Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3348-3352, April 1995

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 pendent type 2 antigens, induced activation characterized by ligates the CD40 of the (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 -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 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 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. cells became CD5+ and showed no change in CD23 levels (Fig. K235 LPS was used at a concentration of 5 ,ug/ml. FK520 1). The T cell plus Fab-stimulated cells neither became CD5+ (L-683,590), rapamycin (L-657,862), and L-685,818 were kind nor had increased CD23 (Fig. 1); in fact, CD23 expression went gifts from Merck that were stored as 1 mg/ml stock solutions in down. Therefore, in response to TI-2 and TD antigens, B cells ethanol. Cyclosporin A (CsA) (Sandoz Pharmaceutical) was acquire different surface phenotypes. stored at 2 mg/ml in 71.5% ethanol/28.5% Tween 80. Sp-cAMPS Previous experiments indicated that the CD23 levels of was purchased from Research Biochemicals (Natick, MA). activated B cells could be lowered in response to interleukin 6 Flow Cytometry. Cultures containing CDC35 T cells were (IL-6) or increased by IL-4 treatment (15). To determine if the treated with anti-CD4 plus complement before staining. In changes in B cells activated cells resulted from some experiments living cells were enriched by centrifugation by T-helper only over a Ficoll gradient (Lympholyte-M, Cedarlane Laborato- direct contact with T-cell membranes or were dependent in ries) prior to staining. Cells were stained as described (15). whole or in part on antigen ligation and/or soluble ILs, we Flow cytometry data were obtained on a FACScan using LYSIS cultured B cells with fixed activated CDC35 cells. In the acquisition and analytic software (Becton Dickinson). absence of any additional antigen they also induced prolifer- ation (Table 1, experiments J-180 and J-181), enlargement, and increased CD44 (Fig. 2). As before, B cells activated by RESULTS contact with activated T-helper cells did not express CD5 but We first asked whether the surface phenotype of B cells they now showed increased levels of CD23 (Fig. 2). The activated by the same nominal antigen in TI-2 and TD forms supernate from activated T cells did not induce proliferation SSC/FSC CD44 CD5 CD23

Medium

FIG. 1. Flow cytometry analysis -- _ ..4y .. of B cells. Top row, small resting splenic B cells at time 0; middle row, cells 3.5 days after coculture with F(ab')2 fragments of anti- IgM; bottom row, cells after stim- Anti 1gM ulation with Fab fragments of the same antibody plus irradiated T- helper cells from the line CDC35. The lefthand column shows for- ward (FSC) and side (SSC) scatter. Smaller, less complex cells are in the far left corner; the larger and more complex cells are in the T Help far right corner. CD44, CD5, CD23, and negative control stain- ing is also shown. Downloaded by guest on September 29, 2021 3350 Immunology: Wortis et aL Proc. Natl. Acad. Sci. USA 92 (1995)

or increased levels of CD23 or CD5 but, when combined with SSC/FSC CD44 CD5 CD23 fixed T cells, augmented proliferation (Table 1) and lowered CD23 levels (Fig. 2). Next we asked whether ligation of CD40 by gp39, the CD40L on activated T-helper cell membranes (10), was sufficient to Medium produce any of these changes in B cells. We consistently found that CD40L in the form of a soluble fusion molecule (CD40L- k CD8) induced proliferation and an increase in cell size and increased surface CD44 and CD23 expression without altering CD5 levels (Table 1 and Fig. 3). Therefore the signals gener- CD40L ated by crosslinking sIgM on the one hand and ligation of CD40 on the other are sufficient to induce different activated B-cell phenotypes. We considered the possibility that the CD5+ CD23 medium cells on the one hand and the CD5- CD23 high B cells on the Anti IgM other might represent two populations arising from different precursors. To test this possibility we cultured B cells with a combination of CD40L and F(ab')2. The cells responded by incorporating even higher levels of thymidine (Table 1) than with either reagent alone. Flow cytometry revealed a single LPS population of cells with little increase in either CD5 or CD23 surface expression (Fig. 4), consistent with there being a single population of multipotential precursors. If some B cells could respond only to slg crosslinking, while others could respond FIG. 3. Flow cytometry of small resting B cells after 42 hr of only to CD40 ligation, then two populations of activated cells stimulation. Top row, small resting splenic B cells after culture in should have been generated: CD5+ CD23 medium and CD5- medium only; second row, cells after-coculture with CD40L; third row, CD23hi. cells after stimulation with F(ab')2 fragments of anti-IgM; fourth row, In a similar experiment, we cultured B cells with F(ab')2 cells after coculture with LPS. The lefthand column shows forward with T cells. In this situation only (FSC) and side (SSC) light scatter. The second column shows CD44 (rather than Fab) together levels; the third, CD5; the fourth, CD23 levels. Negative control 11% of the B cells became CD5+ while cells cultured with is also shown. F(ab')2 in the absence of T-helper cells were 56% CD5+. This staining supports the notion that simultaneous TD- and TI-2-type levels were low on all B cells in cultures with T cells, but stimulation causes modulation of the induction of CD5. CD23 because activated T-cell supernate is sufficient to down- modulate CD23 induction, this observation does not provide SSC/FSC CD5 CD23 information on the possible effects of sIg ligation on the induction of CD23. I p We wished to determine if CD40L and sIg-induced B-cell Medium signaling utilized precisely the same elements (7). To test this 1 2% we took advantage of the fact that anti-IgM activation can be blocked by the immunosuppressants CsA and FK520 (16). L2zz .....i^These agents have many effects, but their ability to immuno- suppress T cells is contingent on their binding to one of several known collectively as immunophilins (16, 17). The resulting complex then inhibits the Ser/Thr phosphatase cal- T - SN

i 8% <1% Anti IgM CD40L Anti IgM + CD40L SSC/FSC

T fixed 44% 13% 7% I LO c]

T fixed + SN CD23 FIG. 4. Effects of stimulation of B cells with a combination of CD40L and F(ab')2 for 40 hr. Two-color flow cytometry shown as contour diagrams. First column, cells stimulated with F(ab')2 of anti-IgM; second column, B cells after culture with CD40L; third FIG. 2. Flow cytometry analysis of B cells. Top row, small resting column, cytographs of B cells cultured with both stimuli. The top row splenic B cells after coculture for 2.5 days without stimulation; second shows forward (FSC) and side (SSC) scatter. In the second row the x row, B cells in activated T-cell supernate (SN) alone; third row, cells axis shows CD23 staining (fluorescein isothiocyanate) and the y axis after culture with activated and fixed T-helper 2 CDC35 cells; fourth shows CD5 (phycoerythrin). The quadrants were drawn on control row, cells in the combination of supemate and fixed T cells. The (unstained) cells. The percentage of cells in each quadrant is shown. lefthand column shows forward (FSC) and side (SSC) light scatter. In this experiment 20% of the untreated B cells were CD5+ after The second column shows levels of CD5; the third, CD23. culture in medium alone. One of two similar experiments is shown. Downloaded by guest on September 29, 2021 Immunology: Wortis et at Proc. Natl. Acad. Sci. USA 92 (1995) 3351 cineurin (18). The phosphatase activity of is re- quired for the transport into the nucleus of several transcrip- 140- tion most NFAT, required for expression factors, particularly 120- of IL-2 and IL-4 (19, 20). Although not characterized in such E 100= detail, in B cells the actions of these immunosuppressants .0 a) appear to involve similar mechanisms. Fig. 5 shows that CsA 2 80- and FK520 blocked anti-IgM-induced thymidine incorporation 14- 60- but that CD40L-induced proliferation was relatively insensi- 40- \ _ tive to these drugs. 20 To determine if this immunosuppression required binding to 0- an immunophilin we performed an experiment using the Medium 3 10 30 100 200 FK520 analogue L685,818. This molecule behaves as an an- Sp-cAMPS uM tagonist of FK520 by competing for binding sites on FK506 Anti Ig-1 Anti Ig-2 s Anti Ig-3 binding protein (FKBP) forming a complex that does not I CD40L-1 CD40L-2 X- CD40L-3 inhibit calcineurin (21). Unlike FK520, CsA binds to cyclo- FIG. 6. Effect of the cAMP agonist Sp-cAMPS on B-cell incor- philin molecules, immunophilins that do not bind L685,818 poration of thymidine as measured after 42 hr of stimulation. Results (22). Thus, if activation is blocked by binding of drug to shown are from three separate experiments, each expressed as a immunophilins, then CsA, but not FK520, should suppress B percent of the values obtained in the absence of Sp-cAMPS. Loga- cells in the presence of L685,818. We observed that in the rithmic mean control (x 10-3) values for these three experiments: presence of L685,818 CsA blocked the thymidine uptake anti-IgM, 99, 25, 49; CD40L, 44, 43, 49; medium (no stimulation), 3, induced by anti-IgM but FK520 did not (Fig. 5). 3, 3. As a second test of possible differences in the signal DISCUSSION pathways activated by slg and CD40 we asked whether addition of Sp-cAMPS, a potent membrane-permeabPle cAMP ana- Klaus (2) showed that CsA inhibits B-cell production Qf logue (23), would differentially affect activ;ation (24, 25). antibody in response to TI-2 but not TI-1 or TD antigens. The Concentrations of Sp-cAMPS that inhibited anti-IgM activa- existence of alternative B-cell signal pathways was proposed tion had the effect of augmenting CD40L-mediated activation but did not exclude the possibility of two progenitors. Our (Fig. 6). observations on the combined effects of minimal TI-2 and TD stimuli are also most simply explained as due to individual cells A responding via different signal pathways and argue against the idea of separate precursors for TI-2 and TD responses. How- 100_ ever, we cannot rule out the formal possibility that there are

E two responsive B-cell populations and that activated TI-2 80- responders cause a down-regulation of CD23 on the TD 0 60- responders while at the same time the activated TD responders

0 cause a down-regulation of CD5 on the TI-2 responders. 40- Considerable evidence has accumulated indicating that the 20------OA- m signal pathways activated by minimal TI-2 and TD stimuli are different. The Klaus experiments mentioned above are im- portant, as are other more recent direct tests. First, it was 0-Medium 1 3 1 30 100 established that T-cell-dependent activation did not require sIg CsA ng/ml crosslinking (9), although in itself this did not show that the B signal pathways were different. Then it was shown that the activation through membrane immunoglobulin was more sen- sitive to the depletion of PKC by phorbol esters than that E induced by fixed, activated T-helper cells (12, 25). Experiments ._ 0 to demonstrate the relative dependency of anti-Ig and T-helper membranes on PKC and PKA have generated con- 0 tradictory results (12, 25-27). The sIg response appears to be PKC dependent and PKA independent, while the CD40 re- sponse is PKC independent. In any case, since these inhibitors 1 are only relatively specific, the interpretation of these results Medium 0.1 0.3 1 3 10 should be viewed with caution. Overall, the evidence generally FK520 ng/mi favors a dichotomy between the TD and TI induction pathways

Anti Ig >< CD40L Anti Ig + L685 -4 CD40L + L685 and our own experiments provide additional support for this FIG. 5. (A) Effect of CsA on proliferation of B cczlls assessed after view. stimulation for 42 hr. Also shown are the effects of CsA in the presence Agonists of cAMP down-modulate sIgM-mediated activa- of 300 ng of L685,818 per ml. Control thymidine inc:orporation (cpm tion (12) and we found that Sp-cAMPs had a similar effect. X 10-3) values: anti-IgM, 55.5; CD40L, 31.5; anti-Ig:M plus L615,818, Two groups reported an increase in intracellular cAMP levels 47.7; CD40L plus L615,818, 24.8; medium (no sstimulation), 1.3; in response to activated T-helper cells (24, 25), but this is not medium plus L615,818 (no stimulation), 1.2. One of three similar a uniform finding (27). If cAMP is increased by CD40 ligation, experiments is shown. (B) Effect of FK520 on prolifieration of B cells and if cAMP dampens the proliferation induced by anti-IgM, assessed after stimulation for 42 hr. Also shown aIre the effects of then the total proliferation induced by CD40L and anti-IgM in FKS20 in the presence of 300 ng of L685,818 per ml. IResults shown are combination might be expected to be no greater than that expressed as a percentage of the values obtained i induced either alone. the FK520. Control thymidine incorporation (cpm x 1(0-3) values: anti- by agent Therefore, augmented IgM, 58.9; CD40L, 35.8; anti-IgM plus L615,818, 416.4; CD40L plus proliferation following combined sIgM and CD40 stimulation L615,818, 30.1; medium (no stimulation), 1.4; mediuim plus L615,818 suggests crosstalk between the sIgM and CD40 signal path- (no stimulation), 1.4. One of three similar experime-nts is shown. ways. Downloaded by guest on September 29, 2021 3352 Immunology: Wortis et at Proc. Natl. Acad Sci USA 92 (1995) Previous reports indicate that ligation of CD40 in mice (28) 6. Mosier, D. E., Mond, J. J. & Goldings, E. A. (1977) J. Immunol. 119, and humans (29) does not generate an immediate Ca2+ flux as 1874-1878. 7. Cambier, J. C., Pleiman, C. M. & Clark, M. R. (1994) Annu. Rev. does slg ligation. Importantly, because L685,818 blocks the Immunol. 12, 457-486. rotamase activity of FKBP, but does not inhibit B-cell activa- 8. Parker, D. C., Wadsworth, D. C. & Schneider, G. B. (1980) J. Exp. tion, rotamase activity is not required for B-cell activation. Med. 152, 138-151. Although CsA and FK520 only partially blocked CD40- 9. Tony, H. P., Phillips, N. E. & Parker, D. C. (1985) J. Exp. Med. 162, mediated activation, this partial suppression is likely to be of 1695-1708. physiological because 10. Armitage, R. J., Fanslow, W. C., Strockbine, L., Sato, T. A., Clifford, significance the effects of FK520 but not K. N., Macduff, B. M., Anderson, D. M., Gimpel, S. D., Davis-Smith, CsA were reversed by L685,818 treatment. Our experiments T., Maliszewski, C. R., Clark, E. A., Smith, C. A., Grabstein, K. H., with CsA and FK520, drugs that suppress Ca2+-dependent Cosman, D. & Spriggs, M. K. (1992) Nature (London) 357, 80-82. signaling (17), indicate that signaling via CD40 may not require 11. Tony, H. P. & Parker, D. C. (1985) J. Exp. Med. 161, 223-241. utilization ofsuch a pathway. Possible counter evidence for this 12. Kawakami, K. & Parker, D. C. (1993) Eur. J. Immunol. 23, 77-84. 13. Lane, P., Brocker, T., Hubele, S., Padovan, E., Lanzavecchia, A. & conclusion is a report that CD40-mediated induction of NF- McConnell, F. (1993) J. Exp. Med. 177, 1209-1213. ATp expression is completely blocked by CsA treatment (30). 14. Burkly, L. C. & Wortis, H. H. (1985) J. Immunol. 135, 1577-1581. However, there is as yet no evidence that NF-ATp is required 15. Cong, Y.-Z., Rabin, E. & Wortis, H. H. (1991) Int. Immunol. 3, for the activation of B cells. It is also reasonable to consider 467-476. that an overlap or redundancy of signaling intermediates is 16. Sigal, N. H. & Dumont, F. J. (1992)Annu. Rev. Immunol. 10, 519-560. 17. Schreiber, S. L. & Crabtree, G. R. (1992) Immunol. Today 13, 136-142. utilized after stimulation of CD40 or IgM (31). 18. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I. & Our results establish that the minimum activating inducers Schreiber, S. L. (1991) Cell 66, 807-815. are sufficient to induce differences in B-cell phenotypes. The 19. Clipstone, N. A. & Crabtree, G. A. (1992) Nature (London) 357, significance of the induction of CD5 expression after TI-2 695-697. activation of B cells is not known. This molecule is found on 20. Jain, J., McCaffrey, P. G., Miner, Z., Kerppola, T. K., Lambert, J. N., Verdine, G. L., Curran, T. & Rao, A. (1993) Nature (London) 365, all mature T cells and a limited population of B cells, the 352-355. so-called B-la subset (32), and appears to be associated with 21. Dumont, F. J., Staruch, M. J., Koprak, S. L., Siekierka, J. J., Lin, C. S., the T-cell receptor (TCR) (33) and B-cell receptor (34). Yet, Harrison, R., Sewell, T., Kindt, V. M., Beattie, T. R., Wyvratt, M. & a recent report suggests that mice expressing a CD5 deletion Sigal, N. H. (1992) J. Exp. Med. 176, 751-760. mutant had no defect in immune responses (35), although the 22. Bram, R. J., Hung, D. T., Martin, P. K., Schreiber, S. L. & Crabtree, G. R. (1993) Mol. Cell. Biol. 13, 4760-4769. authors raised the possibility that lack of CD5 expression might 23. Van Haastert, P. J., Van Driel, R., Jastorff, B., Baraniak, J., Stec, increase responses to TCR stimulation. The origin W. J. & De Wit, R. J. (1984) J. Biol. Chem. 259, 10020-10024. of CD5+ B cells is a matter of controversy; some investigators 24. Pollok, K. E., O'Brien, V. O., Marshall, L., Olson, J. W., Noelle, R. J. believe that they derive from a precommitted pool of fetal/ & Snow, E. C. (1991) J. Immunol. 146, 1633-1641. neonatal precursors (32). Others, including ourselves, believe 25. Kato, T., Kokuho, T., Tamura, T. & Nariuchi, H. (1994) J. Immunol. 152, 2130-2138. that any naive [newly differentiated, "B-0" (36)] B cell can 26. Lalmanach-Girard, A. C., Chiles, T. C., Parker, D. C. & Rothstein, become CD5+ as a consequence of sIgM ligation (37, 38). T. L. (1993) J. Exp. Med. 177, 1215-1219. CD23, the low-affinity receptor for the Fc of IgE (Fc£RII), 27. Marshall, L. S., Shepherd, D. M., Ledbetter, J. A., Aruffo, A. & is a 45-kDa surface glycoprotein found on hematopoietic and Noelle, R. J. (1994) J. Immunol. 152, 4816-4825. follicular dendritic cells (39) but limited to the B-2 population 28. Hasbold, J. & Klaus, G. G. (1994) Eur. J. Immunol. 24, 152-158. 29. Knox, K. A. & Gordon, J. (1993) Eur. J. Immunol. 23, 2578-2584. of B cells (40). Up-regulation of CD23 occurs in mice and 30. Choi, M. S. K., Brines, R. D., Holman, M. J. & Klaus, G. G. B. (1994) humans following CD40 ligation by specific antibody or Immunity 1, 179-187. ligand (28, 41-43). Although CD23 is up-regulated on 31. Klaus, G. G. B., Choi, M. S. K. & Holman, M. (1994) Eur. J. Immunol. previously activated B cells by IL-4, it is not expressed on 24, 3229-3232. recently activated B cells within germinal centers (39), 32. Herzenberg, L. A. & Kantor, A. B. (1993) Immunol. Today 14,79-83. 33. Beyers, A. D., Spruyt, L. L. & Williams, A. F. (1992) Proc. Natl. Acad. perhaps as a consequence of IL-6 activity (15). Interestingly, Sci. USA 89, 2945-2949. IL-4, an inducer of CD23, suppresses CD5 (44-47), and, as 34. Lankester, A. C., van Schijndel, G. M. W., Cordell, J. L., van Noesel, shown here, CD40 ligation, another inducer of high levels of C. J. M. & van Lier, R. A. W. (1994) Eur. J. Immunol. 24, 812-816. CD23, also down-modulates the induction of CD5 by anti- 35. Tarakhovsky, A., Muller, W. & Rajewsky, K (1994) Eur. J. Immunol. IgM. Thus the reciprocal induction of CD5 and CD23 on B 24, 1678-1684. 36. Haughton, G., Arnold, L. W., Whitmore, A. C. & Clarke, S. H. (1993) cells appears to be a general characteristic of normal B-cell Immunol. Today 14, 84. signaling. 37. Arnold, L. W., Pennell, C. A., McCray, S. K. & Clarke, S. H. (1994) Previously it was suggested that a generally held view of J. Eap. Med. 179,1585-1596. B-cell activation, that antigen-mediated slg crosslinking is a 38. Teutsch, M., Higer, M., Wang, D. & Wortis, H. H. (1995) Int. necessary first step for TD activation (48), may not be true (9). Immunol. 7, 381-392. 39. Conrad, D. H. (1990) Annu. Rev. Immunol. 8, 623-645. We now propose that the quality of the initial activation event 40. Waldschmidt, T. J., Kroese, F. G. M., Tygrett, L. T., Conrad, D. H. & may commit a B cell to a particular differentiation pathway and Lynch, R. G. (1991) Int. Immunol. 3, 305-315. a distinct set of functions. 41. Saeland, S., Duvert, V., Moreau, I. & Banchereau, J. (1993)J. Exp. Med. 178, 113-120. We acknowledge with thanks the experimental contributions of 42. Katira, A., Knox, K. A., Finney, M., Michell, R. H., Wakelam, M. & Simon Anderson, Dana Schmidt, Robert Seward, Damien Slater, and Gordon, J. (1993) Clin. Exp. Immunol. 92, 347-352. Camellia Symonowicz. This research was supported, in part, by 43. Maliszewski, C. R., Grabstein, K., Fanslow, W. C., Armitage, R., National Institutes of Spriggs, M. K. & Sato, T. A. (1993) Eur. J. Immunol. 23, 1044-1049. Health Grants P01 A133180, R01 A115803, 44. Hidaka, T., Kitani, A., Hara, M., Harigai, M., Suzuki, K., Kawaguchi, lF32AI0918301, and R01 AI24303. Y., Ishizuka, T., Kawagoe, M. & Nakamura, H. (1992) Clin. Exp. Immunol. 89, 223-229. 1. Sharon, R., McMaster, P. R. B., Kask, A. M., Owens, J. D. & Paul, 45. Jyonouchi, H., Voss, R. M. & Good, R. A. (1990) Cell. Immunol. 130, W. E. (1975) J. Immunol. 114, 1585-1589. 66-78. 2. Klaus, G. G. B. (1988) Transplantation 46, Suppl., 11-14. 46. Freedman, A. S., Freeman, G., Whitman, J., Segil, J., Daley, J., 3. Mongini, P. K. A., Paul, W. E. & Metcalf, E. S. (1982) J. Exp. Med. Levine, H. & Nadler, L. M. (1989) Eur. J. Immunol. 19, 849-855. 155, 884-902. 47. Zupo, S., Dono, M., Massara, R., Taborelli, G., Chiorazzi, N. & 4. Umetsu, D. T., Chapman-Alexander, J. M. & Thorbecke, G. J. (1979) Ferrarini, M. (1994) Eur. 1. Immunol. 24, 1426-1433. J. Immunol. 123, 396-404. 48. Abbas, A. K., Lichtman, A. H. & Pober, J. S. (1994) Cellular and 5. Forum, M. S. & Pure, E. (1991) J. Exp. Med. 173, 687-697. Molecular Immunology (Saunders, Philadelphia). Downloaded by guest on September 29, 2021