Activation of human B cell proliferation through surface Bp35 (CD20) polypeptides or immunoglobulin receptors

Human B cells can be activated with monoclonal antibodies (mAb) to surface IgM receptors or mAb to a 35-kilodalton B cell differentiation antigen, Bp35 (CD20). We compared anti-Ig-induced B cell activation with B cell triggering by anti-Bp35. Both anti-Ig- and anti-Bp35-dependent proliferation were augmented by the same co-stimulants, including a partially purified BCGF, recombinant IL 1, TPA, or each other. When anti-Bp35 and anti-Ig were used together to induce proliferation of tonsillar B cells, the strongest response was observed when anti-Bp35 was added 12 to 24 hr before anti-Ig. Anti-Bp35 also was found to act most effectively when added before the BCGF. Blood and tonsillar B cells differed in their proliferative response to anti-Ig or anti-Bp35: unlike dense tonsillar B cells, which consistently proliferated in response to either stimulus, blood B cells from many donors proliferated in response to anti-Ig but not to anti-Bp35 even in the presence of other co-stimuli. Dense tonsillar B cells that proliferate in response to anti-Bp35 appeared to be at a more activated stage than unresponsive blood B cells because they expressed higher levels of HLA class II molecules than blood B cells. Pretreatment of blood B cells with anti-Bp35 converted them to an HLA-DR(bri) phenotype and made them more responsive to anti-Ig-induced proliferation. These results suggest that B cells at different stages of differentiation differ in their response to anti-Bp35 and anti-Ig. The Bp35 surface polypeptide may play an early role in the activation of B cells prior to antigen or other signals.     

B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

Signal requirements for growth and differentiation of activated murine B lymphocytes

Mouse B lymphocytes were stimulated at high cell concentrations with goat anti-IgM antibodies, which leads to the induction of B cell proliferation without the addition of any growth factors. After 48 hr, blast cells were purified and cultured at low cell concentrations. Proliferation and differentiation of purified B lymphocyte blasts is then dependent on the addition of either mitogens (e.g., LPS) or certain lymphokines derived from activated T cells or macrophages. One such lymphokine was isolated from supernatants of various activated T cells and characterized by gel filtration as a material with an apparent m.w. of 40,000 to 50,000, similar to BCGF II. It supports the proliferation of the B cell blasts and induces their differentiation into plaque-forming cells. Lymphokines such as BCGF I, interleukin 2, and BCDF gamma could neither maintain growth nor induce differentiation of B lymphocytes preactivated by goat anti-IgM.     

T cell-derived B cell growth factor(s) can induce stimulation of both resting and activated B cells

The effects of a preparation containing partially purified, EL4-derived B cell growth factor(s) (BCGF) on B cell growth and proliferation have been examined by using B lymphocyte subpopulations separated on the basis of size. BCGF was found to maintain and enhance proliferation of a significant proportion of large activated B cells. In contrast, small resting B cells required the presence of BCGF and a second stimulus such as anti-IgM antibody (anti-mu) to be induced to proliferate. This disparity was not due to a lack of an effect of BCGF on small resting B cells. A factor contained within the partially purified EL4 supernatant produced time-dependent increases in cell size and RNA content in all subpopulations. These effects were independent of possible effects due to contaminating lymphokines such as interleukin 2 (IL 2), concanavalin A (Con A), and phorbol myristate acetate (PMA). Nonmitogenic doses of lipopolysaccharide (LPS) failed to show similar effects. Our data suggest that B cells at all levels of in vivo activation are responsive to stimulation by a growth factor present in EL4 supernatant, as manifested by cell growth and RNA synthesis. This activity has not previously been described for BCGF preparations. However, because the partially purified, EL4-derived supernatant used as BCGF in these studies has not been purified to homogeneity, we cannot conclude whether the factors that induce resting B cells to increase in size are the same as the growth factors that synergize with anti-mu to induce B cell proliferation or that maintain the proliferation of activated B cells.     

Inhibition of anti-IgM and growth factor-induced proliferation of guinea pig B cells by one of two distinct Fc gamma receptors on the cells

Guinea pig B cells were found to proliferate when co-stimulated with F(ab')2 of rabbit anti-guinea pig IgM and human 12-kDa B cell growth factor (BCGF), though the proliferation did not occur with the replacement of the F(ab')2 by its parent IgG antibody. In addition, the intact antibody inhibited the proliferation induced by F(ab')2 of anti-IgM and BCGF. Because both two distinct types of FcR for IgG on the B cells, one specific for IgG2 (Fc gamma 2R) and the other for both IgG2 and IgG1 (Fc gamma 1/gamma 2R), can bind rabbit IgG, we determined whether they participate in the inhibition of the B cell proliferation by intact anti-guinea pig IgM antibody. Blocking Fc gamma 1/gamma 2R by F(ab')2 of anti-Fc gamma 1/gamma 2R mAb significantly reversed the inhibitory effect of intact anti-IgM antibody. F(ab')2 of anti-Fc gamma 2R mAb, however, was not effective. Furthermore, guinea pig IgG1 and IgG2 anti-rabbit IgG antibodies suppressed similarly the B cell proliferation induced by F(ab')2 of rabbit anti-IgM and BCGF. These results show that between these two types of Fc gamma R on B cells, Fc gamma 1/gamma 2R alone is involved in the regulation of anti-IgM and BCGF-induced B cell proliferation, and inhibits the response when cross-linked to the surface IgM.     

Activity of a partially purified human BCGF on murine assays for B-cell stimulatory factors. I. BCGF II-like activity of human BCGF

To further characterize a human B-cell growth factor (BCGF) produced by phytohemagglutinin (PHA) P-stimulated peripheral blood T cells, a partially purified preparation of this material was tested in a number of murine assays for B-cell stimulatory factors (BSF). Human BCGF lacked murine BSF-1 activity as assessed via the induction of polyclonal proliferation of anti-IgM-stimulated murine B cells; however, this material consistently augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1. Human BCGF also induced proliferation in unstimulated murine B cells, and augmented the proliferative response of dextran sulfate activated murine B cells. Human BCGF is therefore capable of causing proliferation of unstimulated and activated murine B cells, and by these criteria closely resembles murine BCGF II. In contrast to murine BCGF II, however, human BCGF failed to stimulate proliferation or immunoglobulin (Ig) secretion by murine BCL1 B lymphoma cells. A murine analog of this human BCGF showing the same pattern of biological responses was found in concanavalin A-stimulated supernatants of the murine MB2.1 T-cell line and D9-Cl T-cell hybridoma. The active component of the human BCGF preparation was not due to contaminating PHA, interleukin 1, interleukin 2; interferon-gamma, or endotoxin. Comparison between the above human BCGF and a commonly used source of murine BCGF II, i.e., supernatant from antigen-stimulated D10.G4.1 T cells, provided information suggestive of BCGF II heterogeneity. Both human BCGF and D10.G4.1 supernatant caused proliferation of unstimulated and dextran sulfate-stimulated murine B cells; however, only the human BCGF preparation augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1, and only the D10.G4.1 supernatant stimulated BCL1 cell proliferation and immunoglobulin secretion. The data therefore indicate that the different assays for BCGF II used in this study respond to different factors, and suggest the existence of two BCGF II-like activities.     

Human B cell responsiveness to B cell growth factor after activation by phorbol ester and monoclonal anti-mu antibody

The effect of phorbol ester on human B cell activation was examined. Picomolar to nanomolar concentrations of phorbol ester induced a high level of proliferation in small IgM-positive B cells isolated from peripheral blood by fluorescence-activated cell sorting. The addition of optimal doses of anti-mu antibody resulted in enhanced proliferation of phorbol ester-activated B cells. The addition of B cell growth factor (BCGF) to phorbol ester-activated B cells also resulted in a dose-dependent synergistic effect and maximal enhancement on day 3. BCGF activity could be absorbed with either phorbol ester- or anti-mu-activated B cells, but not with resting B cells, thus confirming the induction of functional BCGF receptor expression. Cell proliferation was not necessary for the induction of functional BCGF receptors. Phorbol ester was a more efficient inducer of BCGF receptor expression than was anti-mu antibody; gamma-interferon treatment had no effect. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. The results suggest that phorbol ester-activated small B cells can be used to monitor BCGF activity, and this synergistic combination may be useful in establishing BCGF-dependent B cell clones in culture.     

Amplification of human B cell activation by a monoclonal antibody to the B cell-specific antigen CD22, Bp 130/140

The B cell-specific antigen CD22 is a 130/140-Kd complex and is unique among human B cell antigens, since its surface expression is restricted to a subpopulation of Ig+ B cells. Here the function of the CD22 antigen was evaluated by using the mAb HD6, directed against one of the epitopes on the molecule. The HD6 antibody was constimulatory with anti-Ig in inducing small, dense tonsillar cells to proliferate; however, the antibody by itself was devoid of stimulatory activity. Anti-CD22 antibody also induced more anti-Ig-treated B cells to leave G0 and enter the G1 phase of the cell cycle. It also was constimulatory with low-m.w. BCGF and with an antibody to a 50-Kd polypeptide, Bp50, which mediates a BCGF-like activity. Results of kinetic experiments and analysis of different B cell fractions suggested that anti-CD22 acts during an early phase of B cell activation, probably by amplifying the anti-Ig signal. F(ab')2 fragments of anti-CD22 HD6 were as effective as the whole antibody in inducing augmentation of B cell proliferation, showing that the Fc portion of the molecule was not required for the activity. The results of these experiments, together with the intriguing distribution of the Bp 130/140 antigen in B cell ontogeny, suggest that this molecule plays an important role in the process that leads to B cell activation and proliferation.     

Expression of CD9 antigen on normal activated human B cells

The expression of the CD9 pre-B acute lymphoblastic leukemia (ALL)-associated antigen was studied. CD9-positive B cells were enriched in the in vivo-activated buoyant B cell population isolated from tonsils. Small tonsil B cells activated in vitro with either PWM, phorbol 12-myristate 13-acetate (TPA), or anti-Ig plus low Mr B cell growth factor (BCGF) also demonstrated increased CD9 expression. The peak of CD9 expression (30-40% positive cells) occurred after 4-6 days of activation. The kinetics of increased CD9 expression was similar to that of the 4F2 activation antigen. CD9 antigen expression on tonsillar B cells as well as on pre-B leukemic cell lines was associated with protein kinase C activation. Two phorbols that activate protein kinase C (TPA; phorbol 12,13-dibutyrate) induced expression of the CD9 antigen whereas a phorbol analogue that does not activate C kinase (4-alpha-phorbol 12,13-didecanoate) and an analogue that is a very weak agonist (phorbol 12-myristate 13-acetate-4-0-methyl ether) were unable to induce CD9 expression on tonsil B cells or on the cell lines. The effect of the anti-CD9 monoclonal antibody, DU-ALL-1, on B cell mitogenesis was studied. Dense or buoyant tonsillar B cells were cultured in the presence or absence of DU-ALL-1 antibody plus PWM, anti-Ig, and BCGF, DU-ALL-1 antibody did not inhibit or augment the mitogenic response of resting or activated B cells. These results indicate that the CD9 pre-B ALL antigen is present on a population of normal activated tonsillar B cells and that its induction of expression is associated with protein kinase C activation.     

Human B cell proliferation in response to IL-4 is associated with enhanced production of B cell-derived growth factors

To investigate the capacity of human IL-4 to function as a B cell growth factor (BCGF), we studied its ability to promote proliferation of a selected B cell line. We show that the cell line, designated A4, proliferated in response to IL-4 in a dose-dependent manner. The A4 cells also proliferated in response to their own B cell derived growth factor (B. BCGF), suggesting autocrine-mediated growth. The ability of IL-4 to induce proliferation of the A4 cell line was dependent on the level of autocrine growth. At low cell density, IL-4 induced marked dose-dependent proliferation. However, as A4 cell density increased, the ability of IL-4 to induce proliferation was diminished. The possibility that IL-4 may be mediating the autocrine growth of A4 cells was ruled out, because A4 cell-derived BCGF failed to induce CD23/low affinity receptors for the Fc region of IgE on activated tonsillar B cells and anti-IL-4 antibody did not block B. BCGF activity. We found that IL-4 stimulation of A4 cells and activated tonsillar B cells is associated with enhanced production of B. BCGF. These data indicate that human IL-4 has the capacity to promote proliferation of the B cell line A4, and that the ability of IL-4 to function as BCGF is associated with enhanced autocrine growth of activated B cells.     

Identification of a novel human B cell activation antigen involved in B cell growth factor-dependent proliferation

The B6.4 mAb we present here identifies a novel activation Ag of B cell lineage. The B6.4 mAb was generated by immunizing mice with B cell growth factor (BCGF)-responding BA3 cells, and selected by its capability to block BCGF-induced proliferation of BA3 cells. The inhibitory effect of this antibody on BCGF-dependent proliferation was further confirmed by using normal activated B cells in the presence of exogenous BCGF derived from T cells or B cells. Furthermore, it did not affect IL-2-dependent proliferation of B cells. The expression of the B6.4 Ag, as analyzed by FACS, is restricted to in vitro activated B cells, and remains undetectable on resting B or T cells, PHA-activated T cells, and monocytes. The B6.4 Ag is also expressed on some lymphoblastoid B cell lines and most of the lymphomas and CLL of B cell origin; however, it did not express on pre-B cell ALL and plasma cell leukemias. The B6.4 Ag has a Mr of 35,000 Da, as determined by SDS-PAGE of radiolabeled immunoprecipitates from activated B cells. The B6.4 Ag is detectable on B cells as early as 8 h after activation, and precedes that of the IL-2R or transferrin R. All of these results suggest that the B6.4 Ag is an early activation Ag of B cells and is functionally related to a receptor of BCGF, but not IL-2.     

Reactivity of Leu-1+ tonsillar B cells to a high molecular weight B cell growth factor

This work was focused on the responsiveness of B cells from blood and tonsils to typical B cell growth factors (BCGF) in the absence of anti-mu antibody. This direct responsiveness was not observed with small dense B cells from either organ. Large tonsillar B cells but not large blood B cells did respond directly to a high m.w. BCGF (m.w. 50,000 BCGF), whereas large B cells from both organs responded directly to the low m.w. BCGF (m.w. 20,000 BCGF). The tonsillar B cells directly responsive to the m.w. 50,000 BCGF express the 4F2 marker and thus belong to a population of in vivo-preactivated B cells. Moreover, this direct responsiveness to the m.w. 50,000 BCGF is localized in Leu-1+ tonsillar B cells. The Leu-1+ and Leu-1- tonsillar B cell subsets do not differ in their responsiveness to the m.w. 50,000 BCGF upon costimulation with anti-mu antibody and to the m.w. 20,000 BCGF regardless of the presence of anti-mu antibody. Thus, the Leu-1+ subset present in tonsils shows a particular reactivity to a high m.w. BCGF.     

The effects of interleukin 1 on human B cell activation and proliferation

The precise role of B cell surface immunoglobulin (slg) in the activation of B cells is unclear at present. In particular, it is uncertain whether ligands interacting with the B cell slg suffice to induce proliferation, or simply induce a state of activation in which the B cell becomes responsive to growth factors made by accessory cells. We have examined the effects of two ligands, Staphylococcus aureus Cowan strain I (SAC) and antihuman mu chain (anti-mu), which interact with B cell slg on highly purified human peripheral blood and tonsillar B cells cultured at low cell concentrations. The effects on B cell proliferation of these ligands alone or in combination with highly purified interleukin 1 (IL 1) or a supernatant of a human T-T hybridoma containing a B cell growth factor (BCGF) were studied. SAC with its high cell wall content of protein A triggered maximal B cell proliferation which was not increased further by IL 1 or BCGF. High concentrations of soluble F(ab')2 fragments of goat anti-mu chain also induced significant B cell proliferation. Lower concentrations of anti-mu resulted in little or no B cell proliferation but activated the B cell to a state of responsiveness to both IL 1 and BCGF. IL 1 by itself had no effect on the proliferation of unstimulated B cells or on the proliferation of in vivo-activated B cells which responded to BCGF in vitro, but demonstrated clear synergy with low concentrations of anti-mu antibody. BCGF alone augmented the proliferation of unstimulated B cells, presumably by acting on B cells which had undergone some degree of activation in vivo. In addition, it showed marked synergy with anti-mu antibody, which resulted in proliferation similar in magnitude to that induced by SAC. This synergy was far greater than that seen between anti-mu antibody and IL 1, and the resulting proliferative response was only slightly increased by the presence of IL 1. We conclude that the importance of accessory cell factors for the initial rounds of B cell proliferation depends on the strength of the initial slg-mediated activation signal. When this is strong, the response is maximal and independent of accessory cells or accessory cell factors. When it is suboptimal, a moderate synergy is seen with IL 1 and a dramatic synergy with BCGF.     

Identification of a receptor for high molecular weight human B cell growth factor

Regulation of the proliferation of human B lymphocytes is under the control of several different signals. Various B cell growth factors (BCGF) have been described including a 60-kDa BCGF called high m.w. BCGF (HMW-BCGF). In this paper we describe a mAb BA5 that blocks the proliferation of normal activated human B lymphocytes in response to HMW-BCGF and does not affect the proliferation of T cells in response to PHA or IL-2. BA5 shows minimum binding to resting B cells, significantly enhanced binding to resting B cells, significantly enhanced binding to activated B cells and essentially no binding to resting or activated T cells. BA5 recognizes a 90-kDa protein from solubilized membranes of activated B cells. 125I-HMW-BCGF cross-linked to its binding site on activated B cells produces a 150-kDa R-protein complex. Unlabeled HMW-BCGF cross-linked to its binding site on activated B cells produces a 150-kDa band recognized by both BA5 and BCGF/1/C2 (a mAb to HMW-BCGF) using Western blotting. Thus, BA5 recognizes a molecule intimately associated with the receptor for HMW-BCGF which includes a binding site for HMW-BCGF. BA5 can be used to explore the role of HMW-BCGF and B cell proliferation in various aspects of human B cell physiology.     

Activation of dense human tonsilar B cells. Induction of c-myc gene expression via two distinct signal transduction pathways.

Antibodies to surface Ig or to the B cell marker CD20 trigger resting human B cells in similar yet distinct ways. Either antibody induces five-fold increases in the expression of the protooncogene, c-myc, as detected with semi-quantitative Northern blot assays. The induction of c-myc mRNA by anti-IgM or anti-CD20 is blocked by inhibitors of protein kinase C (PKC) such as staurosporine and by pretreatment of B cells with phorbol esters to reduce cellular PKC levels. This suggests that PKC is involved in the pathways stimulated by both anti-IgM and anti-CD20. However, anti-CD20, unlike anti-IgM, does not activate significant increases in inositol triphosphate or intracellular-free calcium. Further, anti-CD20-triggered elevation of c-myc mRNA is inhibited by pertussis and cholera toxins, whereas the pathway initiated by anti-IgM if anything is stimulated by pertussis toxin and unchanged by cholera toxin. Further differences in the nature of these two signals were seen when the expression of adhesion/recognition molecules were examined. Anti-IgM consistently induces increased expression of the adhesion molecules CD54 (I-CAM-1) and B7/BB-1 on B cells, but anti-CD20 does not. Yet both anti-CD20 and anti-IgM increase class II MHC, CD18 (LFA-1 beta-chain) and LFA-3 levels. These data suggest that the way in which B cells are activated may influence their surface phenotype and possibly subsequent migration or cell-cell interactions.     

Activation of human B cells: alternate options for initial triggering and effects of nonmitogenic concentrations of anti-IgM antibodies on resting and activated cells

The monoclonal antibody 1F5, which is reactive with the CD20 (Bp 35) pan-B cell antigen, was shown to activate resting human peripheral blood B cells into the middle to late G1 phase of the cell cycle. However, in contrast to F(ab')2 fragments of polyclonal anti-mu, 1F5 synergized only weakly with B cell growth factor (BCGF) for DNA synthesis in these cells. We provide evidence that the CD20 molecule and surface immunoglobulin represent two alternative activation pathways in resting B cells. We also show that anti-immunoglobulins, during co-stimulation with BCGF, may play an important role in G1 as well as for the initial cell triggering. Thus, anti-mu in nonmitogenic concentrations was shown to provoke distinct effects on 1F5-pretreated G1 cells, as monitored by increases in cellular volumes as well as in cytoplasmic Ca2+ levels. Moreover, anti-mu could increase c-myc mRNA levels in 1F5-primed cells, implying that c-myc expression can be regulated in G1 as well as during the initial G0 to G1 transition. Partially purified human BCGF neither induced G1 entry in resting peripheral blood cells nor primed the cells for DNA synthesis. The finding that BCGF did not influence c-myc mRNA levels in resting or in activated B cells suggests that its mitogenic action does not involve the c-myc function.     

Regulation of cell division of mature B cells by ionomycin and phorbol ester.

The growth of a human B lymphoma cell line B104, an experimental model for mature B cells, was inhibited by ionomycin but not 12-O-tetradecanoylphorbol-13-acetate (TPA). Ionomycin inhibited B104 cells from entering into the M phase of the cell cycle without affecting DNA synthesis. The inhibition of cell division of B104 cells by ionomycin occurred within 24 h after stimulation. Because such a mode of action resembles that of anti-IgM antibodies, signals transduced by Ca2+ may be responsible for the inhibition of cell division of B104 cells by anti-IgM antibodies. Indeed, EGTA suppressed the inhibition of cell division of B104 cells caused not only by ionomycin, but also by anti-IgM antibody. Although TPA itself did not have any ability to promote the growth of B104 cells, it could cancel the inhibition of cell division of B104 cells by ionomycin and increase the proportion of B104 cells entering into the M phase of the cell cycle. Staphylococcus aureus Cowan I causes the greatest proliferation of normal human peripheral blood B cells during the period from 48 to 72 h after stimulation. When ionomycin was added to S. aureus Cowan I-stimulated peripheral blood B cells at 48 h of culture, it inhibited cell division during this period without affecting DNA synthesis. In the presence of TPA, this activity of ionomycin was suppressed, and the proportion of M-phase cells increased. These results suggest that cell division of mature B cells is regulated by the signals mediated by Ca2+ and protein kinase C in a mode quite different from that of regulation of DNA synthesis.     

Effect of gamma radiation on resting B lymphocytes. II. Functional characterization of the antigen-presentation defect

The effect of radiation on three discrete Ag-presentation functions in resting B cells was examined: 1) Ag uptake and processing, 2) expression of processed Ag in the context of functional class II molecules, and 3) provision of necessary co-stimulatory, or "second," signals. Analysis of radiation's effect on B cell presentation of intact vs fragmented Ag or its effect on presentation by Ag-pulsed B cells indicated that damage to Ag uptake and processing could not account for the bulk of the radiation-induced Ag-presentation defect. Experiments with phosphatidylinositol hydrolysis as an indirect measure of TCR occupancy suggested that irradiation caused a fairly rapid (within 1 to 2 h) decrease in the ability of the B cell APC to display a stimulatory combination of Ag and class II molecule. Ag dose-response analyses demonstrated that when presenting a fragment of the Ag pigeon cytochrome c to a T cell clone, 3000 rad-treated B cell APC were able to stimulate approximately 50% as much phosphatidylinositol turnover as unirradiated B cells. It was also found that, in contrast to their inability to initiate T cell proliferation, and similarly to chemically cross-linked splenocytes, heavily irradiated resting B cells plus Ag induced a state of Ag hyporesponsiveness in T cell clones. This effect on T cells had the same Ag- and MHC-specificity as did receptor occupancy required for proliferation, indicating that heavily irradiated resting B cells bear functional class II molecules. Co-culture of T cells with allogeneic B cells and syngeneic heavily irradiated B cells or chemically cross-linked splenic APC plus Ag resulted in T cell proliferation and interfered with the induction of the hyporesponsive state. This co-stimulatory function was radiosensitive in resting allogeneic B cells. Together, these data support the hypothesis that the major functional consequences of radiation to resting B cell APC are a reduction in the effective display of Ag plus class II molecules and, probably what is more important, a loss in the ability to provide APC-derived co-stimulatory signals.     

Production of B cell growth factor by a Leu-7+, OKM1+ non-T cell with the features of large granular lymphocytes (LGL)

The present study reports the characterization of a non-T cell from human peripheral blood which is capable of releasing BCGF. This BCGF-producing non-T cell had a T3-, T8-, Leu-7+, OKM1+, HLA-DR-, Leu-11- surface phenotype and was likely to belong to the so-called large granular lymphocyte (LGL) subset because: after fractionation of non-T cells according to the expression of Leu-7 or HLA-DR markers, it was found in the Leu-7+, HLA-DR- fractions that were particularly enriched in LGL; it co-purified with LGL on Percoll density gradients; and it expressed Leu-7 and OKM1 markers that are shared by a large fraction of LGL. Although co-purified with cells with potent NK capacities, the BCGF-producing cell was not cytotoxic, because treatment of Leu-7+ cells with Leu-11 monoclonal antibody and complement abolished the NK activity but left the BCGF activity unaltered. The factor released by this LGL subset was not IL 1 or IL 2 mistakenly interpreted as BCGF, because: a) cell supernatants particularly rich in BCGF activity contained very little or no IL 1 or IL 2; b) BCGF-induced B cell proliferation was not inhibitable by anti-Tac antibodies (this in spite of the expression of IL 2 receptor by a proportion of activated B cells); and c) BCGF activity was absorbed by B but not T blasts.     

Sequential effect of a high molecular weight B cell growth factor and of interleukin 2 on activated human B cells

A high m.w. B cell growth factor (50,000 BCGF) prepared from lectin-activated human peripheral blood lymphocyte culture supernatants acts only on B cells preactivated by a first signal. This first signal can be delivered in vitro (with anti-mu antibody (Ab)) or in vivo. Upon costimulation with anti-mu Ab the 50,000 BCGF induces an early and transient proliferative response, whereas the response to interleukin 2 (IL-2) develops more progressively. To determine the respective targets of the 50,000 BCGF and of IL-2, B cells were activated with anti-mu Ab and separated according to the expression of the IL-2 receptor (cluster designation (CD)25 antigen). CD25+ B cells do not respond to the 50,000 BCGF and do not acquire this responsiveness after an additional culture with IL-2. CD25- B cells respond to the 50,000 BCGF and not to IL-2. However, when CD25- B cells are cultured for 3 days with the 50,000 BCGF they become responsive to IL-2. These results demonstrate a pathway of B cell activation based on the ordered and sequential action of anti-mu Ab, the 50,000 BCGF, and IL-2.