Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.     

Learning-induced activation of protein kinase C

PKC activation has been shown to mimic the biophysical consequences of classical conditioning in both rabbit hippocampus and Hermissenda type B cells. Furthermore, conditioning in rabbits results in the 24 h translocation of PKC from cytosol to membrane, which is probably responsible for mediating the biophysical consequences of conditioning. A model has been presented that suggests that long-term translocation of PKC occurs via the synergistic activation of a DG dependent pathway that activates PKC and a calcium dependent pathway that activates CaM kinase. Translocation of PKC to the plasma membrane, by altering ion channel properties, could subserve memory lasting for days, whereas translocation to the nuclear membrane could induce cellular change, by genomic regulation, lasting beyond days. We are, therefore, suggesting that protein kinase C may play a critical role in the formation of short, intermediate, and long-term associative memory.     

BSF1 induces membrane protein phosphorylation but not phosphoinositide metabolism, Ca2+ mobilization, protein kinase C translocation, or membrane depolarization in resting murine B lymphocytes

The findings presented in this study provide evidence that BSF1 receptors and mIg transmit signals via dissimilar transduction mechanisms that result in a common biologic response, hyper-Ia expression. Specifically, BSF1-containing supernatant does not induce PtdInsP2 hydrolysis as determined by measurement of PtdOH and InsP3. Additionally, BSF1 does not stimulate Ca2+ mobilization, PKC translocation from cytosol to membrane, or membrane depolarization. All of these metabolic events appear to play a central role in hyper-Ia expression mediated by mIg and are initiated after treatment of resting B cells with anti-Ig antibodies. In vitro phosphorylation studies with partially purified plasma membranes from resting B cells revealed that BSF1 interaction with membrane receptors stimulates a membrane-associated protein kinase that phosphorylates an endogenous protein of 44 KDa. Anti-Ig does not stimulate phosphorylation of the 44 KDa protein, suggesting that it does not activate the membrane-associated protein kinase. This observation provides the first evidence of a signal transduction mechanism associated with BSF1-receptor ligation. It indicates that although BSF1 does not modulate events associated with PKC activation, it may function via activation of a membrane-associated protein kinase. This provides a focal point for further studies directed at elucidating signal transduction resulting from BSF1-receptor interaction.     

Physiologic activation of protein kinase C limits IL-2 secretion

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.     

Signalling events mediating the activation of protein kinase C by interleukin-2 in cytotoxic T cells.

Interleukin-2 (IL-2) plays a vital role in the generation and regulation of the immune response, including important aspects of T cell survival. IL-2-mediated survival of T cells appears to be dependent on the activation of a pool of membrane-associated protein kinase C (PKC) that occurs in the absence of detectable translocation of the enzyme from the cytosol to membranes. In this report we investigate the mechanism(s) responsible for this PKC activation after IL-2 stimulation in the cytotoxic T cell line, CTLL-2. Tyrosine kinase activity, activated after IL-2 stimulation, was found not to be linked to the activation of PKC by the cytokine. On the other hand, a pertussis toxin (PTX)-sensitive G protein did appear coupled to PKC activation since PTX effectively blocked IL-2 stimulated PKC activity. Diacylglycerols (DAG), but not inositol 1,3,5-triphosphate (IP3) and intracellular Ca2+, increased after IL-2 stimulation suggesting that DAGs were generated via the phosphatidylcholine-phospholipase C (PC-PLC) or phosphatidylcholine-phospholipase D (PC-PLD) pathways. The increase in DAG by IL-2 was probably necessary for activation of membrane-resident PKC since exogenously applied DAG stimulated this PKC pool in both intact cells and in isolated membranes. IL-2 also increased arachidonic acid (AA) production in CTLL-2 cells, probably via phospholipase A2 (PLA2) since the PLA2 inhibitors oleoyloxyethyl phosphocholine and AACOCF3 (AACF) effectively blocked IL-2 stimulated PKC activation. Exogenous AA also increased PKC activity in intact cells and isolated membranes, suggesting that AA produced by IL-2 receptor stimulation was probably linked to PKC activation. These results suggest that the activation of membrane-resident PKC by IL-2 involves multiple second messengers, including G proteins, DAG and AA.     

Protein kinase C-dependent and -independent mechanisms of cloned murine T cell proliferation. The role of protein kinase C translocation and protein kinase C activity

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.     

Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation

To investigate the properties which enable type 2 Ag, as exemplified by dextran and Ficoll, to stimulate high levels of antibody responses in the relative absence of T cells, we conjugated anti-IgD and anti-IgM mAb to both dextran and Ficoll and examined their B cell-activating properties. Such conjugated anti-Ig antibodies stimulated both early and later stages of B cell activation at picogram concentrations, which are at least 1000-fold lower than that required for B cell stimulation by unconjugated anti-Ig antibodies, and the level of proliferation they stimulated was on average 10-fold greater. Furthermore, concentrations of anti-Ig dextran (100 pg/ml) which modulated little sIgD from the B cell surface were strong inducers of enhanced B cell expression of MHC class II molecules. Conjugation of Fab fragments of anti-IgD or nonmitogenic anti-IgM mAb to dextran rendered them as mitogenic as dextran conjugated to strongly stimulatory anti-IgD or anti-IgM antibodies. The ability of dextran and Ficoll to serve as effective carrier molecules for anti-IgD was not related solely to their large m.w., because anti-IgD coupled to polymerized BSA (m.w. 1.5 X 10(6), was only 10- to 50-fold more potent than unconjugated anti-IgD antibodies at stimulating B cell DNA synthesis. These results suggest, therefore, that the unique ability of picogram concentrations of haptenated type 2 Ag to stimulate Ig secretion in the absence of T cells may be a function of their ability to promote effective cross-linking without resulting in the modulation of sIg. This would enable such Ag to mediate repetitive B cell signaling, a situation that cannot be achieved by unconjugated anti-Ig antibodies which result in modulation of sIg at their mitogenic concentrations. These compounds therefore may be employed to study B cell activation stimulated by sIg cross-linking at concentrations that may more closely reflect those which are achieved under physiologic conditions by type 2 Ag.     

Activation signals in human lymphocytes. Incorporation of polyunsaturated fatty acids into plasma membrane phospholipids regulates IL-2 synthesis via sustained activation of protein kinase C

Human PBL activated with anti-TCR/CD3 mAb express high affinity receptors for IL-2, synthesize IL-2, and subsequently proliferate. In contrast, lymphocytes activated by dioctanoylglycerol (DiC8) and ionomycin express high affinity receptors; however, no IL-2 synthesis is detectable. Anti-TCR/CD3 antibodies, as well as DiC8 cause translocation of protein kinase C (PKC) from the cytosol to the plasma membrane. In DiC8-stimulated cells translocation of PKC is detectable after 15 min, then it declines to control levels. In lymphocytes activated by antiTCR/CD3 mAb translocation of PKC is detectable after 15 min, then it declines to control levels, followed by a second, long lasting activation of the enzyme up to 4 h. Addition of polyunsaturated fatty acids to DiC8 + ionomycin-treated cells leads to IL-2 synthesis and proliferation. Incorporation of poly-unsaturated fatty acids into plasma membrane phospholipids results in long term activation of PKC. The results suggest that elevated incorporation of polyunsaturated fatty acids and thus continuous activation and translocation of PKC represents a necessary early signal for IL-2 synthesis and proliferation in human lymphocytes.     

Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.     

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.     

Comparison of tyrosine kinase activation by mitogenic and nonmitogenic anti-IgD antibodies.

Low concentrations of anti-Ig dextran conjugates that stimulate very high levels of B cell proliferation and Ig secretion stimulate no detectable increases in tyrosine phosphorylation. To study this point further, we compared tyrosine phosphorylation patterns induced by mitogenic and nonmitogenic anti-Ig antibodies. Whereas the mitogenic, strongly cross-linking, antibody H delta a/1 induced greater levels of tyrosine phosphorylation than did the nonmitogenic antibody FF1-4D5, the pattern of substrate phosphorylation was equivalent. At lower concentrations of H delta a/1, which were still mitogenic, the degree of phosphorylation that was induced was similar to that induced by high concentrations of FF1-4D5. Both antibodies stimulated comparable increases in the kinase activity of the three src-related kinases present in normal B cells and linked to the IgR, i.e., Blk, Fyn, and Lyn. These results suggest that the extent of tyrosine kinase activation is proportional to mIg cross-linking, that induction of B cell DNA synthesis may require little tyrosine kinase activation, and that activation of tyrosine kinase per se does not necessarily lead to B cell DNA synthesis.     

A major myristylated substrate of protein kinase C and protein kinase C itself are differentially regulated during murine B- and T-lymphocyte development and activation.

The regulation and expression of protein kinase C (PKC) and phosphomyristin C (PMC) (a principal substrate of PKC which is the major myristylated protein in lymphocyte and glioma lines that express it) in murine B and T lymphocytes were investigated. Both PMC and PKC are differentially regulated during T-cell development. The level of PMC expression is highest in CD4-8-, intermediate in CD4+8+, and lowest in J11d-, CD4, or CD8 single-positive thymocytes. PKC is equally expressed by all three thymic populations. In striking contrast to thymocytes, resting peripheral lymph node T cells and T-cell clones express little if any PMC and reduced levels of PKC. Neither PKC nor PMC is significantly induced upon the activation of lymph node T cells: treatment with anti-CD3 antibodies or anti-CD3 and interleukin-2 fails to induce PKC, whereas PMC is not induced by anti-CD3 alone and is only slightly induced by anti-CD3 and interleukin-2. In contrast to the situation with T cells, PMC and PKC are constitutively expressed at moderate levels in mature B cells. PMC is greatly increased in B-cell blasts generated by cross-linking the antigen receptor with anti-immunoglobulin. These results demonstrate that PMC and PKC are differentially regulated during the development and activation of B and T cells, suggesting that cellular events that rely upon PKC and PMC may differ during ontogeny and activation of different lymphocyte subsets.     

T cell activation induced by anti-CD3 antibodies requires prolonged stimulation of protein kinase C

Accessory cell-depleted T cells required the presence of a protein kinase C (PKC) stimulating phorbol ester, such as phorbol 12,13-dibutyrate (PDB), to be activated by soluble antibodies to the CD3 molecular complex. To determine the duration of PDB costimulation necessary to induce a proliferative response, highly purified T cells were pulsed with anti-CD3, incubated with PDB for limited periods of time, and then washed and recultured in the absence of PDB. T cells stimulated with anti-CD3 and PDB for 2 hr were unable to proliferate unless IL-2 or PDB was added to the second culture. With more prolonged exposure to PDB (4-18 hr), anti-CD3-pulsed cells exhibited an increased capacity to proliferate in the absence of additional PDB. Proliferation could be augmented by exogenous IL-2, but remained submaximal. Optimal DNA synthetic responses required the presence of PDB throughout the entire culture. Despite this, costimulation with anti-CD3 and PDB induced a significant number of cells to express IL-2 receptors and enter the cell cycle after 18 hr of costimulation with PDB. Moreover, T cells costimulated by anti-CD3 and PDB produced IL-2 within 4 hr. However, T cells that were stimulated with anti-CD3 and PDB for 4 hr, washed, and recultured rapidly lost the ability to continue to produce IL-2, which reflected a decrease in the content of mRNA encoding IL-2. This loss of IL-2 production was prevented by reculturing the cells with PDB. These studies therefore indicate that after initial T cell activation by anti-CD3, continued stimulation of PKC is necessary for ongoing IL-2 production. These results suggest a model of T cell activation in which sustained stimulation of PKC after cell cycle entry is required to maintain growth factor production and continued proliferation.     

Antigen-induced Fc receptor-dependent and -independent B cell desensitization. An elevation in [Ca2+]i is not sufficient and protein kinase C activation is not required for these pathways of surface IgM-mediated desensitization

The interaction of an Ag ligand with its B cell surface Ig (sIg) receptor can occur via an FcR-dependent or -independent pathway. We previously found that transfected TNP-specific B cells undergo both Ca2+ signaling and desensitization upon interaction with the thymus-dependent Ag TNP-OVA. Similarly, we showed that these B cells can also be desensitized by cross-linking sIg to the Fc gamma R via the formation of an Ag-antibody bridge. Thus, Ag-specific B cells can be desensitized by two different Ag-dependent events, one mediated by Ag-sIg interaction and the other by sIg-Fc gamma R cross-linking. Inasmuch as Ag-sIg and sIg-Fc gamma R interactions lead to positive and negative signaling, it was of interest to determine whether B cell desensitization mediated by these interactions occurs by one of the well known signaling pathways in B cells. We found that Ag-induced changes in [Ca2+]i could be readily dissociated from Ag-induced desensitization, indicating that a Ca(2+)-independent pathway is likely responsible for this pathway of desensitization. To determine if PKC plays a role in B cell desensitization mediated by either Ag or sIg-Fc gamma R interaction, PKC was downregulated by long term exposure to 12-O-tetradecanoylphorbol 13-acetate or inhibited by exposure of cells to staurosporine. The PKC down-regulated and inhibited cells underwent similar Ag- and Fc gamma R-dependent desensitization compared to cells containing active PKC. Taken together, these data indicate that Ag-induced desensitization of B cell signaling likely involves an event(s) that occurs either upstream or independent of Ag-induced elevations in [Ca2+]i and PKC activation.     

The role of protein kinase C activation in signal transmission by interleukin 2

Role of protein kinase C (PKC) in interleukin (IL) 2-induced proliferation was investigated by utilizing two murine IL 2-dependent cell lines, CT6 and CTLL-2 cell lines. CT6 cells showed a marked proliferative response to phorbol 12-myristate 13-acetate (PMA), while CTLL-2 did not. PMA induced PKC translocation from cytosol to membrane only in a PMA-responsive cell line. IL 2 failed to stimulate PKC translocation in both cell lines. H-7, a potent and specific PKC inhibitor, however, inhibited the proliferation of both cell lines induced by IL 2. Taken collectively, IL 2 may induce PKC activation without its translocation.     

Cytokine receptors and B cell functions. I. Recombinant soluble receptors specifically inhibit IL-1- and IL-4-induced B cell activities in vitro

IL-1 and IL-4 are important mediators of B cell growth and differentiation. The cell-surface receptors for these cytokines have recently been cloned and recombinant soluble receptors have been produced that bind their respective ligand. The ability of soluble forms of the murine IL-1R (sIL-1R) and IL-4R (sIL-4R) to inhibit B cell functions in vitro was examined. Proliferation of B cells treated with anti-Ig plus IL-1 or IL-4 was inhibited by the appropriate soluble receptor. sIL-4R also inhibited IL-4-dependent B cell differentiation as measured by: induction of IgG1 and IgE secretion by LPS blasts, down-regulation of IgG3 secretion by LPS blasts, increased Ia expression, and increased Fc epsilon R (CD23) expression. The inhibitory effects of the soluble receptors were found to be highly specific in that sIL-4R had no effect on IL-1-induced B cell activity and sIL-1R had no effect on IL-4 activity, further demonstrating the existence of two independent pathways of B cell activation directed by IL-1 and IL-4.     

Lyb-5- B cells of CBA/N mice can be induced to synthesize DNA by culture with insolubilized but not soluble anti-Ig

The use of anti-immunoglobulin (anti-Ig) antibodies to stimulate B cell proliferation (1-4), and to stimulate B cell differentiation in the presence of T cell derived-lymphokines (5-8), has simplified investigations into the mechanisms of B cell growth and maturation that are dependent on the cross-linking of surface Ig (sIg). It is only the ontogenetically late appearing Lyb-5+ murine splenic B cells, however, that proliferate in response to anti-Ig antibodies, whereas B cells of the Lyb-5- phenotype obtained from neonatal mice or from mice with the xid immune defect cannot be induced to proliferate in response to this stimulus (1, 9, 10). Thus, the analysis of B lymphocyte physiology of the Lyb-5- B cell population has been hampered by the unavailability of B cell stimulants that mimic an antigen-induced sIg cross-linking event that leads to B cell activation. The inability of soluble anti-Ig antibodies to induce the proliferation of Lyb-5- cells has been particularly difficult to explain because these cells can be induced to increase in size (11) and to show an increase in their expression of surface Ia (sIa) after exposure to anti-Ig (12). Apparently, therefore, these cells are not entirely refractory to this stimulus but are simply unable to progress to the latter stages of cell activation. In view of our observations that the cells of CBA/N mice cannot respond to soluble trinitrophenyl-(TNP) dextran or TNP-polyacrylamide (13) but can respond to insolubilized forms of these antigens, we evaluated their ability to respond to insolubilized anti-Ig. In this paper we report that B cells from CBA/N mice can be stimulated to proliferate in response to anti-Ig conjugated to Sepharose beads, but in contrast to normal B cells they need to be stimulated with beads expressing a high-epitope density of anti-Ig antibodies.