Biomedical subjects

F W Alt

Publications and source records attributed to F W Alt.

At least 127 records · Page 7Linked to original sources

PubMed1994

An early haematopoietic defect in mice lacking the transcription factor GATA-2.

Blood cell development relies on the expansion and maintenance of haematopoietic stem and progenitor cells in the embryo. By gene targeting in mouse embryonic stem cells, we demonstrate that the transcription factor GATA-2 plays a critical role in haematopoiesis, particularly of an adult type. We propose that GATA-2 regulates genes controlling growth factor responsiveness or the proliferative capacity of early haematopoietic cells.

Anemia↗

PubMed1994

Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.

Animals↗

PubMed1994

A class switch control region at the 3' end of the immunoglobulin heavy chain locus.

We replaced the IgH 3' enhancer (3'EH) region with a neomycin resistance gene in ES cells and generated chimeric mice in which all mature lymphocytes were either heterozygous (3'EH+/-) or homozygous (3'EH-/-) for the mutation. In vitro activated 3'EH-/- B cells responded similarly to 3'EH+/- B cells with respect to proliferation and secretion of IgM and IgG1 but were specifically deficient in IgG2a, IgG2b, IgG3, and IgE secretion. These isotype deficiencies correlated with a deficiency in accumulation of transcripts from and class switching to affected CH genes. In vivo, chimeric mice containing only 3'EH-/- B cells were deficient in serum IgG2a and IgG3. We propose that the 3'EH-/- mutation disrupts the activity of a regulatory region that influences heavy chain class switching to several different CH genes that lie as far as 100 kb upstream of the mutation.

Animals↗

PubMed1994

Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation.

To study the influence of immunoglobulin heavy-chain (HC) and light-chain (LC) expression in promoting B-cell differentiation, we have introduced functional immunoglobulin HC and/or LC transgenes into the recombinase activating gene-2-deficient background (RAG-2-/-). RAG-2-/- mice do not undergo endogenous V(D)J rearrangement events and, therefore, are blocked in B- and T-cell development at the early pro-B- and pro-T-cell stages. Introduction of immunoglobulin HC transgenes into the RAG-2-/- background promotes the development of a B-lineage cell population that phenotypically has the characteristics of pre-B cells. We have shown further that this population has altered growth characteristics as measured by interleukin-7 responsiveness in culture. Bone marrow cells from immunoglobulin HC transgenic RAG-2-/- mice have up-regulated expression of germ-line kappa LC gene transcripts and down-regulated expression of lambda 5 surrogate LCs (SLCs). Although mu HC/SLC complexes are detectable intracellularly in HC/RAG-2-/- pre-B-cell populations, HC expression is not readily detectable on the surface of these cells. lambda LC RAG-2-/- mice had a bone marrow B-lineage cell phenotype indistinguishable from that of RAG-2-/- littermates, indicating that LC expression by itself has no influence on pro-B cell differentiation. Strikingly, simultaneous introduction of mu HC and lambda LC transgenes into RAG-2-/- mice led to the generation of a substantial population of "monoclonal" peripheral B-cells that were functional with regard to immunoglobulin secretion, indicating that T cells or diverse immunoglobulin repertoires are not necessary for peripheral B-cell development.

Animals↗

PubMed1994

The expression of Vpre-B/lambda 5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice.

Precursor B (pre-B) cells in bone marrow of normal and B cell-deficient mutant mice were analyzed for the expression of Vpre-B/lambda 5 surrogate light chain (SL). The surface expression of SL is confined to the early stages (pro-B and pre-B-I) of pre-B cell development and becomes undetectable once mu heavy chain (microH) is produced. The cell-cycle analysis revealed that cytoplasmic microH+ large cells (large pre-B-II), approximately 30% of which coexpressed SL in the cytoplasm, were most actively cycling, whereas cytoplasmic microH+ small cells (small pre-B-II) were SL- and not in cycle. The analysis of pre-B cells in B cell-deficient mice suggests that the large pre-B-II stage is a critical step for the selection and amplification of cells carrying functionally rearranged microH genes.

Animals↗

PubMed1994

A DNA repair defect in Chinese hamster ovary cells affects V(D)J recombination similarly to the murine scid mutation.

Lymphocyte antigen receptor variable regions are encoded by gene segments that are assembled by the site-specific variable (diversity) joining (V(D)J) recombination process. We have assayed the V-3 Chinese hamster ovary cell line, which has a double-strand DNA break repair (dsbr) defect, for the ability to carry out V(D)J recombination following transfection of constructs that encode the RAG-1 and RAG-2 proteins necessary to confer V(D)J recombination activity to non-lymphoid cells. The V-3 cells had substantially impaired ability to undergo V(D)J recombination of transiently introduced test substrates. Although these cells can initiate V(D)J recombination by introducing endonucleolytic scissions at the junctions of V(D)J recombination signal sequences and the flanking coding sequences, they have a greatly impaired ability to rejoin the coding sequences. Detailed characterization of attempted coding joins recovered from these cells indicated that the V(D)J recombination defect in V-3 is similar in phenotype to the murine severe combined immunodeficient (scid) defect and quite distinct from that found in other Chinese hamster ovary dsbr mutant cell lines. Somatic cell complementation analyses between homozygous scid mutant fibroblasts and V-3 cells confirmed that these mutations fall into the same genetic complementation group.

Animals↗

PubMed1994

S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process.

Stimulation of B lymphocytes with a combination of lipopolysaccharide (LPS) and interleukin-4 (IL-4) induces germline transcription of and subsequent switching to the epsilon heavy chain constant region (C epsilon) gene. Mature germline C epsilon transcripts contain a non-coding exon (I epsilon exon) spliced to the C epsilon exons. To distinguish between the potential roles of germline transcription and those of germline transcripts in regulating the class switch process, we replaced the LPS- and IL-4-inducible I epsilon promoter and exon in ES cells with an LPS-inducible E mu enhancer/VH promoter expression cassette. Wildtype, heterozygous or homozygous mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all B cells derived from ES cells. In contrast to normal B cells, heterozygous and homozygous mutant B cells had substantial transcription through the epsilon switch recombination region (S epsilon) following treatment with LPS alone and, under these conditions, both underwent low level switching (10- to 100-fold less than wildtype cells stimulated with LPS + IL-4) to IgE production. Heterozygous mutant cells underwent switching to IgE at essentially wildtype levels when stimulated with LPS and IL-4. However, homozygous mutant cells still showed extremely low levels of switching to IgE upon LPS and IL-4 stimulation. Analyses of hybridomas from heterozygous mutants indicated that the mutation is cis-acting and normal switching to other isotypes indicated that it is specific for IgE. Thus transcription per se generates low levels of class switch recombination in the absence of I region sequences. However, we demonstrate for the first time that, for optimal efficiency, the process requires the presence of the intact I region and/or I region promoter in cis, implicating factors beyond transcription through the S region in the regulation of class switching.

Animals↗

PubMed1994

Mechanisms that control antigen receptor variable region gene assembly.

The somatic assembly of antigen receptor genes--known as V(D)J recombination--is a highly regulated process at several different levels. V(D)J rearrangement is limited to stages of early B and T cell development by the lymphoid specific expression of recombinase activity. A common recombinase is apparently responsible for the rearrangement of all antigen receptor loci. Thus, the tissue specific and ordered rearrangement of each antigen receptor locus must depend on controlling their 'accessibility' to the recombinase. Here, we outline recent work that has contributed to the current understanding of mechanisms that control V(D)J rearrangement.

Alleles↗

PubMed1994

Probing immune functions in RAG-deficient mice.

Inactivation of recombination activating gene (RAG)-1 or RAG-2 in mice results in the inability of developing lymphocytes to initiate V(D)J recombination, leading to the arrest of lymphocyte differentiation at a very early stage. Introduction of functionally assembled antigen-receptor genes or other potentially relevant genes into the RAG-deficient background can bypass the V(D)J recombination block and promote differentiation of the lymphocytes of RAG-deficient mice to various stages. This approach offers new means for analyzing the control of lymphocyte differentiation. In addition, generation of somatic chimeric mice by injecting mutant embryonic stem cells into the RAG-2-deficient blastocysts has also provided a powerful new method for assaying the potential roles of genes or regulatory elements in lymphocyte development or function.

Animals↗

PubMed1994

Generation of normal T and B lymphocytes by c-jun deficient embryonic stem cells.

To determine the potential roles of c-jun in lymphocyte development, we generated somatic chimeric mice by injecting homozygous c-jun mutant embryonic stem (ES) cells into blastocysts from recombination activating gene-2 (RAG-2)-deficient mice. Chimeric mice had poor restoration of thymocytes, but contained substantial numbers of mature T and B lymphocytes in the periphery. Stimulation of c-jun-/- B cells resulted in normal levels of proliferation and immunoglobulin secretion. Likewise, stimulation of c-jun-/- T cells resulted in essentially normal levels of IL-2R alpha expression, IL-2 secretion, and proliferation. We further showed that the relatively normal activation responses of the c-jun-/- T cells probably results from the fact that other members of the Jun family contribute to the bulk of the activator protein-1 (AP-1) complexes in normal T cells and, as a result, AP-1 complexes are found at relatively normal levels in c-jun-/- T cells.

Animals↗

PubMed1994

Expression of I mu-C gamma hybrid germline transcripts subsequent to immunoglobulin heavy chain class switching.

Germline CH transcripts initiate from a non-coding I exon and terminate downstream of the associated CH exons. Ig heavy chain class switch recombination from the VDJ-C mu gene to particular downstream CH genes appears to be regulated by a process that involves mitogen and/or cytokine induction of germline CH transcripts from the downstream genes. We have examined the expression of germline C mu transcripts (I mu-C mu transcripts) in splenic B cells and pre-B cell lines after cytokine and mitogen stimulation. In contrast to the expression of the germline transcripts from downstream CH genes, expression of germline C mu transcripts was constitutive and unaffected by mitogen and cytokine treatment. After a primary switch recombination event, the germline I mu promoter, which is now associated with a downstream CH gene, continues to be active--leading to the generation of a novel germline transcript consisting of the I mu exon spliced to the CH exons of the switched CH gene. We discuss the potential role of the expression of hybrid I mu-containing transcripts in the class switch process. We also describe a novel and sensitive assay, based on the detection of the hybrid I mu-containing transcripts, that allows detection of class switch recombination events even in heterogeneous populations of cells.

Animals↗

PubMed1994

CD3 epsilon-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR beta chain expression.

Recent studies have shown that TCR beta chain expression can effect the differentiation of CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) thymocytes. The TCR beta chain is expressed on the surface of DP thymocytes in association with CD3 gamma, delta and epsilon chains, suggesting a potential role for CD3 components in this signaling process. We now report detection of a very low level of surface expression of CD3 epsilon on adult DN RAG-2-/- thymocytes. This surface CD3 epsilon was associated with CD3 gamma and delta chains, as detected by anti-CD3 epsilon immunoprecipitation analyses. Significantly, injection of anti-CD3 epsilon mAb into RAG-2-/- mice led to the accumulation of an IL-2R alpha- CD2+ DP cell population and a nearly 100-fold increase in thymic cellularity to essentially normal levels. Together, these data strongly indicate that TCR beta chain-mediated developmental signals are transduced by CD3 components and provide potential insights into mechanisms by which TCR beta chain expression may effect this process.

Animals↗

PubMed1994

IL-2 receptor alpha chain expression during early B lymphocyte differentiation.

The IL-2/IL-2 receptor (IL-2R) has been studied intensively because of its potential function in the development and regulation of the immune system. The IL-2R alpha chain has been shown to be expressed on CD4-CD8- thymocytes and activated T and B cells. In this report, we show that IL-2R alpha is also expressed on precursor B cells in the bone marrow. Its expression is initiated by functional rearrangement and expression of Ig mu heavy chain gene and is down-regulated when immature B cells mature and express IgD. Its potential function in early B cell differentiation is discussed in comparison with its role in thymocyte differentiation.

Animals↗

PubMed1993

Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice.

The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into phenotypically mature cells. However, in vitro, the mature T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and gamma-irradiation. In contrast, stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus, and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune system after birth.

Animals↗

PubMed1993

Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes.

During the assembly of immunoglobulin and T cell receptor variable region genes from variable (V), diversity (D), and joining (J) segments, the germline-encoded repertoire is further diversified by processes that include the template-independent addition of nucleotides (N regions) at gene segment junctions. Terminal deoxynucleotidyl transferase (TdT)-deficient lymphocytes had no N regions in their variable region genes, which shows that TdT is responsible for N region addition. In addition, certain variable region genes appeared at increased frequency in TdT-deficient thymocytes, which indicates that N region addition also influences repertoire development by alleviating sequence-specific constraints imposed on the joining of particular V, D, and J segments.

Animals↗

PubMed1993

Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells.

BACKGROUND: Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline. RESULTS: Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function. CONCLUSION: We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.

Journal Article↗

PubMed1993

RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development.

We describe a system to evaluate the function of lymphocyte-specific and generally expressed genes in the differentiation and/or function of lymphocytes. RAG-2 (recombination-activating gene 2)-deficient mice have no mature B and T lymphocytes due to the inability to initiate VDJ recombination. Blastocysts from RAG-2-deficient mice generate animals with no mature B and T cells following implantation into foster mothers. However, injection of normal ES cells into RAG-2-deficient blastocysts leads to the generation of somatic chimeras with mature B and T cells all of which derive from the injected ES cells (referred to as RAG-2-deficient blastocyst complementation). Complementation of RAG-2-deficient blastocysts with mutant ES cells heterozygous for a targeted mutation that deletes all immunoglobulin heavy-chain joining (JH) gene segments (JH+/-) also leads to generation of chimeras with normal B and T cells. However, complementation with ES cells homozygous for the JH mutation (JH-/-) generates animals with normal T cells but no B cells, due to a block in B-cell development at a very early stage. Transfection of a functionally assembled mu heavy-chain gene into the JH-/- ES cells prior to blastocyst injection rescues the JH-/- mutation and allows the generation of both mature T and mature B cells. The rescued B cells express IgM but not IgD and respond normally to bacterial lipopolysaccharide stimulation by proliferating and by secreting IgM.

Animals↗

PubMed1993

Replacement of germ-line epsilon promoter by gene targeting alters control of immunoglobulin heavy chain class switching.

Recent work has shown that the ability of cytokines to direct immunoglobulin heavy chain class-switch recombination to particular heavy chain constant (C) region (CH) genes correlates with the induction of specific germ-line CH transcripts. To test the role of germ-line transcripts in class switching, we have used homologous recombination to mutate the immunoglobulin heavy chain locus of the 18.81A20 murine pre-B-cell line. In the parent cell line, the combination of interleukin-4 (IL-4) and lipopolysaccharide (LPS) induces germ-line epsilon locus transcription prior to class switching to epsilon. The heavy chain locus of the mutated cell line contains the immunoglobulin heavy chain enhancer and variable region gene promoter in place of the LPS/IL-4-responsive germ-line epsilon promoter. The mutant cell line constitutively transcribes the epsilon locus in the absence of IL-4. Strikingly, the mutant cell line also switches to epsilon in the absence of IL-4. This result demonstrates that, at least in the 18.81A20 cell line, germ-line epsilon transcription plays a direct role in class switching to the epsilon locus. In addition, the ability to change the pattern of class switching by altering transcriptional activity indicates that transcription of germ-line CH is mechanistically important in regulation of class switching.

Abelson murine leukemia virus↗