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Biomedical subjects

F W Alt

Publications and source records attributed to F W Alt.

At least 73 records · Page 4Linked to original sources
PubMed1998

Reevaluation of 3'Ekappa function in stage- and lineage-specific rearrangement and somatic hypermutation.

Transgenic studies have led to the conclusion that the 3'Ekappa enhancer functions to suppress kappa variable region gene assembly in T lineage cells and in progenitor B cells and have also implicated 3'Ekappa as a critical element in promoting somatic hypermutation of kappa variable region genes. To assess the role of the endogenous 3'Ekappa, we assayed these processes in mice homozygous for mutations in which the 3'Ekappa sequences were deleted by the loxP/Cre method (3'Ekappa delta/delta mice). In contrast to transgenic findings, we found that deletion of the endogenous 3'Ekappa did not deregulate kappa gene rearrangement in T lineage cells or in pro-B cells. Furthermore, immunization of the 3'Ekappa delta/delta mice led to the generation of specific antibodies with mutation patterns typical of affinity maturation, showing that there is no absolute requirement for the 3'Ekappa with respect to somatic mutation of endogenous kappa genes.

Animals↗
PubMed1998

Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation.

The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.

Animals↗
PubMed1998

A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination.

The DNA-dependent protein kinase (DNA-PK) consists of Ku70, Ku80, and a large catalytic subunit, DNA-PKcs. Targeted inactivation of the Ku70 or Ku80 genes results in elevated ionizing radiation (IR) sensitivity and inability to perform both V(D)J coding-end and signal (RS)-end joining in cells, with severe growth retardation plus immunodeficiency in mice. In contrast, we now demonstrate that DNA-PKcs-null mice generated by gene-targeted mutation, while also severely immunodeficient, exhibit no growth retardation. Furthermore, DNA-PKcs-null cells are blocked for V(D)J coding-end joining, but retain normal RS-end joining. Finally, while DNA-PK-null fibroblasts exhibited increased IR sensitivity, DNA-PKcs-deficient ES cells did not. We conclude that Ku70 and Ku80 may have functions in V(D)J recombination and DNA repair that are independent of DNA-PKcs.

Animals↗
PubMed1998

An expressed neo(r) cassette provides required functions of the 1gamma2b exon for class switching.

Germline CH transcripts initiate from a promoter upstream of a non-coding I exon, proceed through the switch (S) region and terminate downstream of the associated CH exons. To elucidate the role of germline transcription in Ig heavy chain class switch recombination (CSR), we used gene targeting in embryonic stem (ES) cells to replace most of the Igamma2b exon from immediately 3' of the majority of transcription initiation sites to beyond its donor splice site with a PGK-neo(r)gene inserted in the same transcriptional orientation as the endogenous unit. The mutation was introduced into both alleles of ES cell lines (referred to as gamma2-b(N/N)) and the neo(r) gene was deleted (referred to as Igamma2b-/-) by the loxP/Cre method. These mutations were assayed for effects on CSR in B cells derived via RAG-2-deficient blastocyst complementation. Igamma2b-/- B cells lacked ability to switch to IgG2b both in vivo and in vitro, and, correspondingly, showed no germline transcription through the Igamma2b exon, Sgamma2b or the Cgamma2b region. In contrast, Igamma2b(N/N) B cells switched at normal levels to IgG2b and showed substantial transcription through the Sgamma2b and Cgamma2b regions. Taken together, these results show that the Igamma2b sequences, per se, are not necessary for mediating CSR since a transcribed PGK-neo(r) gene can replace its function. However, the deleted portion of the Igamma2b exon and splice donor site apparently contain sequences necessary for efficient germline gene transcription and thus for CSR to IgG2b.

ATP-Binding Cassette Transporters↗
PubMed1998

Ig heavy chain class switching in Rag-deficient mice.

To investigate potential roles of the RAG-1 and RAG-2 gene products in Ig heavy chain class recombination (CSR), we have generated RAG-1(-/-) and RAG-2(-/-) mice which contain both a rearranged Ig HC V(D)J gene (referred to as B1-8) inserted into the endogenous Ig heavy chain (HC) locus in place of the JH segments, and a rearranged lambda1 light chain (LC) transgene (which are referred to as RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice respectively). The B1-8 HC gene and lambda LC genes encode proteins that associate to form a complete Ig molecule, the expression of which leads to substantial reconstitution of the peripheral B cell compartments of RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice. Both RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice have relatively normal levels of the various IgG isotypes, but greatly reduced levels of serum IgM and IgA compared to normal littermates. Furthermore, RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda B cells activated in vitro with lipopolysaccharide (LPS) or LPS plus IL-4 responded similarly to control B cells with respect to surface expression and secretion of IgG3, IgG1, IgG2b, IgG2a and IgE, but again were deficient in the secretion of IgM. Together, these findings indicate that neither RAG-1 nor RAG-2 expression is required for efficient class switching to most HC isotypes in B cells.

Animals↗
PubMed1998

Deletion of the IgH intronic enhancer and associated matrix-attachment regions decreases, but does not abolish, class switching at the mu locus.

The IgH locus intronic enhancer (Emu), located in the intron between the J(H) segments and the Cmu gene, and flanked by two matrix attachment regions (MAR), has been shown to be a major regulator of IgH gene transcription and VDJ recombination. To define the potential role of Emu plus MAR in class switch recombination (CSR), we generated IgG-expressing hybridomas from B cells heterozygous for mutations that delete all of these elements or replace them with a neo(r) gene and analyzed the switch status of the mutated IgH loci. Emu/MAR-deleted IgH loci displayed a highly significant, although not complete, decrease in CSR when compared to unmutated loci in normal hybridomas. Surprisingly, mutant loci with a pgk promoter-driven neo(r) gene replacing the Emu/MAR showed relatively normal switch frequency. These findings indicate that the Emu/MAR region plays a significant, but not necessary role in facilitating class switching at the mu locus. Potential mechanisms for these findings are discussed.

Age Factors↗
PubMed1998

Accessibility control of variable region gene assembly during T-cell development.

T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR beta and TCR alpha/delta loci during T-cell development.

Animals↗
PubMed1997

Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination.

V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities. All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR). Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function. The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme. To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression. We find that the Ku70(-/-) cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels. Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku. Significantly, the Ku70(-/-) embryonic stem cells have markedly increased sensitivity to gamma-irradiation relative to Ku70(+/-) or wild-type embryonic stem cells. Furthermore, the Ku70(-/-) cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression. We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can be classified as a member of the x-ray cross-complementation group 6 (XRCC6). Potential differences between the Ku70(-/-) and Ku80(-/-) V(D)J recombination defects are discussed.

Animals↗
PubMed1997

Commitment of immature CD4+8+ thymocytes to the CD4 lineage requires CD3 signaling but does not require expression of clonotypic T cell receptor (TCR) chains.

As a consequence of positive selection in the thymus, immature CD4(+)8(+) double-positive, [DP] thymocytes selectively terminate synthesis of one coreceptor molecule and, as a result, differentiate into either CD4(+) or CD8(+) T cells. The decision by individual DP thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment. Previously, we reported that the intrathymic signals that induced commitment to the CD4 versus CD8 T cell lineages were markedly asymmetric. Notably, CD8 commitment appeared to require lineage-specific signals, whereas CD4 commitment appeared to occur in the absence of lineage-specific signals by default. Consequently, it was unclear whether CD4 commitment, as revealed by selective termination of CD8 coreceptor synthesis, occurred in all DP thymocytes, or whether CD4 commitment occurred only in T cell receptor (TCR)-CD3-signaled DP thymocytes. Here, we report that selective termination of CD8 coreceptor synthesis does not occur in DP thymocytes spontaneously. Rather, CD4 commitment in DP thymocytes requires signals transduced by either CD3 or zeta chains, which can signal CD4 commitment even in the absence of clonotypic TCR chains.

Animals↗
PubMed1997

RAB22 and RAB163/mouse BRCA2: proteins that specifically interact with the RAD51 protein.

The human RAD51 protein is a homologue of the bacteria RecA and yeast RAD51 proteins that are involved in homologous recombination and DNA repair. RAD51 interacts with proteins involved in recombination and also with tumor suppressor proteins p53 and breast cancer susceptibility gene 1 (BRCA1). We have used the yeast two-hybrid system to clone murine cDNA sequences that encode two RAD51-associated molecules, RAB22 and RAB163. RAB163 encodes the C-terminal portion of mouse BRCA2, the homologue of the second breast cancer susceptibility gene protein in humans, demonstrating an in vitro association between RAD51 and BRCA2. RAB22 is a novel gene product that also interacts with RAD51 in vitro. To detect RAD51 interactions in vivo, we developed a transient nuclear focus assay that was used to demonstrate a complete colocalization of RAB22 with RAD51 in large nuclear foci.

Amino Acid Sequence↗
PubMed1997

Bruton's tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity.

Mast cells derived from Bruton's tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon Fc(epsilon)RI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

Functional responses and apoptosis of CD25 (IL-2R alpha)-deficient T cells expressing a transgenic antigen receptor.

IL-2 was initially defined as a T lymphocyte growth factor, but recent studies have provided evidence that it may also play a role in regulating T cell differentiation, apoptosis, and tolerance. To examine the contribution of IL-2 to these processes, we have bred a class II-restricted TCR transgene into mice deficient in the alpha-chain of the IL-2R, CD25. We show that in response to Ag, T cells from these mice are unable to use IL-2 and, as a result, are less efficient at traversing the cell cycle, and proliferate less than wild-type cells. Furthermore, CD25 -/- T cells exhibit reduced survival in vitro, even in the presence of costimulatory signals. IL-4 and IL-15, a cytokine related to IL-2, enhance the survival and Ag-induced proliferation of CD25 -/- T cells. Activated CD25 -/- T cells are resistant to Fas-mediated activation-induced cell death (AICD), and this defect cannot be corrected by other cytokines. Therefore, IL-2 plays a unique role in regulating AICD, but has redundant roles in T cell survival and proliferation in vitro. The failure of AICD observed with CD25 -/- T cells may explain the unexpected observation that deficiency of IL-2 or of the alpha- or beta-chain of the IL-2R results not in immunodeficiency, but in autoimmune disease.

Animals↗
PubMed1997

Constitutive Bcl-2 expression during immunoglobulin heavy chain-promoted B cell differentiation expands novel precursor B cells.

To test for effects on B cell differentiation, we introduced immunoglobulin mu heavy chain (HC) and Bcl-2 transgenes, separately or together, into recombination-activating gene 2 (RAG-2)-deficient mice. Transgenic Bcl-2 expression led to increased numbers of RAG-deficient pro-B cells, but did not promote their further differentiation. Expression of the mu HC transgene promoted the differentiation of RAG-deficient pro-B cells into pre-B cells that also expressed certain differentiation markers characteristic of even more mature B cell stages. However, the extent of the mu HC-dependent differentiation effects was greatly enhanced by coexpression of the transgenic Bcl-2 gene, and a subset of pre-B cells from both HC and HC, Bcl-2-transgenic RAG-2-deficient animals expressed surface mu HCs that were functional as judged by cross-linking experiments. These experiments demonstrate that the pro-B to pre-B transition in vivo cannot be effected by the expression of Bcl-2 alone, and that nontransformed immature B-lineage cells are competent to receive signals through a surface mu complex.

Animals↗
PubMed1997

Function of the TCR alpha enhancer in alphabeta and gammadelta T cells.

We have used gene targeted mutational approaches to assess the role of the T cell receptor alpha (TCR alpha) enhancer (E alpha) in the control of TCR alpha and TCR delta gene rearrangement and expression. We show that E alpha functions in cis to promote V alpha to J alpha rearrangement across the entire J alpha locus, a distance of greater than 70 kb. We also show that E alpha is required for normal alphabeta T cell development; in this lineage, E alpha is required for germline J alpha expression, for normal expression levels of rearranged V alpha J alpha genes, and for expression of a diverse V alpha repertoire. In gamma delta T cells, E alpha is not required for VdeltaDJdelta rearrangement, but, surprisingly, is required for normal expression levels of mature VdeltaDJdelta transcripts and for expression of germline J alpha transcripts. Our findings imply that E alpha function is not limited to the TCR alpha components of the TCRalpha/delta locus or to the alpha beta lineage; rather, E alpha function is important in both alphabeta and gammadelta lineage T cells.

Animals↗
PubMed1997

Growth retardation and leaky SCID phenotype of Ku70-deficient mice.

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.

Animals↗
PubMed1997

Molecular genetic characterization of XRCC4 function.

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.

Amino Acid Sequence↗
PubMed1997

Impaired B cell maturation in mice lacking Bruton's tyrosine kinase (Btk) and CD40.

Mutations in Bruton's tyrosine kinase (Btk) gene, in mice, result in reduced numbers and responses of peripheral B cells. Surface Ig-mediated signaling is defective in Btk mutant B cells as they do not proliferate upon slg cross-linking and lack thymus-independent (TI) type II responses. Signals through sIg and CD40 play a critical role in B cell maturation. To investigate the consequences of the lack of both Btk and CD40 on B cell development and function, mice were generated that were homozygous for targeted mutations in the Btk and the CD40 genes (BtkMCD40M). The CD40 mutation (CD40M) had a synergistic effect on the BtkM defects. In BtkMCD40M mice the number of B cells was reduced 3- to 4-fold compared to BtkM mice and mature B cells (IgMlow/IgDhigh) were virtually absent; serum levels of all Ig isotypes were diminished; and antibody responses to TI-I TI-II and thymus-dependent antigens were impaired. Furthermore, although wild-type BtkM and CD40M mice produced germinal centers in response to TI-I antigen, the BtkMCD40M mice did not. Maturational and functional B cell defects in BtkMCD40M mice may result from a combination of intrinsic B cell defects, lack of CD40L-dependent T cell help and microenvironmental defects. These data suggest that signals through Btk and CD40 are necessary for the production and maintenance of the mature B cell.

Agammaglobulinaemia Tyrosine Kinase↗