Proc. Natl Acad. Sci. USA Vol. 79, pp. 7455-7459, December 1982 Immunology B-cell growth factor: Distinction from T-cell growth factor and B-cell maturation factor (B lymphocytes/T-cell hybridomas) TOMAS LEANDERSON*, ERIK LUNDGRENt, ERIK RUUTH*, HAKAN BORGt, HAKAN PERSSONt, AND ANTONIO COUTINHOt tLaboratory for Cell and Tissue Culture Research, *Institute of Pathology, and +Department of Immunology, UmeA University, S-901 87 UmeA, Sweden Communicated by Niels K. Jerne, July 12, 1982 ABSTRACT A T-cell hybridoma was derived by somatic cell We have initiated these studies by systematically screening hybridization between concanavalin A-activated BALB/c spleen hybridoma activities in different assays that measure either cells and the AKR thymoma BW 5147. Media conditioned by hy- growth or maturation ofall B lymphocytes, regardless ofclonal bridoma cells, even at high dilutions (1:1,000) support the growth specificity. We chose to study activated rather than resting B oflipopolysaccharide-stimulated B-cell blasts but not that ofT-cell cells because of the-overwhelming evidence that initiation of growth factor (TCGF)-reactive T-cells. This activity, herein des- cooperative B-cell responses requires direct cellular interaction ignated B-cell growth factor (BCGF), has a Mr of =20,000 and it (13, 14). In this report we describe a hybridoma clone secreting can readily be separated from TCGF (Mr "30,000) by gel filtra- a factor that supports growth but not maturation of lipopoly- tion. BCGF is constitutively produced by the hybridoma cells, it saccharide (LPS)-activated B-cell blasts and is devoid of is removed from conditioned media by incubation with target cells T-cell at +4°C, and it is equally effective on B-cell blasts carrying dif- growthfactor(TCGF) andT-cellreplacingfactor(TRF) activities. ferent major histocompatibility complex and Ig haplotypes. BCGF shows no T-cell replacing factor (TRF) activity, and it is poor in MATERIALS AND METHODS supporting the development of Ig-secreting plaque-forming cells Mice. BALB/c, C57BL/6, C3H/Hej, and C3H/Tif mice of in B-cell blast cultures. Terminal maturation, however, can be both sexes were bred in our colony and used at 8-12 weeks of induced in BCGF-dependent blasts by addition of conditioned age. media from normal helper T cell cultures, suggesting that two Cell Culture. BW 5147 cells and hybridoma cultures were distinct factors are involved in the helper cell-dependent growth kept in RPMI-1640 medium supplemented with 10% fetal calf and maturation of B lymphocytes. serum, antibiotics, 50 ,uM 2-mercaptoethanol, and 0.01 M Immune responses are the result ofextended clonal growth and Hepes; they were cultivated at 37°C in a humidified atmo- maturation ofspecific precursors preexisting in lowfrequencies. sphere. Much work has led to the isolation and characterization of Cell Fusion and Hybridoma Selection. Cell fusion was car- growth factors with specificities for cytotoxic T lymphocytes (1, ried out as described (15). Cells were cloned by seeding cells 2). Ever since 1970, various factors have been reported to in- at 1 or0.2 cell per culture inmicrotiter plates with afeeder layer teract directly with B lymphocytes (3, 4). Recently, evidence of peritoneal cells (limiting dilution). has been presented that activated B cells can grow under the Adsorptions. BCGF-containing supernatant was adsorbed influence of "nonspecific" molecules produced by other cells either on LPS-derived or concanavalin A (Con A)-activated (5, 6). However, there is no general agreement on the char- blasts. Shortly thereafter, purified blasts were suspended in acteristics or a medium containing 10% fetal calf serum at a concentration of physiologic significance of B-cell growth factor 108/ml and incubated on ice for 1 hr. The blasts were then spun, (BCGF). This is most likely due to the widespread belief that resuspended at the same concentration in the appropriate di- antigens are the BCGF (7). In addition, most assays of B-cell lutions of either supernatant or medium, and kept on ice an- responses do not measure growth directly but measure terminal other hour. All supernatants were then tested in the BCGF differentiation ofputative clonalprogenies to antibody production. assay as described below, in parallel with unadsorbed super- Another variable ofthis situation is the source ofconditioned natants, at appropriate dilutions, and in the convenient media containing competence factors. These are currently pro- mixtures. duced by complex cell mixtures or, in the best approaches, by Gel Filtration. Hybridoma supernatant or Con A-condi- mixtures of cloned T cells with ill-defined populations of "an- tioned media from normal spleen cells were precipitated at 80% tigen-presenting cells. " Furthermore, it is nowwell established saturation with ammonium sulfate on ice for 2 hr. The precip- that a single cell type can produce various mediators with a wide itate was spun down (10,000 rpm; 15 min) and resuspended in range of target cell specificities and biological effects (8). Suc- NaCl/0.01 M Hepes, pH 7.3. Samples (1 ml) were applied to cessful fusions between tumor cells of T origin and activated a ACA 54 column (LKB, Bromma, Sweden) equilibrated with lymphocytes have been described (9), and established hybri- phosphate-buffered 0.3 M NaCl. The column was eluted with doma clones with T helper cell characteristics have been re- the same buffer at 4°C; fractions (2 ml) were collected, sterilized ported (10-12). In view of these observations, it would appear by filtration through 0.45-,um Millipore filters, and assayed for feasible to select for hybrid lines showing only one type of bio- BCGF and TCGF activity at 1:60 to 1:120 dilutions in different logical activity-namely, either growth or maturation of B lym- experiments. phocytes. Assays. The assay for BCGF was carried out on B-cell blasts The publication costs ofthis article were defrayed in part by page charge Abbreviations: BCGF, B-cell growth factor; BCMF, B-cell maturation payment. This article must therefore be hereby marked "advertise- factor; Con A, concanavalin A; LPS, lipopolysaccharide; PFC, plaque- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. forming cells; TCGF, T-cell growth factor; TRF, T-cell replacing factor. 7455 Downloaded by guest on September 26, 2021 7456 Immunology: Leanderson et aLPProc. Natl. Acad. Sci. USA 79 (1982) purified by density centrifugation from spleen cell cultures activity for B lymphocytes. The results from the subeloning, on stimulated with LPS (50 Ag/ml; Escherichia coli 055; B5, Difco) the other hand, suggest that, after 6-8 weeks, producing hy- for 20-24 hr as described (6). Blasts were extensively washed brids are relatively stable. As shown below, the hybrid de- and subcultured in microtiter plates at 5 x 104/ml (104 per cul- scribed here has maintained biological activity for >6 months, ture) in the same medium as described above. Hybridoma su- both after freezing and thawing and upon continuous culture. pernatants were titrated into the assay wells, with medium or The BCGF-producing hybridoma (TUH 3) used in these ex- BW 5147-conditioned media as negative controls; restimulation periments expressed both alleles of Thy-i, demonstrating that with LPS provided the positive control. At various periods of it stems from fusion of BW 5147 with a BALB/c T cell. These subculture, the cells were pulsed with [3H]dThd (1 tCi per results also demonstrate that the productions of growth-sup- culture; 1 Ci = 3.7 x 1010 becquerels) for 4 hr, harvested, and porting activities for activated T and B lymphocytes segregate processed for assay of radioactivity. The assay for B-cell matur- in different clones and therefore appear to be distinct molecular ation factor (BCMF) was essentially the same but total IgM-se- entities. creting plaque-forming cells (PFC) were determined in the pro- BCGF: Growth-Promoting Activity for Activated B Cells. tein A plaque assay (16). TCGF assays were performed on T-cell As shown previously (19), activated B cell blasts require the blasts (105/ml), obtained from Con A-stimulated cultures, by continuous presence of a mitotic stimulus for maintenance of titrating the hybridoma supernatants into the assay wells with growth; this stimulus can be provided by nonspecific factors a standard TCGF preparation as positive control (17). Interferon generated by unrelated helper cell activity (5, 6). We have char- assays were performed by titrating the supernatants on a sub- acterized the ability of media conditioned by hybridoma cells clone of mouse L-929 cells with vesicular stomatitis virus as to maintain activated B cell blasts (LPS-derived) in exponential challenge; NIH G 002-902-026 reference preparation was used growth. LPS-activated blasts did not maintain growth when re- as a standard. Assay for TRF activity was carried out by adding .cultured in normal medium, but they did so ifreexposed to LPS the hybridoma supernatant to Mishell-Dutton type cultures or to conditioned medium from TUH 3 hybridoma (Fig. 1). Blast modified to microculture conditions (18) and using anti-Thy-i cell growth often was comparable to that induced by LPS, but and complement-treated spleen cells (106 per culture). it also could be considerably lower. This variation most likely is due to heterogeneity in the LPS blast preparation, and it RESULTS could indicate that not all cycling B cells are reactive to the Cell Fusion and Screening for Factor-Producing Cells. hybridoma-derived activity. Alternatively, the lower response From a fusion between BW 5147 and splenocytes activated for observed with hybridoma supernatants might be due to deple- 18 hr with Con A (5 Ag/ml), 9 growing cultures of 48 seeded tion ofnutrients in the spent medium. We conclude from these were retrieved after the selection procedure. Supernatants from results that TUH 3 cells constitutively produce factors that these nine cultures were tested for different biological activities maintain growth in activated B cells, a biological activity we call (Table 1).