Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1

The role of tumor necrosis factor-alpha (TNF-alpha) in human B cell responses was examined and compared with that of interleukin (IL) 1 by assessing the ability of each cytokine to support proliferation and differentiation. Recombinant TNF-alpha (rTNF-alpha) and recombinant IL-1 (rIL-1) each enhanced the generation of immunoglobulin-secreting cells (ISC) in cultures of pokeweed mitogen-stimulated B cells incubated with T cells. To examine the direct effect of rTNF-alpha and rIL-1 on the responding B cell, highly purified peripheral blood B cells were stimulated with Cowan I Staphylococcus aureus (SA). In the absence of T cell factors, proliferation was minimal and there was no generation of ISC. Recombinant IL-2 (rIL-2) supported both responses. Although rTNF-alpha alone did not support SA-stimulated generation of ISC, it did increase SA-stimulated B cell DNA synthesis by two- to eightfold. In addition, rTNF-alpha augmented B cell proliferation in rIL-2 supported SA-stimulated cultures. Moreover, rTNF-alpha enhanced the generation of ISC stimulated by rIL-2 alone or rIL-2 and SA. rIL-1 also augmented DNA synthesis and generation of ISC by B cells stimulated with SA and rIL-2. However, rTNF-alpha enhanced proliferation and ISC generation in SA + rIL-2-stimulated cultures even when they were supplemented with saturating concentrations of rIL-1. Utilizing a two-stage culture system, it was found that the major effect of rTNF-alpha was to enhance responsiveness of SA-activated B cells to rIL-2, whereas it exerted only a minimal effect during initial stimulation. These results indicate that TNF-alpha as well as IL-1 augment B cell responsiveness. Moreover, the ability of rTNF-alpha to enhance B cell responsiveness was not an indirect effect resulting from the induction of Il-1 production by contaminating monocytes, but rather resulted from the delivery of a signal by rTNF-alpha directly to the responding B cell that promoted both proliferation and differentiation after initial activation. The data therefore indicate that human B cell responsiveness can be independently regulated by the action of two separate monocyte-derived cytokines.     

IL-2 dependence of the promotion of human B cell differentiation by IL-6 (BSF-2)

The effect of rIL-6 on the growth and differentiation of highly purified human peripheral blood B cells was examined. IL-6 alone induced minimal incorporation of [3H]thymidine by unstimulated or Staphylococcus aureus (SA)-stimulated B cells and did not augment proliferation induced by SA and IL-2. Similarly, IL-6 alone did not support the generation of Ig-secreting cells (ISC) or induce the secretion of Ig by unstimulated or SA-stimulated B cells. However, IL-6 did augment the generation of ISC and the secretion of all isotypes of Ig induced by SA and IL-2. Maximal enhancement of B cell responsiveness by IL-6 required its presence from the initiation of culture. Delaying the addition of IL-6 to B cells stimulated with SA and IL-2 beyond 24 h diminished its effect on ISC generation. However, increased Ig production but not ISC generation was observed when IL-6 was added to B cells that had been preactivated for 48 h with SA and IL-2. This effect was most marked when the activated B cells were also stimulated with IL-2. IL-6 in combination with other cytokines such as IL-1 and IL-4 did not induce the secretion of Ig or generation of ISC in the absence of IL-2. Moreover, antibody to IL-6 did not inhibit the effect of IL-2 on the growth and differentiation of B cells stimulated with SA, but did inhibit the IL-6-induced augmentation of Ig secretion by B cells stimulated with SA and IL-2. IL-6 alone enhanced T cell dependent induction of B cell differentiation stimulated by PWM. Part of this enhancement was related to its capacity to increase the production of IL-2 in these cultures. These results indicate that IL-6 has several direct enhancing effects on the differentiation of B cells, all of which are at least in part dependent on the presence of IL-2. In addition, IL-6 can indirectly increase B cell differentiation by increasing IL-2 production by T cells.     

Inhibitory influence of IL-4 on human B cell responsiveness

The role of IL-4 in human B cell activation, proliferation, and differentiation was examined. rIL-2, but not rIL-4, was able to promote maximum proliferation and generation of Ig-secreting cells in cultures of highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA). Addition of rIL-4 to rIL-2-supported cultures of SA-stimulated peripheral blood, spleen, or lymph node B cells dramatically suppressed both proliferation and differentiation. Results from experiments in which rIL-4 was added to culture at progressively later times indicated a requirement for rIL-4 to be present during the first 2 days of a 5-day incubation to cause inhibition of responsiveness. When a two-stage culture system was utilized, rIL-4 was found to support proliferation or differentiation of B cells initially activated with SA for 2 days only minimally. However, rIL-4 did not inhibit responses of SA preactivated B cells supported by IL-2. The presence of rIL-4 during the initial 48-h activation of B cells with SA and rIL-2 resulted in a profound inhibition of the ability of the activated B cells to respond subsequently to rIL-2 or lymphokine-rich T cell supernatants. A similar 48-h incubation with rIL-4 alone without SA had no effect on subsequent B cell responsiveness. The presence of rIFN-gamma during B cell activation decreased the inhibitory effect of IL-4. Other cytokines including IFN-alpha, IL-1, and commercially available low m.w. B cell growth factor also diminished the inhibitory effect of IL-4. These results indicate that IL-4 inhibits the capacity of human B cells to be activated maximally by SA and rIL-2 and therefore suggest a new immunomodulatory role for this cytokine.     

Immunomodulatory role of IL-4 on the secretion of Ig by human B cells

The effect of IL-4 on the production of Ig by human B cells was examined. Highly purified B cells were stimulated with Staphylococcus aureus (SA) and IL-4 alone or in combination with various other cytokines and the supernatants assayed for Ig by isotype-specific ELISA. IL-4 (10 to 100 U/ml) did not support Ig secretion by SA-stimulated blood, spleen, or lymph node B cells, whereas IL-2 supported the production of all isotypes including IgE. Moreover, IL-4 suppressed the production of all isotypes of Ig by B cells stimulated with SA and IL-2 including IgG1, IgG2, and IgE. IL-4-mediated suppression was partially reversed by IFN-gamma or -alpha and low m.w. B cell growth factor. TNF-alpha and IL-6 did not reverse the IL-4-induced suppression of Ig production. The inhibitory action of IL-4 on Ig production appeared to depend on the polyclonal activator used to stimulate the B cells. Thus, Ig secretion by B cells activated by LPS and supported by IL-2 was not inhibited by IL-4. Whereas IL-4 alone supported minimal Ig production by LPS-activated B cells, it augmented production of all Ig isotypes in cultures stimulated with LPS and supported by IL-2. IFN-gamma further enhanced production of Ig in these cultures. When the effect of IL-4 on the responsiveness of B cells preactivated with SA and IL-2 was examined, it was found not to inhibit but rather to promote Ig production modestly. A direct effect of IL-4 on the terminal differentiation of B cells was demonstrated using B lymphoblastoid cell lines. IL-4 was able to enhance the Ig secreted by an IgA-secreting hybridoma, 219 and by SKW6-CL-4, an IL-6-responsive IgM-secreting EBV transformed B cell line. These results indicate that IL-4 exerts a number of immunoregulatory actions on human B cell differentiation. It interferes with the activation of B cells by SA and IL-2, but promotes the differentiation of preactivated B cells, B cell lines, and B cells activated by LPS without apparent isotype specificity.     

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.     

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.     

Effect of recombinant IL 2 and gamma-IFN on proliferation and differentiation of human B cells

The effects of IL 2 and gamma-IFN on the activation of human B cells was studied with recombinant IL 2 and gamma-IFN. BCDF-responsive B lymphoblastoid cell lines and highly purified human B cells were employed as target B cells. IL 2 or gamma-IFN did not induce any IgG or IgM secretion in the B cell lines CESS and SKW6-CL4, in which IgG and IgM were inducible with conventional T cell factor(s). IL 2 alone did not induce the optimum production of Ig, but did induce proliferation in the SAC-stimulated B cell population. No Leu-1-, Leu-4-, or Leu-7-positive cells were detected in B cell populations that had been stimulated with SAC for 3 days. FACS analysis showed that a portion of the SAC-stimulated B cells (30%) were in the G2 or M stages by IL 2 stimulation. The addition of gamma-IFN together with IL 2 induced IgM and IgG secretion in SAC-stimulated B cells that was comparable with that induced by a conventional T cell factor(s). IL 2 induced proliferation not only in SAC-stimulated B cells but also in an anti-mu-stimulated B cell population. Stimulation of T cell populations with anti-mu and IL 2 did not induce significant proliferation, suggesting the direct effect of IL 2 on B cells. Double staining of anti-mu-stimulated B cells with anti-Ig and anti-Tac antibodies demonstrated that anti-mu stimulation induced an increased expression of Tac antigen on surface Ig-positive B cells. All of these results strongly supported the notion that IL 2 was one of the growth factors for B cells, and gamma-IFN was one of the differentiation factors for B cells.     

Identification and characterization of a B cell growth inhibitory factor (BIF) on BCGF-dependent B cell proliferation

The culture supernatants of Con A-activated human peripheral blood mononuclear cells (PBM) contained at least two regulatory factors upon B cell proliferation. One was B cell growth factor (BCGF), which activated antigen-stimulated B cells to proliferation and clonal expansion, and the other was its inhibitory factor, arbitrarily named B cell growth inhibitory factor (BIF). This BIF inhibited the effect of BCGF on anti-mu-stimulated B cells or the monoclonal mature B cell line (CLL-T.H.) obtained from the peripheral blood lymphocytes of B cell-type chronic lymphocytic leukemia patients, which were activated only with BCGF and without adding other proliferating stimuli (e.g., anti-mu). BIF activity was detected in the 24 hr culture supernatants of Con A-activated human PBM in FCS containing medium and also in serum-free RPMI 1640 medium. This substance with BIF activity could not be derived from FCS. Con A-induced BIF (m.w. of 80,000 and an isoelectric point of pH 5.4) was analyzed by Sephadex G-200 gel filtration and chromatofocusing. BIF was stable at pH 2.0 and at 56 degrees C for 30 min. Partially purified BIF had no effect on cell viability and almost no interferon activity (less than 1 IU/ml). BIF with high titer had a slight but significant inhibition on TCGF-dependent T cell growth and on PHA or Con A responses, but the extent of these inhibitions was far less than that of BCGF-dependent B cell growth. Absorption of BIF with Con A blasts made its inhibition on T cell growth even less. On the other hand, BIF activity could not be absorbed with Con A blasts but was almost absorbed with large numbers of CLL-T.H. cells. BIF had almost no inhibitory effect on the proliferation of a mouse fibroblast cell line (NIH 3T3), a mouse myeloma cell line (NS-1), human lymphoid cell lines (MOLT-4, HSB-2, and Daudi), or a human myeloid cell line (K-562). BIF-producing cells were estimated to be T cells and were identified as T8+ T cells. On the other hand, Con A-induced BCGF was demonstrated to be produced predominantly by T4+ T cells. These results show that human B cell proliferation is regulated by interaction between T4+ and T8+ cells via soluble factors, namely BCGF and BIF, respectively.     

Selective suppressive effects of glucocorticoids on the early events in the human B cell activation process

The effect of hydrocortisone on the induction of human B cell activation and proliferation has been described. Hydrocortisone prevents the anti-mu-induced cell enlargement of small tonsillar B cells, blocks expression of the activation markers 4F2 and 5E9 induced by anti-mu, inhibits RNA synthesis of small B cells stimulated by anti-mu with or without BCGF, and suppresses B cell proliferation in response to anti-mu and BCGF or to Staphylococcus aureus Cowan I. In contrast, hydrocortisone does not affect the proliferative response of in vitro or in vivo preactivated B cells. Therefore, hydrocortisone has a selective inhibitory effect on early events in the human B cell cycle that subsequently leads to inhibition of total RNA and DNA synthesis. Possible mechanisms of this action are discussed. These studies further define the nature of glucocorticoid-induced modulation of human B cell activation and proliferation.     

The modulation of membrane Ia on human B lymphocytes

Using flow cytometry techniques, changes in surface Ia (DR and DS) expression on human B lymphocytes were correlated with changes in the cell cycle following stimulation with anti-mu. The effect of interleukin (IL)-1, IL-2, B-cell growth factor (BCGF), and interferons on Ia expression on resting B cells was also examined. A population of resting B lymphocytes was cultured in vitro with 100 micrograms/ml of anti-mu and immunofluorescently stained for DR and DS at various times following stimulation. Detectable increases in DR and DS expression were found within 8 hr, and the major increases (twofold and fourfold) in DR and DS expression occurred over the next 48 hr. Using cell cycle inhibitors and propidium iodide staining, it was demonstrated that the enhanced DR and DS expression following anti-mu stimulation began during G0 to G1 transition and increased as the cells progressed through G1 phase. During S and G2/M phases, there were minimal further increases in surface Ia. Although prolonged exposure of B cells to anti-mu was required for cellular activation, cell size enlargement, and progression into S phase, a brief exposure to anti-mu, insufficient for cellular activation, markedly enhanced Ia expression. Thus anti-mu-stimulated resting human B lymphocytes rapidly increase their surface Ia expression. This increase occurs predominantly prior to entrance into S phase and can occur in the absence of significant cellular activation. Interferons have been reported to modulate surface Ia expression on a human lymphoid cell line and on monocytes and supernatants with BCGF activity to enhance surface Ia expression on murine B cells; however, neither alpha-interferon, gamma-interferon, IL-1, IL-2, nor BCGF modified surface DR expression on normal resting human B cells.     

Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells

A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.     

B lymphocyte differentiation induced by lipopolysaccharide. III. Suppression of B cell maturation by anti-mouse immunoglobulin antibodies.

Lipopolysaccharide-(LPS) induced differentiation of mouse B lymphocytes to cells synthesizing large amounts of cytoplasmic IgM and IgG2 could be suppressed by antibodies to mu-chains. Maximal inhibition of LPS-induced differentiation was associated with increased cellular proliferation as measured by incorporation of 3H-thymidine, whereas treatment with anti-mu alone over a wide dosage range did not stimulate cellular proliferation. Spleen cells from newborn mice were suppressed by concentrations of anti-mu several hundred-fold lower than required for adult spleen cells; the adult pattern of susceptibility to suppression was acquired by 1 week of age. No significant differences in susceptibility to anti-mu were found in comparisons of adult spleen, lymph node, bone marrow, and Peyer's patch lymphocytes.     

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.     

Inhibition of human B cell responsiveness by prostaglandin E2

The capacity of prostaglandin E2 (PCE2) to modulate human peripheral blood B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) stimulated by Cowan 1 strain Staphylococcus aureus and mitogen-stimulated T cell supernatant was examined. PGE2 significantly inhibited both responses, whereas PGF2 alpha had no inhibitory effect. Responses of highly purified B cells obtained from spleen, lymph node, and tonsil were also inhibited. In addition PGE2 suppressed B cell responses supported by recombinant interleukin 2 rather than T cell supernatant. PGE2-mediated inhibition was mimicked by forskolin, a direct activator of adenylate cyclase. Kinetic studies indicated that PGE2 inhibited B cell responses by a progressively greater increment as cultures were prolonged. Evaluation by flow cytometry after staining with acridine orange or mithramycin indicated that PGE2 had no effect on initial B cell entry into the G1 phase of the cell cycle, passage through G1, and entry into S, G2, and M. Rather, PGE2 inhibited responses of postdivisional daughter cells. PGE2 inhibited responses in cultures stimulated by the calcium ionophore ionomycin and T cell supernatant but had minimal effects in cultures stimulated by the combination of ionomycin and phorbol myristate acetate. Moreover, phorbol myristate acetate reversed PGE2-mediated inhibition of proliferation stimulated by S. aureus or S. aureus + T cell supernatant. These results indicate that PGE2 suppresses the continued growth and differentiation of human B cells, although it has no effect on initial B cell activation and suggest that PGE2 may play a role in regulating human B cell responses in vivo.     

Functional properties of two human B cell growth factor species separated by lectin affinity column

The supernatants from PHA-activated normal human T cells (conditioned medium) were fractionated by differential adsorption on Con A-Sepharose columns. The effluent contained both IL 2 activity (tested on the CTL-L2 cell line) and BCGF activity (tested on anti-mu-activated normal B-enriched cells). Absorption of such effluent on PHA-activated T cell blasts removed the IL 2 activity without affecting the BCGF activity. The eluate also displayed BCGF activity (tested on anti-mu-activated B-enriched cells) without detectable IL 2 activity. The two BCGF species isolated by this criteria synergize with semipurified IL 1 to support the anti-mu-induced proliferation of highly monocyte-depleted B cells. Both BCGF species required the presence of anti-mu Ab to support the proliferation of small B cells (at the G0 stage). However, in contrast to crude conditioned medium and to the BCGF present in the effluent, the BCGF present in the eluate was unable to induce the proliferation of unfractionated or large (at the G1 stage) B cells in the absence of anti-mu Ab. These results support the existence of two functionally different human BCGF species, differing at least in their relative sugar content and distinct from IL 2.     

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.     

Human peripheral blood B lymphocyte subpopulations: functional and phenotypic analysis of surface IgD positive and negative subsets

Highly purified human peripheral blood B cells stimulated with Cowan I Staphylococcus aureus (SA) and mitogen-activated T cell supernatants (T supt) generated large numbers of immunoglobulin (Ig)-secreting cells (ISC), whereas fewer ISC developed in cultures containing T supt in the absence of SA. To determine whether surface Ig isotype expression defined responsive B cell subsets, IgD+ and IgD- B cells were prepared with the fluorescence-activated cell sorter. Whereas both the IgD+ and IgD- B cells responded to SA + T supt, only the IgD- subset generated substantial numbers of ISC in response to T supt alone. Analysis of secreted Ig revealed that IgG and IgA were the predominant isotypes secreted by IgD- B cells in response to T supt or SA + T supt. By contrast, the IgD+ cells secreted predominantly IgM in response to SA + T supt but not to T supt alone. When responsiveness to pokeweed mitogen (PWM) was examined in the presence of supplemental T cells, the IgD- subset was found to be greatly enriched for responsive cells, and again, IgG and IgA were the predominant isotypes secreted, although these cells were also capable of secreting some IgM. The magnitude of the response induced by PWM from IgD- B cells was usually greater than that induced by SA + T supt. Although IgD+ B cells responded poorly to PWM, the differentiation of a small number of IgM-secreting cells was routinely stimulated by this polyclonal activator in the presence of T cells. The magnitude of the PWM response by IgD+ B cells was always greatly diminished compared with that stimulated by SA + T supt. Cell cycle analysis after acridine orange staining, cell volume measurement, and staining for expression of activation antigens (transferrin receptor and 4F2) indicated that the IgD- B cells were largely resting, but did contain a population of activated cells. Removal of activated 4F2+ cells from the IgD- subset diminished but did not abolish their capacity to generate ISC in response to SA + T supt or PWM in the presence of T cells. These results suggest that the IgD- population contains both an activated 4F2+ and a resting 4F2- subset. The data emphasize that multiple subpopulations of peripheral blood B cells contain precursors of ISC. Moreover, the responsiveness of the subsets to various stimuli and the Ig isotype subsequently secreted appear to be intrinsic features of each subset.     

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.     

The roles of T cell factors in activation, cell cycle progression, and differentiation of human B cells

The relationship of the T cell influences involved in human B cell activation and differentiation into immunoglobulin-secreting cells (ISC) was investigated. T cell supernatants (T supt) generated by stimulating T cells with phytohemagglutinin and phorbol myristate acetate contained activities capable of augmenting DNA synthesis and the growth of mitogen-stimulated B cells and supporting the differentiation of ISC. To examine the role of T supt in B cell activation and the progression through the cell cycle, T cell- and monocyte-depleted B cells were stimulated with formalinized Cowan I strain Staphylococcus aureus (SA), and the percentages of cells in G1, S, and G2 + M were determined by acridine orange staining and analysis. In all experiments, a similar percentage of cells entered G1 during the first 24 to 36 hr of culture when stimulated with SA or SA + T supt. Similar results were seen when B cell activation was analyzed by acquisition of a number of other markers of cell activation. Analysis of cell cycle progression with mithramycin staining of cellular DNA in the presence or absence of vinblastine to arrest mitosis indicated that SA-activated B cells were able to complete S and divide in the absence of T supt. Although an effect of T supt on the progression of B cells through the S phase was evident during the first cell cycle, the major effect only became apparent after the first round of cell division. Although T supt was not necessary for initial B cell activation, T cell influences were absolutely necessary for the differentiation of ISC. T supt did not need to be present during the initial 24 to 36 hr of incubation to permit subsequent generation of ISC. However, when T supt was present initially, an increased number of ISC were produced. Hydroxyurea elimination of cells traversing the G1-S interphase indicated that reception of the differentiation signal occurred before the S phase, but that the generation of ISC required subsequent DNA synthesis and/or cell division. Although precursors of ISC were entirely contained within the population triggered to divide by SA alone, there was no preferential expansion of such precursors as a result of SA stimulation. These results indicate that T cell signals are not absolutely necessary for initial B cell activation and progression through the first cell cycle, although T cell factors promote DNA synthesis by some activated B cells. In contrast, differentiation into ISC is completely dependent on T cell influences.(ABSTRACT TRUNCATED AT 400 WORDS)