Cross-linking Of Surface Ig Research Articles

MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.

Follicular Lymphoma-associated BTK Mutations are Inactivating Resulting in Augmented AKT Activation.

On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor. Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.See related commentary by Afaghani and Taylor, p. 2123.

Effects of Epstein-Barr virus on host gene expression in Burkitt's lymphoma cell lines

Epstein-Barr virus (EBV) is present in Burkitt's lymphoma (BL) cells in a latent form, showing a highly restricted pattern of gene expression with few tumor cells undergoing viral lytic replication. BL cell lines can be induced to enter the viral lytic cycle and initiate replication by stimulating surface immunoglobulin molecules. During this process many EBV genes are expressed that have the potential to influence host gene expression. We aimed to identify host genes that are regulated by EBV in BL cells and those that are regulated following ligation of surface IgG. The differentially expressed genes in EBV-positive Akata cells and EBV-negative AK31 cells were detected by microarray. A total of 91 human genes were differentially expressed between Akata and AK31 cells and 198 were differentially expressed when cells were stimulated to enter lytic replication. The differential expression of one gene, myd88, was correlated with disrupted TLR9 signaling. EBV down-regulates most of the genes regulated by surface Ig cross-linking in the early stages of lytic cycle activation. These include genes involved in cell survival, signal transduction, transcription control and the immune response that may mediate EBV transformation of B-lymphocytes and others such as HDAC4 that may affect virus replication.

Antinuclear Antigen B Cells That Down-Regulate Surface B Cell Receptor during Development to Mature, Follicular Phenotype Do Not Display Features of Anergy In Vitro

We previously demonstrated that B cells expressing a transgenic BCR with "dual reactivity" for the hapten arsonate and nuclear autoantigens efficiently complete development to follicular phenotype and stably reside in follicles in vivo. These B cells express very low levels of surface IgM and IgD, suggesting that they avoid central deletion and peripheral anergy by reducing their avidity for autoantigen via surface BCR (sBCR) down-regulation. Since a variety of states of B cell anergy have been previously described, a thorough examination of the functional capabilities of these B cells was required to test this hypothesis. In this study, we show that surface Ig cross-linking induces amounts of proximal BCR signaling in these B cells commensurate with their reduced sBCR levels. Functionally, however, they are comparable to nonautoreactive B cells in cell cycle progression, up-regulation of activation and costimulatory molecules, and Ab-forming cell differentiation when treated with a variety of stimuli in vitro. In addition, these B cells can efficiently process and present Ag and are capable of undergoing cognate interaction with naive TCR-transgenic T cells, resulting in robust IL-2 production. Together, these data reveal a lack of intrinsic anergy involving any known mechanism, supporting the idea that this type of antinuclear Ag B cell becomes indifferent to cognate autoantigen by down-regulating sBCR.

Transgenic expression of a human polyreactive Ig expressed in chronic lymphocytic leukemia generates memory-type B cells that respond to nonspecific immune activation.

We generated transgenic mice, designated SMI, expressing unmutated H and L chain Ig genes encoding a low-affinity, polyreactive human (h)IgM/kappa rheumatoid factor. These animals were compared with control AB29 transgenic mice expressing a hIgM/kappa rheumatoid factor specific for human IgG, with no detectable reactivity with mouse proteins. SMI B cells expressed significantly lower levels of surface hIgM/kappa than did the B cells of AB29 mice, but still could be induced to proliferate by surface Ig cross-linking in vitro and could be deleted with anti-Id mAb in vivo. Transgene-expressing B cells of AB29 mice had a B-2 phenotype and were located in the primary follicle. In contrast, a relatively high proportion of hIgM-expressing B cells of SMI mice had the phenotype of B-1 B cells in the peritoneum or marginal zone B cells in the spleen, where they were located in the periarteriolar sheath, marginal zone, and interfollicular areas that typically are populated by memory-type B cells. Although the relative proportions of transgene-expressing B cells in both types of transgenic mice declined with aging, SMI mice experienced progressive increases in the serum levels of IgM transgene protein over time. Finally, SMI transgene-expressing B cells, but not AB29 transgene-expressing B cells, were induced to secrete Ab when cultured with alloreactive T cells. These results indicate that expression of polyreactive autoantibodies can allow for development of B cells that are neither deleted nor rendered anergic, but instead have a phenotype of memory-type or Ag-experienced B cells that respond to nonspecific immune activation.

Role of OCA-B in 3'-IgH enhancer function.

OCA-B (alternately called Bob1 and OBF-1) is a B cell-specific coactivator that interacts with the ubiquitously expressed Oct-1 and the B cell-restricted Oct-2 to activate transcription via the octamer site (5'-ATGCAAAT-3'). OCA-B-/- mice appear to undergo normal Ag-independent B cell maturation. However, Ag-dependent B cell differentiation, including germinal center formation, production of secondary Ig isotypes, and proliferation in response to surface Ig cross-linking, is greatly affected. We demonstrate that the observed reductions in expression of class-switched isotypes in OCA-B-/- mice may be due in part to deficiencies in the function of the 3'-IgH enhancer elements. Furthermore, we find that surface Ig cross-linking represses all the Ig enhancers and that this repression is absent in OCA-B-/- B cells. These results suggest an important role for OCA-B in Ig enhancer function in vivo.

Induction of the Igkappa 3' enhancer by distinct pathways can be inhibited by cross-linking of the CD40 receptor.

Upon treatment with lipopolysaccharide (LPS), primary B cells proliferate and differentiate into plasma cells with concomitant up-regulation of immunoglobulin (Ig) gene expression. Here we examine the role of the Igkappa 3' enhancer in this process using a kappa3'-enhancer-driven beta-globin reporter gene in transgenic mice. We find that LPS treatment up-regulates kappa3' enhancer activity as a function of differentiation rather than proliferation, since proliferation only (induced by cross-linking of CD40) is insufficient to activate the element, whilst differentiation with only limited proliferation (LPS + transforming growth factor-beta) does allow activation to occur. The Igkappa 3' enhancer is also induced by cross-linking of surface Ig and this signal can synergize with LPS activation, suggesting that distinct activation pathways are used. Nevertheless, both of these pathways can be inhibited by co-cross-linking of CD40. Thus Ig enhancers in the heavy and light chain loci are differentially regulated in response to CD40.

PMA/ionomycin induces Ig kappa 3' enhancer activity which is in part mediated by a unique NFAT transcription complex.

The Ig kappa 3' enhancer is required for high levels of Ig kappa gene expression. We now show that kappa 3' enhancer function increases five- to eightfold after stimulation of primary murine B cells with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. In the presence of cyclosporin A this induction is almost halved, suggesting that transcription factors of the NFAT family contribute to kappa 3' enhancer induction. Indeed, we identify a novel NFAT binding site which is required for full enhancer function. We find that this site is transcriptionally active in stimulated B cells, T cells and fibroblasts and that both PMA and ionomycin are required for maximal induction. Time course analysis of the components of the protein-DNA complex in primary lymphocytes reveals that both NFATp and NFATc are present in the complex after 15 min, while only NFATc is detectable after 4 h. This suggests that NFATc plays the dominant role in controlling long-term responses of this transcription factor family. Furthermore, JunB, JunD, FosB and cFos form part of the DNA-protein complex in Bal-17 B cells. Complex formation as well as transcriptional activity can also be induced by crosslinking of surface Ig. We have, thus, identified a unique NFAT complex in B cells that contributes to Ig kappa gene expression.

Characterization of murine CD70 by molecular cloning and mAb.

CD27, a member of the tumor necrosis factor (TNF) receptor family, has been implicated in T cell activation, T cell development and T-dependent antibody production by B cells. Its ligand CD70 has been identified only in humans, and, thus, physiological and pathological roles of the CD70-CD27 interaction remain to be determined in an experimental animal system. In the present study, we identified murine (m) CD70 by molecular cloning, and characterized its expression and function by generating an anti-mCD70 mAb. The mCD70 cDNA encoded a type II transmembrane glycoprotein of the TNF family, having 56.5% identity to the human CD70 amino acid sequence. The mCd70 gene was assigned in the central region of chromosome 17. To explore the expression and function of mCD70, we generated cDNA transfectants and anti-mCD70 mAb (FR70), which inhibited binding of a murine CD27-Fc fusion protein (mCD27-Ig) to mCD70 transfectants. FR70, as well as mCD27-Ig, immunoprecipitated a 30-33 kDa surface protein from A20 and mCD70-P815 cells but not from P815 cells. The mCD70 transfectants exhibited a potent co-stimulatory activity for anti-CD3-stimulated T cell proliferation, which was blocked by FR70 far more efficiently than mCD27-Ig. FR70 also abrogated the CD28-independent co-stimulatory activity of A20 cells. The expression of mCD70 was detected on splenic T cells after stimulation with anti-CD3 and anti-CD28 mAb, and on splenic B cells after stimulation with anti-CD40 mAb. Cross-linking of surface Ig by anti-IgM mAb did not induce the mCD70 expression but enhanced the anti-CD40-induced mCD70 expression on splenic B cells. These results suggest a contribution of CD70 to murine T-B cognate interaction as proposed in the human system. FR70 will be useful for further investigating the physiological and pathological roles of the CD70-CD27 interaction in T cell development, T-dependent antibody production and various disease models in the murine system.

Syk is required for BCR-mediated activation of p90Rsk, but not p70S6k, via a mitogen-activated protein kinase-independent pathway in B cells.

The tyrosine kinases Syk and Lyn are activated in B lymphocytes following antibody induced cross-linking of the B cell receptor for antigen (BCR). It has been suggested that activation of Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules in parental DT40, DT40Syk- and DT40Lyn- B cells. The phosphorylation and activation of p90Rsk was ablated in Syk-deficient B cells but unaffected in Lyn-deficient B cells while the phosphorylation/activation of Ras GTPase activating protein (Ras GAP) and mitogen activated protein (MAP) kinase required both Syk and Lyn. Thus, these data indicate that Syk can be activated in the absence of Lyn after BCR cross-linking and results in the activation of p90Rsk via a MAP kinase-independent pathway in DT40Lyn- cells. We also demonstrated that BCR mediates the activation of p70S6k. However, activation of p70S6k in DT40Syk- and DT40Lyn- cells was comparable with that observed in parental cells. Thus, either Syk or Lyn may be sufficient for activation of p70S6k, or activation of p70S6k occurs independently of both Syk and Lyn. The kinase activity of Syk was required for the phosphorylation/activation of each of these downstream effector molecules but only the phosphorylation of Ras GAP was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines.

Induction of somatic hypermutation in a human B cell line upon surface Ig cross-linking and coculture with anti-CD3 activated T cells

Induction of somatic hypermutation in a human B cell line upon surface Ig cross-linking and coculture with anti-CD3 activated T cells

Induction of somatic hypermutation in a human B cell line upon surface Ig cross-linking and coculture with anti-CD3 activated T cells

Induction of somatic hypermutation in a human B cell line upon surface Ig cross-linking and coculture with anti-CD3 activated T cells

Overexpression of c-fos inhibits down-regulation of a cyclin-dependent kinase-2 inhibitor p27Kip1 in splenic B cells activated by surface Ig cross-linking.

Splenic B cells activated by surface Ig (sIg) cross-linking transiently express the c-fos gene within 0.5 h and then enter into S phase of the cell cycle within 48 h. To investigate a role of c-fos in cell cycle progression, we used splenic B cells from IFN-alphabeta-inducible c-fos transgenic mice (Mx-c-fos). In the absence of IFN, the cell cycle progression of Mx-c-fos B cells stimulated with anti-IgM Ab was similar to that in control B cells. The cell cycle was arrested in G1 phase when we added IFN to the culture within 12 h after anti-IgM Ab stimulation, suggesting that overexpression of c-fos until mid-G1 phase perturbs activation of the cell cycle regulatory machinery. In control B cells, cyclin E and cdk2 were induced within 24 to 48 h after stimulation, and this induction was accompanied by down-regulation of a cdk2 inhibitor p27Kip1. As a consequence of these activation processes, cdk2 kinase activity was induced in B cells in the late G1 phase. However, kinase activity was not detected in Mx-c-fos B cells, presumably because the down-regulation of p27 was perturbed. These data suggest that c-Fos can negatively control cell cycle regulatory machinery in sIg-stimulated B cells.

Tumor Necrosis Factor-α (TNF-α) and Interleukin-6 (IL-6) in B-Lymphocyte Function

Two cytokines important in the regulation of B-cell function are tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). They act at different steps in B-cell differentiation and can be produced by the B cells themselves upon appropriate stimulation. Crosslinking of surface Ig and signaling through CD22 or CD40 lead to increased secretion of both cytokines. Neutralization of TNF-α or IL-6 biologic activity in B-cell cultures results in a significant reduction in B-cell proliferation and Ig secretion. Increased production of these cytokines is found in several diseases associated with aberrant B-cell function. This review will focus on the role of TNF-α and IL-6 in normal and pathophysiological conditions of B-cell function.

Delayed tyrosine phosphorylation and nuclear expression of STAT1 following antigen receptor stimulation of B lymphocytes.

The regulation of the STAT1 alpha transcription factor was assessed during B cell activation induced by cross-linking the surface IgM Ag receptor. Surface Ig ligation or pharmacologic stimulation with PMA and ionomycin resulted in the delayed (2-3 h after stimulation) nuclear appearance of tyrosine-phosphorylated STAT1 alpha, in contrast to the rapid induction that follows cytokine treatment. Nuclear expression of phosphorylated STAT1 alpha was abrogated by co-incubation of anti-Ig-treated B cells with the protein synthesis inhibitor cycloheximide (CHX), with the protein kinase inhibitor H-7, or with the immunosuppressive drug rapamycin. Tyrosine-phosphorylated STAT1 alpha was found to be recruited to the STAT binding site of the IFN regulatory factor-1 (IRF-1) gene promoter only after 2 to 3 h, and this association was also inhibitable by CHX and rapamycin. The arrival of STAT1 alpha coincided with attenuation of anti-Ig-induced STAT-binding activity specific for the IRF-1 promoter site, and both rapamycin and CHX treatment counteracted the loss of this activity. Furthermore, basal transcription of the endogenous IRF-1 gene was decreased as a result of anti-Ig treatment, and this effect of anti-Ig was blocked by co-incubation with rapamycin. Thus, STAT1 alpha plays a dynamic role in the composition of IRF-1 promoter-specific DNA binding complexes stimulated by B cell Ag receptor ligation, and nuclear expression of phosphorylated STAT1 alpha is regulated in a unique fashion by Ag receptor engagement. In addition, surface Ig cross-linking imparts negative regulatory control of IRF-1 gene expression, possibly through activation of STAT1 alpha.

Apoptosis of ileal Peyer's patch B cells is increased by glucocorticoids or anti-immunoglobulin antibodies.

The ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (< 5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24 h, 60 +/- 1% of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose-dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2-2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 +/- 6%, compared with 2.4 +/- 0.1% in untreated lambs. Anti-immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti-Ig (PIg47A), DNA fragmentation was 74 +/- 2% and 75 +/- 3%, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca2+, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti-Ig. We speculate that the apoptosis of ileal PP B cells in situ may be modulated by glucocorticoids and by the cross-linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self-reactive B cells during the expansion of the Ig repertoire in the ileal PP.

Differential susceptibility to tolerance induction in vitro of splenic B cells from several transgenic mouse lines. Role of B1 cells.

Spleen cells from transgenic mice, whose rearranged Ig receptors reflect the repertoires of B1 (CD5+ and "sister" B cells) or normal B2 cells, were examined for their ability to be rendered unresponsive. By using an anti-Ig tolerance protocol that is independent of receptor specificity, we previously reported that peritoneal B cells, containing primarily CD5+ and "sister" B cells, were not susceptible to unresponsiveness. Herein, we show that splenic B cells from two separate transgenic mouse lines, each expressing a B1-type receptor, are resistant to tolerance induction in vitro. In contrast, splenic B cells from two other transgenic mouse lines with a large representation of conventional B cells were sensitive to anti-lg-mediated unresponsiveness. This difference does not reside in the surface Ig density, cell cycle, or activation stage of these cells, but is reflected in the initial calcium mobilization and tyrosine phosphorylation after surface Ig cross-linking. Therefore, these results support the hypothesis that the antigenic specificity of B cell receptors may drive cells toward the B1 subset, as suggested by Cong et al. (Cong, Y-Z., E. Rabin, and H. H. Wortis. 1991. Int. Immunol. 3:467-476), and that B1 cell characteristics confer the ability of B cells to withstand in vitro tolerance induction, irrespective of their anatomical location. The possibility that this results from previous antigenic experience is discussed.

Protein kinase C mediates activation of nuclear cAMP response element-binding protein (CREB) in B lymphocytes stimulated through surface Ig.

The cAMP response element-binding protein (CREB) is generally considered to be responsive to elevation of cAMP through the activity of protein kinase A (PKA). Although it is well known that cAMP-raising agents can strongly influence B cell stimulation, the regulation of CREB has been little studied. Recently, cross-linking of surface Ig (sIg) was shown to result in trans-activation of a cAMP response element (CRE)-dependent promoter to which bound B cell CREB. In this study, we explored the mechanism underlying this unexpected linkage between sIg and CREB. We found that sIg cross-linking results in phosphorylation of CREB at Ser133. Although this phosphorylation step is mediated by PKA in pheochromocytoma cells, it depends on protein kinase C (PKC) in B lymphocytes. This conclusion is based on abrogation of sIg-induced CREB Ser133 phosphorylation by long-term phorbol-ester treatment to deplete PKC, and mimicking of sIg-induced CREB phosphorylation and CRE-dependent gene expression by short-term PKC agonism. Furthermore, CD40 ligand (CD40L) and LPS, two PKC-independent forms of B cell stimulation, failed to induce phosphorylation of CREB Ser133. These results suggest that CREB responds to specific surface-receptor signals in B cells and that this response is mediated by PKC. Interestingly, forskolin failed to induce phosphorylation of CREB Ser133 in B cells, although it did so in PC12 pheochromocytoma cells. Taken together with PKC mediation of CREB Ser133 phosphorylation in B cells, these results suggest that the dominant mode of CREB regulation is cell-type specific.

Cross-linking of Fc gamma receptor to surface immunoglobulin on B cells provides an inhibitory signal that closes the plasma membrane calcium channel

Stimulation of B lymphocytes by the cross-linking of surface Ig (sIg) with an F(ab')2 antibody fragment leads to the rapid activation of several tyrosine kinases. This gives rise to the activation of phospholipase C gamma (PLC gamma) and the generation of inositol phosphates. These, in turn, lead to a prolonged elevation of intracellular Ca2+ ([Ca2+]i) consisting of a rapid release of Ca2+ from intracellular stores and a sustained influx of extracellular Ca2+. In contrast, co-cross-linking sIg to Fc gamma receptor (Fc gamma RII) with intact anti-sIg induces a much more transient increase in [Ca2+]i. Stimulation of the murine B cell lymphoma, A20, with F(ab')2 anti-sIgG leads to the production of high levels of IL-2, while co-cross-linking of sIgG with Fc gamma RII blocks this response. In studies reported here, we show that co-cross-linking of Fc gamma RII with sIg prevents the influx of extracellular Ca2+ without significantly affecting the tyrosine phosphorylation of substrates including PLC gamma 1, PLC gamma 2, and Syk or the mobilization of Ca2+ from intracellular stores. In cells that had been previously activated with F(ab')2 anti-IgG, co-cross-linking of sIg to Fc gamma RII rapidly abrogated the influx of extracellular Ca2+ by closing the plasma membrane Ca2+ channel. Additionally, even 2-3 h after stimulation of the cells with F(ab')2 fragment, addition of intact anti-IgG to the cells, or removal of extracellular Ca2+, markedly inhibited (> 90%) IL-2 production. These results indicate that co-cross-linking sIg with Fc gamma RII both prevented the opening of and actively closed the Ca2+ channel, and, through this mechanism, Fc gamma RII was able to control production of IL-2. Overall, since influx of extracellular Ca2+ has been found to be necessary for the proliferation and differentiation of B cells, Fc gamma RII may play a critical role in controlling these responses by regulating the opening of the Ca2+ channel.

P72syk tyrosine kinase is activated by oxidizing conditions that induce lymphocyte tyrosine phosphorylation and Ca2+ signals

We have used H2O2 as a pharmacologic agent to examine the effects of oxidizing conditions on lymphocyte signal pathways. Treatment of Ramos cells with 5-10 mM H2O2 gave rapid and strong tyrosine phosphorylation of multiple cellular proteins and activation of p72syk to levels equal to or greater than that observed upon surface Ig cross-linking. Strong Ca2+ signals that could be blocked by the tyrosine kinase inhibitor herbimycin A were also observed under these conditions. However, there was no increase in activity for the Src family kinases p56lck, p59fyn, or p56/p53lyn. Our findings that the p72syk tyrosine kinase responds to H2O2 treatment of cells suggest that this kinase is likely to contribute to cellular tyrosine phosphorylation and calcium signaling induced by oxidizing conditions. Furthermore, H2O2 may be useful as a pharmacologic agent to distinguish the effects of p72syk-related kinases from those of Src family kinases.