Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3348-3352, April 1995 Immunology B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes HENRY H. WORTIS*t, MARK TEUTSCH*, MINDY HIGER*, JENNY ZHENG*, AND DAVID C. PARKERt§ *Department of Pathology, Tufts University School of Medicine, and Graduate Program in Immunology, Sackler School of Graduate Biomedical Sciences, Boston, MA 02111; and tDepartment of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655 Communicated by Salome G. Waelsch, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY December 7, 1994 ABSTRACT Treatment of small resting B cells with sol- direct contact with an activated T-helper cell such that the uble F(ab')2 fragments of anti-IgM, an analogue of T-inde- surface molecule gp39 [CD40 ligand (CD40L)] of the T cell pendent type 2 antigens, induced activation characterized by ligates the CD40 of the B cell (10). proliferation and the expression of surface CD5. In contrast, Our strategy to determine if minimal TD and TI-2 signals B cells induced to proliferate in response to thymus-dependent were sufficient to induce phenotypic differences in B cells was inductive signals provided by either fixed activated T-helper 2 to use an antigen that could be modified so as to initiate either cells or soluble CD40 ligand-CD8 (CD40L) recombinant pro- a TD or a TI-2 response. To produce a TD response we used tein displayed elevated levels of CD23 (FcJII receptor) and no monovalent Fab of rabbit anti-IgM and provided help in the surface CD5. Treatment with anti-IgM and CD40L induced form of the clone CDC35, a T-helper 2 line known to provide higher levels of proliferation and generated a single popula- help to B cells that have bound, processed, and presented tion ofB cells coexpressing minimal amounts ofCD5 and only rabbit Fab in the context of IAd (9, 11). To provide the a slight elevation ofCD23. Anti-IgM- but not CD40L-mediated equivalent of a TI-2 signal we used the divalent F(ab')2 form activation was highly sensitive to inhibition by cyclosporin A of the same antibody. We then compared the surface pheno- and FK520. Sp-cAMPS, an analogue of cAMP, augmented types of the responding B cells. CD40L and suppressed surface IgM-mediated activation. We followed up these initial experiments with others using Taken together these results are interpreted to mean that fixed, preactivated helper T cells as B-cell activators (12). This there is a single population of small resting B cells that can allowed us to examine the phenotype of B cells activated by respond"to either T-independent type 2 (surface IgM)- or T-helper cells in the absence of interleukins. Finally, we T-dependent (CD40)-mediated activation. In response to dif- examined the phenotype of B cells stimulated by a soluble ferent intracellular signals these cells are induced to enter fusion protein containing the CD40L (13) so as to directly alternative differentiation pathways. compare the phenotypes and the signaling pathways involved in minimal TI-2 [F(ab')2 anti-IgM] and TD (CD40L) re- Though it has been suggested that T-cell-independent (TI) and sponses. Our experiments led us to the conclusion that a single thymus-dependent (TD) responses (1) might be generated population of B cells can respond to either sIgM or CD40 from different B-cell precursors (2), the weight of evidence ligation using alternative signal pathways to generate activated suggests that a given B cell can enter either the TI or TD B cells of different phenotypes. pathway (3-5). We wanted to directly determine if B cells exist as two precursor populations or if there is a single population MATERIALS AND METHODS- that can be induced to enter alternative differentiation path- ways. If the latter proved to be the case, we wanted to Mice. Experiments involving CDC35 cells were done with determine if the minimal inductive events were sufficient to cells from BALB/c/J mice purchased from The Jackson induce phenotypic differences in responding cells. To study Laboratory. Others were done using cells from CBA/HHW or this we chose to use anti-IgM antibody as an experimental CBA/HHW.Ighb mice (14). analogue of one type of TI antigen. TI antigens are divided into Cells. Cells of the T-helper 2 line CDC35 were used 2-3 type 1, which are intrinsically mitogenic [such as lipopolysac- weeks after their last stimulation. B cells were partially purified charide (LPS) in the mouse], and type 2, which are nonmito- as described (15). Activated CDC35 T cells were generated by genic repeating unit molecules such as polysaccharides or their incubating cells at 1 x 106 per ml for 24 hr on plates precoated haptenated derivatives (6). TI-2 activation is initiated by the with anti-CD3 (145-2C11). The activated cells were then crosslinking of surface immunoglobulin (slg) followed by a washed three times in phosphate-buffered saline and fixed with cascade of events that includes cytoplasmic tyrosine kinases, a 0.4% fresh paraformaldehyde for 5 min (12). Fixed cells were Ca2+ flux, and the catalytic activity of protein kinase C (PKC) kept in tissue culture medium at 4°C. The supernate generated (reviewed in ref. 7). The F(ab')2 fragments of antibody to by these activated T cells was harvested and stored frozen. mouse 1gM can bind to B-cell sIgM so as to effect its B-Cell Culture and Stimulation. Cells were cultured in RPMI crosslinking and thereby can provide a polyclonal B-cell acti- 1640 medium supplemented as described (15). A polyclonal vating signal equivalent to a TI-2 antigen (8). rabbit anti-mouse IgM was prepared as Fab and F(ab')2 frag- Unlike TI-2 responses, a TD response does not require sIg ments as described (9). For experiments involving immunosup- crosslinking, as even monomeric antigen can be bound, endo- pressants we prepared F(ab')2 fragments from goat anti-(MOPC- cytosed, processed, and presented in the context of major histocompatibility complex class II for T-helper cell recogni- Abbreviations: CsA, cyclosporin A; LPS, lipopolysaccharide; FKBP, tion and activation followed ultimately by T-cell-mediated FK506 binding protein; CD40L, CD40 ligand; TD, thymus-dependent; activation of B cells (9). The crucial TD signal is provided by TI, T-cell-independent; sIg, surface Ig; IL, interleukin; PK, protein kinase. tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge §Present address: Department of Molecular Microbiology and Immu- payment. This article must therefore be hereby marked "advertisement" in nology, Oregon Health Science University, 3181 S.W. Sam Jackson accordance with 18 U.S.C. §1734 solely to indicate this fact. Park Road, L220-Portland, OR 97201. 3348 Downloaded by guest on September 29, 2021 Immunology: Wortis et al Proc. Natl. Acad. Sci. USA 92 (1995) 3349 Table 1. Proliferation of small resting splenic B cells Fixed CD40L Fab F(ab')2 T T cells Fixed T cells + Exp. Medium anti-IgM anti-IgM cells + Fab SN T cells + SN CD40L LPS F(ab')2 DS-4 0.3* 1.2 17.7 42.0 DS-9 1.2 5.8 45.2 J180 [2.3] 2.7 12.7 96.5 [4.6] 22.9 42.9 96.6 J185 8.1 12.5 14.9 83.1 129.6 J181 0.2 0.2 1.6 25.1 15.1 85.6 117t 3.2 78.0 42.5 108.6 123t 1.6 13.8 37.6 85.8 SA 3.1 101.3 52.0 120.8 200.2 SN, supernate. *[3H]Thymidine at 60 hr (42 hr in experiments marked t) expressed as the geometric mean of triplicate values of cpm x 10-3. SEM were -x/1.2 for all values -2 x 103 cpm except for the values in experiment J180 (shown in brackets), in which the SEM values were x/1.5. 104E, TEPC-183). By a Limulus amebocyte lysate method (Asso- would express identical surface phenotypes. Small resting ciates of Cape Cod) the levels of endotoxin were -10 ng per 30 BALB/c splenic B cells were placed in culture with either 10 ,ug of F(ab')2 for goat anti-mouse and rabbit anti-mouse prepa- ,ug of F(ab')2 of rabbit anti-IgM per ml or 10 ng of Fab rations. Cells producing the fusion protein CD40L-CD8 (13) were fragments of the same antibody per ml together with CDC35 from Peter Lane (Basel Institute for Immunology). Supernate T-helper 2 cells specific for Fab. Both treatments induced containing recombinant CD40L produced optimal proliferation B-cell proliferation (experiments DS-4 and DS-9, Table 1), cell when used at a final dilution of 1:2. For thymidine incorporation cells were cultured in 96-microwell plates in 200 ,ul of medium. enlargement, and increased levels of surface CD44 (Fig. 1), For cytometric analysis the cells were cultured in bulk. In both events previously described in association with lymphocyte cases they were established at a density of 1 x 106 per ml. Fresh activation (15). Culture with Fab or T cells alone did not or fixed T cells were added at 1 x 106 cells per ml. Fresh T cells induce proliferation (Table 1), nor was any change in B-cell were irradiated to a total dose of3600 rads (1 rad = 0.01 Gy) from surface CD5 or CD23 seen (data not shown). F(ab')2-treated a cesium source before being placed in culture with the B cells.