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Beta transforming growth factors are potential regulators of B lymphopoiesis.

Members of the transforming growth factor beta (TGF-beta) family of polypeptides were found to be potent in vitro inhibitors of kappa light chain expression on normal bone marrow-derived and transformed cloned pre-B cells, and of the maturation of these cells to mitogen responsiveness. The inhibition by TGF-beta was selective in that Ia expression was not blocked. Together with the observations that LPS, IL- 1, NZB serum factors, IL-4, and IFN-gamma preferentially induced either kappa or Ia, or both, on a pre-B cell line, these results further suggest that acquisition of Ig and class II molecules is independently controlled by different antagonists as well as agonists. In addition, kappa chain induction by IFN-gamma does not appear to be as sensitive to TGF-beta downregulation as that stimulated by other factors tested, and this raises the possibility that activation of the same gene may result from different transmembrane signaling pathways. In contrast to the inhibitory effects of TGF-beta on kappa acquisition by pre-B cells and on kappa increase after exposure of mature B cells to LPS, as measured by kappa RNA levels and/or surface fluorescence, no inhibition was observed on unstimulated spleen B cells or on two cloned B cell lines that constitutively produce kappa. Thus, TGF-beta may function during specific stages of B cell differentiation by inhibiting initiation of, or increased transcription of Ig genes, and therefore, may be an important negative regulator of B lymphopoiesis. It is the first natural substance found to have this effect.

suspensions of adult B6DIF, (The Jackson Laboratory, Bar Harbor, ME) spleen or bone marrow were made as described (18). B cells were enriched from the spleens of adult mice by depletion of adherent cells on Sephadex G-10 columns (18), followed by passage through nylon wool (19). B cells retained in the column were shaken loose from the nylon wool and found to be 92% surface is light chain-positive . Bone marrow B cell precursors were enriched by depletion of B cells on anti-Ig-coated plates (20) and of adherent cells on a G-10 column as above . B cell-depleted preparations contained <1% K+ cells . For some experiments, highly enriched B cell precursors were isolated by positive selection on anti-Ly-5(220) (14 .8) coated plates after removal of surface Ig+ (sIg+) cells (20).
Assays. Bone marrow or spleen cells were assayed for clonable B cells in semi-solid agar medium containing 25 Ag/ml LPS as described (18). 70Z/3 and bone marrow cells were analyzed for surface K or class II molecules before and after incubation with soluble mediators . Cells were stained for 20 min with FITC-anti-a (Southern Biotechnology Associates, Inc., Birmingham, AL) or unlabeled anti-la' (Becton Dickinson & Co., Mountain View, CA) followed by FITC-anti-IgG2a (Pandex Laboratories, Inc., Mundelein, IL) for an additional 20 min after two washes. Propidium iodide (P-5264 ; Sigma Chemical Co., St. Louis, MO) was added during the last 5 min of labeling to detect dead cells . Labeled cells were analyzed on an EPICS V (Coulter Electronics Inc., Hialeah, FL) using Omega 590 SP, BP 527, and BP 630 filters . Dead cells and erythrocytes were gated out on the basis of propidium iodide uptake and forward angle light scatter . The presence of TGF-/31 was detected in fixed cells using an anti-TGF-#1 antiserum (21) with an immunoperoxidase kit (Zymed Laboratories, San Francisco, CA), or on viable cells by immunofluorescence analysis with FITC-goat anti-rabbit Ig (F-0511 ; Sigma Chemical Co.) as second antibody . This antiserum does not crossreact with TGF-,82 . Cytoplasmic ji+ pre-B cells were enumerated as described (20). The phenotypic and functional maturation of B lineage progenitors in short-term culture was monitored as in our previous reports (18). RNA Analysis. Total RNA was prepared from treated cells, slot-blotted, and probed for K and j chain RNA as described (22). Relative amounts of RNA bands were determined by densitometric readings .

Results
Inhibitory Effects of TGF-Q on Cloned Pre-B Cells. The pre-13 lymphoma cell line, 70Z/3, produces P heavy chains, has one rearranged K light chain allele, and can be stimulated to express K by LPS (25)(26)(27)(28) . A small percentage, 1-7%, of 70Z/3 cells maintained in culture medium have detectable surface a, and typically, incubation with LPS for 48 h resulted in an increase of K+ cells to 77-90% as determined by channel-by-channel subtraction of cells nonspecifically stained by FITC-goat-Ig from cells labeled with FITC-goat anti-K. This induction of x acquisition by LPS was identically inhibited by TGF-,Q1 and TGF-a2 in a dose-dependent fashion (Fig. 1) . Viability of cells in all cultures after 48 h incubation was >90% . Cultures containing LPS alone showed some growth inhibition_ and cell recoveries, determined by counting cells with trypan blue under a light microscope, were 78-89% of medium controls. Little effect on cell recoveries of 70Z/3 was noted in cultures containing TGF-al or TGF-,82 alone (91-105% of medium controls), or together with LPS (96-112% of LPS con- Inhibitidn of LPS induction of cell surface K expression . 70Z/3 cells were cultured for 48 h at 0.1 x 106 cells/ml with LPS (10 FAg/ml) and various concentrations of TGF-S1 (Q) or TGF-02 ("). Cells were then labeled with FITC-goat anti-a or FITC-goat Ig (background control) and analyzed by flow cytometry . After subtracting background staining, 81 % of LPS-induced cells and 4% of untreated cells were K' . Percent inhibition = [(percent a' with LPS) -(percent a' with LPS + TGF) -(percent K' with medium)]/[(percent K' TGF Concentration (ng/mL) with LPS) -(percent K with medium)] .
Light chain expression in response to three other stimuli, IFN-'y, IL-1, and young NZB mouse serum-derived factors (NZB-F), was also inhibitable by TGF-0 ( Fig. 2). However, while K induction by IL-1, NZB-F, or LPS was completely abolished, that elicited by IFN-y was not as sensitive to inhibition by either form of TGF-#. This was also the case when higher concentrations of inhibitors were combined with lower doses of IFN-y (data not shown) .
Like LPS, IFN-y did not affect cell viability but was somewhat inhibitory to cell growth and in several different experiments, cell recoveries after culture were 80-88% of medium controls . Cell recoveries for TGF-/31 or -02 plus IFN-7 cultures were similar to those for cultures containing only IFN-'r (92-98% of IFN-y controls) . IL-1 and NZB serum factors alone or in combination with TGF-# did not affect cell growth or viability . K RNA Levels in TGF-,Q-treated Cells. Hybridization with a K gene probe to slot blots of total RNA isolated from 70Z/3 cells suggests that inhibition of K FIGURE 3. Inhibition of K RNA levels . 70Z/3 cells were incubated overnight with medium alone (control), LPS (10 wg/ml), NZB-F (100 U/ml), IL-1 (25 U/ml), IFN-y (50 U/ml), or IL-4 (labeled as BSF-1 in figure, 400 U/ml) with or without TGF-01 (10 ng/ml). W279 and W231 cells were similarly cultured overnight with or without TGF-fl1 (10 ng/ml) . Total RNA was extracted, slot-blotted, and probed for K RNA (A and B) or ju RNA (C and D) . TGF-S2 was also tested and gave identical results (not shown). expression by TGF-0 may occur by prevention of rc RNA transcription or accumulation (Fig. 3). Induction of K RNA in response to LPS, IL-1, and NZB-F was inhibited by 82, 91, and 95%, respectively, while that mediated by IFN-1' was decreased by only 21 %. Identical results were obtained using either TGF-01 or TGF-#2. In contrast, equivalent amounts of RNA from the same samples hybridized with a u heavy chain probe showed no effect of TFG-,B on levels of' RNA (Fig. 3).
Enhancement of Class II Antigen Expression . That TGF-0 is selectively inhibitory was further demonstrated by its effects on class II MHC molecules (la). Some subclones of the 70Z/3 line are responsive to IL-4 (formerly called BSF-I) (29,30). Consistent with reported observations (30), transcription of class II MHC molecules was markedly increased while Ig gene expression was unaffected (Figs. 3 and 4). Our recently cloned 70Z/3 line was also inducible for la by IFN-.y (Fig. 4) but not by IL-1, NZB serum factors, or LPS (data not shown) . Acquisition of la was not blocked and, in fact, was slightly enhanced by either form of TGF-a (Fig. 4). Elevation of la expression by IL-4 on spleen B cells was similarly not inhibited by TGF-0 (data not shown) .
Inhibition of Bone Marrow B Lineage Cell Maturation . The inhibitory effects of TGF-0 were also demonstrable with normal bone marrow cells (Table I) spontaneous emergence in short-term liquid cultures of mitogen responsive, surface K+ B cells was stimulated by adherent accessory cells in bone marrow, or by inclusion of NZB serum factors to adherent cell-depleted bone marrow (18,23). This was strikingly diminished by TGF-al or TGF-,82 (data not shown for TGF-a2). In addition, numbers of surface Ig, cytoplasmic A+ cells were significantly reduced, indicating that TGF-# may also inhibit generation of pre-B cells. TGF-# did not significantly affect total cell recoveries after culture as compared with the controls in each group.
Effects of TGF-,0 on Mature B Cells. As might be anticipated from a recent report (11), TGF-,81 and TGF-#2 completely inhibited mitogen-stimulated proliferation of mature B cells when cultured with the B cells for 6 d in the presence of LPS in a semi-solid agar cloning assay (Table II). Short-term (1 or 2 h) exposure of spleen or bone marrow cells to the factors, followed by a wash, had no effect (data not shown) . In addition, mature B cells in unseparated marrow suspensions that had been exposed to TGF-,B1 or TGF-a2 for 48 h and washed still retained functional capability (Table 1).
In contrast to the effects on pre-B cells, no reduction in the density of surface Ig or is RNA was observed after incubating mature splenic B cells or B lymphoma cells with these factors (Figs. 3 and 5). The average increase in x that follows LPS stimulation was inhibited (Fig. 5).
Presence of TGF-0 on B Lineage Cells. TGF-# was easily demonstrable by  Single cell suspensions of spleen or bone marrow from 8-wk-old mice were cultured in semi-solid agar medium containing LPS (25 ug/ml) with or without (control) various concentrations of TGF-B1 or TGF-B2 . B cell colonies (CFU-B) were enumerated after incubation for 6 d . immunofluorescence, using an antibody recognizing the TGF-,01 form, on the surface of 70Z/3, WEHI 231 and WEHI 279 cells, and a majority of spleen and bone marrow nucleated cells. An analysis of adult bone marrow in four separate determinations revealed that 59-71% of nucleated cells were positive and this included populations of B lymphocyte lineage cells. Thus, 70-76% of cells that express Ly-5(220) but not surface Ig (total B lineage precursors) and at least 75% of pre-B cells were positive . TGF-# was also demonstrable by immunoperoxidase staining on lymphocytes and macrophages but not stromal cells in longterm bone marrow cultures established according to the Whitlock-Witte procedure, and TGF-0 activity was detectable in these culture supernatants (data not shown) .
This study demonstrates that members of the TGF-,Q family are potent inhibitors of an important step during pre-B cell maturation, acquisition of Ig light chains, and prevent emergence of surface K+, mitogen-responsive B lymphocytes after in vitro culture of precursor cells. The factors are also antiproliferative agents for mature B lymphocytes and inhibited the average increase in K displayed by splenic B cells that followed LPS stimulation. However, in contrast to the inhibition observed in stimulated pre-B and B cells, no reduction in the density of surface Ig or K RNA was detected after incubating unstimulated, mature splenic B cells or B lymphoma cells with either TGF-ail or TGF-02. In addition, levels of u heavy chain RNA were not reduced in pre-B or B cell lines already constitutively producing j chains. Therefore, initiation of light chain gene transcription in pre-B cells, and its increase after activation of mature B cells, but not ongoing Ig RNA synthesis and translation, appear to be TGF-0 sensitive. Our findings suggest that members of the TGF-0 family may selectively influence gene expression at particular stages of B lineage differentiation . Under conditions that promote the formation of pre-B and B cells in vitro, TGF-,Q also decreased the numbers of surface K -, cytoplasmic A' pre-B cells after short-term culture of bone marrow cells enriched for B lineage precursors . However, no inhibition was observed in levels of 14 RNA in pre-B or B cell lines. These observations suggest the possibility that TGF-0 might inhibit an earlier stage of B cell differentiation, initiation of heavy chain gene expression . Experiments are planned to determine if TGF-,Qs have a role in regulation of Ig gene rearrangement, and/or activation of heavy chain genes.
It will be interesting to investigate the mechanism through which TGF-Os prevent the accumulation of K mRNA in pre-B cells. While destabilization or increased degradation of the message is possible, there are several reasons for suspecting that this is unlikely . There were significant amounts of K RNA in pre-B cells induced by IFN -,y in the presence of TGF-,0, and levels of message in established B cell lines were unchanged by these factors. Thus, a very selective process would have to be invoked, such that message lifespan is dramatically reduced, but only in cells that receive a particular induction stimulus . Changes in chromatin state accompanying K transcription have been documented with our models (28) and TGF-# could be inhibitory at that level. Similarly, activation of proteins that bind to the is enhancer (22,31,32) might be aborted.
The selective inhibitory activity of TGF-0 was further demonstrated by its inability to block class II antigen expression . Neither form of TGF-0 induced la, but they synergized with IL-4 or IFN--y to enhance class II expression on 70Z/3 pre-B cells. IL-4 did not induce K on 70Z/3 cells (Fig. 3) or enhance acquisition of K by bone marrow B cell precursors (Lee, G., unpublished observations), while LPS, IL-1, and NZB serum factors induced a but not la, and IFN-,y enhanced expression of both molecules on 70Z/3 cells. A recent report (30) indicates that negative regulation of class II expression by maturing pre-B cells may be achieved with prostaglandins . Taken together with our results, this suggests that the acquisition of these two functionally significant B cell surface markers, Ig and la, is independently controlled by particular agonists and antagonists. It is also noteworthy that K induced by immune interferon was more refractory to downregulation by TGF-0 than that induced by other stimuli. Variants of the 70Z/3 cell line have been isolated that are inducible for K by IFN-, y but not LPS (33). Therefore, activation of the same gene may result from different transmembrane signaling pathways .
An autocrine regulatory function has been proposed for TGF-0 (34). Activated B and T cells have increased message and receptors for TGF-a, and the differ-entiation and/or proliferation of these cells can be inhibited by TGF-# (10,11). Most of the TGF-,B produced by various cell types is in a latent, and presumably precursor form, which becomes functionally potent after acidification (7). Latent TGF-,61 had no effect on K or Ia induction in our assays (Lee, G., unpublished observations) and it is not known how the conversion to an active form is achieved in vivo . TGF-0 was readily detectable by immunofluorescence on the surface of 70Z/3, WEHI 231, and WEHI 279 cells, a majority of spleen cells, bone marrow cells, and B cell precursors in bone marrow . Interestingly, TGF-0 activity was also found in supernatants from long-term bone marrow cultures established according to the Whitlock-Witte procedure (35, and Ellingsworth, L. R., unpublished observations). The material seemed to be most closely associated with macrophages and lymphocytes and it may be significant that pre-B cells in that situation are refractory to maturation-inducing agents (4, 36, and Lee, G., unpublished observations). While we did not distinguish passively acquired from endogenously produced TGF-,B, these findings suggest that maturing precursors may auto-regulate their maturation in some circumstances . Certainly, the factors are intimately associated with cells that are potential targets for their action .

Summary
Members of the transforming growth factor (3 (TGF-a) family of polypeptides were found to be potent in vitro inhibitors of K light chain expression on normal bone marrow-derived and transformed cloned pre-B cells, and of the maturation of these cells to mitogen responsiveness . The inhibition by TGF-0 was selective in that la expression was not blocked. Together with the observations that LPS, IL-1, NZB serum factors, IL-4, and IFN-y preferentially induced either K or la, or both, on a pre-B cell line, these results further suggest that acquisition of Ig and class II molecules is independently controlled by different antagonists as well as agonists . In addition, K chain induction by IFN-y does not appear to be as sensitive to TGF-# downregulation as that stimulated by other factors tested, and this raises the possibility that activation of the same gene may result from different transm6mbrane signaling pathways . In contrast to the inhibitory effects of TGF-0 on K acquisition by pre-B cells and on K increase after exposure of mature B cells to LPS, as measured by K RNA levels and/or surface fluorescence, no inhibition was observed on unstimulated spleen B cells or on two cloned B cell lines that constitutively produce K . Thus, TGF-0 may function during specific stages of B cell differentiation by inhibiting initiation of, or increased transcription of Ig genes, and therefore, may be an important negative regulator of B lymphopoiesis . It is the first natural substance found to have this effect .