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Biomedical subjects

R R Hardy

Publications and source records attributed to R R Hardy.

At least 37 records · Page 2Linked to original sources
PubMed1998

Regulation of anti-DNA B cells in recombination-activating gene-deficient mice.

Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti-double-stranded (ds)DNA and anti-single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B cells seem to be functionally silenced. Here we have investigated how anti-DNA B cells are regulated in recombination- activating gene (RAG)-2-/- mice. In this setting, anti-dsDNA B cells are eliminated by apoptosis in the bone marrow and anti-ssDNA B cells are partially activated.

Animals↗
PubMed1998

HD mice: a novel mouse mutant with a specific defect in the generation of CD4(+) T cells.

We have identified a spontaneous mutation in mice, which we term HD for "helper T cell deficient." This mouse is distinguished by the virtual absence of peripheral T cells of the CD4(+)8(-) major histocompatibility complex (MHC) class II-restricted T helper subset due to a specific block in thymic development. The developmental defect is selective for CD4(+)8(-) cells; the maturation of CD4(-)8(+) and gamma delta T cells is normal. The autosomal recessive mutation underlying the HD phenotype is unrelated to MHC class II, since it segregates independently of the MHC class II locus. Moreover, the HD phenotype is not caused by a defect of the CD4 gene. Bone marrow transfer experiments demonstrate that the defect is intrinsic to cells of the hematopoietic lineage, i.e., most likely to developing thymocytes themselves. The frequency of CD4(+)8(low) intermediate cells is markedly increased in HD mice, suggesting that class II-restricted thymocytes are arrested at this stage. This is the first genetic defect of its kind to be described in the mouse and may prove highly informative in understanding the molecular pathways underlying lineage commitment.

Animals↗
PubMed1998

The evolution of B precursor leukemia in the Emu-ret mouse.

Emu-ret mice carrying an RFP/RET fusion gene under the transcriptional control of the immunoglobulin heavy chain enhancer develop B lineage leukemias/lymphomas. We have characterized B-cell development in these mice before the onset of clinical disease to determine the steps involved in leukemogenesis. Flow cytometry reveals that the CD45R+CD43(+)CD24(+)BP-1(+) late pro-B-cell population is markedly expanded in the bone marrow of 3- to 5-week-old Emu-ret mice. Compared with late pro-B cells from transgene-negative mice, Emu-ret late pro-B cells have a limited capacity to differentiate in interleukin (IL)-7 and a higher incidence of VDJ rearrangements, but a similar cell cycle profile. In contrast, CD45R+CD43(+)CD24(+)BP-1(-) early pro-B cells from 3- to 5-week-old Emu-ret mice, which also express the RFP/RET transgene, differentiate in IL-7 similarly to their normal counterparts. Furthermore, early pro-B cells from Emu-ret and transgene-negative mice have an identical pattern of growth inhibition when exposed to interferons (IFNs)-alpha/beta and -gamma, whereas, pro-B-cell leukemia lines derived from Emu-ret mice are markedly less sensitive to growth inhibition by these IFNs. In 13-week-old well-appearing Emu-ret mice, late pro-B cells upregulate CYCLIN D1 expression and downregulate CASPASE-1 expression in a pattern that correlates with the emergence of B precursor cells in the peripheral blood and the loss of other B lineage subsets in the bone marrow. Taken together, these results suggest that the expression of the RFP/RET transgene initially prevents the normal elimination of late pro-B cells with nonproductive rearrangements. Secondary events that simultaneously disturb the normal transcriptional regulation of genes involved in the control of the cell cycle and apoptosis may allow for subsequent malignant transformation within the expanded late pro-B-cell population.

Animals↗
PubMed1998

A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly.

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a long-standing puzzle in immunology. Recently it has become clear that production of immunoglobulin mu heavy chain and subsequent assembly with a surrogate light chain to form the pre-B cell receptor complex is critical for development of B cells. Here we show that instead of promoting pre-B cell progression as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver. Curiously, we identify a fetal-associated VH11 mu heavy chain that allows continued pre-B proliferation in fetal liver. Interestingly, this heavy chain does not associate efficiently with a surrogate light chain, providing a previously unrecognized mechanism for skewing the expression of distinctive VH genes toward fetal through early neonatal life.

Animals↗
PubMed1998

c-Rel is essential for B lymphocyte survival and cell cycle progression.

c-Rel is a lymphoid-specific member of the NF-kappaB/Rel family of transcriptional factors. To investigate the role of c-Rel in B lymphocyte function, we generated a c-Rel(-/-) mouse via a gene targeting approach. Although early lymphocyte development is normal in c-Rel(-/-) mice, there are significantly fewer B cells displaying a memory (IgM/IgD-) phenotype. Upon immunization, c-Rel(-/-) mice generate fewer B cells with a germinal center (PNAhi) phenotype. In vitro, c-Rel(-/-) B cells proliferate poorly upon ligation of their surface IgM or CD40 receptors or when stimulated with either lipopolysaccharide (LPS) or T cell help. Early molecular events that precede proliferation, such as increases in RNA synthesis as well as IL-2 receptor alpha chain expression, are greatly diminished in c-Rel(-/-) B cells. Furthermore, c-Rel(-/-) B cells are impaired in the ability to receive survival signals generated by anti-IgM or LPS. In contrast, CD40-mediated cell survival is normal in c-Rel(-/-) B cells, suggesting the involvement of a survival-signaling pathway that is independent of c-Rel. When c-Rel (-/-) B cells are co-stimulated with either anti-IgM and CD40 or LPS and CD40, they are rendered capable of progressing through the cell cycle. Finally, co-culture experiments suggest that the defects observed in c-Rel(-/-) B cells are intrinsic to the cell and can not be rescued through either cell-cell contact or addition of soluble factors. Thus, c-Rel is requisite for differentiation to the germinal center and memory B cells in vivo and is required for the transduction of survival and cell cycle progression signals mediated by anti-IgM and LPS in vitro. Furthermore, while c-Rel is involved in CD40-induced proliferation, it is apparently dispensable for the survival signals transduced by CD40.

Animals↗
PubMed1997

The human PD-1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors.

We report the complete cDNA sequence and the genomic structure of the human PD-1 homologue. An analysis of the expression pattern of the human PD-1 gene (hPD-1) and the murine PD-1 gene (mPD-1) in developing bone marrow B-lineage cells was also undertaken. The full length hPD-1 cDNA is 2106 nucleotides long and encodes a predicted protein of 288 amino acid residues. The hPD-1 and mPD-1 genes share 70% homology at the nucleotide level and 60% homology at the amino acid level. Four potential sites for N-linked glycosylation are conserved, as are a stretch of amino acids between two cysteine residues resembling a V-set immunoglobulin domain, and another region containing a motif similar to an immunoreceptor tyrosine-based inhibitory motif. Isolation of the genomic locus of the hPD-1 gene reveals that the gene is composed of five exons located on human chromosome 2 at band q37. The 5' flanking region lacks TATA and CAAT cis-acting elements, but includes a number of potential transcription factor binding sites and a dominant transcription start site. The mPD-1 gene was preferentially expressed in pro-B cells from murine adult bone marrow. Although hPD-1 was not preferentially expressed in pro-B cells from human fetal bone marrow, treatment of isolated pro-B cells with interleukin-7 resulted in a dramatic increase in expression. These data suggest that PD-1 may play a role in B-cell differentiation during the pro-B cell stage.

Amino Acid Sequence↗
PubMed1997

The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells.

The expressed human Ig repertoire is not an equal representation of all V(H) segments present in genomic DNA. Studies have shown that a restricted set of V(H) gene segments are over-represented in Ab repertoires of fetal/neonatal and adult B cells. Additionally, this restricted set of V(H) genes is frequently expressed by autoimmune and tumor B cells. To investigate at which developmental stage a bias in the repertoire begins, we compared the V(H)3 and V(H)4 family repertoires of pre-B and immature B cells from bone marrow and mature B cells from peripheral blood of two adults. We found that the V4-34 and V4-59 gene segments of the V(H)4 family and the V3-23 gene segment of the V(H)3 family dominate the repertoires of the surface Ig-negative early pre-B as well as immature and mature B cells. Furthermore, the pattern of utilization of other V(H)3 family members suggests that certain genes that are frequently rearranged during early stages of B cell development are subsequently disfavored during later stages of B cell maturation. We conclude that the over-representation of certain V genes could arise from sequential mechanisms operating at both early and later stages of B cell development. These V(H)-mediated mechanisms might include preferential rearrangement and/or efficiency of pairing with the surrogate light chain at the surface Ig-negative, early pre-B cell stage and ligand selection at more mature, surface Ig-positive, B cell stages.

Adult↗
PubMed1997

Down-regulation of terminal deoxynucleotidyl transferase by Ig heavy chain in B lineage cells.

The enzyme terminal deoxynucleotidyl transferase (TdT) adds nontemplate-derived nucleotides (N regions) to the junctions between recombining variable, diversity, and joining segments of Ig genes. The relative paucity of N regions in Ig light chains, together with the down-regulation of TdT transcription in pre-B cells (prior to light chain production), suggested that production of IgM heavy chain (mu) protein might negatively regulate TdT expression. In this study, we examined the effect of mu production on TdT gene expression in B lineage subsets from normal mice, from recombination-deficient mice (SCID and Rag-1-) carrying mu transgenes, and in transformed pro-B cell lines transfected with mu constructs. In normal mice, TdT is sharply down-regulated at the early pre-B stage in which cells have just completed productive mu rearrangement. Furthermore, the expression of mu transgenes in pro-B stage cells from recombination-deficient mice results in a similar decrease. Finally, transfection of genomic constructs encoding mu into pro-B cell lines results in a marked reduction of TdT expression. Taken together, these findings indicate that mu protein production results in the down-regulation of TdT. The ability of mu transgenes to alter TdT expression in cell lines also suggests that signaling through the pre-B receptor does not necessarily require interaction with an external stromal cell-derived ligand.

Animals↗
PubMed1997

Both E12 and E47 allow commitment to the B cell lineage.

The E2A gene products, E12 and E47, are required for proper B cell development. Mice lacking the E2A gene products generate only a very small number of B220+ cells, which lack immunoglobulin DJ(H) rearrangements. We have now generated mice expressing either E12 or E47. B cell development in mice expressing E12 but lacking E47 is perturbed at the pro-B cell stage, and these mice lack IgM+B220+ B cells in both bone marrow and spleen. IgM+B220+ B cells can be detected, albeit at significantly reduced levels, in the bone marrow and spleen of mice lacking E12. Ectopic expression of both E12 and E47 in a null mutant background shows that E12 and E47 act in concert to promote B lineage development. Taken together, the data indicate that both E12 and E47 allow commitment to the B cell lineage and act synergistically to promote B lymphocyte maturation.

Animals↗
PubMed1996

Transcription factor B cell lineage-specific activator protein regulates the gene for human X-box binding protein 1.

The transcription factor human X-box binding protein 1 (hXBP-1) is a basic region-leucine zipper protein implicated in the regulation of major histocompatibility complex class II gene expression as well as in exocrine gland and skeletal development. Multiple regulatory elements in the hXBP-1 promoter lie 3' to the transcription start site, including the hX2 site, whose core sequence is an AP-1-like element identical to the hXBP-1 target sequence in the HLA-DRA promoter. One complex identified by electrophoretic mobility shift assay (EMSA), complex 3, was previously shown to protect the hX2 site and more 3' bases. Sequence analysis now shows that this region contains a consensus binding site for transcription factor BSAP (B cell lineage-specific activator protein). Complex 3 and BSAP have identical cell-type specificities, as they are found only in pre-B and mature B cell lines. In EMSAs, BSAP antibody specifically recognized complex 3, and in vitro translated BSAP could bind to an hXBP promoter fragment. Cotransfections using an hXBP-1 reporter construct indicated that BSAP downregulates the hXBP-1 promoter. The highest levels of hXBP-1 mRNA were found when BSAP was not expressed, in pre-Pro-B cells and in plasma cell lines. In addition, hXBP-1 and BSAP levels were inversely correlated along the early stages of B cell development. In the regulation of the hXBP-1 promoter, a strong positive transcriptional influence at the hX2 site is opposed by the downregulatory actions of BSAP.

Antibody Specificity↗
PubMed1996

Self-renewal of B-1 lymphocytes is dependent on CD19.

The B-1 subset of B lymphocytes is maintained by self-renewal of mature cells, and this process may involve signaling through membrane immunoglobulin (mIg). We determined whether CD19, a membrane protein that co-stimulates B cells by mIg, has a role in this process. Pre-natal treatment of mice with 1D3, a rat anti-mouse CD19 monoclonal antibody, down-regulated CD19 expression and reduced by sixfold the number of B-1a cells at birth; B-2 cells were relatively unaffected. Prolonged treatment of adult mice with 1D3 caused the loss of approximately 2% per day of peritoneal B-1a cells, without diminishing the recovery of splenic B-2 cells. The loss of B-1a cells was associated with inhibition of their replication rather than with accelerated turnover. Therefore, CD19 is involved in the development and self-renewal of B-1a cells, perhaps through its ability to amplify signaling through mIgM.

Animals↗
PubMed1996

Distinctive developmental origins and specificities of the CD5+ B-cell subset.

In this review we present our recent work defining the pathway of B-cell development in the adult bone marrow of the mouse. We next present similarities and contrasts between fetal and adult B lymphopoiesis. Finally, considering the striking biases in specificity and V-gene usage between the progeny of fetal B lymphopoiesis, CD5+ B cells, and the bulk of adult-derived CD5- B cells, we suggest a model for developmentally distinct generation of this subset.

Animals↗
PubMed1996

Identification of the earliest B lineage stage in mouse bone marrow.

We have identified a very early stage of B lineage cells in the CD45R (B220)+CD24 (HSA) pre-pro-B fraction of mouse bone marrow delineated by expression of AA4.1, a molecule found on stem cells and early B lineage cells. These cells are B lineage precursors based on their capacity to generate B lineage cells rapidly in stromal-dependent culture and their expression of high levels of germline IgH transcripts in the absence of Rag-1/2. Half of these AA4.1+ precursors express low levels of CD4, characteristic of lymphoid progenitors, but few if any have up-regulated CD19, a molecule expressed very early in the B lineage. Furthermore, expression of genes encoding pre-B and B cell receptor components (mb-1, B29, and lambda 5) and transcription factors necessary for B lineage differentiation (BSAP, E12, E47, and Id) provide further support for designating these cells as the earliest B cell precursors.

Animals↗
PubMed1996

Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene.

During B lymphocyte development, pro-B cells that fail to rearrange an immunoglobulin heavy (IgH) chain allele productively are thought to undergo developmental arrest and death, but because these cells are short-lived in vivo they are not well characterized. Transgenic mice expressing the apoptosis regulatory gene bcl-xL in the B lineage developed large expansions of pro-B cells in bone marrow. V(D)J rearrangements in the expanded populations were nearly all nonproductive, and DJH rearrangements were enriched for joints in DH reading frame 2 and for aberrant joints with extensive DH or JH deletions. Thus, the death of pro-B cells with failed immunoglobulin rearrangements occurs by apoptosis, and bcl-xL can deliver a strong survival signal at the pro-B stage. This analysis also demonstrated that immunoglobulin gene rearrangement is less precise than previously appreciated.

Animals↗
PubMed1995

The 3' enhancer region determines the B/T specificity and pro-B/pre-B specificity of immunoglobulin V kappa-J kappa joining.

Using transgenic substrates, we found that the immunoglobulin kappa gene 3' enhancer (E3') acts as a negative regulator in V kappa-J kappa joining. Although the E3' was originally identified as a transcriptional enhancer, it acts in a suppressive manner for recombinational regulation. Base substitution analysis has shown that the PU.1-binding site within the E3' regulates the B/T specificity of V kappa-J kappa joining. In a substrate with a mutated PU.1-binding site (GAGGAA to TCTTCG), V kappa-J kappa joining occurred not only in B cells, but also in T cells. The E3' region is also responsible for determining the pro-B/pre-B specificity of V kappa-J kappa joining. When the E3' region was deleted, kappa gene rearrangement actively occurred at the early pro-B stage of B cell development: nongermline (N) nucleotides were common at recombination junctions.

Animals↗