Antigen Receptor Ligation Research Articles

Bruton tyrosine kinase is tyrosine phosphorylated and activated in pre-B lymphocytes and receptor-ligated B cells.

The gene encoding Bruton tyrosine kinase (Btk) is known to be mutated in human X chromosome-linked agammaglobulinemia and in the Xid mouse. This kinase was examined in B lymphocytes before and after antigen receptor ligation and also in pre-B cells. Btk was found to be catalytically activated and tyrosine phosphorylated in response to anti-IgM stimulation in B cells. This kinase is also constitutively phosphorylated on tyrosine residues in pre-B cells. These findings point to a functional role for Btk in pre-antigen and antigen receptor signaling during B-cell development and provide a biochemical explanation for the X-linked genetic syndromes already linked to this kinase.

The Interplay of Interleukin-10 (IL-10) and Interleukin-2 (IL-2) in Humoral Immune Responses: IL-10 Synergizes with IL-2 to Enhance Responses of Human B Lymphocytes in a Mechanism Which Is Different from Upregulation of CD25 Expression

The role of the interplay of interleukin-2 (IL-2) and interleukin-10 (IL-10) in responses of human peripheral blood B cells stimulated by ligation of antigen receptors was examined in detail. Highly purified peripheral blood B cells from normal human individuals were cultured with Staphylococcus aureus Cowan I (SA) in the presence or absence of IL-10 and IL-2. Although IL-10 alone could modestly induce Ig production by SA-activated B cells, IL-10 markedly enhanced the Ig production in synergism with IL-2. This synergistic effect between IL-2 and IL-10 was completely abrogated by addition of either anti-CD25 mAb or anti-IL-10 mAb, indicating signals through high-affinity IL-2 receptors are involved in the synergism. In fact, IL-10 upregulated the expression of α-chain of IL-2 receptors (CD25) on B cells stimulated by SA alone or SA + IL-2 for 72 hr. However, such upregulation of CD25 expression did not result in significant enhancement of subsequent responses of the activated B cells to IL-2. Of note, during the initial activation phase of B cells, IL-2 was much more effective than IL-10 in enhancing the subsequent responses to either IL-2 or IL-10. By contrast, IL-10 was much more effective than IL-2 in supporting the maturation of B cells following the initial activation. Finally, the synergy between IL-2 and IL-10 was not observed in the initial activation phase, but in the subsequent maturation phase of activated B cells. These results indicate that IL-10 and IL-2 synergistically enhance Ig production of SA-activated B cells in a mechanism which is different from the upregulation of IL-2 receptors. Moreover, the data emphasize the importance of the interplay of IL-2 and IL-10 in determining the outcome of humoral immune responses.

The T-cell antigen receptor utilizes Lck, Raf-1, and MEK-1 for activating mitogen-activated protein kinase. Evidence for the existence of a second protein kinase C-dependent pathway in an Lck-negative Jurkat cell mutant.

T-cell antigen receptor (TCR) ligation of an Lck-deficient Jurkat mutant, J.CaM1, with anti-CD3 or anti-TCR beta monoclonal antibodies failed to induce tyrosine phosphorylation and activation of p42MAPK. The same stimuli activated mitogen-activated protein (MAP) kinase in J.CaM1 cells transfected with Lck, demonstrating that Lck plays a critical role in MAP kinase activation. Utilizing immunocomplex kinase assays, we demonstrated that TCR/CD3 ligation activated a MAP kinase kinase kinase (Raf-1) as well as a MAP kinase kinase (MEK-1) in Jurkat but not in J.CaM1 cells. It was possible, however, to activate Raf-1, MEK-1, and p42MAPK in J.CaM1 cells during treatment with the phorbol ester phorbol 12-myristate 13-acetate, which activates protein kinase C (PKC). This demonstrates the presence of a PKC-dependent pathway which functions independently from Lck in MAP kinase activation. Stimulation of Jurkat cells with either anti-TCR beta or anti-CD3 monoclonal antibody failed to induce substantial tyrosine phosphorylation of Shc proteins or their association with Grb2 which forms a complex with the guanine nucleotide exchange factor hSOS. However, the same stimuli induced tyrosine phosphorylation of another putative guanine nucleotide exchange factor, p95Vav, in Jurkat but not J.CaM1 cells. Moreover, Lck was reversibly co-immunoprecipitated with p95Vav, and the stoichiometry of binding increased in anti-CD3-treated Jurkat cells. Phorbol 12-myristate 13-acetate did not induce tyrosine phosphorylation of p95Vav. These data show that the TCR activates MAP kinase by way of a signaling cascade, which depends upon Lck, and may be mediated by downstream events involving PKC or p95Vav which act on Raf-1 and MEK-1.

Cyclosporin A and FK506 Inhibit Activation-Induced Cell Death in the Murine WEHI-231 B Cell Line

The WEHI-231 B lymphoma cell line expresses the phenotype of immature B cells. Crosslinking of surface IgM induces programmed cell death (PCD) with typical features of apoptosis demonstrated by the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Activation of protein kinase C (PKC) by phorbol esters was reported to protect WEHI-231 cells against apoptosis induced by ligation of antigen receptors. It was therefore hypothesized that PCD could result from a defect in PKC response with an imbalance in the phosphoinositide pathway in favor of Ca 2+ mobilization. In support of this hypothesis, we show here that apoptosis can be readily triggered by the calcium ionophore ionomycin. Furthermore, pretreatment of cells with cyclosporin A or FK506 which inhibit selectively the phosphoprotein calcineurin, a calcium-and calmodulin-dependent serine/threonine phosphatase, protects WEHI-231 cells against apoptosis induced by ionomycin or ligation of surface IgM. Unlike phorbol esters, cyclosporin A did not impair the rise of intracellular Ca 2+ induced by cross-linking of antigen receptors. Altogether, the data indicate that the phosphorylation status of yet undefined key cellular substrates controls the cellular response to calcium-dependent apoptotic signals in this B cell lymphoma.

Lipopolysaccharide-induced IgM production is not suppressed by antigen receptor ligation in B cells from some autoimmune strains of mice.

Ligation of surface immunoglobulin on resting or activated nonautoimmune B cells inhibited lipopolysaccharide (LPS)-induced total IgM production. B cells from NZB, (NZB x NZW)F1, and BXSB mice were relatively resistant, but B cells from NZW or MRL/lpr mice were inhibited. The resistance occurs in B cells from young and old NZB mice, and in both resting and activated splenic NZB B cells. Anti-ssDNA responses induced by lipopolysaccharide occur in the presence of antigen-receptor-ligating antibody in NZB, but not in DBA/2, B cells. Antagonism in signaling between the antigen and LPS receptor is not a general B cell hyporesponsiveness, but defects in antagonism specifically between antigen and LPS signaling may be a predisposing factor to autoimmune disease in some autoimmune strains of mice.

Persistent calcium elevation correlates with the induction of surface immunoglobulin-mediated B cell DNA synthesis.

Surface immunoglobulin (sIg)-mediated stimulation of B lymphocytes induces a tyrosine kinase-dependent sequence of events leading to rapid and large elevations in intracellular ionized calcium ([Ca2+]i). These early biochemical events do not necessarily lead to proliferation of B cells, however, and conversely, the absence of or inhibition of these events does not necessarily prevent cellular proliferation. We now show by digital image analysis of single B cells that conditions which lead to B cell proliferation are associated with low-level but persistent sustained or cyclic elevations in [Ca2+]i. In marked contrast, early and nonsustained elevations in [Ca2+]i are induced in B cells by stimuli that lead to G1 transition but fail to progress to DNA synthesis. Thus, when B cells were stimulated with mitogenic and nonmitogenic anti-IgD antibodies, both of which induce entry of cells into G1 and early calcium transients of comparable magnitude, persistent low-level calcium elevations were only detected in cells stimulated with the mitogenic antibody. Furthermore, persistent calcium elevations were also seen when B cells were stimulated with a multivalent dextran-anti-Ig conjugate which induced very high levels of B cell proliferation in the absence of detectable phosphatidylinositol 4,5-biphosphate hydrolysis or elevations in [Ca2+]i as detected by flow cytometry. Finally, B cells from X-linked B cell-defective mice, which do not proliferate in response to anti-Ig antibody, show marked and early increases in [Ca2+]i, but do not show persistent calcium elevations. These data suggest that the rapid and large increases of [Ca2+]i seen in lymphocytes within seconds after antigen receptor ligation may be associated with entry in G1, whereas low-level but persistent elevations may be the hallmark of a cell destined to synthesize DNA.

T cell-dependent induction of NF-kappa B in B cells.

In comparison to B cell stimulation mediated by surface immunoglobulin (Ig) antigen receptor ligation, little is known about the intracellular events associated with T cell-dependent B cell responses. A model for the efferent phase of T cell-B cell interaction was used to examine the capacity of activated T cells to trigger nuclear expression of the trans-acting transcription factor, NF-kappa B, in B cells. Fixed, activated, but not fixed, resting Th2 cells were found to induce increased binding activity for a kappa B site-containing oligonucleotide in a time-dependent manner. This induction of NF-kappa B was eliminated by an antibody directed against a 39-kD cell interaction protein on activated T cells as well as by a soluble form of B cell CD40. Of particular relevance to intracellular signaling, NF-kappa B induction was not diminished by prior depletion of B cell protein kinase C (PKC) with phorbol myristate acetate. These results strongly suggest that T cell-dependent B cell stimulation is associated with NF-kappa B induction via p39-CD40 interaction and that this is brought about by non-PKC dependent signaling, in marked contrast to the previously documented requirement for PKC in sIg receptor-mediated stimulation. This suggest that NF-kappa B responds to more than one receptor-mediated intracellular signaling pathway in B cells and may be part of a "final common pathway" for B cell stimulation.

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.

T-cell receptor-CD4 physical association in a murine T-cell hybridoma: induction by antigen receptor ligation.

By employing flow cytometric analysis and fluorescence resonance energy transfer (FRET), we examined the physical relationship between the T-cell receptor-CD3 complex (Ti-CD3) and the CD4 molecule on helper T cells. Through the use of an L3T4-negative murine T-cell hybridoma infectant expressing the human CD4 gene and having antigen specificity for HLA-DR, we show that binding of the Ti-CD3 complex with an anti-CD3 monoclonal antibody induces its redistribution proximal to cell-surface CD4. FRET efficiency was 9.4% on cells labeled with rhodaminated anti-CD3 and fluoresceinated anti-CD4. FRET was found to be temperature dependent, since similarly treated cells held at 4 degrees C displayed a FRET efficiency of less than 1%. Energy transfer was evident within 3 min after warming cells to 37 degrees C. Energy transfer was not detected between Ti-CD3 and the abundantly expressed leukocyte common antigen (CD45). Of greater significance was our observation that hybridomas infected with a truncated CD4 gene lacking the cytoplasmic domain failed to transfer energy despite the fact that CD4 was expressed on the cell surface at levels equivalent to or greater than the wild type. These studies suggest that after crosslinking of the Ti-CD3 on CD4+ T cells, a physical association occurs between the antigen receptor complex and CD4 and that the association is dependent upon the presence of the cytoplasmic domain of CD4.

B-cell-stimulatory factor 1 reverses Fc receptor-mediated inhibition of B-lymphocyte activation.

Intact (IgG class) rabbit anti-immunoglobulin antibodies are not mitogenic for mouse B cells but inhibit proliferation induced by F(ab')2 anti-Fab fragment antibodies (anti-Ig). In addition, cross-linkage of Fc and surface immunoglobulin receptors on B cells by intact anti-Ig inhibits inositol phospholipid breakdown (but not Ca2+ flux) resulting from ligation of antigen receptors. This system, therefore, provides a polyclonal model for B-cell inactivation by antigen-antibody complexes. T-cell-derived B-cell-stimulatory factor 1 acts synergistically with submitogenic concentrations of F(ab')2 anti-Ig to induce B-cell proliferation. We show here that B-cell-stimulatory factor 1 and intact anti-Ig also induce B cells to synthesize DNA. However, B-cell-stimulatory factor 1 does not induce inositol phospholipid breakdown, does not mobilize Ca2+ in B cells, nor does it influence the magnitude of these responses provoked by intact anti-Ig.