Proc. Nati. Acad. Sci. USA Vol. 84, pp. 9140-9144, December 1987 Immunology Activation of resting human B cells by helper T-cell clone supernatant: Characterization of a human B-cell-activating factor (T-celi lymphokines/B-ceil stimulation) ANITA DIu*, MARIE-LISE GOUGEON*, JEAN-LOUIS MOREAU*, ELLIS L. REINHERZt, AND JACQUES THtZE* *Unitd d'Immunogdndtique Cellulaire, Institut Pasteur, 25, rue du Docteur Roux 75015 Paris, France; and tDivision of Immunobiology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115 Communicated by Baruj Benacerraf, August 17, 1987 ABSTRACT The effects of helper T-cell clone superna- (Pharmacia, Paris) density-gradient centrifugation of periph- tants on resting human B cells were investigated. Four different eral blood from healthy adult volunteers and were partially helper T-cell clones (two T4' and two T8+) were stimulated by depleted of adherent cells by adherence to plastic culture anti-T3 monoclonal antibodies on Sepharose beads or anti-T112 dishes. Nonadherent cells were submitted to rosette forma- plus anti-T113 monoclonal antibodies. The supernatants from tion with neuraminidase-treated sheep erythrocytes, and these activated clones induced the proliferation of highly nonrosetting cells were recovered after centrifugation on a purified resting B lymphocytes from the peripheral blood. The Lymphoprep gradient (Nyegaard, Oslo). The B cells were B cells exhibited a cell size and a surface-antigen pattern (4F2 then separated by using a discontinuous gradient of Percoll antigen and transferrin receptor) of phase Go B cells, and they (Pharmacia) as described (12), and cells banding at the were functionally resting. In response to T-cell supernatants a interface between Percoll concentrations 50o and 60% were large fraction of the B cells enlarged and expressed 4F2 collected. These preparations (referred to as dense B cells) antigens and transferrin receptors. In gel filtration, the cor- routinely contained <1% T cells, 2-3% monocytes, and responding activity migrated with an apparent Mr of 12,000- >85% B cells as determined by immunofluorescence staining 15,000. Our fwdings strongly support the existence ofa human using anti-T4 plus anti-T8, MO1, and anti-Bi mAbs, respec- B-cell-activating factor acting on resting B cells and causing tively (21, 22). The B-cell preparations showed no prolifer- them to enter phase G1 of the cell cycle. ative response to T-cell mitogens such as phytohemagglutinin (PHA), concanavalin A, or anti-T112 plus anti-T113 mAbs. In T-cell-dependent responses, specific T-cell-B-cell contact Dense B cells (5 x 104 per well) were cultured in 96-well is supposed to control B-cell activation, whereas later stages round-bottomed microtiter plates (Costar, Cambridge, MA) of B-cell maturation are driven by nonspecific T-cell-derived in 200-,lI final volume in RPMI 1640 medium (M.A. Bio- lymphokines (1, 2). In the human model, B-cell proliferation products, Walkersville, MD) containing 10% fetal calf serum is mediated by two B-cell growth factors (BCGF): a low-Mr (Flow Laboratories, Paris), 1 mM L-glutamine, 1% penicil- and a high-Mr BCGF (3-5). Human interleukin 4 (IL-4) also lin/streptomycin, and 10 mM Hepes. Thymidine uptake was acts as a B-cell proliferative factor (6). In vitro assays for the measured after 3 days of culture with a 16-hr exposure to 1 detection of these lymphokines involve submitogenic con- ,uCi of [3H]thymidine per well (1 Ci = 37 GBq). centrations of anti-, heavy chain immunoglobulin (anti-,) T-Cell Clones. P2 and N2 (T8+) were cloned twice by antibodies or Staphylococcus aureus Cowan A strain (SAC) limiting dilution methods, in which T cells from a healthy that are supposed to polyclonally activate resting B cells. volunteer (0.3 cell per well) were plated in the presence of Interleukin 2 (IL-2) also induces proliferation and, in some irradiated autologous feeder layers in IL-2-containing medi- cases, differentiation of SAC or anti-,-activated human B um with 0.25 ,ug of PHA per ml (Wellcome). Tetanus cells (7). In addition, a direct effect ofvery high doses ofIL-2 toxoid-specific T-cell clones TT1 and TT9 (T4+) were ob- on unstimulated B cells (8) was also reported. tained in the same way from an antigen-specific cell line In the mouse model, we have shown that the T cells can be raised from T cells ofa healthy volunteer using tetanus toxoid replaced by their supernatant for mouse resting B-cell acti- at 0.3 floculation unit/ml (Massachusetts State Biological vation, proliferation, and differentiation (9-11). We have Laboratories, Jamaica Plain, MA). T cells were cultured in hypothesized that a new lymphokine called B-cell-activating IL-2-containing medium. Restimulation was done every 4 factor (BCAF) was responsible for resting B-cell activation, weeks in the presence of irradiated feeder cells, PHA (0.25 and we now have evidence that this factor is distinct from ,tg/ml), and IL-2. The T-cell cultures tested negatively for other mouse lymphokines. In this report, we extend these mycoplasma at least twice during the course of this work. observations to the human model. Using helper T-cell clones Production ofT-Cell Supernatants. T-cell clones were taken stimulated by anti-T3 or anti-T11 monoclonal antibodies 15 days after restimulation, washed once, and incubated in 5 (mAbs), we have produced supernatants that could polyclon- ml of polypropylene tubes (Falcon) at 106 cells/ml for 6 hr or ally activate highly purified resting human B cells from the 24 hr at 37°C in medium alone, or in the presence of anti-T3 peripheral blood. Our findings strongly support the existence mAbs on Sepharose beads (1/50) (13), or a combination of of a human BCAF with an apparent Mr of 12,000- anti-T112 plus anti-T113 mAbs (1/200 dilution of ascitic fluid) 15,000. (14). The supernatants were then collected, filtered through a 0.22-,um Millipore membrane, and used immediately or MATERIALS AND METHODS stored at -20°C. B-Cell Preparation and Proliferation Assay. Peripheral blood mononuclear cells were obtained by Ficoll/Hypaque Abbreviations: anti-A, anti-, heavy chain immunoglobulin antibod- ies; BCAF, B-cell-activating factor; BCGF, B-cell growth factor; IL-2, interleukin 2; IL-4, interleukin 4; mAb, monoclonal antibody; The publication costs of this article were defrayed in part by page charge PHA, phytohemagglutinin; P2(T11)-SN, P2(T11) supernatant; FITC, payment. This article must therefore be hereby marked "advertisement" fluorescein isothiocyanate; SAC, Staphylococcus aureus Cowan A in accordance with 18 U.S.C. §1734 solely to indicate this fact. strain. 9140 Downloaded by guest on September 26, 2021 Immunology: Diu et al. Proc. Natl. Acad. Sci. USA 84 (1987) 9141 RESULTS IL-2 Is not Involved in the B-Cell Response to T-Cell Supernatant. Because high doses of IL-2 were able to induce Supernatants from Activated T-Cell Clones Aire Sufficient to some proliferation of resting B cells and because T-cell Induce the Proliferation of Unstimulated B Ccells. Two T4' supernatants did contain some IL-2 (as measured on an T-cell clones (TT1 and TT9) and two T8' clone1s (P2 and N2) IL-2-dependent T-cell line), it was of interest to evaluate the were activated for 24 hr by anti-T3 mAbs 4on Sepharose contribution of IL-2 to our results. The final concentration of beads, or by a combination of anti-T112 and aniti-T113 mAbs. IL-2 from P2(Tll)-SN in the B-cell assays (between 0.6 and The culture supernatants were collected and as&sayed in B-cell 1.25 ng/ml) was too low to account by itselffor the observed cultures. As shown in Fig. 1, dense B cells are driven to response (Table 1). We further excluded the participation of proliferate in response to these helper T-cell suipernatants, in IL-2 in the effects ofthe T-cell supernatant by either blocking the absence of any other added stimulus, andI this effect is the IL-2 receptor or neutralizing the IL-2 activity with dose dependent. The release of this proliferrative activity appropriate mAbs. As shown in Table 2, when resting B cells needs stimulation of the T-cell clones becauwse little or no were cultured with anti-pt and recombinant IL-2, both 33B3-1 activity is detectable in supernatants from uinstimulated T (anti-IL-2R IgG2a, ref. 15) and DMS-1 (anti-IL-2 IgG1, ref. cells. In addition, the proliferation of B cellIs induced by 16) mAbs were able to inhibit the B-cell response to IL-2. On T-cell supernatant is not major histocompatiboility complex- the contrary, the response to P2(T11)-SN was not signifi- iit comf cantly affected by either mAb. As a control, we checked that restricted. Supernatants obtained after stimul o ploex 33B3-1 and DMS-1 mAbs did not affect the BCGF-induced P2 with anti-T112 plus anti-T113 mAbs [P2(Ta11tSN'11)-SN] weree response of anti-,u activated B cells (data not shown). studied in greater detail. Size Analysis of Resting B Cells Stimulated with P2(Tll)-SN. The Responding B-Cell Population Consists c sf Functionally The change in size of small B cells was analyzed at different Resting B Cells. The B cells used in the culturels consisted of times after stimulation with P2(T11)-SN. Fig. 2 shows that at dense B cells with a mean volume correspondling to cells in initiation of the culture, Percoll gradient-purified B cells phase Go of the cell cycle (Fig. 2A). To verify Ithat they were exhibited a mean cell volume ranging between 140 and 160 also functionally resting and to exclude that our results could ,1Mm3.