and a research grant from the New York Chapter of the Arthritis Foundation. A.P. was ..... IgM, IgG, and IgA standards (Rockland, Gilbertsville, PA) or IgE stan-.
T-BAM/CD40-1 on Helper T Lymphocytes Augments Lymphokine-Induced B Cell lg lsotype Switch Recombination and Rescues B Cells from Programmed Cell Death’ Seth Lederman,** Michael J.Yellin,* Aileen M. Cleary,* Alessandra Pernis,* Giorgio Inghirami,+ Lauren E. Cohn,* Lori R. Covey,* Julie J. Lee,* Paul Rothman,* and Leonard Chess* *Departments of Medicine and ‘Pathology, Columbia University, College of Physicians and Surgeons, New York, NY 10032
An important component of T cell help for B lymphocyte differentiation is the contact-dependent signaling mediated by the T cell-B cell activating molecule (T-BAM/CD40-L), an activation-induced surface membrane protein on CD4’ T helper cells in lymphoid follicles that interacts with the B cell surface molecule, CD40. The present study dissects the roles of T-BAMICD40-L inhelper function by means of a neutralizing anti-T-BAM/CD40-L mAb (5c8), a T-BAM/CD40-L-expressing T cell tumor subclone (Jurkat D l .l), and a T-BAM/CD40-L-responsive lgM’ B cell tumor of germinal center origin (RAMOS 266). Like activated T cells, Dl .1 cells induce B cells to synthesize IgG, IgA, and IgE in a process that is specifically inhibited by the mAb 5c8. Although rlL-4 alone, but not Jurkat D l .l,induces IgH C y mRNA transcripts in RAMOS 266, theT-BAM/CD40-L moleculeon D l .1 acts on rll-4-primed RAMOS B cells to augment expression of C y transcripts. In addition, IgG’ RAMOS 266 clones were expanded from D l .1- and rlL-4-stimulated cultures that had undergone deletional IgH isotype switch recombination events. Furthermore, T-BAM/CD40-L signals delivered by the D l .1 clone dramatically rescue RAMOS 266 from mAb anti-lgM-induced apoptosis. Taken together, these data support the idea that T-BAM/CD40-L plays important roles in inducing Ig isotype switch recombination and the clonal selection of isotype-switched B cells. Journal of Immunology, 1994, 1 52: 21 63.
T
lymphocytes provide critical signals, termed “help,” to B cells that induce and refine the humoral (Ab-mediated) immune response by participating in the clonal selection and differentiation of Agspecific B cells(1, 2). The T-dependentselection and differentiation of B cells are also associated with the rearrangement of the productive Ig heavy chain gene locus Received for publication May19, 1993. Accepted for publication December4, 1993.
of The costs of publication of this article were defrayed in part by the payment page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
’
This work was supported in part by National Institutes of Health Grants RO1-CA-55713 to S.L.; Pol-AI-26886 and RO-1-AI-14969 to L.C.; and RO-1A133450 to P.R. S.L. is an Arthritis Investigator of the Arthritis Foundation. M.Y. is the recipient of a postdoctoral award from the National Arthritis Foundation and a research grant from the New York Chapter of the Arthritis Foundation. A.P. was supported by a National Institutes of Health Immunology Training Scholar Award. Grant. P.R. is the recipient ofa Pfizer Scholar Award and a Pew
’Address correspondence and reprint requests to Seth Lederman, Black Building 81 2, Columbia University, 630 W. 168th St., New York, NY 10032. Copyright 0 1994 by The American Association of Immunologists
to express Abs with constant (C) domains that encode distinct effector functions in a process termed “isotype switching” (3-6). The T-dependent selection and differentiation of B cells occurs in vivoon a population of germinal center B cells undergoing programmed cell death (apoptosis) (7). Theprecisemechanisms by which T cellsinduce isotypeswitching and participate in the selection of Ag-specific Bcellsare not well understood but involvebothcontact-dependent and lymphokine-mediated signals (8-13). Recently, an activation-induced surfacemolecule on CD4+ T cells, which directs B cell activation, proliferation, and differentiation into Ab-forming cells, was identified by a mAb (mAb 5c8) and termed T-BAM3 (14, 15). T-BAM was found to be expressed nearly exclusively by
Abbreviations used in this paper: T-BAM or T-BAM/CD40-L, activatingmolecule;HIM,X-linkedhyper-lgMsyndrome;IMDM, Modified Dulbecco Medium.
T cell-B cell Iscove’s
0022-1 767/94/$02.00
21 64
ROLES OF T-BAM/CD40-L SIGNALS IN B CELL DIFFERENTIATION
CD4+ T cells located in the mantle and centrocytic zones of lymphoid follicles, where helper function and isotype switching occur in vivo (6, 16, 17). In addition, T-BAM was found to be identical to a recently described T cell ligand for the B cell CD40 molecule, termed “CD40-ligand” (CD40-L), TRAP, or gp39 (18-22) (Covey et al.).4 The functional effects of T-BAM/CD40-L interactions with CD40 are incompletely known, but several potential roles for this interaction in B cell differentiation are suggested by experiments with anti-CD40 mAb(23-25). First, anti-CD40 mAbs, in association with other signals, induce B cell activation and proliferation (23,25). Second, anti-CD40 mAbs presented on human FcRyII-expressing mouse fibroblasts (L cells), in conjunction with lymphokinessuch as rIL-4, induce proliferation and longterm growth ofhuman B cells secreting multiple Ig isotypes (26, 27). Third, anti-CD40 prevents spontaneous programmed cell death (apoptosis) of cultured human germinal center B cells (7, 28), as well as the anti-IgM-induced apoptosis of germinal center B cell tumor lines (29, 30). The relationship of these effects of anti-CD40 mAb to the functional roles of T-BAM/CD40-L is unclear because the valency and/or cellular association of anti-CD40 mAb are known to contribute importantly to their observed effects on B cells. For example, soluble anti-CD40 mAb induce IgE (31, 32) and inhibit apoptosis (7, 28), whereas only cell-associated anti-CD40 mAbs induce IgG and IgA secretion (26, 27). Moreover, anti-CD40 mAbs generally induce B cell functions, such as theinduction of IgE, whereas these same “stimulatory” anti-CD40 mAbs inhibit T-B contact-dependentB cell activation measured by CD23 expression (15). Whatever the precise roles of T-BAM/CD40-L signalling in B cell differentiation, T-BAM/CD40-L molecules were found to be necessary for isotype switching in man because the T-BAM/CD40-L gene encodes dysfunctional proteins in patients withtheX-linkedhyper-IgM syndrome(HIM) (33-36), a serious genetic immunodeficiency in which affected individuals have elevated serum IgM but low or absent serum IgG, IgA, and IgE (37, 38) related to defective T cell help (39,40). How the defective expression of T-BAM/CD40-L results in the phenotype of HIM is not clear, however, and may relate either to the absence of germinal centers in HIM, where isotypeswitching and clonal selection of B cells normally occurs (7), or to specific roles of T-BAM/CD40-L in these processes. In the present report we studied the role of T-BAMI CD40-L signaling in the inductionof Ab isotype secretion, isotype switching, and apoptosis of B cells. Ab inhibition studies with the mAb 5c8 show that T-BAM/CD40-L is involved inthe induction of B cells to synthesize IgG, IgA, and IgE driven by normal T cells or specifically by the Covey, L. R., A.M. Cleary, M. J.Yellin, R. W. Ware, G. Sullivan, I. Belko, M. Parker, J. ]. Lee, P. Rothrnan, L. Chess,and s. Lederman. 1994. Isolation of cDNAs encoding T-BAM, a surface protein on T lymphocytes mediating contact-dependent helper functionfor B cells: identity with the CD4O-ligand. Mol. Irnrnunol. In press.
T-BAM/CD40-L+D1.l cell line. The effects of T-BAM/ CD40-L on distinct steps of B cell differentiation were studied by means of the RAMOS 266 B cell clone, which had previously been shown to respond to T-BAM/ CD40-L- and/or rIL-4-mediated signals (15). In this report, RAMOS 266 cells are shown to respond to rIL-4 by expressing low levels of Cy germ-line and productive mRNA transcripts and to respond to anti-IgM by undergoing apoptosis. D1.l cells are shown to augment rIL-4induced germ-line and productive Cy mRNA in a process that seems to result in deletional IgH class switch recombination. In addition, T-BAM/CD40-L signals are shown to rescue anti-IgM-induced RAMOS 266 cells from apoptosis. Critical rolesfor T-BAM/CD40-L inthe outgrowth of switched cells and the rescue fromapoptosis are demonstrated by the findings that the mAb 5c8 inhibits the effects of D1.l on boththe synthesis of germ-line and productive Cy mRNA and on the rescue from apoptosis. Taken together, thesedata suggest roles for T cell T-BAM/ CD40-L in thematuration of the humoralimmuneresponse by providing signals that induce the differentiation of B cells at several distinct steps.
Materials and Methods Cell lines The Jurkat clones D1.l and B2.7have been described (14, 41). RAMOS 266, 4CN 3F10 (RAMOS 266) was a kind gift of Dr. Jay Siegle, Food and Drug Administration, Bethesda, MD (42).
A bs The anti-T-BAM/CD40-L mAb (5c8) is a murine IgG2a that has been described (14). The anti-CD4mAb (OKT4), anti-MHC Class I mAb (W6/32), anti+ light chain (HP6053), anti-A light chain (HP6054), antiIgM (DA4-4), anti-IgG (C3-124) and anti-IgG1 (HP-6001) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). mAbs werepurified from ascites fluid on protein A (Biorad Corp., Rockville Center, NY) or protein G columns (Pharmacia, Uppsala, Sweden). Goat anti-IgM-biotin and goat anti-IgG-biotin were purchased from TAG0 (Burlingame, CA). The anti-CD40 mAb (G28.5) was the gift of Dr. Ed Clark, University of Washington, Seattle, WA.
Isolation of cell populations PBLs were obtained from the freshly drawn blood of healthy volunteers by centrifugation on Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO). T cells were positively selected with neuraminidase-treated SRBC. B cells were derived from the population of cells that did not pellet through Ficoll-Hypaque after two rounds of rosetting with neuraminidase-treated SRBC. B cells were further separated from monocytes by overnight adherence in polystyrene flasks (37% 5% CO, ).
T-B cultures 250,000 B cells werecultured together with an equal number of T cells in 17 x 17 polystyrene tubes in a total volume of 1 mlof Iscove’s Modified Dulbecco Medium (IMDM) with 10% FCS at 37°C and 5% CO, for 7 to 14 days. PWM was added at a final concentration of 1:400. For mAb inhibition studies, mAb 5c8 or OKT4 was added at a final concentration of 500 ngiml. In experiments involving Jurkat D1.l or B2.7 cells, the Jurkat cells were added at a ratio of 0.6:l Jurkat:B cells. To study IgE production, 250,000 B cells were cocultured with 250,000 T cells in a total volume of 1 ml for 14 days at 37°C and 5% CO,. The cultures were supplemented with 300 U/ml of rIL-4 (rIL-4 was the kind gift of Dr. Satwant Narula, Schering-Plough Corp.). The positive control consisted of anti-CD40 mAb G28.5 at final concentration of 1 &ml and
21 65
Journal of Immunology
rIL4 (300 Uiml). In the IgE experiments, D1.l or B2.7 cells were cultured at a 1:l ratio with B cells.
Mitomycin-C treatment Jurkat cells (lo'iml) were treated with 50 pg/ml mitomycin-C (Sigma) for 45 minutes at 37°C. The mitomycin-treated cells were then washed three times, resuspended in mitomycin-free media, then cultured for 1 h at 37°C. The cells were washed three additional times before use in cell culture experiments.
Induction and cloning of IgG+ RAMOS 266 RAMOS 266 (30cells/well) were seeded onto 5000 mitomycin-C-treated D1.l or B2.7 cells in 96-well round bottom plates (NUNC, Draperville, IL) in the presence or absence of rIL4 (300 U/ml). Control plates did not contain any D1.l or B2.7. Cultures were incubated at 37°C in a 5% CO, atmosphere and wells with significant cell growth were initially screened by IgG ELISA. Any well that gave a value >2 ngiml was expanded in 24-well plates, then retested by isotype-specific ELISA and FACS analysis by use of anti-IgG or anti-IgM Abs. Positive clones wereconsidered those that gave repeated values >2 ngiml on Ig ELISA. By FACS analysis, such cultures typically gave a bimodal distribution for sIgM staining. IgG' and IgM+ RAMOS 266 clones werederived by several rounds of cloning at limiting dilution (0.3 cellsiwell) and screening growing cultures for IgG secretion by ELISA.
Measurement of lg production Ig secretion was measured by ELISA. Briefly, 96-well flat bottom microtiter plates (Dynatech, Chantilly, VA) were coated overnight at 4°C with affinity-purified goat anti-human IgG (at 1/1000), IgM or I g A (both at 1/5000)(Tago, Burlingame, CA) in 0.1 Mcarbonate buffer pH 9.6. The plates were washed 5 times, then blocked with 1% BSA in PBS for 1 h at room temperature. The samples (100 pl) were then incubated at room temperature for 2h. After washing, goat anti-human IgM, IgG, or IgA (at li5000) antiserum conjugated to alkaline phosphatase was added for 2 h at room temperature. The plates were then washed and the substrate solution, p-nitrophenyl phosphate, was added. In experiments measuring IgG subclasses, supernatants were studied with a quantitative IgG subclass kit (Binding Site, Birmingham, UK). IgE measurements were derived by use of an amplified ELISA method. Ninety-six-well flat bottom plates were coated with goat antihuman IgE F(ab'), at 11500 dilution (DIAMED, Windam, MN) overnight at 4°C in 0.1 M carbonate buffer, pH 9.6. After washing, the plates were blocked with 1%BSA in PBS at room temperature for 1 h, then samples (100 p1) were added and incubated for 2 h at room temperature. Plates were washed and a goat anti-human IgE F(ab'), antiserum conjugated to alkaline phosphatase (1/500) (Diamed, ME) was added for 2 h at room temperature. Plates were then washed in 0.05 M Tris buffered saline. The ELISA amplification system (GIBCO BRL, Grand Island, N.Y.) was reconstituted according to manufacturer's instructions. Fifty p1 of the alkaline phosphatase substrate (NADPH) was added for 12 min at room temperature, then 50 pl of the amplifier (alcohol dehydrogenase and diaphorase) was added for12 min at room temperature. The sensitivity of the assay was typically 250 pgiml IgE. The optical density (OD) was read at 405 nm (IgG, IgA, IgM) and at 490 nm (IgE) on a Molecular Devices ELISA reader (Menlo Park, CA). Experimental samples were studied in triplicate and Ig concentrations were determined by extrapolation from standard curves of dilutions of IgM, IgG, and IgA standards (Rockland, Gilbertsville, PA) or IgE standards (Chemicon, Temecula, CA). Background (B) Ig levels (induced by B cells + PWM) were subtracted from the experimental means (A). The standard deviation (SD) of the subtracted means (A-B) was obtained by the formula SD(A-B) = SQRT (SD(A)' + SD(B)').
Apoptosis To cross-link sIgM, 20 X lo6 RAMOS 266 cellsiml were resuspended in anti-IgM supernatant (mAb DA4-4) (1:4) or control mAb W6132 (10 pgiml) for 0.5 h at 4°C. Cells were then pelleted by centrifugation and washed three times in MEM. Cells were then treated with 0.25 ml beads (Dynabeads, Advanced Magnetics, Cambridge, MA) coated with goat anti-mouse IgG for 0.5 h at 4°C. After treatment, cells bound to heads were collected by a magnet and resuspended at 6 X lo6 celliml in IMDM/10% FCS.
To measure DNA fragmentation, 3 x lo6 RAMOS 266 cells (either pre-cross-linked or control) and/or 3 X lo6 Jurkat T cells (Jurkat D1.l or B2.7) were added to indicated wells in IMDM/10% FCS in a final volume of 1 ml. Where indicated, rIL-4 (DNAX) was added to 300 Uiml. For Ab inhibition studies, D1.l cells were preincubated with either mAb 5c8 (10 pgiml) or OKT4 (10 pgjml) for 30 min at room temperature before addition to cultures containing cross-linked RAMOS 266. After 44 h at 3 7 T , cells were harvested, pelleted by centrifugation (2000 rpm for 5 min in an Eppendorf microfuge) and washed twice in HBSS. Cell pellets were then resuspended in lysis buffer (10 mM EDTA, 200 mM NaCI, 0.1 rngiml proteinase K, 0.5% SDS, 50 mM Tris, pH 8.0) and incubated for 1 h at 50°C. Genomic DNA was extracted from lysed cells by phenolisevag, precipitated with ethanol, and removed by a glasshook. Fragmented DNA was reprecipitated by the addition of 1/10 volume of 3 M sodium acetate (pH 7.2) followed by ethanol and cooled to - 80°C for 3 h. Samples were then centrifuged at 13,000 X g for 30 min at 4°C (microfuge). Pelleted DNA was washed with 70% ethanol, dried, and resuspended in 20 p1 RNAse buffer (0.5 mg/ml RNAse A, 15 mM NaCI, 10 mm Tris, pH 7.5) for 1 h at 50°C. DNA was then diluted 1:2 in gel loading buffer (25 mM EDTA, 40% sucrose, 0.5% bromphenol blue), heated for 10 min at 68"C, and loaded on a 2% agarose gel containing 0.1 pgiml ethidium bromide, and electrophoresed, and UV fluorescence was photographed.
Southern blot hybridization analysis Genomic DNA was prepared from mononuclear cell suspensions by cesium chloride as previously described (43) or, alternatively, by a saltingout procedure not requiring organic extraction (44). Briefly, the cells were resuspended in 3 ml of nucleic lysis buffer containing 10 mM TrisHCI, 400 mM NaCI, and 2 mM EDTA 200 p1of 10% SDS and 500 &I of proteinase K solution (1 mg proteinase K in 1% SDS and 2 mM EDTA) were subsequently added. After an overnight digestion at 37"C, 1 ml of saturated NaCl was added, This mixture was centrifuged at 2500 rpm for 20 min and 2 vol of ethanol was added to the supernatant to precipitate the DNA, which was washed several times in 70% ethanol and resuspended in Tris-EDTA. Aliquots (5 pg) of genomic DNAs were digested with the appropriate endonucleases according to the manufacturer's recommendation (Boehringer Mannheim), and electrophoresed on 0.8% agarose gels before transfer to nitrocellulose. The filters were hybridized in 50% formamidei3X SSC at 3 7 T , washed in 0.2X SSC/0.5% SDS at60°C for 2 h, and autoradiographed. DNA fragments were labeled with [ L X - ~ ~ P ] ~by C TaPrandom priming technique for use as probes. The Ig heavy chain (IgH) joining region gene (JH) was investigated by hybridization of Hind111 and BamHI digested DNAs to a JHprobe (451, and the IgH constant region genes (Cp or Cy) were studied by hybridization of BamHl digested DNAs to C p (50) or Cy (46) gene region probes.
RNA protection assays RAMOS 266 cells (1.2 X lo5 cells) were cultured in 1 ml of IMDM/lO% FCS, either alone, or with mitomycin-C-treated Jurkat cells (D1.l or B2.7) at a RAM0S:Jurkat ratio of 1:lO in the presence or absence of rIL-4 (400 Uiml). In addition, in indicated cultures, mAbs were added to the wells before RAMOS 266 for a final concentration of 500 ngiml. Cultures were replated every three days with freshly prepared mitomycin-C-treated Jurkat cells, rIL-4, and mAb, each at the original concentration. At 3, 6, and 10 days, cultures were harvested and mRNA was isolated by an LiCl extraction method. The prohe used in the RNA protection assays was generated by using a 440-bp Sac1 fragment of a germline y3 cDNA (Sxy3 was generously provided by T. Honjo) which was subcloned into pBluescript SK I1 (Stratagem, La Jolla, CA). This probe contains contiguous sequences of the I (germ line) region and the CH1 domain of Cy3 and protects mRNA from Iyl and Iy3 regions, as well as all of the four human Cy genes due to sequence homology in the CHI domains of these genes. A s R N A probe was synthesized with T3 polymerase, uniformly labeled with 3zP, and 100,000 counts of the probe were hybridized to 10 p g of total cellular RNA for 15 h in 80% formamide, 40 mM Pipes, 1 mM EDTA, 400 mM NaCI, at 55" to 65°C. Hybrids were then incubated for 1 h with 100 U of S1 nuclease and subjected to electrophoresis on a 6% polyacrylamide-urea gel.
21 66
ROLES OF T-BAM/CD40-L SIGNALS IN B CELL DIFFERENTIATION
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FIGURE 1 . Effect of T-BAM/CD40-L on secretion of Ig isotypes. Shown are the Ig isotypes: (a) IgG, (b) IgM, (c) IgA, and ( d ) IgE produced by cultures of B cells driven by normalT cells (cross-hatched bars) or by T-BAM' Dl .1 cells (rising striped bars). Ig isotypes were determined by quantitative ELISA. B cells were stimulatedwith (a-c) PWM or (d)rlL-4 (300 U/ml). Background (a-c) (B + PWM) or ( d )(B + rlL-4) was subtracted. The amount of Ig induced by theT-BAM/CD40-L- Jurkat clone 82.7 inthe presence of PWM was: IgG, 132 (f37) pg/ml; IgM, 171 (+-25)pg/rnl; IgA, 9.5 ( k8.0) pg/ml. The amount of IgE induced by the T-BAM/CD40-L- lurkat clone B2.7 in the presence of rlL-4 was as follows: media, 720 ( k 2 4 ) pg/ml; mAb 5c8, 283 ( k13) pg/ml; and OKT4, 128 ( k 13) pg/ml. Errors represent S E M from triplicate cultures and interpolation from standard curves.
Results Effect of mAb 5c8 (anti-T-BAM/CD40-L) on the secretion of lg isotypes Activated CD4' Tlymphocytes provide contact-dependent and lymphokine-mediated signals that direct B lymphocytes to differentiate into Ab-forming cells expressing different Ig isotypes. We previously reported the generation of an mAb, termed 5c8, that inhibits contact-dependent helper processes and immunoprecipitates the T-BAM/CD40molecule(14). To address the role of T-BAM/CD40-L signaling on the induction of B cell Ig isotype switching, we extended previous studies (14) and cultured normal T and B lymphocytes with PWM or IL-4 for 7 to 14 d in the presence or absence of the neutralizing anti-T-BAM/CD40-L mAb, 5c8. In the presence of PWM, T cells induce synthesis of IgG, IgM, and IgA (Fig. L4-C). The mAb 5c8, but not control mAb, inhibits the secretion of IgG (Fig. 1A) and IgA (Fig. l e ) , but has no effect on the secretion of IgM(Fig. 1B). Similarly, the role of T-BAM/CD40-L in T-dependent, rIL-4 driven IgE synthesis was studied with the mAb 5c8. The mAb 5c8, but not control mAb, inhibits the T-dependent induction of IgE synthesis(Fig. l o ) . These data suggest that T-BAM/
CD40-L plays an important role in the in vitro induction of IgG, IgA, and IgE synthesis, but that T cells can induce IgM secretion in a non-T-BAMKD40-L-dependent manner at least from populationsof peripheral B cells containing both resting and activated cells. Effects of T-BAM/CDLFO-L signals on B ceff secretion of lg isotypes
To further define the role of T-BAM/CD40-L in isotype switching, we studied the T-BAMICD40-L specificeffects on B cell isotype secretion by supplying T-BAM/CD40-L signals by the T-BAM/CD40-L' Jurkat T cell lymphoma D1.1 (14, 41). Previously, we showed that the D1.l clone, but not the T-BAM/CD40-L- B2.7 clone, induces B cell differentiation into Ab-forming cells in a manner that is inhibited by the mAb 5c8 (14, 41). We have recently extended the molecular analysis of D1.l and B2.7 by analyzing their expression of T-BAM/CD40-L mRNA. The D1.l clone, which expresses T-BAM/CD40-L protein, expressesT-BAM/CD40-L-specificmRNA,whereas the B2.7 clone,whichlacksT-BAM/CD40-L protein, also lacks T-BAM/CD40-L mRNA (Covey et
Journal of Immunology
The next series of experiments utilized theseclones (D1.l and B2.7) to further define therole of T-BAM/ CD40-L in the induction of Ab synthesis by normalB cells. The D1.l clone, but not the T-BAM/CD40-L- B2.7 clone, induces IgM,IgG, and I g A in PWM-stimulated cultures (Fig. la-c) and IgE secretion in IL-4 stimulated cultures (Fig. I d ) . The effect of D1.l on IgG, IgA, and IgE secretion is inhibitedby the mAb 5c8, but not by a control mAb, demonstrating a role for T-BAM/CD40-L in the induction of IgG, IgA, and IgE synthesis (Fig. 1). Induction of IgM secretionby D1.l is also inhibitedby the mAb 5c8, is in contrast to T cell-induced IgM secretion, which is not affected by mAb 5c8 (Fig. lb). The differential effects of mAb 5c8 on IgM secretion are not unexpected because, whereas D1.l may be relatively limited to signalingB cells via T-BAM/CD40-L, it is known that T cells induce I3 cells (particularly circulatinglowdensityBcells) to secrete IgM by multiplemechanisms(reviewed in Ref. 47).Together,theseobservationsindicate that T-BAM/ CD40-L plays a role in the T cell-driven secretion of IgG, IgA, and IgE and appears to have a less critical role in IgM induction.These in vitro data areconsistent with the in vivo phenotype of T-BAM/CD40-L defects in the Xlinked HIM syndrome, in which IgG, IgA,and IgE are low or absent, but IgM is elevated (38). Studies on T-BAM/CD40-L functions in a tumor cell model of T-B interactions
In order to studythemechanism by whichT-BAM/ CD40-L induces the secretion of multiple Ig isotypes, the next series of experiments addressed the role of T-BAM/ CD40-L and rIL-4 in isotype switching. We have previously shown that the CD40' RAMOS 266 B cell tumor subclone is responsive to both rIL-4 signals and T-BAM/ CD40-Lsignals(15).Therefore,theinitialexperiments asked whether rIL-4 signals, T-BAM/CD40-L signals, or combinations of these signals would induce isotype switching in RAMOS 266 B cells. We utilized an mRNA protection assay to study the roles of T-BAM/CD40-L and rIL-4 signals on Iy (germ line) and VDJ-Cy (productive) transcripts in RAMOS 266cells.Intheseexperiments, RAMOS 266cellswerecoculturedwitheither D1.l or B2.7 cells in the presence or absence of rIL-4 (Fig. 24). Independent of T cell contact, rIL-4 induces low levels of both I-y RNA and VDJ-Cy RNA (Fig. 24, lane 3). In contrast, in the absence of rIL-4, neither D1.l (Fig. 2A, lane 5 ) nor B2.7 (Fig. 24, lane 4 ) induces synthesis of y-hybridizing RNA. In the presenceof rIL-4, however, the D1.l clone (Fig. 2 A , lane 7), but not the B2.7 clone (Fig. 2 A , lune 6), augments the level of rIL-4-induced germ line and productive Cy RNA. Further, the D1.l effects on augmenting RAMOS 266 Cy transcription are specifically inhibited by the mAb 5c8 (Fig. 2 B , lane 3) and not by control mAbs (Fig. 2 B , lane 4). These studies indicate that T-BAMKD40-L augments the expression of both germ-line Cy and productive IgG-encoding transcripts.
21 67
Cloning and characterization of T-BAM/CD40-L induced /gG+ RAMOS 266 cells
To determinetherelationship of the T-BAM/CD40-Linduced Cy mRNAto isotype switching, thenext series of experimentssought to expand and characterize Cy-expressing RAMOS 266 clones. In these experiments, multiply subcloned IgM' RAMOS 266 B cells wereseeded (at 30 cells/well) in empty wells, or wellscontaining a feeder layer of mitomycin-C treated D1.l or B2.7 T cells in the presence or the absence of rIL-4. The RAMOS 266 cultures were analyzed forisotype switching by measurement of supernatant Ig by isotype-specific ELISA over a 44-day period during which medium was periodically replenished but no additional rIL-4 or Jurkat feeder cells were added. The combination of D1.l feeder cells and rIL-4 resulted in an augmentation of the number of IgG-secreting RAMOS 266cultures, and RAMOS266cells were cloned from these D1.l and rIL4-induced cultures that grew without exogenous rIL4 or D1.1 cells and were found to express either surface IgG or surface IgM (data not shown). To define molecularly whetherthestablyswitched, IgG' RAMOS 266 cells had undergone class switching by gene recombination, Southern analysis was performed on genomic DNA from 3 IgG' RAMOS clones, 3 IgM' control clones, and 3 IgM' parental lines with probes for the C p and Cy exons (Fig. 3). All 3 IgG' clones express surface IgGl by FACS and secrete IgGl by isotype-specific shown). ELISA Southern (not blotting revealed that the coding Cp exon is deleted in three of three IgG' clones but not in six IgM' clones examined (Fig.3). Further, the Cy1 exons in the parental RAMOS 266 clone (or control, D1.1/ rIL-4 cultured, unswitched controls) had been rearranged in all IgG' clones, but in none of the IgM' clones examined (Fig. 3). In addition, cDNAs from two IgG+ clones were sequenced and found to contain the clonotypic V,1 in framewiththe Cy1 region encoding the F, domain demonstrating conclusively that the switch recombination events result in the expression of IgG protein (data not shown). Further, the fact that both Southern and sequence analysis reveal distinct recombination events, supports the idea that class switch recombination was occurring during the culture period and was not a result of D1.l inducing the outgrowth of a single IgG' RAMOS that might have contaminated the initial IgM' RAMOS266 clone. Together, these data suggest that the T-BAM/CD40-L signals provided by D1.l cells, which augment rIL4-induced Cy mRNA, seem to also drive the process of deletional class switch recombination. Effect o f T-BAM/CD40-L signals on anti-lgM induced apoptosis o f RAMOS 266 cells
Because it is known that apoptosis is a general feature of germinal center B cells undergoing Ag selection, and because anti-CD40 mAbs inhibit apoptosis of germinal center Bcells (7, 28), we directly asked whether T-BAM/ CD40-L interactions with CD40 modulate apoptosis. The
ROLES OF T-BAM/CD40-L SIGNALS IN B CELL DIFFERENTIATION
2168
A i cp Probe
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-
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3
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C germllney cDNA
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300
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Cy-hybrldlzlng RNA
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-300
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FIGURE 2. Effect of T-BAWCD40-L and rlL-4 on expression of y transcripts in the RAMOS 266 cell line. Shown are the results of S1 nuclease protection assays (A, B) of bulk cultures of RAMOS 266 using a labeled probe to detect germline or productive Cy transcripts (0.(A) RAMOS 266 cells were cultured with or without rlL-4 in the presence or absence of different Jurkatcells (clones D l .1 or B2.7). RNA was harvested at day 6 of culture and analyzed by RNA protection assay. pBR322IMspl markers are shown. Lane I, probe alone; lane 2, RAMOS 266 cultured alone; lane 3, RAMOS 266 cells cultured with rlL-4; lane 4, RAMOS 266 cells cultured with 82.7; lane 5, RAMOS 266 cells cultured with D l .l;lane 6, RAMOS 266 cells cultured with rlL-4 and B2.7; lane 7, RAMOS 266 cells cultured with rlL-4 and D l .l. (6) Effect of mAb 5c8 on Cy transcripts. Lane 7, probe alone; lanes 2-4, RAMOS 266 cells cultured with rlL-4 and D l .l; lane 2, control (no mAb); lane 3, mAb 5c8; and lane 4, mAb OKT4. (0 Diagram depicting the sizes of S1 nuclease-protected fragments of 440 bp uniformly labeled probe derived from the human Sxy3 germline transcript cDNA. RNA was
IgM+
IgG
+
FIGURE 3. Genotypic characterization of IgG+ and IgM' RAMOS 266 clones. RepresentativeIgG- and IgM-expressing RAMOS 266 clones were analyzed with respect to Ig heavy chain gene rearrangement by Southern blotting. Shown are Southern blot analyses of RAMOS 266 clones (I-D9-M and LD8-G) that were obtained from limiting dilution culture of RAMOS 266 on mitomycin-C treated D l .1 cells. All clones express surface CD40 by FACS (not shown). Southern blots were performed on BamHl digested DNA from IgG' and IgM+ RAMOS 266 clones hybridized to (top)a C p probe and (bottom)a y probe, as indicated. Clones LD8-G and LC3-G were subcloned from a single IgG-expressing well. The following IgMand IgG clones derived from the same subcloned wells (IgM/lgG pairs): I-C7-M/I-D8-G; I-D9-M/I-D7-G and I-E4"/I-C3-G.
RAMOS 266 subclone, like other germinal center B cell tumors (29,30), responds to anti-IgM signals by undergoingapoptosis(Fig. 4). Therefore,toaddresstheroleof T-BAMICDIO-L signaling in the regulation of B cell apoptosis, the effect of D1.l cells was studied on anti-IgMinduced apoptosis in RAMOS 266 cells measured by an assessment of the DNA fragmentation that characterizes apoptosis(Fig. 4). Interestingly,theT-BAM/CD40-L+ D1.l clone, but not the T-BAM/CD40-L- B2.7 clone, dramatically inhibits anti-IgM-driven apoptosisin RAMOS 266 B cells (Fig. 4). Further, the mAb5c8 selectively inhibits the
quantified both by optical density (OD) and by Northern blotting with a GAPDH probe and found to be equivalent within the experiments shown in (A) and (B).
Journal
21 69
directed by the T-BAM/CD40-L-expressingclones are inhibited by the mAb 5c8, which is known to bind to and I . ' . , inhibitthe function of theT-BAM/CD4O-Lmolecule. Taken together, these data implicate the T-BAM/CD40-L molecule in several distinct mechanisms important in B cell differentiation. Because lymphokines and T cell contact-dependent signals are known to be important in T cell helper function, these experiments specifically addressed theroles in B cell differentiation of IL-4andT-BAM/CD40-L,whichare known to be expressed by germinal center T cells (15,48, 49). In the Dl.l-RAMOS 266 system studied here, lymphokine-mediated signals alone induce low level isotype switching and the T-contact signal, mediated by T-BAM/ CD40-L, augments this effect butis not sufficient to induce germline transcription or switching in the absence of lymphokines. Therefore, these data suggest thatT-BAM/ CD40-LandrIL-4 provide distinct signals to B cells, which are complementary in inducing germ-line and productive IgH Cy transcripts. These data are consistent with the idea that germ-line transcription may be a limiting facFIGURE 4. Effect of T-BAWCD40-L or rlL-4 on IgMtor in isotype switch recombination and that the primary induced apoptosis of RAMOS 266 cells.Shownare DNA effect of T-BAM/CD40-L on isotype switching in this sysfragmentationpatterns of cultures of RAMOS 266 cellsor tem is on the induction of germ-line transcripts (50, 51). Jurkat clones in the presence ofanti-IgM (or control mAb) or However, the data do not exclude the possibilities that UV fluoresanti-TBAWCD40-L (orcontrols)visualizedby T-BAM/CD40-L signals may also induce IgH recombinacence of ethidium bromide-stained agarose gels. The experimental conditions are indicated in the grid caption. tion,itself, and/or promote theoutgrowth of stably switched B cells. In this paper it is shown thatin the RAMOS266 system, apoptosis is specifically induced by ligationof the surface protective effect of D1.l cells, but not that of rIL-4, which Ag receptor by a model Ag (anti-IgM) and that T-BAM/ also inhibits the anti-IgM-induced apoptosis RAMOS of 266 CD40-L signals rescue RAMOS 266 cells from anti-IgMcells (Fig. 4). These observations demonstrate that T-BAM/ induced apoptosis. The role of Ag signals in inducing apCD40-L signals rescueRAMOS 266 B cells from anti-IgMoptosis in germinal center B cells isunclear,however, induced apoptosis and suggest that T-BAM/CD40-L interacin contrast to the effect of anti-Ig on inducing because, tions with CD40 may play roles in inhibiting the apoptosis of apoptosis in RAMOS, anti-Ig generally activates B cells Ag-specific B cells in lymphoid follicles. and has been shown to temporarily prevent the spontaneous apoptosis of cultured germinal center B cells (11,52). Discussion Therefore, apoptosis in germinal centers is either drivenby other signals (aside from sIg) or more likely B cell AgThe present studies indicate that T-BAM/CD40-L is intriggering may result in either apoptosis or rescue, dependvolved atmultiple levels in helper function for B cells. The roles of T-BAM/CD40-L were studied both by specifically ing on the context of the Ag trigger. Regardless of the in vivo trigger for apoptosis in germinal centers, positive seblocking T-BAM/CD40-L functions with a neutralizing lection of Ag-specific B cells occurs under the negative mAb (5c8) and by supplying T-BAM/CD40-L signals to B selective pressure of apoptosis (52), and the data in this cells on a selectively T-BAM/CD40-L-expressing Jurkat T paper suggest that T-BAM/CD40-L signals may rescue B lymphoma clone. The principal findings in this report are: cells from apoptosis in vivo, and thus may play a critical 1) T-BAM/CD40-L signals mediated by T-B contact play role in the selection of B cells. a critical role in directing the synthesis of IgG, IgA, and The role of CD40 in rescuing B cells from apoptosis is IgE by peripheral B cells; 2) T-BAM/CD40-L+D1.l cells actonrIL-4-primedRAMOS 266 cells to augment the interesting given the structural homology of CD40 with level of germ-line and productive Cy transcripts, which other receptors that regulate apoptosis, including:Fas, TNF, and nerve growth factor receptor (53). Fas is a lymcorrelated later in culture withthe presence of stably switched RAMOS 266 B cells that had undergone delephocyte receptor for apoptosis signals, and Fas abnormaltional class switch recombination events to Cyl. 3) ities encode an autoimmune phenotype in mice (54, 55). TNF-a and TNF-p (lymphotoxin) interact with the T-BAM/CD40-L signals inhibit anti-IgM-drivenapoptosis TNF-aR to mediate apoptosis of targets.Nervegrowth of RAMOS 266 B cells. Importantly, the major functions
21 70
ROLES OF T-BAM/CD40-L SIGNALS IN 6 CELL DIFFERENTIATION
factor receptor rescues neuronal cell lines from apoptotic death (56). It is intriguing that in addition to the relationship of CD40 with these molecules, TNF-a, lymphotoxin (TNF-P), and nervegrowthfactorarehomologous to T-BAM/CD40-L,suggesting that homologous receptorligand pairs regulate cell death, both positively and negatively. How this familyof molecules regulates cell death is becoming understood. In this regard, it will be of interest to determine whether T-BAM/CD40-L signals induce B cell bcl-2 expression, because expression of this cellular transforming gene product is known to inhibit B cell apoptosis and bel-2 has been reported to be induced by antiCD40 mAb (52). These data suggest more precise interpretations of how defects in the T-BAM/CD40-L gene result in the phenotype of the X-linked HIMsyndrome (33-36). X-linked HIM patients manifest high levels of serum IgM and low or absent IgG or I g A (37, 38). A pathogenic feature of HIM has been shown to be a defect in T cell helper function (39) with intact capacity for B cells to switch (40). In this light, the absence of functional T-BAM/CD40-L in this syndrome has been interpreted to suggest a role for T-BAM/CD40-L in “isotypeswitching” (33-35). Our dataextendthefunctionalanalysis of T-BAM/CD40-L signals and suggest that T-BAM/CD40-Lplaysseveral distinctive roles in discrete steps of isotype switching that may relate to features of X-linked HIM. Importantly, T-BAM/CD40-L plays a critical role in the T-directed secretion of IgG, IgA, and IgE, but is not essential for IgM secretion. These data relate to the elevated IgM and low levels of IgG, IgA, and IgE in HIM. The presence of elevated levels of IgM in HIM may relate to lymphokineinduced switching in the absence of the T-BAM/CD40-L signal and emphasizes the importance of T-BAM/CD40-L in augmenting germ-line transcription, isotype switching, and-possibly-in promoting the growth of switched B cells. Taken together, the profound defects in humoral immunity in X-linked HIM demonstrate an apparent lack of redundant effector pathways, which may result from the involvement of T-BAM/CD40-L signaling at several levels of B cell differentiation. The obligatory role of T-BAM/ CD40-L in proper B cell differentiation suggests that modulation of T-BAM/CD40-L expression may be a critical target of physiologic immunoregulatory mechanisms. Moreover, the T-BAM/CD40-L interactions with CD40 may provide a target for pharmacologic interventions of T-B interactions.
Acknowledgments The authors thank Eva Glickman-Nir for excellent technical assistance. rIL-4 was the kind gift of Dr. Satwant Narula, Schering-Plough COT.; the anti-CD40 mAb (G28.5) was the gift of Dr. Ed Clark, University of Washington; and the Sxy3 probe was generously provided by Dr. Tasuku Honjo, Kyoto University.
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