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M R Kehry

Publications and source records attributed to M R Kehry.

At least 37 records · Page 2Linked to original sources
PubMed1996

BCL-6 expression during B-cell activation.

Translocations involving the BCL-6 gene are common in the diffuse large cell subtype of non-Hodgkin's lymphoma. Invariably, the BCL-6 coding region is intact, but its 5' untranslated region is replaced with sequences from the translocation partner. The present study shows that BCL-6 expression is regulated in lymphocytes during mitogenic stimulation. Resting B and T lymphocytes contain high levels of BCL-6 mRNA. Stimulation of mouse B cells with anti-IgM or IgD antibodies, bacterial lipopolysaccharide, phorbol 12-myristate 13-acetate plus ionomycin, or CD40 ligand led to a five-fold to 35-fold decrease in BCL-6 mRNA levels. Similar downregulation of BCL-6 mRNA was seen in human B cells stimulated with Staphylococcus aureus plus interleukin-2 or anti-IgM antibodies and in human T lymphocytes stimulated with phytohemagglutinin. BCL-6 mRNA levels began to decrease 8 to 16 hours after stimulation, before cells entered S phase. Although polyclonal activation of B cells in vitro invariably decreased BCL-6 MRNA expression, activated B cells from human germinal centers expressed BCL-6 mRNA at levels comparable to the levels in resting B cells. Despite these similar mRNA levels, BCL-6 protein expression was threefold to 34-fold higher in germinal center B cells than in resting B cells, suggesting that BCL-6 protein levels are controlled by translational or posttranslational mechanisms. These observations suggest that the germinal center reaction provides unique activation signals to B cells that allow for continued, high-level BCL-6 expression.

Amino Acid Sequence↗
PubMed1996

CD40-mediated signaling in B cells. Balancing cell survival, growth, and death.

Multimerization of CD40 molecules on B cells by binding CD40 ligand on activated T cells initiates signaling events essential for B cell differentiation. In mature B cells, CD40 mediates stimulation and costimulation of cell growth, switch recombination, and transcriptional regulation. CD40-mediated signaling also regulates cell death, rescuing B cells from anti-Ig-induced apoptosis and inducing the expression of the Fas surface molecule. Recent efforts to elucidate the biochemistry of CD40-mediated signaling pathways have identified members of the TRAF protein family that are associated with the cytoplasmic tail of CD40. CD40 cross-linking probably multimerizes TRAF proteins which may act as direct transcriptional regulators. Modulation of protein tyrosine kinase and protein tyrosine phosphatase activity also occurs after CD40-mediated signaling; however, a connection to the TRAF pathway has not been established. Multiple pathways of B cell triggering are probably integrated at the level of transcriptional activation to produce differentiation stage-specific B cell responses.

Animals↗
PubMed1996

B cells from p50/NF-kappa B knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching.

To better understand the role of NF-kappaB in normal B cell physiology, we used a purified population of resting B cells from p50/NF-kappa B knockout (p50-/-) mice to determine their ability to proliferate, secrete Ig, express germ-line CHRNA, and undergo Ig isotype switching in vitro in response to a number of distinct stimuli. p50-/- B cells proliferated normally in response to dextran-anti-IgD Abs (alpha delta-dex) and membrane-bound, but not soluble, CD40 ligand (CD40), and they were virtually unresponsive to LPS when compared with control B cells. p50-/- B cells secreted markedly reduced Ig in response to alpha delta-dex or mCD40L in the presence of IL-4 + IL-5, despite their relatively normal proliferative rates, whereas normal Ig secretion was restored by the combination of alpha delta-dex and CD40L. p50-/- B cells expressed normal steady-state levels of germ-line CH gamma 1 and CH alpha RNA but markedly reduced germ-line CH gamma 3 and CH epsilon RNA upon appropriate stimulation. Although p50-/- B cells underwent substantial switching to IgG1, a marked reduction in the switch to IgG3 and IgE, as IgA, was observed. These data are the first to demonstrate key, independent roles for p50/NF-kappaB in normal B cell maturation to Ig secretion, germ-line CH gene activation, and Ig class switching, as well as mitogenesis, and provide a powerful and well-defined in vitro model system for studying the role of p50/NF-kappaB in a wide range of normal cellular functions.

Animals↗
PubMed1996

IL-10 selectively regulates murine Ig isotype switching.

A role for IL-10 in regulating Ig isotype switching directly at the level of the murine B cell has not been previously reported. In this report we show that IL-10 selectively up-regulated IgM to IgG3 class switching in lipopolysaccharide (LPS)-activated cultures through a direct effect on membrane (m) IgM+IgG3(-)B cells in vitro. IL-10 stimulated a 3- to 4-fold enhancement (from 6-8 to 20-30%) in membrane mIgG3(+) cells and a significant increase in Smu-Sgamma3 DNA rearrangement events as measured by digestion-circularization PCR (DC-PCR) over that observed with LPS alone. IL-10 induction of switching to IgG3 was not accompanied by a corresponding increase in the steady-state levels of germline CHgamma3 RNA. By contrast, IL-10 strongly inhibited the transforming growth factor-beta-mediated generation of mIgA+ cells and Smu-Salpha DNA rearrangement events in LPS-, but not CD40 ligand (CD40L)-activated B cells. This effect was not accompanied by changes in the steady-state levels of germline CHalpha RNA. IL-10 had no effect on IL-4-mediated switching to either IgG1 or IgE in either LPS- or CD40L-activated B cells. Thus, IL-10 can either enhance or suppress switching to particular murine Ig isotypes but it differs from most other murine cytokines in that its effects on switching do not appear to be associated with changes in the corresponding steady-state levels of germline CH RNA.

Animals↗
PubMed1996

IFN-gamma is a potent inducer of Ig secretion by sort-purified murine B cells activated through the mIg, but not the CD40, signaling pathway.

IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.

Animals↗
PubMed1995

Bacterial lipoproteins may substitute for cytokines in the humoral immune response to T cell-independent type II antigens.

Bacterial lipoproteins share a common structural motif that has been shown to stimulate proliferation and Ig secretion of murine B cells, in a manner distinct from that mediated by LPSs. Studies of lipoprotein-mediated B cell activation utilized heterogeneous populations of lymphoid cells, leaving unresolved their ability to directly activate resting B cells, as well as their ability to interact with other B cell stimuli. Using highly enriched and/or sort-purified resting murine B cells, we demonstrate that, in contrast to previous reports, lipoproteins (lipoprotein-D, lipoprotein-OspA, and/or the synthetic analogue Pam3Cys) stimulate little, if any, proliferation or Ig secretion in resting B cells. However, when combined with a multivalent membrane (m)Ig-mediated cross-linking signal, dextran-conjugated anti-IgD Abs (alpha delta-dex), lipoproteins mediate up to 10,000-fold inductions in IgM secretion and up to 25-fold enhancements in cellular proliferation relative to that observed with alpha delta-dex alone, in the absence of added cytokines. This mIg-mediated enhancement of Ig secretion was not observed when B cells were stimulated with bivalent, unconjugated anti-Ig. CD40 ligand (CD40L), shows a similar, although somewhat more moderate, synergy with lipoproteins for induction of proliferation and IgM secretion. By contrast, lipoproteins by themselves are relatively ineffective at costimulating Ig secretion in the presence of various combinations of cytokines. These data suggest that bacteria may induce Ag-specific humoral immunity through the action of bacterial polysaccharides that mediate an Ag-specific multivalent mIg signal, in concert with bacterial lipoproteins that deliver ancillary signals, without a requirement for recruitment of non-B cell types.

Animals↗
PubMed1995

IL-3 and granulocyte-macrophage colony-stimulating factor strongly induce Ig secretion by sort-purified murine B cell activated through the membrane Ig, but not the CD40, signaling pathway.

Sort-purified resting murine B cells proliferate in response to dextran-conjugated anti-IgD Abs (alpha delta-dex) but fail to secrete significant amounts of Ig even after the addition of IL-1 + IL-2. We show that either IL-3 or granulocyte-macrophage CSF (GM-CSF) stimulates 10- to 50-fold enhancements in IgM secretion by sort-purified B cells treated with alpha delta-dex + IL-1 + IL-2, and that the combined actions of IL-3 and GM-CSF are typically greater than additive. Both IL-3 and GM-CSF act primarily as B cell differentiation factors, although IL-3 induces a modest enhancement in cellular outgrowth. The enhancing effects of IL-3 and GM-CSF require multivalent Ag receptor cross-linkage, mediated by alpha delta-dex, as neither cytokine induces IgM secretion in the presence of unconjugated anti-IgD Abs. Although both alpha delta-dex and IL-1 + IL-2 are required for optimal IL-3- and GM-CSF-mediated IgM secretion, both IL-3 and GM-CSF stimulate a modest IgM secretory response by cells activated with alpha delta-dex alone. In this regard, supernatant from either an activated CD4+ Th1 or Th2 clone potently induces IgM secretion by alpha delta-dex + IL-1 + IL-2-activated B cells and this is due, in large part, to the presence in these supernatants of either IL-3 and/or GM-CSF. Neither IL-3 nor GM-CSF stimulates significant IgM secretion by B cells activated through the CD40 signaling pathway alone, although the combination of CD40 and membrane Ig signaling leads to a strong enhancement of the IL-3 + GM-CSF-mediated IgM synthesis above that obtained with membrane Ig signaling alone. The demonstration that IL-3 and GM-CSF act directly as differentiation factors for B cells activated through their Ag receptor establishes a novel cytokine pathway for induction of humoral immunity.

Animals↗
PubMed1995

Novel in vitro model for high-rate IgA class switching.

The parameters necessary for induction of high-rate IgA class switching are unknown. Thus, although TGF-beta is switch factor for the IgA class, the percentage of membrane (m)IgA+ cells generated in vitro in response to TGF-beta and various individual modes of B cell activation is limited to 1 to 2% of the total B cell population, a percentage far below that observed within Peyer's patches. In this report we determined a set of parameters that act synergistically to generate up to 15 to 20% mIgA+ cells in vitro. A dual mode of B cell activation is required whereby signaling through CD40 or in response to LPS stimulation must occur in concert with multivalent Ag receptor crosslinking. A complex cytokine requirement is also revealed in that both IL-4 and IL-5 must be present with TGF-beta for high-rate IgA class switching to occur. By contrast, IFN-gamma, a known antagonist of IL-4, strongly suppresses the induction of mIgA+ cells in response to these stimuli. This novel cellular system should serve as a powerful tool for studying the molecular mechanisms that underly the IgA class switch and may provide insight into the physiologic parameters that induce it.

Animals↗
PubMed1995

Multivalent, but not divalent, antigen receptor cross-linkers synergize with CD40 ligand for induction of Ig synthesis and class switching in normal murine B cells. A redefinition of the TI-2 vs T cell-dependent antigen dichotomy.

A number of previous studies have suggested that cross-linkage of the B cell Ag receptor may be critical for induction of humoral immune responses to T cell-dependent (TD) Ags in vivo. Previous work also indicated a critical role, in these responses, for CD40-mediated signaling mediated by binding of the inducible T cell membrane protein, CD40 ligand (CD40L). Data in this manuscript demonstrate that concentrations of bivalent anti-IgD or anti-IgM Ab as high as 30 micrograms/ml induced little if any enhancement of CD40-dependent Ig secretion by resting murine B cells. In contrast, concentrations as low as 3 pg/ml of multivalent, dextran-conjugated, anti-IgD (alpha delta-dex) or anti-IgM (alpha mu-dex) were strongly synergistic with CD40L for induction of B cell proliferation, viable cell outgrowth, Ig isotype switching, and maturation to Ig secretion. As many as 30% of the B cells became membrane IgG1+ after stimulation with CD40L, anti-Ig-dextran, and IL-4 + IL-5, with a concomitant three- to fivefold increase in numbers of viable cells as compared with control cultures. High Ig secretory responses were obtained in response to the combined actions of CD40L and alpha delta-dex or alpha mu-dex, utilizing concentrations of B cell activator that when acting alone induced only modest Ig secretion. Surprisingly, although we previously demonstrated that alpha delta-dex selectively and strongly suppressed IgE production by T cell-activated B cells, it strikingly augmented IgE expression by CD40L-activated B cells. These data suggest 1) a key role for Ag receptor cross-linkage in CD40-dependent induction of humoral immune responses, 2) that to achieve a membrane Ig-dependent enhancing effect in the presence of activated T cells, TD Ags must be displayed to the B cell as a multivalent array of epitopes, 3) that picomolar concentrations of Ag can mediate this effect, and 4) that at least for induction of IgE responses, B cell stimulation via CD40L or via activated T cells may lead to a qualitatively different pathway of activation.

Animals↗
PubMed1994

Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigen-receptor-mediated functions.

B-cell tolerance to soluble protein self antigens such as hen egg lysozyme (HEL) is mediated by clonal anergy. Anergic B cells fail to mount antibody responses even in the presence of carrier-primed T cells, suggesting an inability to activate or respond to T helper cells. To investigate the nature of this defect, B cells from tolerant HEL/anti-HEL double-transgenic mice were incubated with a membrane preparation from activated T-cell clones expressing the CD40 ligand. These membranes, together with interleukin 4 and 5 deliver the downstream antigen-independent CD40-dependent B-cell-activating signals required for productive T-B collaboration. Anergic B cells responded to this stimulus by proliferating and secreting antibody at levels comparable to or better than control B cells. Furthermore, anergic B cells presented HEL acquired in vivo and could present the unrelated antigen, conalbumin, targeted for processing via surface IgD. In contrast, the low immunoglobulin receptor levels on anergic B cells were associated with reduced de novo presentation of HEL and a failure to upregulate costimulatory ligands for CD28. These defects in immunoglobulin-receptor-mediated functions could be overcome in vivo, suggesting a number of mechanisms for induction of autoantibody responses.

Animals↗
PubMed1994

B cell differentiation induced by helper T cell membranes: evidence for sequential isotype switching and a requirement for lymphokines during proliferation.

Small dense B cells are stimulated to proliferate by membranes prepared from activated helper T (Th) cell clones. In combination with Th2 lymphokines, Th membranes stimulate B cells to differentiate to secrete predominantly immunoglobulin (Ig)M, IgG1 and IgE. The activity in Th membrane requires the expression of CD40 ligand by the T cells, and initiation of the B cell response occurs through the ligation of CD40 on the B cell surface. We have further characterized the properties of the B cell response and found that Th membranes stimulated B cell proliferation and Ig secretion in a cell density independent manner and the majority of the stimulated B cells underwent a limited number of division rounds between day 2 and 5 of culture. IgM-secreting cells appeared in culture by day 3 and increased to reach a maximum, comprised of 30% of the viable cells, on day 5. IgG1-secreting cells appeared 12-24 h after IgM-secreting cells, and IgE-secreting cells did not appear until day 5. These data are consistent with a sequential model of isotype switching related to cell division. As lymphokines were absolutely required for antibody production we were able to determine when during culture they were essential. Lymphokines needed to be present prior to and during B cell proliferation for differentiation to Ig-secreting cells to proceed. This period corresponded closely to the time of maximum DNA synthesis. Consistent with sequential switching of Ig isotypes, differentiation to IgM secretion required the shortest exposure to lymphokines and IgE the longest. These experiments strongly suggest that the lymphokine-induced commitment to differentiate is made before DNA synthesis begins, although the lymphokine-delivered signals are necessary during DNA synthesis to support Ig class switching.

Animals↗
PubMed1994

Regulation of CD40 ligand expression and use of recombinant CD40 ligand for studying B cell growth and differentiation.

Helper T cell activation leads to transient expression of a ligand for the B cell surface protein, CD40. CD40 ligand can deliver helper T cell-derived contact signals to B lymphocytes that drive B cell activation and proliferation. Regulation of expression of CD40 ligand is analogous to that of other helper T cell-derived lymphokines. A soluble form of CD40 ligand is capable of delivering proliferative signals to B cells only when cross-linked or when IL-4 is added. Recombinant CD40 ligand in membrane vesicles can be used to develop in vitro systems for studying class switching, clonal selection, and affinity maturation in normal B lymphocytes.

Animals↗
PubMed1994

B-cell activation by helper T-cell membranes.

Resting B cells can be stimulated to proliferate and differentiate to antibody-producing cells by the combination of cell contact and soluble signals provided by activated primed helper T (Th) cells. The ability of purified plasma membranes from activated Th cell clones and recombinant lymphokines to reconstitute B cell proliferation and differentiation has allowed an increased understanding of B cell activation and characterization of the molecules involved. B cell-Th cell contact appears sufficient for delivering the proliferative signal to B cells in the absence of lymphokines. A receptor ligand pair that plays a critical role in delivery of the contact signal is CD40 on the B cell surface and the ligand for CD40 on activated Th cells. Lymphokines alone do not drive resting B cell differentiation, however, when these soluble signals are delivered during the time of B cell DNA replication, they effect B cell differentiation and isotype switching. Delivery of the CD40-dependent contact signal to resting B cells appears to require a high degree of CD40 crosslinking on the B cell surface. Providing contact signals to naive B cells with recombinant molecules in membrane fractions may allow the generation of methodology to support the production of novel antibodies in vitro.

Animals↗
PubMed1993

Regulation of expression of the ligand for CD40 on T helper lymphocytes.

Activated Th cells deliver contact-dependent signals to resting B lymphocytes that initiate and drive B cell proliferation. Recently, a ligand for the B lymphocyte membrane protein, CD40, has been identified that delivers contact-dependent Th cell signals to B cells. A dimeric soluble form of CD40 was produced and used to further characterize the regulation of expression of the CD40 ligand. Expression of the CD40 ligand was rapidly induced after Th lymphocyte activation, and its stability depended upon whether Th cells were activated with soluble or plastic-bound stimuli. Th cells activated with soluble stimuli rapidly turned over cell-surface CD40 ligand whereas Th cells activated with plastic-bound stimuli exhibited more stable CD40 ligand expression for up to 48 h. Removal of activated Th cells from the plastic-bound stimulus resulted in a rapid turnover of CD40 ligand, suggesting that continuous stimulation could maintain CD40 ligand expression. Ligation by soluble CD40 could also stabilize expression of CD40 ligand on the Th cell surface. Both CD40 ligand and IL-2 were transiently synthesized from 1 to 12 h after Th cell activation and had similar kinetics of synthesis. In Con A-activated Th cells newly synthesized CD40 ligand exhibited an initial high turnover (1.5 h t1/2) and after 5 h of Th cell activation became more stable (10-h t1/2). In Th cells activated with plastic-bound anti-CD3, CD40 ligand exhibited a similar biphasic turnover except that the rapid turnover phase began significantly later. This delay could allow more time for newly synthesized CD40 ligand to assemble or associate with other molecules and thus become stabilized on the cell surface. Newly synthesized CD40 ligand in Con A-activated Th cells appeared to not be efficient in delivering Th cell-dependent contact signals to resting B cells, implying the need for assembly or accessory proteins. Regulation of CD40 ligand expression was consistent with all the characteristics of Th cell-delivered contact signals to B cells and may contribute to the high degree of specificity in B cell responses.

Animals↗
PubMed1993

Helper T cells: delivery of cell contact and lymphokine-dependent signals to B cells.

In the effector phase of delivering T cell help for B cell differentiation, proteins induced following helper T cell activation deliver cell contact signals to the B cell that drive its activation and proliferation. Activation induced soluble lymphokines also deliver signals that, in conjunction with the contact signals, drive B cell differentiation and immunoglobulin class switching. Helper T cell activation leads to transient expression of a ligand for the constitutively expressed B cell surface protein, CD40. The CD40 ligand is involved in delivering the T cell induced contact signals. Regulation of synthesis of the CD40 ligand by activated helper T cells contributes to the maintenance of specificity of the antibody response. Knowledge of the molecules involved in delivering T cell help allows a clearer understanding of events regulating antibody affinity maturation, selection and class switching in vivo.

Animals↗
PubMed1991

Membranes from both Th1 and Th2 T cell clones stimulate B cell proliferation and prepare B cells for lymphokine-induced differentiation to secrete Ig.

Plasma membranes from the mitogen-activated mouse Th2 cell clone D10.G4.1 have recently been shown to provide the cell contact-dependent signals necessary for the induction of small B cell proliferation. Together with the Th2-derived lymphokines IL-4 and IL-5, these membranes stimulate production of Ig isotypes identical to those produced when B cells were stimulated by intact Th2 cells. In contrast, Th1 clones are poor inducers of Ig production in vitro. This could be solely due to differences in the lymphokines released by Th1 and Th2 cells or to differences in the cell-cell contact signals delivered by activated Th1 and Th2 cells. We report that membranes from three different activated Th1 clones induced strong Ag-independent proliferation of small dense B cells. The level of B cell proliferation was enhanced approximately fourfold by the addition of lymphokine-containing supernatant from Con A-activated Th2 cells and was unaffected by any of the lymphokine-containing supernatants from Con A-activated Th1 clones. As with D10.G4.1 membranes, Th1 membranes alone induced B cell proliferation but not secretion of Ig. However, addition of supernatant from Con A-activated D10.G41 cells, but not any supernatants from Con A-activated Th1 cells, induced Ig secretion of all isotypes. These effects were shown to not simply result from increased B cell numbers after stimulation with Th2 lymphokines. Thus, Th1 cell clones seem to poorly induce antibody responses entirely because of their lymphokine repertoire and not because of differences or deficiencies in the ability of these cells to deliver cell contact-dependent signals to B cells.

Animals↗