B-Cell Growth Factor

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). Low interferon activity was detected in two super- BCGF. natants; threshold BCGF activity was detected in three other Fig. 2 shows a number ofexperiments in which various TUH supernatants. In this particular fusion, no cultures were iden- 3 supernatants were titrated in parallel with media conditioned tified that showed either TCGF or TRF activity. by BW 5147 cells or by a similar hybridoma devoid of BCGF One of the hybridoma cultures from this experiment (AC 6) activity. High concentrations ofTUH 3 supernatants often were was cloned, and the clones isolated were screened for TCGF somewhat inhibitory (see below) but between 12% and 1% (vol/ and BCGF production. Of 34 clones, 3 showed BCGF activity and 1 showed low, but detectable, TCGF activity. One of the Exp. 1 Exp. 2 Exp. 3 three BCGF-producing clones was recloned; of 14 subelones A tested, all showed constitutive production of BCGF activity and 0 , none showed TCGF activity. One ofthese (TUH 3) showed high production and favorable growth properties, and it was selected for further studies. A~~~~~ The results of cloning indicated that, early after fusion, hybridoma cells are very unstable in regard to the production of 104- -4 biologically active material. Thus, only 3 of 34 clones isolated $. 0. from a primary culture that should not be expected to contain 0 AL Al a high.;multiplicity of different hybrids (9 positive among 48 tH cultures) were found to continue producing growth promoting A 0 Table 1. Screening of hybridoma culture supernatants for c0 \ o A 0 various biological activities 0 Culture IFN* TCGF TRF BCGF Primary: AC5 - - - 103 , AB3 - - - + AC1 - - - - 0 AD6 - - - - AB2 - - - - 1 3 1 3 1 3 AC4 + - - - BB5 - - - - Days after subculture AA4 + + - - AC6 - - - + FIG. 1. B-cell blasts (104 per culture) purified from 24-hr LPS- stimulated cultures were reactivated with medium (o), LPS (o), or 5% * IFN, interferon. TUH 3 supernatant (A). Downloaded by guest on September 26, 2021 Immunology: Leanderson et al. Proc. Natl. Acad. Sci. USA 79 (1982) 7457

A B C D S 0

s. 104 0 0 0. 104 FIG. 2. Proliferation of LPS-acti- 0 vated B-cell.blasts recultured (104 cells perculture) in the presence of medium 0 (o), LPS (e), or the indicated mixture of conditioned media prepared from * - TUH 3 hybridoma cells ( 0, inA, C, and D), an irrelevant hybridoma isolated from the same fusion protocol 0 3 (A, in B), or BW 5147 thymoma cells 103 (a, in C andD). Supernatants were prepared from TUH 3 cells continuously 256 1.5 6 1.5 03 256 1.5 6 1.5 0.3 maintained in culture (A) early after or later or fro- 50 12.5 3 12.5 3 0.75 50 12.5 3 12.5 3 0.75 fusion (A) 6 months (C) zen and thawed ( 0 ) at two different Supernatant in medium, % (vol/vol) time points (D).

vol), a plateau of activity was observed. The activity decreased the two supernatants, these results demonstrate that BCGF and- at higher dilutions to reach background levels at 0.02-0.01% TCGF are distinct molecular entities as previously suggested (not shown). BCGF activity was characteristic of the superna- by some reports but denied by others (10, 11). tants from the TUH 3 cells because neither BW 5147 nor an Preliminary experiments aiming at the biochemical charac- irrelevant hybridoma produced growth-supporting activity. terization of BCGF had revealed that all biological activity could TUH 3 cells kept in continuous culture produced BCGF activity be recovered from sizing columns in a single peak of Mr similar to that obtained from cells frozen and thawed at different =20,000. Because murine TCGF has been previously assigned times. a Mr of 30,000 (20), we sought to separate these two biological Growth factors for activated B cells have been described as activities biochemically. We mixed equal volumes of BCGF- major histocompatibility complex-unrestricted and nonspecific containing hybridoma supernatant and TCGF-rich Con A-con- (5, 6). In our experiments measuring the activity of a clonally ditioned medium. This mixture was then chromatographed, and produced factor on polyclonally activated B cells, it was evident the profile of activities recovered from the eluent is shown in that BCGF is not antigen specific and it acted equally well in Fig. 3. BCGF and TCGF activities eluted at two distinct peaks syngeneic conditions as on targets differing both in major his- corresponding to Mrs -20,000 and =30,000, respectively. tocompatibility complex and Ig haplotypes (Table 2). These observations establish that the B-lymphocyte and cyto- BCGF and TCGF Activities Are Carried by Distinct Mo- toxic T-lymphocyte specific growth factors are two distinct molecular Entities. TCGF is the product ofactivated T-helper cells lecular entities. that appears to be a selective growth factor for cytotoxic T lymphocytes (20, 21). Because the TUH 3 hybridoma is derived from populations containing TCGF-producing cells that have Table 3. Target cell specificity of BCGF and TCGF previously been used to derive TCGF-producing hybrids (11), Growth, cpm/culture it was important to analyze the TUH 3-conditioned media for T-cell blasts B-cell blasts TCGF activity. The same supernatant preparation that showed high mitotic activity for B-cell blasts failed to maintain prolif- Restimulation Exp. 1 Exp. 2 Exp. 1 Exp. 2 eration of TCGF-reactive, activated T cells (Table 3). Since LPS (50 ,ug/ml) ND ND 29,758 26,560 mixing experiments excluded mutually inhibitory activities in TUH 3 supernatant (5%)* 1,050 434 27,047 23,523 Table 2. Lack of genetic restriction in the activity of BCGF for TCGF CM (35%)t 42,233 66,155 2,024 4,564 various types of B-cell blasts Medium 1,106 628 4,422 3,350 Donor blasts TUH 3 supernatant (5%) + Haplotypes Response, cpm/culture TCGF CM (25%) ND 52,500 ND 19,547 Strain MHC Ig-1 Medium 5% TUH 3 sup. LPS T-cell blasts (2 x 104 per culture) were activated with Con A for 48 BALB/c d a 2,142 10,514 11,503 hr; B-cell blasts (104 per culture) were stimulated with LPS for 24 hr. C3H/Tif k 1 1,575 10,527 8,030 The cells were restimulated as shown and pulsed for 4 hr with 1 tkCi C57BL/6 b b 1,650 14,365 15,279 of [3H]dThd on day 2 of culture. Results are means of triplicate cultures. ND, not done; CM, conditioned medium. Blasts purified after 24 hr of LPS stimulation of spleen cells of the * Conditioned medium from TUH 3 hybridoma cells (105/ml) in ex- indicated strains were recultured as indicated (104 cells per culture). ponential growth for 24 hr. All cultures were pulsed with 1 puCi of [3H]dThd per culture for 4 hr t Standard TCGF-containing conditioned medium (supernatant from on day 2 of subculture. The results, shown as cpm, represent the mean 24-hr cultures of 5 x 106 rat spleen cells per ml with 5 pug of Con A responses upon restimulation of triplicate cultures. MHC, major his- per ml) supplemented with 10 mg/ml of a-methyl D-mannoside (10 tocompatibility complex; Ig-1, Ig-1 heavy chain. mg/ml) for inactivation of the lectin. Downloaded by guest on September 26, 2021 7458 Immunology: Leanderson et al. Proc. Natl. Acad. Sci. USA 79 (1982)

20 - 67 43 25 13.7 co X 10- x A

. . A AAA1

.....A......

pF-1 ce

Q--. 1 10 20 30 40 50 c). Fraction FIG. 5. Ig-secreting PFC upon restimulation of LPS-activated 'FIG. 3. Gel filtration on ACA 54 of mixture of equal volumes of B-cell blasts with LPS (e), 10% conditioned media from TUH 3 hybridoma and Con A-stimulated nor- TUH 3 supernatant (A), or me- mal mouse spleen cells. Fractions were assayed in parallel on B-cell dium (o). (Left) At 104 cells per blasts (A) and T-cell blasts (A) with medium (0,0); TCGF-containing culture. (Right) At 2 x 104 cells medium (.), and LPS (mnas controls. Mr standards (shown x 10-3) were per culture. Parallel proliferative bovine serum albumin (67,000), ovalbumin (43,000), chymotrypsino- assays on day 2 of restimulation gen A (25,000), and RNase A (13,700) all obtained from Pharmacia yielded (cpm perculture): for 104 (Uppsala, Sweden). cells per culture, medium, 1,497; LPS, 5 413; and BCGF, 4,178; for 2 x 0w perculture, BCGF is Specifically Adsorbed onto Activated B Lympho- 1 3 1 3 cells medium, cytes. The B-cell specificity of the TUH 3 activity was further 1,150; LPS, 17,742; and BCGF, probed by specific binding ofthe growth-promoting activity to Days after subculture 7,293. target cells. As shown previously, TCGF can be adsorbed readily on target cytotoxic T-cell blasts (22) which express 1,000-fold the stimulatory effects of BCGF. The nature of this inhibitory more specific receptors for growth factors than do irrelevant activity in crude supernatants is unknown, but its presence cells (23). BCGF-containing supernatants were incubated in the might explain why optimal dilutions of TUH 3 supernatant are cold with the same number ofeither LPS-activated B-cell blasts always <25%. or Con A-stimulated T cells. There was a specific loss ofgrowth- Requirement-for Additional BCMF in PFC 'Responses of promoting activity upon incubation with activated B cells but BCGF-Dependent B Cells. When PFC responses of activated notwith activated Tcells (Fig. 4). This was not due to production B-cell blasts maintained in BCGF or LPS were compared, a of inhibitory substances by target B cells because mixtures of marked deficiency in the ability of BCGF to induce high-rate unadsorbed BCGF-containing supernatants and medium in- Ig secretion in target cells was observed (Fig. 5). In most ex- cubated with activated B cells under identical conditions re- periments, BCGF- and LPS-dependent proliferative responses tained full activity. On the other hand, removal of BCGF from were comparable (Fig. 1), but there was 1 order of magnitude TUH 3 supernatants revealed a marked inhibitory effect that difference in the numbers of PFC induced by either type of also must be present in crude supernatants but is masked by growth-promoting stimulus. It therefore would appear that .BCGF does not provide to responding B cells the necessary signals for.induction of terminal maturation and high-rate antibody secretion. 0 We considered the possibility that the two biologically distinct responses are induced by distinct factors, in particular because TRF, an activity defined by increased antibody production, has previously been claimed to be devoid of growth-

W Table 4. Failure of BCGF-containing supernatants to support PFC development in B-cell cultures can be corrected by crude 4- 0 1 helper cell. supernatants Supernatants added Is4 FIG. 4. Appropriate dilu- TUH 3 TH PFC/105 cells tions of TUH 3.supernatant were cO (BCGF) 1 2 incubated in the cold for 1 hr with (BCMF) Exp. Exp. 0 either B or T blasts and then ti- - - 285 650 trated in the BCGF assay. The + _ 570 760 data show the proliferation of 104 + + (day 1) 7,200 4,600 LPS-derived blasts after 48 hr of + + (day 2) 6,600 3,200 reculture with medium (o), LPS (e), unadsorbed TUH 3 superna- Purified LPS-activated B-cell blasts were cultured in 10% TUH 3 103 tant (A),' TUH 3 supernatant ad- supernatant (104 or 2 x 104 cells per culture in Exps. 1 and 2, respec- sorbed on B-cell blasts (0), TUH tively). All cultures were assayed on day 3 after subculture. After 24 3 supernatant adsorbed on T-cell or48 hr as indicated, a 15% dilution (vol/vol) ofa supernatant collected O 0.25 0.075 0.01 blasts (*), and unadsorbed TUH at 48 hr from cultures of specific helper cells and irradiated stimulator 0.5 0.125 0.03 3 supernatant mixed with me- cells (9) was added to parallel cultures. Control response to LPS or to dium adsorbed on B-cell blasts the helper supernatant alone added at 24 hr were 29,500 or 2,090 and Supernatant, % (vol/vol) (0). 14,300 or 920 PFC in Exps. 1 and 2, respectively. Downloaded by guest on September 26, 2021 Immunology: Leanderson et al. Proc. Natl. Acad. Sci. USA 79 (1982) 7459 promoting activity (24). To assess this possibility, we exposed phocytes, the phases of growth and maturation in B-cell LPS-derived B-cell blasts to either BCGF alone or to BCGF responses are nonspecific, driven by polyclonally active factors. and dilutions of supernatants produced in cultures of helper It follows that the restriction in T/B cell collaboration, as well cells known to induce antibody formation (6). Even a short ex- as the antigen selection of the B-cell clonal specificities, must posure to "helper" supernatants induced a marked increase in take place at the level ofinduction ofthese responses. Induction the numbers of Ig-secreting PFC in BCGF-dependent blasts is known to require cell-to-cell contact and antigen-bridges (13, (Table 4). 14) and it would appear that such specific major histocompat- These results demonstrate that factors competent to maintain ibility complex-restricted inductive interactions activate reac- B-lymphocyte proliferation lack the ability of inducing matur- tivity to nonspecific factors in resting B lymphocytes. ation to high-rate Ig secretion. We conclude that helper cell- After submission of this manuscript, we read a report by dependent growth and maturation ofB cells are induced by two Howard et al (27) describing the detection of BCGF with sim- different factors that could be designated as BCGF and BCMF. ilar properties in supernatants from an established cell line that also produces TCGF and CSF. DISCUSSION We thank Lena Bergkvistfor assistance in typing the manuscript. This The results presented here describe a biological activity in me- study was supported by grants from the Medical Research Council, dia conditioned by a T-cell hybridoma that is capable of main- Sweden, and the Unit of Applied Microbiology, UmeA University. taining exponential growth in B-cell blast populations. The 1. Morgan, D. A., Rosetti, F. W. & Gallo, R. (1976) Science 193, growth-promoting activity is carried by molecules homoge- 1007-1008. neous as to molecular weight, suggesting that a single factor is 2. Gillis, J. & Smith, K. A. (1977) Nature (London) 268, 154-156. responsible for all the biological activity. The present experi- 3. Dutton, R. W., Falkoff, R., Hirst, J. A., Hoffman, M., Kappler, ments distinguish between BCGF and TCGF not only on the J. W., Kettman, J. R., Lesley, J. F. & Vann, D. (1971) in Prog- ress in Immunology, ed. Amos, B. (Academic, New York), pp. basis of their molecular weight and adsorption to activated tar- 355-364. get cells but also by the demonstration of hybridoma clones 4. Schimpl, A. & Wecker, E. (1972) Nature (London) 237, 15-17. producing only one of them. 5. Schreier, M. H., Andersson, J., Lernhardt, W. & Melchers, F. Previous experiments describing growth-promoting activi- (1980) J. Exp. Med. 151, 194-203. ties for B lymphocytes used conditioned media from enriched 6. Martinez-A, C. & Coutinho, A. (1981) Nature (London) 290, 60- or cloned T helper cells stimulated by the appropriate antigen 61. 7. Moller, G. (1975) Transplant. Rev. 23. on presenting cells (5, 6). Supernatants from Con A-stimulated 8. Dennert, G., Weiss, S. & Warner, J. (1981) Proc. Natl. Acad. Sci. spleen cells, on the other hand, appear to contain little growth- USA 78, 4540-4543. promoting activityfor B lymphocytes, ifany (Table 2), indicating 9. Kohler, G., Lefkovits, I., Elliott, B. & Coutinho, A. (1977) Eur. that BCGF-producing T cells are activated in low frequencies J. Immunol 7, 758-761. or with low efficiency in those cultures. However, the identity 10. Harwell, L., Skidmore, B., Marrack, P. & Kappler, J. (1980) J. ofthe hybridoma-produced BCGF and its relationship to "phys- Exp. Med. 152, 893-904. 11. Larsson, E.-L., Bernab6, R. R., Martinez-A, C., Leanderson, T. iological" BCGFs must await further characterization. & Coutinho, A. (1980) Behring Inst. Mitt. 67, 18-25. It is apparent that hybridoma-derived BCGF displays a poor 12. Takatsu, K., Tanaka, K., Tominaga, A., Kumahura, Y. & Ha- ability to induce high-rate Ig secretion by the responding B lym- maoka, T. (1980)J. Immunol 125, 2646-2653. phoblasts. The fact that PFC responses can be enhanced by 13. Katz, D., Hamaoka, T., Dorf, M. E. & Benacerraf, B. (1973) addition ofanother type ofhelper cell-conditioned media offers Proc. Natl Acad. Sci. USA 70, 2024-2029. strong support to the idea that distinct helper cell-derived fac- 14. Sprent, J. (1978)J. Exp. Med. 147, 1159-1174. 15. Fazekas de St. Groth, S. & Scheidegger, D. (1980)J. Immunol. tors control proliferation and maturation, respectively, of acti- Methods 35, 1-31. vated B cells. Independent confirmation of this suggestion has 16. Gronowics, E., Coutinho, A. & Melchers, F. (1976) Eur. J. Im- recently been obtained by isolating a clone ofT helper cells that munol 6, 588-590. are competent to activate target B-lymphocyte expansion with 17. Gullberg, M., Ivars, F., Coutinho, A. & Larsson, E.-L. (1981)j. little generation of PFC. Furthermore, antibody responses Immunol. 127, 407-411. could be reconstituted readily with crude helper cell-condi- 18. Bernab6, R. R., Martinez-A, C. & Coutinho, A. (1979) Eur. J. Immunol 9, 546-552. tioned media produced by other helpercell clones (unpublished 19. Andersson, J., Coutinho, A. & Melchers, F. (1979) J. Exp. Med. data). 149, 553-564. Previously, B lymphocyte-specific growth factors have not 20. Larsson, E.-L., Iscove, N. N. & Coutinho, A. (1980) Nature been distinguished from maturation factors except by the ina- (London) 283, 664-666. bility ofTRF to support proliferation. Thus, recent observations 21. Larsson, E.-L., Fischer-Lindahl, K., Langhorne, J. & Coutinho, have been interpreted as if the same factor (BRMF) would be A. (1981) J. Immunol 127, 1081-1085. 22. Coutinho, A., Larsson, E.-L., Gronvik, K.-O. & Andersson, J. growth promoting for activated B cells but maturation inducing (1979) Eur. J. Immunol 9, 587-592. in resting, small B lymphocytes (25, 26). Our findings separate 23. Robb, R., Munck, A. & Smith, K. A. (1981) J. Exp. Med. 154, these entities and, in view ofthe lack of TRF activity in BCGF- 1455-1474. containing supernatants, they also support previous interpre- 24. Hunig, T., Schimpl, A. & Wecker, E. (1974) J. Exp. Med. 139, tations suggesting that TRF might be a pure maturation factor 754-760. (24). 25. Melchers, F., Andersson, J., Lernhardt, W. & Schreier, M. H. (1980) Eur. J. Immunol 10, 679-685. Our experiments confirm previous observations on the lack 26. Andersson, J. & Melchers, F. (1981) Proc. Natl Acad. Sci. USA ofspecificity ofgrowth and maturation factors in regard to rec- 78, 2497-2501. ognition ofantigen and target cell major histocompatibility com- 27. Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, K., plex or Ig determinants. It appears that, as for cytotoxic T lym- Hamaoka, T. & Paul, W. (1982)J. Exp. Med. 155, 914-923. Downloaded by guest on September 26, 2021