STAT3-mediated up-regulation of BLIMP1 Is coordinated with BCL6 down-regulation to control human plasma cell differentiation

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

Identification of two distinct factors, B151-TRF1 and B151-TRF2, inducing differentiation of activated B cells and small resting B cells into antibody-producing cells

We demonstrated previously that cellfree supernatant of the B151K12 T cell hybridoma (B151-CFS) contained T cell-replacing factor (here in after referred to as B151-TRF1) capable of inducing growth and differentiation of antigen-activated B cells into antigen-specific plaque-forming cells (PFC). In the present study, we have identified in B151-CFS another unique lymphokine activity (referred to as B151-TRF2), which induces polyclonal differentiation of unstimulated B cells into IgM-secreting cells without concomitant stimulation of antigen, mitogen, or anti-Ig antibody. The B151-TRF2 activity induced polyclonal IgM PFC responses via the action on surface Ig-positive small resting B cells from normal unprimed mice. This activation was effective across an H-2 barrier, and apparently independent of the presence of T cells and accessory cells. Interestingly, the B151-TRF2 activity notably stimulated B cells of neonatal and mutant DBA/2Ha mice, which are nonresponders to B151-TRF1, whereas it failed to activate the xid B cells from CBA/N mice. To substantiate that B151-TRF1 and B151-TRF2 activities are mediated by mutually distinguishable molecules, an absorption experiment of B151-CFS was performed by utilizing DBA/2Ha B cells which are lacking in B151-TRF1 receptor. It was found that DBA/2Ha B cells could absorb B151-TRF2 activity but not B151-TRF1 activity. In contrast, murine chronic B cell leukemia BCL1 cells, which were shown to differentiate into IgM-secreting cells by stimulation with B151-CFS, selectively removed B151-TRF1 activity but not B151-TRF2 activity. Furthermore, biochemical analysis revealed that the B151-TRF2 was a heat (56 degrees C for 30 min)-sensitive protein with an apparent m.w. of 30,000 by gel filtration, whereas B151-TRF1 was a heat-resistant glycoprotein with m.w. of 50,000. In addition, it was shown that prostaglandin E2 selectively inhibited B151-TRF2-mediated B cell responses. These results demonstrate clearly that B151-TRF1 and B151-TRF2 are distinct B cell differentiation factors involved in the different activation pathways of distinct B cell subpopulations. The immunologic implication of B151-TRF2 activity in B cell differentiation is discussed in comparison with other lymphokines so far reported to activate small resting B cells.     

Both B151-T cell replacing factor 1 and IL-5 regulate Ig secretion and IL-2 receptor expression on a cloned B lymphoma line

In the present study, we have demonstrated that both B151-T cell-replacing factor 1 and rIL-5 are responsible for the activity to partially induce CL-3 cells into IgM-synthesizing cells and also to synergize with IL-2 to augment IL-2R expression on and IgM synthesis in CL-3 cells. These actions of rIL-5 on a homogeneous cloned line (BCL1-CL-3 cells) allow us to identify and characterize the two alternated B cell developmental pathways. One is an IL-2-independent, IL-5-driven differentiation pathway without preceding up-regulated IL-2R expression, and the other is an IL-5 plus IL-2-dependent augmented differentiation pathway with preceding up-regulated IL-2R expression. We have also demonstrated the functional difference of two distinct B cell growth-promoting factors, B cell-stimulating factor 1 (rIL-4) and rIL-5. CL-3 cells are equally stimulated to grow by rIL-4 and rIL-5, whereas only rIL-5 can render CL-3 cells responsive to rIL-2, indicating that these two lymphokines affect B cells in a strikingly different manner.     

Role of BCGFII in the differentiation to antibody secretion normal and tumor B cells

This report describes the effects of B cell growth factor (BCGFII) and other lymphokines in the differentiation of normal and tumor B cells. We compared BCL1 tumor B cells, normal B cells giving rise to a polyclonal response without the intentional addition of antigen, and an antigen-driven, SRBC-specific response. BCL1 tumor B cells gave maximum PFC responses when partially purified BCGFII was added or when suboptimal doses of BCGFII were mixed with one of several putative terminal differentiation factors we call B cell differentiation factors BCDF. IFN-gamma was not active as any of these factors. Maximum polyclonal responses of B cells were seen when either IL 2 or BCGFII were mixed with BCDF. In contrast, SRBC-specific responses showed a strict requirement for IL 2, and BCGFII and BCDF synergized with IL 2 to give a maximum response. The involvement of BCGFII in all of these responses suggests that BCGFII acts as a growth factor for a population of B cells that has differentiated much of the way towards Ig secretion, and that many B cells become responsive to this growth factor. In addition, the fact that different lymphokine requirements were seen in the different experimental systems raises the possibility that there are multiple pathways to Ig secretion, and suggests that different subpopulations of B cells defined either by different lineages or by different stages of development within a single lineage have requirements for distinct lymphokines that regulate their growth and differentiation.     

Stimulation of immunoglobulin secretion in human B lymphocytes as a direct effect of high concentrations of IL 2

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.     

Antigen-driven T cell clones can proliferate in vivo, eradicate disseminated leukemia, and provide specific immunologic memory

The aim of the current study was to determine the ability of antigen-driven cloned helper cell independent cytotoxic T lymphocytes (HITc) to proliferate and to survive in vivo and to mediate tumor therapy. The HITc clone utilized (denoted 1.B6) was specifically cytolytic to FBL-3, a syngeneic Friend virus-induced murine leukemia. Activation in vitro (48 hr) with FBL-3 induced secretion of interleukin 2 (IL 2), expression of IL 2 receptors (IL 2R), and in vitro proliferation. These cells could be "rested" for several weeks without stimulation, which resulted in reduced expression of IL 2R; however, restimulation with antigen resulted in reinduction of IL 2R and proliferation. The ability of cloned HITc to proliferate and to survive in vivo was examined in cyclophosphamide (CY) pretreated donor mice congenic for the Thy-1 gene. Adoptively transferred cloned HITc could be found in large numbers, and were widely distributed in vivo 1 wk after transfer. In tumor therapy, 1.B6 cells when injected into a site of tumor (i.p.) and used as an adjunct to CY were effective against disseminated FBL-3. In this circumstance, cloned 1.B6 cells could be recovered from cured mice 125 days after transfer and were shown to specifically lyse tumor and proliferate in vitro in response to FBL-3. Thus as an adjunct to CY, tumor-specific cloned HITc are capable of eradicating disseminated leukemia, persisting long-term in vivo, and providing specific immunologic memory.     

Polyclonal B cell activation by a B cell differentiation factor, B151-TRF2. II. Evidence for interaction of B151-TRF2 with glycoprotein on B cell membrane via recognition of terminal N-acetyl-D-glucosamine residue(s)

We investigated the role of carbohydrates in the interaction of a B cell differentiation factor designated as B151-TRF2 derived from B151K12 T cell hybridoma with the corresponding receptor on B cells. Induction of polyclonal differentiation of unprimed B cells into IgM-secreting cells by B151-TRF2 was specifically inhibited by addition of N-acetyl-D-glucosamine (GlcNAc) but not by structurally unrelated monosaccharides such as D-galactose, D-glucose, and N-acetyl-D-galactosamine (GalNAc). Absorption of B151-TRF2 activity with spleen cells was specifically inhibited by the presence of GlcNAc. These results indicate that GlcNAc residues are involved in the interaction of B151-TRF2 with the receptor on B cells. To gain insight into mechanism by which GlcNAc inhibits B151-TRF2-mediated B cell responses, the existence of GlcNAc residues was examined on the B151-TRF2 molecule and the corresponding receptor on the B cell surface. The results revealed that B151-TRF2 molecule was not bound to various lectin-coupled agarose beads so far tested, suggesting absence of carbohydrate moieties on the B151-TRF2 molecule. By contrast, pretreatment of spleen cells with trypsin or glycosidase mixture abolished their ability to absorb B151-TRF2 activity. Moreover, B151-TRF2-absorbing ability of spleen cells disappeared by the pretreatment with beta-N-acetylglucosaminidase, which cleaves terminal GlcNAc. The fact that pnitrophenyl (PNP)-GlcNAc specifically inhibited such enzyme activity on target cells indicates that terminal GlcNAc on the B cell surface plays a crucial role in the interaction with B151-TRF2 molecule. Interestingly, it was also found that B151-TRF2 activity was trapped and eluted from GlcNAc-coupled agarose beads. Taken collectively, these results strongly suggest that B cell membrane receptors for B151-TRF2 comprise glycoproteins with a terminal GlcNAc residue(s), and that binding of B151-TRF2 with terminal GlcNAc on the receptor is important for the subsequent activation of B cells.     

Establishment of an assay system for the detection of translated materials of T-cell-replacing factor mRNA in Xenopus oocytes

We established an assay system for detecting T cell-replacing factor (TRF) activity of translated materials in Xenopus oocytes of poly (A)-positive mRNA extracted from a T cell hybrid cell line, B151K12 (B151) which constitutively produces TRF. Since it was difficult to detect TRF activity of the translated products of B151-mRNA, partly because of low TRF activities, we developed the following two systems. First, RNA was prepared from B151 cells stimulated with phorbol myristate acetate and calcium ionophore A23187 because such stimulations augmented TRF production by approximately three to five-fold. Second, interleukin 2 (IL-2, 125 U/ml) was added to the culture of BCL1 cells to detect a small amount of TRF-active materials since IL-2 synergizes with a suboptimal dose of TRF to induce IgM secretion in TRF-responding BCL1 cells (chronic B cell leukemic cells). Here we describe TRF activity of translation products of B151-mRNA in Xenopus oocytes. B151-TRF mRNA was detected in the fractions sedimented between 15 and 18S by analysis using sucrose density gradient centrifugation.     

Activity of a partially purified human BCGF on murine assays for B cell stimulatory factors. II. Synergy between two distinct BCGF II-like factors in promoting B cell differentiation

We have previously identified two BCGF II-like factors which can be distinguished by their differential reactivity in several murine BCGF II assays. Prototype sources of these two factors are a partially purified preparation derived from PHA-P-stimulated human peripheral blood T lymphocytes (designated human BCGF), and supernatant from antigen-stimulated D10.G4.1 murine T cells (designated D10 sup). Extending the characterization of these two factors, we show here that human BCGF and D10 sup both cause tritiated thymidine ([3H]TdR) incorporation and IgM secretion by T cell-depleted, in vivo-activated, large murine B cells. In contrast, only the human BCGF consistently induced proliferation and IgM secretion by T cell-depleted, small murine B cells. When simultaneously added to cultures, D10 sup and human BCGF synergized to produce optimal IgM secretion by large murine B cells and murine BCL1 B lymphoma cells. The same factors were tested in an IgM-specific plaque assay, and a similar synergistic response was observed for the large B cells, but not for the BCL1 cells. The combination of factors also produced maximal [3H]TdR incorporation by large murine B cells. In contrast, the addition of human BCGF totally abrogated D10 sup-induced BCL1 proliferation. Together, these data suggest that the synergies observed in IgM secretion result from an increased production of plaque-forming cells (PFC) in cultures of large B cells and an increase in IgM production per responding cell in BCL1 cells. Kinetic analysis of the time of action of the two BCGF II-like lymphokines in the induction of the PFC response by large B cells indicated that human BCGF was required within the first 24 hr of a 4-day culture period, while D10 sup could be added as late as the final 15 hr without significant diminution of the response. In summary, these data provide further support for the existence of two distinct B cell stimulatory factors which cause growth and differentiation of activated B cells, and indicate that these two factors synergize to produce optimal Ig secretion. For ease of discussion, the activity in the human BCGF preparation is referred to as BCGF IIA, and the activity in D10 sup is referred to as BCGF IIB.     

Lymphokine-regulated differential expression of mRNA for p75kDa-IL-2R and p55kDa-IL-2R in a cloned B lymphoma line (BLC1-CL-3 cells)

IL-5 renders BCL1-CL-3 (CL-3) cells responsive to IL-2 by increasing the number of high affinity IL-2R, whereas IL-4 prohibits such action of IL-5 to prepare CL-3 cells responsive to IL-2. Here we have found that genes for p75kDa-IL-2R and p55kDa-IL-2R are differentially regulated by IL-4 and IL-5. Nonstimulated CL-3 cells constitutively express mRNA for p75kDa-IL-2R and p55kDa-IL-2R. IL-5 stimulation principally augments the expression of p75kDa-IL-2R mRNA (4- to 8-fold), although modestly increasing the expression of p55kDa-IL-2R mRNA. Kinetic studies have revealed a maximal increase in p75kDa-IL-2R mRNA expression at 12 h and a decline thereafter, substantiating our previous kinetic study of the expression of high affinity IL-2R after the IL-5 stimulation. By contrast, IL-4 stimulation modestly increases the expression of p75kDa-IL-2R mRNA, whereas markedly reducing the expression of p55kDa-IL-2R mRNA, irrespective of whether CL-3 cells were stimulated with IL-4 alone or together with IL-5 and IL-2. Moreover, addition of IL-4 into the culture containing IL-5 and IL-2 causes striking reduction in the level of J-chain mRNA, which otherwise is markedly induced by stimulation with IL-5 and IL-2. These results clearly illustrate the differential regulation of p75kDa- and p55kDa-IL-2R-gene expression by IL-5 and IL-4, and reinforce our notion that increased expression of high affinity IL-2R induced by IL-5 is responsible for the IL-2 competent state, and decreased expression of p55kDa-IL-2R by IL-4 is responsible for IL-2 unresponsive state.     

The role of interleukin 2 in inducing Ig production in a pokeweed mitogen-stimulated mononuclear cell system

Cyclosporin A (CsA) has been found previously to block mitogen-stimulated T cell proliferation and production of discrete T cell-derived lymphokines such as interleukin 2 (IL 2) and interferon (IFN)-gamma. In addition, CsA blocks pokeweed mitogen (PWM)-driven T cell-dependent differentiation of B cells into immunoglobulin (Ig)-secreting cells. Recently, we reported that CsA (1 microgram/ml) inhibited PWM-induced T cell production of IL 2 and IFN-gamma, but supernatants retained B cell differentiation factor (BCDF)-like activity. The present study demonstrates the ability of CsA to suppress T cell functions in PWM-driven Ig production in mononuclear cells (MNC), and the capacity of exogenous T cell lymphokines to reverse CsA-induced suppression. CsA profoundly suppressed PWM-driven PFC formation (greater than 95%). However, Ig production was substantially reconstituted by the addition of IL 2 at concentrations of 10 to 50 U/ml. In contrast, no effects were observed by the addition of IFN-gamma or BCGF. The kinetics of CsA inhibition of Ig production and IL 2 secretion were found to be closely related. In addition, to obtain effective reconstitution in the CsA-treated PWM-MNC system it was necessary to add IL 2 at the initiation of culture. T cells themselves were also required for B cell differentiation in this system. However, surface Ig+ cells obtained by cell sorting after 3 days of culture could differentiate in the absence of T cells but only in response to IL 2, not in response to IFN-gamma or BCDF. Thus, in PWM-driven B cell differentiation T cells are necessary early in culture, whereas IL 2 is essential from the initial stage of B cell activation through the final stage of B cell differentiation.     

B cell response pathways regulated by IL-5 and IL-2. Secretory microH chain-mRNA and J chain mRNA expression are separately controlled events

We have examined the effect of IL-5 and/or IL-2 on the expression of the secretory form of microH chain (microsecond) and J chain mRNA in a homogeneous neoplastic B cell clone, in which proliferation, IL-2R up-regulation and entry into the IgM secretory state are separately controlled events. The IL-5 signal triggers a partial induction of CL-3 cells into the IgM secretory state, characterized by a striking increase of microsecond mRNA expression and an increase in the ratio of the secretory to membrane forms of microH chain mRNA, with a modest increase of J chain mRNA. In contrast, amplification of J chain mRNA is accomplished by the late-acting B cell differentiation stimulus, IL-2, acting on IL-5-pretreated CL-3 cells or acting simultaneously with IL-5 on CL-3 cells. Such dually stimulated cells now are fully induced into IgM secreting cells. These results define the relative roles of IL-5 and IL-2 in B cell differentiation by showing important regulatory effects at the mRNA level. In addition, these results substantiate that appearance of mRNA for J chain, a molecule key to the formation of pentameric IgM, is a limiting factor for high level IgM secretion. The separate control of microsecond and J chain mRNA found in CL-3 cells stimulated with IL-5 and IL-2 elucidates a molecular mechanism by which these two lymphokines synergize in the development of CL-3 cells into IgM secreting cells.     

Immunoregulation in the rat: cellular and molecular requirements for B cell responses to types 1, 2, and T-dependent antigens

The requirements for primary in vitro plaque-forming cell (PFC) development in cultures of purified rat splenic B cells have been examined. Rat B cells were directly responsive to the type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA), but both T cells and adherent accessory cells were required for B cell responses to the type 2 antigen TNP-Ficoll and the T cell-dependent (TD) antigen sheep erythrocytes (SRBC). However, the cellfree supernatants from concanavalin A-induced spleen cells of rat or mouse origin replaced the requirement for T cells and macrophages, and resulted in PFC development in response to TNP-Ficoll and SRBC and augmented PFC numbers in response to TNP-BA. Culture supernatants from induced murine T cell and macrophage cell lines were used to partially deduce the molecular requirements for the support of PFC development by rat B cells to these three antigens. Supernatants from the EL-4 (EL-4 sup) and B151 K12 (B15 sup) T cell lines augmented TNP-BA responses, suggesting that B cell growth factor II (BCGF-II) mediated this effect. An admixture of purified interleukin 2 (IL 2) and B15 sup supported PFC development to SRBC; indicating that IL 2, BCGF-II, and the T cell-replacing factor in B15 sup (B15-TRF) were sufficient to support this response. In addition, the IL 2 plus B15 sup-supported anti-SRBC PFC response was increased by the addition of an interleukin 1-containing fraction from the supernatant of the macrophage line P388D1. PFC development in response to TNP-Ficoll had the most stringent requirements and only occurred in the presence of EL-4 sup and B15 sup (IL 2, BCGF-I, BCGF-II, EL-TRF, B15-TRF). These data indicate that different cellular and molecular requirements exist for PFC development in response to types 1, 2, and TD antigens by rat B cells.     

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes

The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.     

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.     

Purification and physicochemical characterization of murine T cell replacing factor (TRF)

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.     

Structural and functional characterization of IL 2 receptors on activated human B cells

After activation, B cells express the IL 2 receptor as determined by their reactivity with monoclonal anti-IL 2 receptor antibodies. In this report we show that anti-IL 2 receptor antibodies precipitated comparable 60,000 to 65,000 dalton proteins from highly purified B and T cells. Limited peptide mapping suggested that the receptors on B and T cells were identical. Moreover, activated B cells could be induced to proliferate by IL 2, but not to secrete Ig. Anti-IL 2R antibody blocked the effect of IL 2 but not the proliferative response induced by B cell growth factor (BCGF), suggesting independent growth factor receptors. Investigation of the kinetics of the B cell response to growth factor indicated that BCGF acts within 24 hr, whereas IL 2 was virtually devoid of activity for 48 hr. Nevertheless, after 72 to 96 hr, the effect of IL 2 was equal to or greater than that obtained with BCGF. These studies suggest that the initial stages of B cell proliferation involves a sequential interaction of BCGF and IL 2 with their respective receptors.     

Elevated expression of proto-oncogenes during interleukin-5-induced growth and differentiation of murine B lineage cells

Interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. To elucidate IL-5-mediated intracellular mechanisms, we have established IL-5-dependent and -independent murine early B cell lines, J6 and MJ88-1, respectively, and examined the effect of IL-5 on the expression of proto-oncogenes during proliferation. Two- to 3.5-fold increases in the levels of c-myb, c-myc, c-fos, and c-fms mRNA were observed in J6 cells, compared with those in MJ88-1 cells. Further, a role of IL-5 in the proto-oncogene expression during differentiation was examined by using thymidine-treated murine B-cell chronic leukemia BCL1-B20 cells with growth arrest. After 4-day culture, the amount of IgM secreted from BCL1-B20 cells was augmented 4-6 fold in the presence of IL-5. Although expression of c-myb, c-fos, and c-fms mRNA did not change, only c-myc mRNA expression was elevated within 30 min of stimulation with IL-5 and reached a maximal level by 1 hr. Addition of phorbol 12-myristate 13-acetate (PMA) or IL-4 to the culture of BCL1-B20 cells inhibited both the IL-5-mediated augmentation of IgM secretion and the elevated expression of c-myc mRNA. These findings suggest that the IL-5 signal may be associated with the up-regulation of c-myc expression.