Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11633-11636, December 1995 Immunology

Characterization of a 23-kDa associated with CD40 TOMOHIRO MORIO, SILVA HANISSIAN, AND RAIF S. GEHA* Division of Immunology, Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115 Communicated by Mary Ellen Avery, Harvard Medical School, Boston, MA, August 30, 1995 (received for review May 1, 1995)

ABSTRACT CD40 is a 45-kDa member ofthe MATERIALS AND METHODS tumor necrosis factor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carci- Cells. The human B-cell lines Raji, BJAB, and Ramos and the human bladder cell line T24 were obtained from noma cells. The unique capacity of CD40 to trigger immuno- American Type Culture Collection. The human B-cell line globulin isotype switching is dependent on the activation of Jijoye which expresses TNFR p80 (CD120b) was a kind gift protein- kinases, yet CD40 possesses no kinase do- from G. Mosialos and E. Kieff (Harvard Medical School). main and no known consensus sequences for binding to Human tonsil B cells were purified as described (13). protein-tyrosine kinases. Recently, an intracellular protein Monoclonal (mAbs) and Reagents. Mouse anti- (CD40bp/LAP-1/CRAF-1) which belongs to the family of human CD40 mAb 626.1 (IgGl) was a gift from S. M. Fu TNFR-associated was reported to associate with (University of Virginia, Richmond). Murine anti-major histo- CD40. We describe a 23-kDa cell surface protein (p23) which compatibility complex (MHC) class I mAb W6/32 (IgGl), and is specifically associated with CD40 on B cells and on urinary anti-HLA-DR mAb L243 (IgG2a) were obtained through bladder transitional carcinoma cells. Protein microsequenc- American Type Culture Collection. Anti-MHC class II mAb ing revealed that p23 shows no homology to any known 3B12 (IgGl) has been described (14). Anti-human Fas mAb protein. A rabbit raised against a peptide derived ZB-4 (IgGl) was obtained from Kamiya Biomedical (Thou- from p23 recognized a 23-kDa protein in CD40 immunopre- sand Oaks, CA). Rat anti-human TNFR p80 mAb was pur- cipitates. In contrast to CD40bp/LAP-1/CRAF-1, p23 was chased from Genzyme. Anti-CD81 mAb 5A6 (IgGl) was not associated with TNFR p80 (CD120b). These findings kindly provided by S. Levy (Stanford University). Tunicamycin suggest that p23 is a novel member of the CD40 receptor was obtained from Boehringer Mannheim. Phosphatidylinos- complex. itol-specific phospholipase C (PI-PLC) was purchased from Calbiochem. Anti-p23 peptide polyclonal antibody was raised Interaction of the B-cell surface CD40 with its ligand in rabbits against a 17-aa peptide derived from p23 and (CD40L) expressed on activated T cells plays a critical role in conjugated to keyhole limpet hemocyanin (KLH) and was T- collaboration, particularly in immunoglobulin isotype absorbed with KLH as described (15). switching, in the induction of expression of costimulatory Surface lodination and Immunoprecipitation. Surface io- survival and in dination and immunoprecipitation were carried out with Bolt- molecules, in B-cell the formation of germinal- on-Hunter reagent (16). In brief, 5 x 107 cells were washed center B cells (1-3). This is evidenced by the observations that with phosphate-buffered saline, and labeled with 2 mCi (74 mice with a disrupted CD40 gene fail to undergo isotype MBq) of 1251 in the presence of Bolton-Hunter reagent for 30 switching and to develop germinal centers in response to min on ice. The cells were washed with PBS and lysed on ice T-cell-dependent (4, 5) and that B cells from CD40- for 30 min in 1% (vol/vol) Brij 96/150 mM NaCl/20 mM null mice fail to elicit an allogeneic response (G. Hollander, E. Hepes, pH 7.4/1 mM Na3VO4/50 mM NaF/1 mM phenyl- Castigli, and R.S.G., unpublished work). methanesulfonyl fluoride containing leupeptin at 5 ,ug/ml and The unique capacity of CD40 to trigger immunoglobulin antipain, chymostatin, and pepstatin each at 1 ,ug/ml. The cell class switching in B cells suggests that CD40 ligation activates lysates were precleared with protein G-Sepharose beads (Phar- a unique signaling pathway(s). Our studies and those of others macia) precoupled with normal mouse immunoglobulin and indicate that CD40 ligation activates protein-tyrosine kinases then were immunoprecipitated with protein G beads preab- (PTKs), including Lyn and Syk and results in tyrosine phos- sorbed with the indicated antibody. The immunoprecipitates phorylation of multiple substrates, including phosphatidylino- were resolved by SDS/12% PAGE and the bands were visu- sitol 3-kinase, phospholipase C-,y2, and a 28-kDa phospho- alized by autoradiography. protein (6, 7). More importantly, PTK inhibitors and crosslink- 355 Metabolic Labeling. BJAB cells were first washed in ing of CD45 to CD40 inhibit CD40-mediated isotype switching minimal essential medium deficient in Met/Cys (GIBCO/ (8, 9). However, CD40 has no kinase domain and no known BRL) and incubated for 1 hr in the same medium supple- consensus sequence for binding to PTK, suggesting that CD40 mented with 5% fetal bovine serum that had been dialyzed uses associated molecules for transducing intracellular signals. against 150 mM NaCl/20 mM Hepes, pH 7.4. The cells were Recently, a member of the tumor necrosis factor receptor then incubated with [35S]methionine/[35S]cysteine (Tran35S- (TNFR)-associated factor (TRAF) family of proteins was label; ICN) for 5 hr, washed, and lysed in 1% Brij 96 lysis found to bind to the cytoplasmic region of CD40 and to play buffer. Immunoprecipitation was carried out as described a role in CD40-mediated induction of CD23 expression (10- above. 12). We have identified and biochemically characterized a Two-Dimensional Electrophoresis. Two-dimensional gel 23-kDa cell surface protein (p23) associated with human electrophoresis was carried out with a Bio-Rad in accord CD40. p23 was not associated with CD95 (Fas) or with TNFR with the manufacturer's guidance. For first-dimension isoelec- p80 (CD120b), suggesting that it could be important in CD40- tric focusing, radiolabeled immunoprecipitates were solubi- specific signaling. Abbreviations: mAb, ; MHC, major histocom- patibility complex; NMS, normal mouse serum; PI-PLC, phosphati- The publication costs of this article were defrayed in part by page charge dylinositol-specific phospholipase C; TNFR, tumor necrosis factor payment. This article must therefore be hereby marked "advertisement" in receptor; TRAF, TNFR-associated factor. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11633 Downloaded by guest on September 27, 2021 11634 Immunology: Morio et al. Proc. Natl. Acad. Sci. USA 92 (1995)

lized in a 9 M urea/2% (vol/vol) Nonidet P-40/5% (vol/vol) Raji BJAB Tonsil B T24 2-mercaptoethanol and focused for 16 hr at 800 V in tube gels

consisting of 9 M urea, 4% (wt/vol) acrylamide/N,N'- c_ CZ) C() -Z C0 0c methylenebisacrylamide (30:0.8 weight ratio), 2% (vol/vol) 0c .- ampholytes, and 2% Nonidet P-40. A 20 mM NaOH cathode kDa buffer and a 10 mM phosphoric acid anode buffer were used. Q- The second-dimension electrophoresis was carried out in an -46 SDS/12% polyacrylamide gel. The isoelectric point (pl) and 0 the molecular weight were calibrated with standard 2D marker (Bio-Rad). -30 Purification and Microsequencing of p23. The 1% Brij 96 lysate from 8 x 1010 BJAB B cells was passed through a column packed with protein G-Sepharose crosslinked to anti-CD40 mAb 626.1 by dimethyl pimelimidate dihydrochloride. The column was washed, and bound proteins were eluted with dimethylamine (pH 11.5) and then concentrated by ultrafil- -1 4 10 kDa). After SDS/12% tration with Centricon-10 (cutoff, FIG. 1. p23 is coprecipitated with CD40 in human B cells and in PAGE, proteins were electrotransferred to a poly(vinylidene urinary bladder transitional carcinoma cells. Raji and BJAB (Epstein- difluoride) (PVDF) membrane and visualized with Ponceau S Barr virus-negative) human B- cells, normal human tonsil B stain. Two major bands were observed: a 46-kDa band that cells, and T24 human urinary bladder carcinoma cells were radiola- corresponded to CD40 and a 23-kDa band. The area of the beled with 1251 and immunoprecipitated with normal mouse serum PVDF membrane containing p23 was excised for in situ (NMS), L243 (anti-HLA-DR) (anti-human MHC class II), or 626.1 digestion with trypsin. The resultant peptide mixture was (anti-CD40). The immunoprecipitates (equivalent to 2 x 107 cells per separated by narrow-bore high-performance liquid chroma- lane) were resolved by SDS/12% PAGE under reducing conditions tography on a Vydac C18 reverse-phase column (2.1 mm x 150 and the gels were autoradiographed for 5 days, 2 days, 2 days, and 5 acetonitrile in 0.057% triflu- days for Raji cells, BJAB cells, tonsil B cells, and T24 cells, respectively. mm) with a gradient of 0-50% Arrows at left indicate the position of CD40 and p23. Molecular size oroacetic acid on a Hewlett-Packard 1090 chromatograph with markers are at right. a 1040 diode array detector. Optimal fractions from the chromatogram were chosen for sequencing on the basis of cholamidopropyl)dimethylammonio]- 1 -propanesulfonate different UV absorbance at 210, 277, and 292 nm, peak (CHAPS) or 1% Tween 20 lysates (data not shown). symmetry resolution, and molecular weight information from Members of the TNFR family, which includes CD40, display laser desorption mass spectrometry. sequence was considerable homology in their extracellular regions (18). We obtained in the Harvard Protein Microsequencing Facility for therefore examined for the association of p23 with Fas (CD95) three different peptide peaks by automated Edman degrada- was detected in tion on an Applied Biosystems 477A protein sequencer using and TNFR p80 (CD120b). No 23-kDa protein microcartridge and cycles optimized for a 30-min cycle time. Fas immunoprecipitates from the Fas+ BJAB cell line (Fig. For all three peptides, which were 7, 8, and 17 aa long, 2A) or in TNFR p80 immunoprecipitates from the TNFR p80+ unambiguous amino acid sequences were obtained from two Jijoye B-cell line (Fig. 2B) even after prolonged exposure of independent samples analyzed. the autoradiographs (up to 10 days). Western Blotting. Immunoblotting was carried out as de- One potential candidate for p23 is the 22- to 24-kDa scribed (17). tetraspan molecule TAPA-1 (CD81), a cell surface protein associated with MHC class II molecules and with CD19 on B cells (19, 20). However, p23 and TAPA-1 migrated differently RESULTS in SDS/polyacrylamide gels (Fig. 3A). More importantly, A 23-kDa Protein Coprecipitates with CD40. CD40 immu- noprecipitates prepared from '25I-labeled Raji B cells lysed in BJAB B Jijoye 1% Brij 96 buffer contained a prominent 23-kDa protein (p23), A in addition to a prominent 45-kDa protein which corresponds to CD40 (Fig. 1). The association of p23 with CD40 was o r 0P C.Q specific, since p23 was not detected in anti-HLA-DR immu- I; kD, noprecipitates (mAb L243) (Fig. 1, lane 3) or in anti-CD45, kDa )a anti-MHC class I, or anti-IgM immunoprecipitates (data not ". -97-TNFRp80 shown) prepared from Raji B cells. The association of p23 with -69 -69 CD40 was not restricted to Raji cells, as p23 was detected in -46 CD40 immunoprecipitates from the Epstein-Barr virus- 0 F D40- negative BJAB cell line and, more importantly, from normal CD40--aF tonsil B cells (Fig. 1). The association of p23 with CD40 was - 30 not restricted to B-lineage cells, since p23 was also detected in - 30 CD40 immunoprecipitates from the transitional bladder car- cinoma cell line T24 (21) (Fig. 1). Densitometric scanning of p23-- p23-'- the intensity of the p23 and CD40 bands revealed a consistently higher ratio of intensity of p23 to CD40 bands in BJAB and T24 -14 cell lines than in Raji and tonsil B cells. This suggests that the -14 strength of the p23-CD40 association and/or the fraction of CD40 molecules associated with p23 varies in different cell lines. FIG. 2. p23 is not associated with Fas or with TNFR p80. Human The migration of p23 in SDS/polyacrylamide gels was similar in BJAB cells (A) and Jijoye cells (B) were surface labeled with 1251, not indicat- lysed, and immunoprecipitated with the indicated antibodies. The reducing and nonreducing conditions (data shown), immunoprecipitates were resolved by SDS/12% PAGE with lysate ing that p23 does not exist in a disulfide-linked complex. The from 3 x 107 cells loaded per lane and the gels were autoradiographed association of p23 protein with CD40 was not detected in 1% for 5 days. Arrows indicate the position of CD40, Fas, TNFR p80, and Nonidet P-40 cell lysates and was poorly detected in 1% 3-[(3- p23. Molecular size markers (kDa) are at right. Downloaded by guest on September 27, 2021 Immunology: Morio et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11635

A BC A B pl 4.5 5.2 5.5 kDa p1 4.5 5.2 5.5 6.0 kDa 7fo 9' -35 -35 -29 -29 XkkDaAz * 4 -20 -20

FIG. 5. Two-dimensional electrophoresis of p23. BJAB cells were 30 _ _ m .... either surface radiolabeled with 125I (A) or metabolically labeled with [35S]methionine/[35S]cysteine (B). CD40 immunoprecipitates pre- p23_ pared from Brij 96 cell lysates (2 x 107 cells) were solubilized with urea TAPA-1 and Nonidet P-40 and subjected to two-dimensional electrophoresis...... pI values and molecular mass are indicated. The spot(s) corresponding to p23 is marked by an arrow. Spots migrating at 28 kDa and marked -14 by a star in B are probably derived from the nonspecific 28-kDa band present in both NMS and CD40 precipitates from 35S-labeled cells as depicted in Fig. 4A. FIG. 3. p23 is not TAPA-1 (CD81). (A) Human Raji B cells were surface iodinated, lysed, and immunoprecipitated with the indicated indicating that p23 is a cellular protein and not a serum protein mAbs. (B) Western blot of CD40 immunoprecipitates with anti- that adsorbed to CD40. Incubation with tunicamycin at 10 TAPA-1 mAb. Cells were lysed in 1% Brij 96 and TAPA-1, CD40, and Jg/ml for 1 hr prior to and throughout the 35S-labeling period MHC class I and class II immunoprecipitates were resolved by SDS/12% PAGE (5 x 107 cells per lane), transferred onto nitrocel- did not result in a detectable shift in the migration of p23, lulose membrane, and probed with mAb 5A6 at 2 Ag/ml. The suggesting that p23 may not be N-glycosylated. In contrast, as membrane was then incubated with biotinylated rabbit anti-mouse previously described (1), the migration of CD40 was shifted immunoglobulin, followed by horseradish peroxidase-conjugated from 45 kDa to 39 kDa in tunicamycin-treated cells. Treatment streptavidin. The blot was developed by a chemiluminescence system. of Raji cells with PI-PLC 0.25 unit/ml for 90 min prior to 125I-labeling did not alter the intensity of the p23 band detected Western blotting revealed the presence of TAPA-1 in in CD40 immunoprecipitates, suggesting that p23 is not a HLA-DR immunoprecipitates but not in CD40 or MHC class glycosylphosphatidylinositol-linked protein. PI-PLC treatment I immunoprecipitates (Fig. 3B). strongly reduced the density of the glycosylphosphatidylinosi- Biochemical Characterization of p23. A labeled 23-kDa tol-linked surface protein CD14 on (data not band was detected in CD40 immunoprecipitates from lysates shown). of B cells labeled with [35S]methionine/[35S]cysteine (Fig. 4A), In an attempt to determine whether p23 consisted of more than one protein, we performed two-dimensional electro- A Tunicamycin B PI-PLC phoresis on CD40 immunoprecipitates from BJAB cells. The p23 from 1251-surface-labeled cells was detected as two closely - f located spots with pl values of 5.3 and 5.4 (Fig. SA). Western 0C 00 blotting revealed that both spots reacted with antibody raised against the 17-aa peptide derived from p23 (data not shown). p23 intrinsically labeled with 35S was detected as a broad spot kDa IT kDa with a pl range of 5.3-5.4 (Fig. SB). These data are compatible with the existence of a single 23-kDa protein with minor posttranslational modification. -46 - 46 We sequenced three internal tryptic peptides derived from p23 that were 7, 9, and 17 aa long. No significant matches were - found with proteins in the National Biomedical Research -30 - 30 Foundation data bank (as of March 1995). Rabbit polyclonal antibody against the 17-aa peptide detected a 23-kDa protein in Western blots of CD40 immunoprecipitates but not of MHC class II or MHC class I immunoprecipitates of BJAB cells (Fig. 6). Western blot analysis of lysates from the Jurkat T-cell line and the myelomonocytic cell lines U-937 and THP-1 revealed the presence of p23 in all these lines (data not shown). This - 14 observation suggests that p23 can be expressed independently -14 of CD40. FIG. 4. (A) p23 is metabolically labeled but is poorly N- glycosylated. BJAB cells were preincubated in the presence or absence DISCUSSION of tunicamycin (10 ,ug/ml) in methionine- and cysteine-free minimal essential medium for 1 hr and then incubated with [35S]methionine/ In this paper we report the association of a 23-kDa cell surface [35S]cysteine for an additional 5 hr. The labeled proteins were analyzed protein, p23, with CD40. p23 was found associated with CD40 (2 x 107 cells per lane) as described in Fig. 1. The top arrow and the in all B-cell lines examined, which included the Raji, BJAB arrow with the star indicate the position of CD40 precipitated from (Fig. 1), Jijoye (Fig. 2), and Ramos cell lines and Epstein-Barr untreated cells and from tunicamycin-treated cells, respectively. The virus-transformed normal B cells (data not shown). p23 was bottom arrow indicates p23. (B) p23 is not a glycosylphosphatidyli- nositol-linked protein. Raji B cells were incubated with or without also associated with CD40 in the single epithelial cell line PI-PLC (0.25 unit/ml) for 90 min in PBS, washed, labeled with 1251, examined. and analyzed (107 cells per lane) as described in Fig. 1. The lower The association of p23 with CD40 in B cells was specific and arrow indicates p23. was not a peculiarity of transformed cell lines, since it was Downloaded by guest on September 27, 2021 11636 Immunology: Morio et al. Proc. Natl. Acad. Sci. USA 92 (1995) lation of an 28-kDa protein which could represent a phos- .co CID phorylated form of p23. Generation of an immunoprecipitat- 0 ing antibody to p23 is required to address this question. Recently, a 63-kDa intracellular protein (CD40bp/LAP-1/ ; Z~I CRAF-1) belonging to the TRAF family has been found to IT kDa associate with the cytoplasmic domain of CD40 (10-12). Like p23, this protein can be expressed independently of CD40 (11, 12). In one report CD40bp/LAP-1/CRAF-1 was found to

- 53 associate with TNFR p80 and the lymphotoxin ,3 receptor (1 1), raising the possibility that other components of the CD40 -35 complex may be required for CD40-specific functions such as isotype switching. p23 was not found to associate with TNFR

p23 .- p8O or with Fas, another member of the TNFR family (Fig. 2). -20 The exact role of p23 in CD40 signaling and function will require the cloning of this molecule.

We thank Drs. George Mosialos and Elliot Kieff for providing the Jijoye cell line, Dr. Shoshana Levy for the 5A6 mAb, and Dr. Carl FIG. 6. Western blotting of p23 with anti-peptide antibody. Brij 96 Ware for his suggestion to use the TNFR p80+ Jijoye cell line. This were with lysates of BJAB cells precleared and immunoprecipitated work was supported by National Institutes of Health Grant A131541 The were resolved by the indicated antibodies. immunoprecipitates to R.S.G. and by grants from Baxter Healthcare and Alpha Thera- PAGE and transferred to nitrocellulose membrane. The SDS/12% peutics. T.M. was a recipient of an Allen & Hanburys Respiratory was of rabbit polyclonal membrane incubated with 1:200 dilution a P. Markey Physician Scientist Award. anti-p23 peptide antiserum and then with horseradish peroxidase- Diseases Award and of Lucille conjugated protein A. Bands were visualized with a chemilumines- J., Bazan, F., Blanchard, D., Briere, F., Galizzi, J. P., cence system. 1. Bancherau, van Kooten, C., Liu, Y. J., Rousset, F. & Saeland, S. (1994)Annu. detected on normal tonsil B cells (Fig. 1). The association of Rev. Immunol. 12, 881-922. 2. Foy, T. M., Laman, J. D., Ledbetter, J. A., Aruffo, A., Claasen, p23 and CD40 appears to be relatively weak, since it was E. & Noelle, R. J. (1994) J. Exp. Med. 180, 157-163. disrupted by the strong detergent Nonidet P-40. p23 is likely 3. Yellin, J. M., Sinning, J., Corey, L. R., Sherman, W., Lee, J. J., to be a cell surface protein, since it was readily labeled by cell Blickman-Nir, E., Sippel, K. C., Rogers, J., Cleary, A. M., Parker, surface iodination, and is likely to be a transmembrane protein, M., Chess, L. & Lederman, S. (1994) J. Immunol. 153, 666-674. since it was resistant to PI-PLC (Fig. 4B). p23 was judged to be 4. Kawabe, T., Yoshida, K., Tanaka, T., Fujiwara, H., Suematsu, S., a cellular protein because it was labeled in cells incubated with Yoshida, N., Kishimoto, T. & Kikutani, H. (1994) Immunity 423, 35S-labeled amino acids. It did not appear to be N-glycosylated, 167-178. since its mobility did not change following treatment with 5. Castigli, E., Alt, F., Mizoguchi, E., Bahn, A. K. & Geha, R. S. tunicamycin (Fig. 4A). (1994) Proc. Natl. Acad. Sci. USA 91, 12135-12139. J. Several lines of evidence indicate that p23 is a novel protein. 6. Ren, C. L., Morio, T., Fu, S. M. & Geha, R. S. (1994) Exp. Med. is close to that of 179, 673-680. Although its apparent molecular weight 7. Faris, M., Gaskin, F., Parsons, J. T. & Fu, S. M. (1994) J. Exp. TAPA-1, p23 is clearly distinct from TAPA-1 because it Med. 179, 1923-1931. migrated slightly slower than TAPA-1 and more importantly, 8. Loh, R., Ren, C., Fu, S. M., Jabara, H. & Geha, R. S. (1994) J. it did not react in Western blots with anti-TAPA-1 mAb (Fig. Clin. Immunol. 94, 784-792. 3). The results of two-dimensional gel electrophoresis were 9. Loh, R., Ren, C., Fu, S. M., Jabara, H. & Geha, R. S. (1995) compatible with p23 consisting of a single species with minor Immunol. Lett. 45, 99-106. posttranslational modification (Fig. 5), although we cannot 10. Hu, H. M., O'Rourke, K., Boguski, M. S. & Dixit, V. M. (1994) completely rule out the presence of two protein species. The J. Biol. Chem. 269, 30069-30072. strongest evidence for p23 being a novel protein was provided 11. Mosialos, G., Birkenbach, M., Yalamanchili, R., VanArsdale, T., by the unique amino acid sequences obtained for all three Ware, C. & Kieff, E. (1995) Cell 80, 389-399. 12. Chen, G., Cleary, A. M., Ye, Z.-S., Hong, D. I., Lederman, S. & internal derived from a tryptic peptides p23. Furthermore, Baltimore, D. (1995) Science 267, 1494-1498. nonprecipitating rabbit antibody raised against one of these 13. Jabara, H., Fu, S. M., Geha, R. S. & Vercelli, D. (1990) J. Exp. peptides recognized in Western blots a single 23-kDa protein Med. 172, 1861-1864. in CD40 immunoprecipitates but not in MHC class I or MHC 14. Spertini, F., Chatila, T. & Geha, R. (1992) J. Immunol. 149, class II immunoprecipitates (Fig. 6). Taken together, these 65-70. results strongly suggest that p23 is neither an already identified 15. Morio, T., Geha, R. S. & Chatila, T. A. (1994) Eur. J. Immunol. protein nor a breakdown product of CD40. 24, 651-658. The mode of association of p23 with CD40 and the role of 16. Scholl, P. R. & Geha, R. S. (1994) Proc. Natl. Acad. Sci. USA 90, p23 in CD40 signaling remain to be determined. Preliminary 8847-8850. experiments using chimeric molecules consisting of the extra- 17. Wang, A. V., Scholl, P. R. & Geha, R. S. (1994) J. Exp. Med. 180, cellular domain of CD8 and of the transmembrane and intra- 1165-1170. 18. Mallett, S. & Berclay, A. N. (1991) Immunol. Today 12, 220. cellular domains of CD40 transfected into BJAB cells indicate 19. Schick, M. R. & Levy, S. (1993) J. Immunol. 151, 4090-4097. that p23 associates with the extracellular domain of CD40 and 20. Bradbury, L. E., Goldmacher, V. S. & Tedder, T. F. (1993) J. is important for CD40-mediated cell aggregation. This sug- Immunol. 151, 2915-2917. gests that p23 plays a role in CD40 signaling. Interestingly, 21. Koho, H., Paulie, S., Ben-Aissa, H., Jonsdottir, I., Hansson, Y., Faris et al. (7) reported, and we have confirmed, that CD40 Lundblad, M. L. & Perlman, P. (1984) Cancer Immunol. Immu- ligation on B cells leads to the transient tyrosine phosphory- nother. 17, 165-172. Downloaded by guest on September 27, 2021