Decreased expression of Ii-derived p25 and of P67/69 in hairy leukemic cells.

Hairy leukemic cells had altered expressions of accessory proteins (Ii, Ip, p70, p25) which associated intracellularly with class II MHC molecules. Immunoprecipitates from [35S]methionine, pulse-chase labeled leukemic cells, B-lymphoblastoid cells, and Burkitt's lymphoma cells were examined after 2-dimensional, polyacrylamide gel electrophoresis. The class II MHC-associated Ii chain was present in all three cell types, but the Ii-derived p25 protein was not found in HCL cells. Ip, the sialic acid-derivatized form of Ii, was well expressed in BLCL and Raji cells, but weakly in HCL cells. P70 was strongly expressed in BLCL and Raji cells, but negligibly in HCL cells. These alterations might reflect deficient mechanisms for antigen processing and presentation by these leukemic cells.

[Induction of differentiation of human T-cell leukemic cell line (CEM) by phorbol ester].

In the present study, we investigated the effect of phorbol 12-myristate 13-acetate (PMA) on induction of differentiation of human T-cell leukemic cell line (CEM). The results showed that uninduced cells were T1+, T4+, T9+, T10+, T3- and TcR-, representing thymus stage II differentiation. When culturing in vitro with PMA for 5 to 7 days, CEM cells expressed mature T-cell marker (T3) whereas the percentage of cells expressing proliferation-association antigen (T9) was markedly decreased. In addition, PMA could significantly inhibit the growth of CEM cells. We also found that after co-culture with PMA for 2 to 3 days, CEM cells showed distinct morphologic changes. These findings indicate that PMA may induce CEM cells to differentiate towards mature stage.

Inhibition of protein tyrosine kinase activity by herbimycin A prevents anti-mu but not LPS-mediated cell cycle progression and differentiation of splenic B lymphocytes.

Protein tyrosine kinases (PTKs) have been implicated in signal transduction in a variety of cell types. B lymphocytes express the genes encoding for eight members of the src family of nonreceptor PTKs. Four of these PTKs (p55blk, p53/56lyn, p59fyn, and p56lck) are activated by the ligation of mIg receptors. The functional roles of these PTKs in membrane-bound immunoglobulins (mIg) receptor-mediated activation of resting B lymphocytes were examined using the PTK inhibitor, herbimycin A. Here we show that mIg receptor-mediated B-cell proliferation and differentiation were inhibited by treatment with herbimycin A, while inhibitor-treated B cells retained LPS (mitogen) responsiveness for proliferation and antibody formation. Further studies demonstrated that herbimycin A blocked the G0 to G1 transition during B-cell activation. When the effects of herbimycin A were directly examined by a kinase activity assay, the enzymatic activity of each PTK was inhibited to varying degrees. The inhibition of PTK activity was also reflected by reduced tyrosine phosphorylation of intracellular substrates, including phospholipase C-gamma. These results implicate PTK-dependent signaling pathways in the mIg receptor-mediated functional activation of B lymphocytes.

Expression of protein tyrosine kinases in the Ig complex of anti-mu-sensitive and anti-mu-resistant B-cell lymphomas: role of the p55blk kinase in signaling growth arrest and apoptosis.

The src family of non-receptor protein tyrosine kinases (PTKs), including the blk, fyn, lyn and lck kinases, is expressed in B-lineage cells, may associate with the immunoglobulin receptor complex and, therefore, play a role in signal transduction via membrane IgM. To establish which of these PTKs is involved in growth inhibition of B-cell lymphomas by anti-mu, we examined the expression pattern and state of activation of these kinases in nine B-cell lymphomas. Tyrosine-phosphorylated p55blk was constitutively expressed in all growth-inhibitable lymphomas; furthermore, anti-mu caused a relative increase of tyrosine phosphorylation in p55blk and a 2- to 3-fold increase in its kinase activity in these cells within minutes. In contrast, p55blk was not present in three of five anti-mu-resistant lymphomas and there was no detectable increase of blk activity in one of the resistant cell lines tested. Thus, we proposed that activatable blk kinase in the IgM complex is essential for the growth inhibitory effect of anti-mu. To test this hypothesis, CH31 lymphoma cells were treated with antisense oligos for the blk kinase and found to be resistant to anti-mu-mediated growth inhibition and subsequent apoptosis. These studies implicate the blk kinase as an integral part of the growth inhibitory pathway leading to arrest and apoptosis. Transfectants of blk gene constructs are being generated to further test this hypothesis.

Effect of priming with a thymus-independent antigen on susceptibility to B-cell tolerance.

The effects of priming on the susceptibility of B-cell subsets to tolerance induction have been tested in a model system in which anti-immunoglobulin (anti-Ig) has been employed as a surrogate for tolerogen. T-cell-depleted B cells were primed in vitro with fluorescein or trinitrophenylated Ficoll (a thymus-independent (TI) antigen) and then exposed overnight to anti-Ig to attempt to induce B-cell anergy. Primed cells were relatively resistant to this tolerance protocol and resistance was hapten specific. The dose response and kinetics suggested that this process was not due to receptor blockade or modulation, but was an active process. Moreover, this priming for resistance to tolerance was reproduced in vivo upon intraperitoneal treatment with haptenated Ficoll. Such in vivo priming for tolerance resistance was long-lasting and did not occur with a thymus-dependent priming protocol with fluoresceinated hemocyanin. These results are discussed in terms of TI priming to drive B cells into cycle and express novel functional and phenotypic properties.

Antisense oligodeoxynucleotides to the blk tyrosine kinase prevent anti-mu-chain-mediated growth inhibition and apoptosis in a B-cell lymphoma.

Crosslinking of membrane immunoglobulin (mIg) receptors by anti-Ig causes growth inhibition and subsequent cell death due to apoptosis in a murine B-cell lymphoma model. The earliest signal transduction via mIg has recently been shown to be dependent on the activation of one or more protein tyrosine kinases (PTKs). In this study, we utilized the CH31 lymphoma, which is extremely sensitive to growth inhibition by anti-Ig, to examine the role of PTKs in cell cycle arrest. This cell line expresses multiple PTKs, whose activities are stimulated by crosslinking mIg. To determine whether PTK activity is essential for the inhibition of cell growth, we exposed CH31 cells to antisense oligodeoxynucleotides for the blk PTK prior to the growth inhibition assay. We found that exposure of CH31 cells to blk antisense effectively prevented anti-mu-chain-mediated growth inhibition and subsequent apoptosis. Corresponding blk sense or antisense oligonucleotides for other PTKs had no protective effect against anti-mu. Moreover, antisense blk oligonucleotides had no effect on transforming growth factor beta-mediated growth arrest and apoptosis. Further experiments showed significantly reduced endogenous p55blk in blk antisense-treated cells. In addition, anti-mu stimulation of antisense-treated cells failed to induce any detectable increase in kinase activity of p55blk, a result suggesting the uncoupling of blk proteins from normal signal pathways that are essential for growth inhibition. These results implicate a role of blk kinase in anti-mu-mediated pathway to cell cycle arrest.

B cell tolerance induction by cross-linking of membrane IgM, but not IgD, and synergy by cross-linking of both isotypes.

Previous studies in our laboratory demonstrated that overnight exposure of adult splenic B cells to anti-Ig resulted in an unresponsive state characterized by decreased antibody synthesis but normal mitogen-driven proliferation (i.e., energy). Because both anti-F(ab')2 and anti-mu were equally effective at inducing tolerance, it was important to determine whether cross-linking of IgD together with or separately from IgM influenced the induction of unresponsiveness. Although anti-mu induced significant unresponsiveness, treatment of adult splenic B cells with anti-delta alone generally failed to reduce the subsequent response to either LPS or fluoresceinated Brucella abortus. Interestingly, anti-delta synergized with suboptimal concentrations of anti-mu to induce tolerance. Synergy could be observed in this system when anti-delta was added either simultaneously with or before (but not after) anti-mu; moreover, anti-delta was effective in a pretreatment (wash-out) protocol. To investigate the role of protein tyrosine kinase (PTK) activity in tolerance induction, splenic B cells were treated with tyrphostin before treatment with either anti-mu or anti-delta. We found that pretreatment with tyrphostin for 2 h before the addition of anti-mu prevented the induction of unresponsiveness with this antibody, whereas this PTK inhibitor facilitated tolerance when used with anti-delta treatment only. We propose that cross-linking of surface IgM directly or indirectly invokes a tyrphostin-sensitive, PTK-dependent pathway leading to the early events in tolerance induction, which can be augmented under limiting conditions by anti-IgD. Because cross-linking of either receptor initiates several common pathways, simultaneous cross-linking can lead to synergy and a dominance of the IgM signal. In contrast, IgD alone may fail to elicit tolerance because this isotype may also be associated with different PTK that cause positive signaling.

Immunoreceptor tyrosine-based activation motif is required to signal pathways of receptor-mediated growth arrest and apoptosis in murine B lymphoma cells.

The B cell Ag receptor is a multimeric protein complex consisting of the ligand binding mlg and the Ig alpha/lg beta heterodimer. The cytoplasmic tails of Ig alpha and Ig beta both contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif is believed to play a critical role in the receptor-mediated signal transduction. To explore the role of ITAM in signaling for B cell death (apoptosis), we transfected CH31 cells, an immature B lymphoma cell line, with expression vectors encoding for the CD8 extracellular/transmembrane domains and the cytoplasmic signal-transducing domain (ITAM) of Ig alpha or Ig beta, respectively. Here, we demonstrate that cross-linking of CD8:Ig alpha or CD:Ig beta with anti-CD8 mAb effectively induced cell growth arrest and apoptosis characterized by [3H]thymidine release and DNA fragmentation; in contrast, CD8:gamma 2a or truncated CD8:Ig alpha lacking the ITAM could not do so. Moreover, selective point mutation of either of the two conserved tyrosine residues within the ITAM, but not the nonconserved tyrosine, completely abrogated the ability of this motif to mediate cell death signals. These findings clearly indicate that ITAM is a critical component required for transmitting growth arrest and apoptotic signals, and that these functions of ITAM are positively regulated by tyrosine phosphorylation.