1 to 2 per 10(6) with the B lymphoblastoid line. Fifteen cloned IgM-secreting hybridomas have been isolated from five different CVI patients. These hybridomas are tetraploid and have been stable in culture for 6 to 12 mo. All of the hybridoma lines that were examined contain a functionally rearranged IgM heavy chain gene from the B cell parent of the CVI patients. These human-human B cell hybridoma lines will enable a more thorough characterization of the B cell defects involved in CVI at the cellular and molecular levels.

Lymphoid bone marrow cultures can reconstitute heterogeneous B and T cell-dependent responses in severe combined immunodeficient mice.

Long-term lymphoid bone marrow cultures (LBMC) produce B lymphocytes and their precursors for several months in vitro. To assess their differentiative potential and determine their capacity to function as immune effectors, cells from the cultures were transplanted into mice with severe combined immune deficiency disease (SCID). SCID mice are deficient in T and B lymphocytes and are serum immunoglobulin (Ig) negative, but grafts of normal lymphoid precursors can expand and differentiate in them, thereby restoring immunocompetence. The results of these studies indicate that cells from LBMC are able to reconstitute splenic B lymphocytes in the SCID mice. Upon in vivo transfer, LBMC cells secreted Ig that displayed isotype distribution and a pattern of heterogeneity comparable with normal BALB/c mice, as determined by two-dimensional gel electrophoresis. The transplanted LBMC cells were functional, because reconstituted mice could respond to immunization with the T-independent antigen TNP-Ficoll. The results also indicate that cultured cells could reconstitute T cell activity in SCID mice. Splenocytes from approximately one-third of the recipients could generate a cytotoxic response to alloantigens after 5 days of sensitization in a mixed lymphocyte culture, and all reconstituted SCID mice could respond to immunization with the T cell-dependent antigen TNP-BSA. These results demonstrate that B cells, as well as T cell activity, are present in LBMC-reconstituted SCID mice, and show that LBMC cells have the capacity to mediate an immune response.

Expression of an immunoglobulin heavy chain gene transfected into lymphocytes.

We determined that mouse lymphoid cell lines can be transfected at high efficiencies (10-70%) by a polyoma virus shuttle vector. With this vector, we obtained expression of a cloned mouse alpha heavy chain gene transfected into cell lines representative of all stages in B-lymphocyte development, a T-cell lymphoma line, and 3T3 fibroblasts. Heavy chain gene expression in transfected light chain-producing myeloma cells occurred at levels comparable to those in IgA-secreting myeloma cells. Heavy chains produced in transfected myeloma cells were associated with light chains in membrane-bound IgA. While T-lymphoma cells and fibroblasts were transfected at similar efficiencies to B cells, significantly lower levels of alpha heavy chains were produced. This immunoglobulin gene transfection system provides a powerful approach for defining important regulatory regions in immunoglobulin genes and for identifying lymphoid cell factors involved in immunoglobulin gene expression in B-lymphocyte development.

Pre-B cells in kappa-transgenic mice.

Recent experiments have shown that the microinjected kappa-chain gene of transgenic mice is expressed in a tissue-specific fashion only in B lymphocytes. The next step was to determine whether, within the B-lymphocyte lineage, the kappa-chain gene was expressed in a normal developmental fashion. Normally, only mu heavy(H)-chain genes, and not kappa-chain genes, are expressed in pre-B cells. To obtain cloned cell lines derived from early cells of the B-cell lineage, we transformed bone marrow cells from kappa-transgenic mice with Abelson murine leukaemia virus (A-MuLV) and tested the resultant cell lines for the retention of the kappa transgene and its expression in RNA and protein. We found that cells with the pre-B phenotype exist in kappa-transgenic mice. We further observed that in A-MuLV-transformed cell lines from a kappa-transgenic mouse with a high copy number of the transgene, the proportion of cell lines expressing kappa (transgenic kappa) was higher than in cell lines from normal or low copy number transgenic mice.

Delay of early B-lymphocyte development by gamma 2b immunoglobulin transgene: effect on differentiation-specific molecules.

Mice transgenic for gamma 2b Ig heavy chain were examined for alterations in B-cell differentiation and endogenous Ig gene rearrangement and expression. Fresh bone marrow from these mice was markedly reduced in BP-1+ cells and there were small reductions in B220+ and sIg+ cells. A-MuLV (Abelson murine leukemia virus) transformants from these bone marrow cells showed little alteration in Ig gene rearrangement and expression when compared to controls, however. Isolation of the B-lymphoid compartment from these mice in vitro using LBMC (lymphoid bone marrow cultures) enabled more detailed characterization of the effects of the transgene. LBMC derived from gamma 2b transgenic mice had similar growth kinetics, but a 4-5-week delay in the expression of endogenous mu Ig in comparison to control cultures. Nucleic acids derived from these early cultures prior to endogenous mu Ig expression showed reduced Ig JH rearrangements, some sterile mu transcription, low levels of BP-1 expression, and virtually undetectable TdT (terminal deoxynucleotidyl transferase) expression. Thus, this gamma 2b transgene appears able to affect early B-lymphocyte development.

Severity of infections in IgA deficiency: correlation with decreased serum antibodies to pneumococcal polysaccharides and decreased serum IgG2 and/or IgG4.

In order to define abnormalities of humoral immunity which determine susceptibility to respiratory tract infections in IgA-deficient adults, serum IgG subclass concentrations, and serum concentrations of pneumococcal antibodies and Haemophilus influenzae type B (Hib) antibodies sera from IgA-deficient adults with and without susceptibility to respiratory tract infections were compared. Infection susceptibility was not related to the degree of IgA deficiency, but was related to deficiency of IgG4 and, to a lesser extent, IgG2, as well as to low basal serum concentrations of pneumococcal polysaccharide antibodies. The combination of IgG2 and/or IgG4 deficiency and a non-protective basal serum concentration of antibody against two or more pneumococcal polysaccharides was present in the serum of six of 12 (50%) patients with severe infections, but only one of 44 (2%) patients without infections. Furthermore, the preservation of antibody responses against the most immunogenic pneumococcal polysaccharide type 3, but not against the less immunogenic types 7F, 9N and 14, in patients with severe infections suggested that abnormalities of pneumococcal polysaccharide antibody responses might include defects of affinity maturation.

Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement.

We have analyzed the configuration of the H chain locus of 41 hybridomas by Southern blot analysis. Each H chain switch region was determined to be germ line, rearranged, or deleted. Including 13 previously analyzed hybridomas, 60% of those with rearrangements on both alleles showed a correlation of the two alleles, i.e., both the expressed and the nonexpressed alleles have rearranged to the same H chain constant region gene segment. When the two H chain alleles did not rearrange to the same gene, they often rearranged to neighboring H chain genes. These results support a role for isotype-specific factors in H chain switch recombination. The action of these isotype-specific factors may be propagated to some extent along the chromosome, which would lead to rearrangements to neighboring genes.

Differential expression of myc family genes during murine development.

The myc family of cellular oncogenes contains three known members. The N-myc and c-myc genes have 5'-noncoding exons, strikingly homologous coding regions, and display similar oncogenic potential in an in vitro transformation assay. The L-myc gene is less well characterized, but shows homology to N-myc and c-myc (ref. 6; also see below). c-myc is expressed in most dividing cells, and deregulated expression of this gene has been implicated in the development of many classes of tumours. In contrast, expression of N-myc has been found only in a restricted set of tumours, most of which show neural characteristics; these include human neuroblastoma, retinoblastoma and small cell lung carcinoma (SCLC). L-myc expression has so far been found only in SCLC. Activated N-myc and L-myc expression has been implicated in oncogenesis; for example, although N-myc expression has been found in all neuroblastomas tested, activated (greatly increased) N-myc expression, resulting from gene amplification, is correlated with progression of the tumour. We now report that high-level expression of N- and L-myc is very restricted with respect to tissue and stage in the developing mouse, while that of c-myc is more generalized. Furthermore, we demonstrate that N-myc is not simply a neuroectoderm-specific gene; both N- and L-myc seem to be involved in the early stages of multiple differentiation pathways. Our findings suggest that differential myc gene expression has a role in mammalian development and that the normal expression patterns of these genes generally predict the types of tumours in which they are expressed or activated.