B-cell activation stimulated by monoclonal anti-Lyb2.1 antibody is mediated through a receptor distinct from the BSF-1 receptor

The experiments in this paper demonstrate that monoclonal anti-Lyb2.1 antibody enhances the proliferative response of anti-immunoglobulin (anti-Ig)-stimulated but not of dextran sulfate-stimulated B cells. The magnitude of this enhanced B-cell proliferation is comparable to that induced by BSF-1 on anti-Ig-stimulated cells. The ability of this antibody to enhance B-cell proliferation does not result from its ability to neutralize the suppressive effects on B-cell activation that is mediated by the Fc fragment of anti-Ig antibody as it is equally as effective in enhancing B-cell proliferative responses stimulated by F(ab')2 fragments of anti-Ig. BSF-1 and Anti-Lyb2.1 appear to stimulate nonoverlapping pathways leading to B-cell activation since the enhanced responses induced by the combination of BSF-1 and anti-Lyb2.1 on anti-Ig-stimulated cells are additive even when maximum quantities of these activators are employed. There is also a marked difference in their activity on T cells; while BSF-1 can enhance T-cell proliferation in synergy with phorbol ester, anti-Lyb2.1 is ineffective in this regard. These data, while consistent with the suggestion that the Lyb2 surface determinant on B cells may be involved in B-cell activation, indicate that it is distinct from the receptors for BSF-1 or BCGF-II.     

Induction of B lymphocyte proliferation by monoclonal anti-Lyb 2 antibody

Monoclonal antibody to Lyb 2, a differentiation antigen present on all B cells, has been used to study the role of Lyb 2 molecules in B cell activation. Monoclonal anti-Lyb 2 antibody (m-anti-Lyb 2) transforms resting B cells into blast cells and induces proliferation in these activated B cells. The proliferative response to anti-Lyb 2 is a property of the Lyb 5+ subset of B cells, since the antibody fails to stimulate B cells from mice expressing the CBA/N immune defect. B cells activated by anti-Lyb 2 mature into antibody-secreting cells in the added presence of T cell-replacing factors contained in the supernatants from concanavalin A-activated T cells. Thus, Lyb 2 molecules may participate in the delivery of stimulatory signals during the early phases of B lymphocyte activation.     

Biochemical analysis of inhibitory effects of a lymphokine suppressive B-cell factor on the activation process of resting B cells

Suppressive B-cell factor (SBF) is elaborated by FcR gamma (Fc receptor for IgG)-bearing small, resting B cells after the stimulation of immune complexes and is known to inhibit humoral immune responses by acting on resting B cells. In order to elucidate where and how SBF interferes with B-cell activation in the course of transmembrane signaling, we examined the effect of SBF on the several sequential events which B cells undergo after crosslinking surface immunoglobulin (sIg). Hyper-Ia expression, plasma membrane depolarization, and activation of phosphatidylinositol (PI) hydrolysis of resting B cells, all of which were induced by the stimulation with anti-mu antibody, were significantly suppressed by the pretreatment of cells with SBF. However, SBF had no effect on the intracytoplasmic cyclic AMP level of either activated or resting B cells. Another inhibitory effect of SBF on the activation process of resting B cells by anti-mu antibody was to suppress the transient elevation of intracytoplasmic free Ca2+ only in the initial phase after triggering with anti-mu antibody. This seems to be due to a decrease in the release of inositol triphosphate into the cytoplasm by suppressing the activation of PI hydrolysis. Considering all the data, the suppressive effect of SBF on the transmembrane signaling by sIg crosslinking is ascribed to the selective suppression of the activation of PI hydrolysis. This provides a concept on a molecular basis that feedback regulation of humoral immune response is, at least partly, regulated by SBF.     

Properties of anti-Lyb-2-mediated B-cell activation and the relationship between Lyb-2 molecules and receptors for B-cell stimulatory factor-1 on murine B lymphocytes

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to synergize with anti-mu in induction of B-cell proliferation. The relation between Lyb-2 molecules and BSF-1 receptors was tested in assays that measure binding of anti-Lyb-2 or BSF-1 in B cells and were found not to compete with each other. It appears that the two B-cell agonists anti-Lyb-2 and BSF-1 may exert their effects on B cells through different cell surface moieties as well as different intracellular pathways.     

Analysis of B-cell subpopulations. I. Relationships among splenic xid, Ly 1+, and Lyb 5+ B cells

The phenotypic and functional relationships among the various B-cell subsets is of importance for better understanding studies of the immune system. In this report, the realm of B cells encompassed by Lyb 5, Ly 1, and xid has been examined through the use of the alloantiserum anti-Lyb 5, the unique functional properties of Ly 1+ B cells, and the spontaneous autoantibody producing congenic xid mice, NZB.xid. By functional and phenotypic analysis, we have shown that (i) Lyb 5- B cells and B cells of xid mice are largely, but not completely, overlapping; (ii) xid spleen cells contain a population which is Lyb 5+; (iii) Ly 1+ B cells fall largely in the Lyb 5+ compartment; and (iv) the autoantibody-producing Ly 1+ B cells are predominantly Lyb 5+.     

Requirement for BSF-1 in the induction of antigen-specific B cell proliferation by a thymus-dependent antigen and carrier-reactive T cell line

We report here a role of B cell stimulatory factor 1 (BSF-1) in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes by a thymus-dependent antigen and a carrier-reactive T cell line. By using an ovalbumin-reactive T cell line (designated Hen-1), which does not produce BSF-1 following activation, it was possible to demonstrate that the antigen-specific proliferative response of trinitrophenyl (TNP)-binding B cells to TNP-ovalbumin required exogenous BSF-1 in addition to direct interaction with irradiated Hen-1 T cells. The activation obtained under these conditions was highly efficient, being sensitive to antigen doses as low as 0.001 microgram/ml. The addition of saturating amounts of BSF-1 did not alter the antigen-specificity or the requirements for hapten-carrier linkage or major histocompatibility complex-restricted T-B interaction in this system. The involvement of BSF-1 was confirmed by the ability of 11B11 anti-BSF-1 antibody to specifically suppress the response of TNP-binding B cells to TNP-ovalbumin, BSF-1, and irradiated Hen-1 T cells. Finally, this response was augmented by addition of the monokine interleukin 1. These data indicate that the proliferative response of small B cells to the thymus-dependent antigen and carrier-reactive T cell line used in our experiments can be regulated by the same factors that govern B cell proliferation induced by thymus-independent type 2 antigens or anti-IgM antibodies.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

A role for Lyb-2 in B cell activation mediated by a B cell stimulatory factor

The Lyb-2 system of the mouse is involved in regulation of a proliferative step in the differentiation of B cells responding to T-dependent antigen. The present study concerns the role of Lyb-2 in an early phase of B cell activation with respect to B cell receptor functions for activation factors. It is shown that interaction of monoclonal anti (alpha)-Lyb-2 antibody with Lyb-2 on the B cell surface induces B cell proliferation by synergistic action with B cell growth factor II-containing factor or interleukin 1. In contrast, alpha-Lyb-2 antibody could not synergize with the Con A-induced culture supernatant of T cell hybridoma FS6-14.13 (FS6) containing B cell stimulatory factor-1 (BSF-1; formerly called BCGF I), and the effect of combining the two was only additive on B cell proliferation. Absorption studies showed that BSF-1 in FS6 could be absorbed by unstimulated B cells, about 95% of which were at Go phase of the cell cycle, but not by thymocytes, and more importantly that alpha-Lyb-2 antibody blocked the absorption in an Lyb-2-specific manner, possibly by competing with BSF-1. It is thus likely that alpha-Lyb-2 antibody may interact with a BSF-1 receptor on B cells or a molecule closely associated with it. Interestingly, alpha-Lyb-2 antibody mimicked the action of BSF-1 in a costimulator assay with affinity-purified goat alpha-mouse IgM antibody, but could not replace all the activities ascribed to BSF-1. Possible mechanisms involved are discussed.     

Regulation of murine T cell proliferation by B cell stimulatory factor-1

The proliferation of mitogen-activated primary T cells, antigen-activated memory T cells from mixed leukocyte culture, and antigen-dependent alloreactive T cell clones in response to purified murine recombinant B cell stimulatory factor-1 (also known as interleukin 4) was examined. We found that B cell stimulatory factor-1 (BSF-1) stimulated optimal proliferation of these T cells only after their recent activation by antigen or mitogen. Analysis of cell surface BSF-1 receptor expression indicated that although T cell activation is accompanied by a small increase in BSF-1 receptor expression, the cells also express BSF-1 receptors prior to activation at a time when they do not proliferate in response to BSF-1. BSF-1 was as effective a stimulus as interleukin 2 for inducing proliferation of the Lyt-2+ subpopulation of concanavalin A-activated murine spleen cells and an alloreactive cytolytic T cell clone. However, the L3T4+ subpopulation of concanavalin A-activated spleen and an alloreactive helper T cell clone were less responsive to BSF-1 than to interleukin 2. Taken together, the data indicate an important role for BSF-1 in the regulation of normal T cell proliferation.     

Differential regulation by Ly-5 and Lyb-2 of IgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4)

We have previously shown that mAb Ly-5 which on B cells recognizes a 220,000-Da (B220) molecule, inhibits LPS-induced IgG responses without affecting IgM or proliferative responses, whereas mAb Lyb-2 which modulates B cell activation processes induced by B cell stimulatory factor-1 (BSF-1) or IL-4, has no effect on LPS-induced B cell responses. In this report we further examined the cellular mechanisms of Ly-5 antibody action and the effect of Lyb-2 antibody in IgG responses induced by LPS and BSF-1. The results presented demonstrated that the inhibitory effect of Ly-5 antibody seems to be restricted to the IgG class and is observed in all IgG subclasses induced by LPS. Limiting dilution analysis showed that the Ly-5 antibody reduces primarily the precursor frequency of IgG-secreting cells and that the effect on the clone size is partial. Lyb-2 antibody, on the other hand, greatly inhibited IgG1 induction initiated by LPS and BSF-1 by the action on processes triggered by BSF-1, although it could not reverse the reduced IgG2b or IgG3 responses. Limiting dilution analysis revealed that Lyb-2 antibody reduces the precursor frequency but not the clone size of BSF-1-induced IgG1-producing cells, supporting our previous proposition that Lyb-2 plays a critical role in the B cell differentiation mediated by BSF-1. Taken together, these results indicate that both Ly-5 and Lyb-2 are important molecules in IgG subclass regulation, each acting on a distinct activation step.     

Differential regulation of surface Ig- and Lyb2-mediated B cell activation by cyclic AMP. I. Evidence for alternative regulation of signaling through two different receptors linked to phosphatidylinositol hydrolysis in murine B cells.

The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5-triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation.     

Enhanced in vitro human immunodeficiency virus type 1 replication in B cells expressing surface antibody to the TM Env protein.

The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 tightly binds CD4 as its principal cellular receptor, explaining the tropism of HIV-1 for CD4+ cells. Nevertheless, reports documenting HIV infection or HIV binding in cells lacking CD4 surface expression have raised the possibility that cellular receptors in addition to CD4 may interact with HIV envelope. Moreover, the lymphocyte adhesion molecule LFA-1 appears to play an important role in augmenting HIV-1 viral spread and cytopathicity in vitro, although the mechanism of this function is still not completely defined. In the course of characterizing a human anti-HIV gp41 monoclonal antibody, we transfected a CD4-negative, LFA-1-negative B-cell line to express an anti-gp41 immunoglobulin receptor (surface immunoglobulin [sIg]/gp41). Despite acquiring the ability to bind HIV envelope, such transfected B cells could not be infected by HIV-1. These cells were not intrinsically defective for supporting HIV-1 infection, because when directed to produce surface CD4 by using retroviral constructs, they acquired the ability to replicate HIV-1. Interestingly, transfected cells expressing both surface CD4 and sIg/gp41 receptors replicated HIV much better than cells expressing only CD4. The enhancement resided specifically in sIg/gp41, because isotype-specific, anti-IgG1 antibodies directed against sIg/gp41 blocked the enhancement. These data directly establish the ability of a cell surface anti-gp41 receptor to enhance HIV-1 replication.     

Differential effects of the stimulation of complement receptors CR1 (CD35) and CR2 (CD21) on cell proliferation and intracellular Ca2+ mobilization of chronic lymphocytic leukemia B cells.

The regulatory role of CR1 and CR2 on B cell activation and proliferation has been investigated by using B cells from patients with chronic lymphocytic leukemia. The chronic lymphocytic leukemia B cells are clonal expansions of B lymphocytes frozen at specific stages of activation. They displayed two patterns of response upon surface Ig (sIg) cross-linking in terms of in vitro proliferation and intracellular free Ca2+ mobilization: cells from patient F (first pattern) proliferated in the presence of mitogenic anti-mu antibodies, whereas cells from patient A (second pattern) did not respond to sIg cross-linking but proliferated in the presence of low m.w. B cell growth factor and IL-2. Coculture of A or F cells with C3b-bearing SRBC led to a two- to four-fold increase in thymidine incorporation in cultures containing low m.w. B cell growth factor but not in cultures containing rIL-2. This enhanced proliferation was inhibited by F(ab')2 polyclonal rabbit antihuman CR1 antibodies. Only cells which proliferated in the presence of anti-mu (cells F) responded to cross-linking of sIg with a rise in intracellular Ca2+. No increase in calcium mobilization was observed after co-cross-linking of CR1 and sIg on A and F cells with mAb or polyclonal anti-CR1 antibodies. Co-cross-linking of CR2 with sIg only led to an enhanced intracellular Ca2+ rise in F cells but not in A cells. The lack of CR2-mediated synergy in Ca2+ rise in A cells indicates that the synergy occurs only if there is a proper coupling of sIg to phospholipase C. CR1-induced proliferation of B cells does not involve the signaling pathways of sIg. These results provide additional evidences for the role of C3 fragments in modulation of human B cell activation.     

Regulation of B-lymphocyte activation, proliferation, and immunoglobulin secretion

Lymphocyte growth and differentiation are controlled by signals resulting from the interaction of antigen and cellular products, such as lymphokines, with specific cell membrane receptors. Resting B lymphocytes can be activated by low concentrations (1-5 micrograms/ml) of antibodies to membrane IgM, which is the B-lymphocyte receptor for antigen. The binding of anti-IgM to B cells causes a rapid increase in intracellular free calcium concentration ([Ca2+]i), in inositol phosphate concentration, and in protein kinase activity. Moreover, the effects of anti-IgM on B cells are mimicked by the combined use of calcium ionophores and phorbol esters. Since phorbol esters activate protein kinase c, this suggests that the increase in [Ca2+]i and in phosphatidylinositol metabolism stimulated by anti-IgM are critical events in B-cell activation. The entry into S phase of B cells stimulated with anti-IgM depends on the action of a T-cell-derived factor designated B-cell stimulatory factor (BSF)-1. This is a 20,000-Da protein which is a powerful inducer of class II major histocompatibility complex molecules. Although an important cofactor for B-cell proliferative responses to anti-IgM, its major locus of action is on resting B cells. B cells stimulated with anti-IgM and BSF-1 do not synthesize secretory IgM. However, if two additional T-cell-derived factors, B151-TRF and interleukin-2, are added to cultures, a substantial proportion of stimulated B cells produce secretory IgM. BSF-1 has also been shown to participate in the "switch" in Ig class expression. Resting B cells cultured with lipopolysaccharide will switch to IgG1 secretion in the presence of purified BSF-1.     

Antigen-induced Ca2+ signaling and desensitization in B cells

Cross-linking of B cell surface Ig (sIg) by anti-Ig results in transmembrane signaling. However, the capacity of a thymus-dependent (TD) Ag to mediate B cell signal transduction has been less well documented. Therefore, we examined Ag-induced intracellular free calcium concentration [( Ca2+]) in B cells by using TD Ag that would be expected to either cross-link or not cross-link sIgM and/or induce the coupling of sIgM to FcR. Stimulation of mouse TA3 hybridoma B cell transfectants that express the SP6 anti-TNP specific sIgM with either TNP-OVA or anti-IgM antibodies resulted in a maximal fourfold increase in [Ca2+]i. The net increase in [Ca2+]i in response to TNP-OVA was dependent upon both the Ag dose and the TNP:OVA molar ratio. Because occupancy of several cell-surface receptor types leads to a loss of response to subsequent stimulation by ligand (homologous desensitization), we examined the ability of Ag to induce homologous desensitization of sIgM in these B cells. TNP1-OVA at all concentrations tested (up to 500 micrograms/ml) did not lead to any change in [Ca2+]i or desensitization. Cross-linking of TNP1-OVA (10 micrograms/ml) with F(ab')2 of anti-OVA antibody induced both a rise in [Ca2+]i and homologous desensitization of sIg, suggesting that cross-linking of sIgM by Ag is sufficient to induce both these processes. TNP6-OVA at a concentration of 10 micrograms/ml induced changes in [Ca2+]i and partially desensitized TNP-specific B cells to stimulation by anti-IgM. Interestingly, a high dose (180 micrograms/ml) of TNP6-OVA stimulated minimal changes in [Ca2+]i yet did not lead to desensitization. However, cross-linking of TNP6-OVA at this high dose with F(ab')2 of rabbit anti-OVA elevated [Ca2+]i and elicited partial desensitization. Complete desensitization of sIgM by Ag was achieved when intact (Fc-containing) anti-OVA antibody was used, suggesting that the FcR can play a role in desensitization. Ag- and antibody-mediated desensitization was not caused by steric hindrance of sIg. Thus, we have observed two forms of Ag-induced desensitization of sIgM, both of which involve sIg cross-linking and one of which is mediated by the physiologic coupling of sIg to FcR.     

The phosphatidylinositol response is an early event in the physiologically relevant activation of antigen-specific B lymphocytes

Receptor ligand-induced turnover of plasma membrane phosphatidylinositol (PI) has been implicated as part of a membrane receptor signal transduction system in a number of mammalian cell types. Signaling through B-lymphocyte surface immunoglobulin (sIg2) has been explored polyclonally through the use of anti-Ig reagents, with the assumption that anti-Ig mimics the process of antigen binding to the antigen-specific cell. We have utilized a method of obtaining trinitrophenyl (TNP)-specific populations of B lymphocytes in order to determine if antigen binding to these antigen-specific cells initiates PI turnover. This method has allowed us to explore the membrane phospholipid events following antigen binding directly, rather than with inference from the anti-Ig system. We have found that both thymus-dependent and thymus-independent antigens (with the exception of TNP-lipopolysaccharide) produced an increase in PI turnover comparable to that generated by anti-IgM stimulation. The lack of increased PI turnover following TNP-LPS stimulation may be attributable to the action of LPS on the biochemical events of the PI cycle. In a B-cell subpopulation depleted of antigen-specific cells, only anti-IgM produced a PI effect. These results represent the first demonstration of PI turnover as an early activation event in a physiologically relevant lymphocyte system.     

Establishment of T-cell hybridoma constitutively producing macrophage-dependent T-cell replacing factor

A T-cell hybridoma was established by the fusion of concanavalin A-stimulated splenic T cells with BW 5147. The hybridoma cells secrete a factor constitutively to support antibody formation of spleen cells depleted of T cells against TNP-Ficoll but not against horse red blood cells. The activity was indicated not to be due to interleukin 2, B-cell growth factor I, B-cell growth factor II, or interferon. The factor-mediated antibody response to TNP-Ficoll required the presence of adherent cells. The adherent cell function could be replaced by the macrophage culture supernatant containing interleukin 1. B cells responding to TNP-Ficoll in the culture with hybridoma factor were indicated to be Lyb 5+ and to bear receptors for third component of complement.     

Human recombinant IL-6/B cell stimulatory factor 2 augments murine antigen-specific antibody responses in vitro and in vivo

The effects of human rIL-6/B cell stimulatory factor 2 (hrIL-6/BSF-2) from Escherichia coli on murine Ag, SRBC-specific antibody responses were examined in vitro and in vivo. HrBSF-2 was effective in augmenting the primary and the anamnestic plaque-forming cells response to SRBC in vitro. The augmentation of the primary response was apparent when B cell-enriched spleen cells (B cells) were cultured with BSF-2 in the presence of IL-2. On the other hand, hrBSF-2 alone strongly enhanced the anamnestic response in a dose-dependent manner when spleen cells from SRBC-immunized mice were used. These effects of BSF-2 were abolished completely by anti-BSF-2 antibody, but not by normal rabbit Ig. Cell depletion experiments indicated that L3T4 (CD4)+ T cells, but not Lyt-2(CD8)+ T cells, and adherent cells (macrophages) have an important role in this BSF-2-induced augmentation of the response. In addition, kinetic studies showed that hrBSF-2 acts on B cells in the anamnestic response even when added relatively late in the culture. Finally, it was determined whether BSF-2 also could be active in modulating antibody responses in vivo. BSF-2 was shown to enhance the primary and secondary antibody responses in mice. The most apparent effect of BSF-2 was observed in the secondary response.     

In vitro translation of B-cell stimulatory factor-1 in Xenopus laevis oocytes

B-cell stimulatory factor-1 (BSF-1) can be translated in vitro in Xenopus laevis oocytes. This activity is blocked by an antibody to BSF-1. The RNA species coding for BSF-1 activity sediments of approximately 8-9S and is separable from RNA coding for interleukin-2 activity which sediments at approximately 11.5S. Finally, the fact that BSF-1 can be translated in vitro confirms that functions attributed to BSF-1 do not depend on contamination with other biologically active molecules such as phorbol myristate acetate.