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Biomedical subjects

R S Geha

Publications and source records attributed to R S Geha.

At least 55 records · Page 3Linked to original sources
PubMed1997

Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice.

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

Animals↗
PubMed1997

Homodimerization of the human interleukin 4 receptor alpha chain induces Cepsilon germline transcripts in B cells in the absence of the interleukin 2 receptor gamma chain.

The cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cepsilon germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Ralpha chain, which is shared with the IL-13R, and the IL-2Rgamma (gammac) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cepsilon germline transcripts and IgE isotype switching in B cells from patients with gammac chain deficiency. Induction of Cepsilon germline transcripts by IL-4 in B cells that lack the gammac chain may involve signaling via the IL-13R. Alternatively, the IL-4Ralpha chain may transduce intracellular signals that lead to Cepsilon gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Ralpha induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cepsilon germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Ralpha abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8alpha/IL-4Ralpha receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cepsilon germline transcripts. These results suggest that homodimerization of the IL-4Ralpha chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching.

Animals↗
PubMed1997

CD30 induction of human immunodeficiency virus gene transcription is mediated by TRAF2.

CD30 is a member of the tumor necrosis factor receptor (TNFR) superfamily expressed on activated T and B lymphocytes and natural killer cells. Ligation of CD30 was previously shown to induce NF-kappaB activation and HIV expression in chronically infected T lymphocytes. In this study, we report that two members of the TNFR-associated factor (TRAF) family of proteins, TRAF1 and TRAF2, independently bind to the intracellular domain of CD30 (CD30IC). Transient overexpression of TRAF2, but not TRAF1, induced NF-kappaB activation and HIV-1-long terminal repeat-driven transcription in the T cell line, KT3. Moreover, dominant negative mutants consisting of the TRAF domain of TRAF1 and TRAF2 inhibited CD30 induction of NF-kappaB activation and HIV-1 transcription. These results suggest that CD30 ligation may enhance the expression of HIV via TRAF-2-mediated activation of NF-kappaB.

Cell Line↗
PubMed1997

Jak3 is associated with CD40 and is critical for CD40 induction of gene expression in B cells.

CD40 is a receptor that is critical for the survival, growth, differentiation, and isotype switching of B lymphocytes. Although CD40 lacks intrinsic tyrosine kinase activity, its ligation induces protein tyrosine phosphorylation, which is necessary for several CD40-mediated events. We show that engagement of CD40 induces tyrosine phosphorylation and activation of Jak3 as well as of STAT3. Jak3 is constitutively associated with CD40, and this interaction requires a proline-rich sequence in the membrane-proximal region of CD40. Deletion of this sequence abolishes the capacity of CD40 to induce expression of CD23, ICAM-1, and lymphotoxin-alpha genes in B cells. These results indicate that signaling through Jak3 is activated by CD40 and plays an important role in CD40-mediated functions.

B-Lymphocytes↗
PubMed1997

HIV glycoprotein gp120 inhibits TCR-CD3-mediated activation of fyn and lck.

HIV major glycoprotein gp120 interacts with CD4 molecules and perturbs signaling through the TCR-CD3 complex. We examined the effects of gp120 on TCR-CD3-induced phosphorylation and activation of the src-type protein tyrosine kinases (PTK), fyn and lck. gp120 caused minimal changes in lck phosphorylation or lck enzymatic activity, but preincubation of Jurkat cells with gp120 for 20 min strongly inhibited TCR-CD3-mediated phosphorylation and activation of lck and fyn, as well as phosphorylation of CD3 zeta. Inhibition of TCR-CD3 signaling in T cells preincubated with gp120 was paralleled by inhibition of T cell proliferation to the antigen tetanus toxoid. Neither surface CD4 expression nor CD4-lck association was affected by gp120. Furthermore, gp120 inhibited lck phosphorylation induced by cross-linking of TCR-CD3 and CD4 suggesting that the inhibition of lck phosphorylation could not be simply accounted for by sequestration of CD4 molecules. gp120 selectively enhanced the phosphorylation of the lck peptide containing the autoinhibitory tyrosine residue Tyr505 relative to the lck peptide containing the positive regulatory residue Tyr394, suggesting that a qualitative alteration in lck may underlie the inhibition of TCR-CD3 signaling by gp120.

CD4 Antigens↗
PubMed1997

Targeted gene disruption of murine CD7.

CD7 is a 40 kDa type I transmembrane glycoprotein member of the Ig superfamily. CD7 is a marker of mature human T cells and NK cells, and is expressed early in their development. Cross-linking CD7 positively modulates T cell and NK cell activity as measured by calcium fluxes, expression of adhesion molecules, cytokine secretion and proliferation. CD7 associates directly with phosphoinositol 3'-kinase, and CD7 ligation induces production of D-3 phosphoinositides and tyrosine phosphorylation. Severe combined immunodeficiency has been associated with a lack of lymphocyte surface CD7. The CD7 ligand is unknown. The murine CD7 homolog is encoded by a single gene on chromosome 11. In order to characterize the role of CD7 in lymphocyte development and function we have eliminated the CD7 gene by targeted disruption. CD7-deficient mice display normal histology of thymus and spleen, normal lymphocyte populations in primary and secondary lymphoid tissues, and normal serum Ig levels. Specific antibody responses after immunization with T-dependent and T-independent antigens are equivalent in wild-type and CD7 knockout mice. CD7-deficient lymphocytes respond normally to T cell mitogenic and allogeneic stimuli, and display normal NK cell cytotoxicity.

Animals↗
PubMed1996

Association of human fas (CD95) with a ubiquitin-conjugating enzyme (UBC-FAP).

A novel human ubiquitin conjugating enzyme (UBC) was found to associate with Fas (CD95). The mRNA for this UBC Fas-associated protein (FAP) was widely expressed in human tissues, and the protein was identified in several mammalian cell lines. UBC-FAP shows strong homology to two recently identified UBCs, Hus5 and Ubc9, which control yeast cell cycle progression. UBC-FAP, but not an active site mutant, complemented ubc9-1(ts) mutants. This suggests that UBC-FAP is a human homologue of Ubc9, possesses ubiquitin conjugating activity, and may play an important role in mammalian cell cycle regulation. A single amino acid substitution in the death domain of Fas that abolishes Fas-mediated apoptosis also abolished Fas association with UBC-FAP, suggesting that UBC-FAP may play a role in Fas signal transduction. The sequence of UBC-FAP is identical to that of HsUbc9, a UBC recently shown to interact with Rad51.

Animals↗
PubMed1996

Affinity maturation without germinal centres in lymphotoxin-alpha-deficient mice.

Affinity maturation by somatic hypermutation is thought to occur within germinal centres. Mice deficient in lymphotoxin-alpha (LT alpha-/- mice) have no lymph nodes or Peyer's patches, and fail to form germinal centres in the spleen. We tested whether germinal centres are essential for maturation of antibody responses to T-cell-dependent antigens. LT alpha-/- mice immunized with low doses of (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) showed dramatically impaired production of high-affinity anti-NP IgG1. However, LT alpha-/- mice immunized with high doses of NP-OVA, even though they failed to produce germinal centres, manifested a high-affinity anti-NP IgG1 response similar to wild-type mice. Furthermore, when LT alpha-/- mice were multiply immunized with high doses of NP-OVA, the predominantly expressed anti-NP VH gene segment VH186.2 showed somatic mutations typical of affinity maturation. Thus, B-cell memory and affinity maturation are not absolutely dependent on the presence of germinal centres.

Amino Acid Sequence↗
PubMed1996

Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.

Animals↗
PubMed1996

The CD40L promoter contains nuclear factor of activated T cells-binding motifs which require AP-1 binding for activation of transcription.

Four nuclear factor of activated T cells (NF-AT) binding motifs were found in the murine CD40 ligand promoter. Electrophoretic mobility shift assays using 18-base pair (bp) long oligonucleotides corresponding to the proximal site and nuclear extracts from activated T cells revealed two complexes which were inhibited by cyclosporin A and contained NF-ATc and NF-ATp. Neither complex contained AP-1 proteins. Multimers of the 18-bp oligonucleotides were not active in transient transfection assays using luciferase reporter gene constructs. In contrast, a 30-bp long oligonucleotide bound AP-1 proteins in addition to NF-AT proteins and its multimers strongly induced luciferase gene expression. These results suggested that NF-AT proteins play an important role in the expression of the CD40L gene and that their transcriptional activity requires AP-1 binding.

Animals↗
PubMed1996

Mechanisms of inhibition of IgE synthesis by nedocromil sodium: nedocromil sodium inhibits deletional switch recombination in human B cells.

IgE synthesis requires IL-4 and a T cell-B cell interaction that involves the B-cell antigen CD40 and its ligand expressed on activated T cells. Nedocromil sodium (NS), an effective prophylactic agent in asthma, inhibits IgE synthesis by human B cells. In this report we examined the mechanisms of this inhibition. NS targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in highly purified B cells (greater than 98% CD19+). NS had no effect on the induction of epsilon-germline transcripts by IL-4 but strongly inhibited CD40-mediated S mu --> S epsilon deletional switch recombination. The effect of NS was not specific for CD40 because it inhibited IgE synthesis in B cells stimulated with hydrocortisone plus IL-4. Moreover, the effect of NS was not specific for IgE because it inhibited CD40/IL-4-driven IgG4 synthesis by B cells sorted for lack of surface expression of IgG4. NS caused only modest inhibition of spontaneous IgE synthesis by B cells from patients with hyper-IgE syndrome, suggesting that it has little effect on B cells that have already undergone isotype switching. These results strongly suggest that NS inhibits IgE isotype switching by inhibiting deletional switch recombination and that NS has a novel potential mechanism for the prevention of asthma and other allergic diseases.

Antibodies, Monoclonal↗
PubMed1996

The superantigen toxic shock syndrome toxin-1 induces CD40 ligand expression and modulates IgE isotype switching.

Isotype switching to IgE requires two signals. The first signal is provided by the cytokines IL-4 or IL-13, and the second signal is delivered by the interaction between the B cell antigen CD40 and its ligand (CD40L) which is expressed on activated T cells. Since superantigens have been shown to activate T cells, we examined the effect of the superantigen toxic shock syndrome toxin-1 (TSST-1) on CD40L expression on T cells. TSST-1 induced expression of CD40L in both freshly isolated T cells and in T cell lines expanded by re-stimulation with TSST-1. CD40L was preferentially expressed in the V beta 2 subset of T cells expanded by TSST-1. We next examined the effect of TSST-1 on IgE synthesis by human peripheral blood mononuclear cells (PBMC). Addition of TSST-1 to PBMC inhibited IL-4-induced IgE synthesis in a dose-dependent manner. This inhibition was reversed partly by adding a neutralizing antibody to IFN-gamma. In contrast, TSST-1 induced high amounts of IgE synthesis in the presence of IL-4 at low T:B cell ratios (0.5:10 to 4:10), a condition which circumvents the inhibitory effect of IFN-gamma. TSST-1 induction of IgE synthesis was inhibited by a mAb to CD40L. These results indicate that superantigens induce CD40L expression in T cells and cause isotype switching in B cells which is mediated by CD40L-CD40 interaction.

B-Lymphocytes↗
PubMed1996

CD40 expression and function in murine B cell ontogeny.

The CD40 antigen, a member of the nerve growth factor/tumor necrosis factor receptor family, is expressed on all mature B lymphocytes and plays a crucial role in B cell activation, T cell-dependent antigen-driven isotype switching and germinal center formation. We have analyzed CD40 expression and function during mouse B cell development by examining B cell precursors in normal mice and in transgenic animals in which B cell development is frozen at discrete stages. These models included RAG-2-/- mice, and transgenic littermates that express a mu heavy chain and/or the bcl-2 proto-oncogene transgene. CD40 was undetectable at the pro-B cell stage, but was expressed, although at low levels, on pre-B cells. However, pre-B cells failed to respond to CD40 triggering either by expression of CD23 or by proliferation in the presence of IL-4. Overexpression of bcl-2 increased the density of CD40 expression on pre-B cells: these cells respond to CD40 ligation by expressing CD23 and by proliferating in the presence of IL-4.

Animals↗
PubMed1995

Characterization of a 23-kDa protein associated with CD40.

CD40 is a 45-kDa glycoprotein member of the tumor necrosis factor receptor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carcinoma cells. The unique capacity of CD40 to trigger immunoglobulin isotype switching is dependent on the activation of protein-tyrosine kinases, yet CD40 possesses no kinase domain and no known consensus sequences for binding to protein-tyrosine kinases. Recently, an intracellular protein (CD40bp/LAP-1/CRAF-1) which belongs to the family of TNFR-associated proteins was reported to associate with CD40. We describe a 23-kDa cell surface protein (p23) which is specifically associated with CD40 on B cells and on urinary bladder transitional carcinoma cells. Protein microsequencing revealed that p23 shows no homology to any known protein. A rabbit antibody raised against a peptide derived from p23 recognized a 23-kDa protein in CD40 immunoprecipitates. In contrast to CD40bp/LAP-1/CRAF-1, p23 was not associated with TNFR p80 (CD120b). These findings suggest that p23 is a novel member of the CD40 receptor complex.

B-Lymphocytes↗
PubMed1995

Transcriptional activation of the human TNF-alpha promoter by superantigen in human monocytic cells: role of NF-kappa B.

We have studied the transcriptional activation of the human TNF-alpha gene by the superantigen staphylococcal enterotoxin A (SEA) in the human premonocytic cell line THP-1. Nuclear proteins from SEA-stimulated THP-1 cells bound strongly to kappa 3, the most proximal of three putative NF-kappa B binding sites (kappa 1-kappa 3) found in the 5' regulatory region of the TNF-alpha gene, but only weakly to kappa 1, the most distal of the NF-kappa B binding sites, and showed no binding to kappa 2. The mobility of the kappa 3-nucleoprotein complex was identical to that of complexes formed between nuclear proteins and the consensus NF-kappa B seuqence. Moreover, both 5' and 3' mutants of kappa 3 were unable to displace kappa 3 binding, suggesting that the kappa 3 binding complex induced by SEA has the characteristics of NF-kappa B. Studies using Abs directed against the NF-kappa B subunits p50 and p65 suggested that both p50 and p65 bind to the kappa 3 sequence. Reporter gene assays showed that deletion of kappa 3 (-99 to -89 bp) and point mutation of the three 5' guanine bases in the kappa 3 sequence reduced the inducibility of the TNF-alpha promoter by SEA and LPS. These results indicate that superantigen induces NF-kappa B in human monocytic cells and suggest that binding of NF-kappa B to the kappa 3 site of the TNF-alpha promoter plays an important role in the transcriptional activation of the TNF-alpha gene by superantigen.

Base Sequence↗
PubMed1995

gamma/delta T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes.

T cells expressing gamma/delta T cell receptors home to epithelial tissue and may play a role in immunity to infectious agents and foreign antigens. In an effort to understand the role of gamma/delta T cells in directing B cell responses, we investigated the capacity of human gamma/delta T cells to express CD40 ligand (CD40L) and to drive immunoglobulin (Ig) isotype switching in B cells. A multiple step purification procedure resulted in the recovery of highly pure populations of peripheral blood CD4-CD8- gamma/delta T cells. Neither CD40L surface expression nor CD40L mRNA were detected in unstimulated gamma/delta T cells. Stimulation with phorbol ester and ionomycin induced CD40L mRNA and surface CD40L expression by gamma/delta T cells. Both the percentage of CD40L+ cells and the cell surface density of CD40L were significantly lower in gamma/delta T cells compared to unselected T cells. We further demonstrated that in the presence of neutralizing monoclonal antibody to interferon gamma (IFN-gamma), gamma/delta T cells could induce IgE synthesis in B cells, albeit to a lesser extent than unselected T cells. Furthermore, IgE synthesis driven by gamma/delta T cells was inhibited by monoclonal antibody to CD40L. These observations demonstrate that activated gamma/delta T cells express CD40L and can induce isotype switching in B cells.

Adult↗