Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8522-8526, September 1992 Immunology

Heterogeneity of immunoglobulin-associated molecules on human B cells identified by monoclonal antibodies TETSUYA NAKAMURA*, HIRoMI KUBAGAWA*,

AND

MAx D. COOPER*t

*Division of Developmental and Clinical Immunology, Departments of Medicine, Pathology, Pediatrics, and Microbiology, University of Alabama at

Birmingam, and tHoward Hughes Medical Institute, Birmingham, AL 35294

Contributed by Max D. Cooper, May 26, 1992

counterpart and induce down-modulation of the antigen receptors on B cells.

Two covalently linked transmembrane molABSTRACT ecules, encoded in mice by the mb-i and B29 genes, have been defined as integral components of the antibody receptor units expressed on B cells. We have produced monoclonal antibodies against an exposed extracellular epitope on the putative human equivalent of the mouse B29 product. These antibodies, CB3-1 and -2, were used to show that cytoplasmic expression of this molecule begs in human pro-B cells (terminal deoxynudeotidyltransferase-poitive, , chain-negative), whereas surface expression coincides strictly with surface immunolobulin expression ofall isotypes. Immunochemical analysis of the human immunoglobulin-associated molecules revealed greater molecular heterogeneity than has been noted for the murine analogues. This molecular heterogeneity of immunoglobulinassociated molecules varied as a function of differentiation stage and the immunoglobulin isotypes expressed by B-lineage cells. Our data support the hypothesis that biochemical heterogeneity of the surface immunoglobulin-assocated molecules may contribute to the variabllity in biological effects of antigen receptor crossllnkage on B cells of different maturational stages. Because the CB3 antibodies are capable of downmodulating the antigen receptors on all B cells, they may prove therapeutically useful as universal B-cell suppressants.

MATERIALS AND METHODS Antibodies and Cells. Anti-human ,u chain (SA-DA44), anti-human 8 (SIA6-2), anti-human K (TB28-2), anti-human A (1-155-2) and anti-mouse K (187-1) mAbs were described previously (15, 16). Anti-CD19, -CD20, and -CD38 mAbs were purchased from Becton Dickinson. Affinity-purified goat antibodies specific for mouse or human Ig, and the appropriate developing reagents, were from Southern Biotechnology Associates (Birmingham, AL). Pre-B cell lines 207 (Ig-) and 697 (cytoplasmic and surface iz+, surrogate light chain+), B cell lines Ramos (surface ,uA+), Daudi (surface ,UK+), and IM9 (surface 'ylK+), and a T cell line, MOLT4, were described previously (17). Preparation of mAbs Specific for Ig-Assocated Molecules. Ramos B cells (4 x 109) were lysed in 1% digitonin (Aldrich) lysis buffer and centrifuged for 30 min at 17,000 X g. The soluble components were incubated with Sepharose 4B copled with the SA-DA4-4 mAb and, after extensive washing, bound proteins were eluted by either Laemmli's sample buffer or 1% Nonidet P-40 (NP-40) lysis buffer. Proteins eluted with sample buffer were subjected to SDS/PAGE under reducing conditions and then stained with Coomassie brilliant blue. Bands corresponding to 36-52 kDa, shown to contain Ig-associated molecules in pilot experiments, were excised and used for the three initial immunizations of BALB/c mice. Proteins eluted with 1% NP40 lysis buffer were injected in two additional immunizations. Finally, 1 day after a booster immunization with viable Ramos cells (2 x 107), lymph node cells were harvested and fused with P3X63-Ag8.653 myeloma variant cells. Culture supernatants of hybridomas were tested in flat-bottomed 96-well plates coated first with goat anti-human ,u antibody and then with a 1% digitonin lysate of Daudi cells as a source of IgMassociated molecules (5 x 105 cells per well). Bound antibodies were detected with (3-galactosidase-labeled goat antimouse Ig antibody, followed by o-nitrophenyl 3-D-galactopyranoside. Each supernatant was also tested for ELISA reactivity with an IgM paraprotein. Immunofluorescence Analyses. Cells (5 x 105) were incubated with antibodies (50 pg/ml) for 20 min at 40C unless otherwise indicated. For single-color surface staining cells were incubated with purified CB3-1, CB3-2, SA-DA44, or TB28-2, followed by a biotinylated goat anti-mouse Ig antibody and then phycoerythrin-conjugated streptavidin (10 lg/ml). For two- or three-color staining, cells were incubated with biotinylated CB3-1, developed with phycoerythrinconjugated streptavidin and then stained with fluoresceinlabeled anti-CD19 or anti-CD38 mAb or a mixture of fluo-

Signal transduction through cell surface immunoglobulin (sIg) receptors requires the presence of physically associated molecules, as the cytoplasmic tail of slg molecules is very short. Two molecules encoded by B-cell-specific genes, mb-i (1) and B29 (2), have been shown to be noncovalently associated with sIgM and sIgD to form antigen receptor units on B cells that resemble the CD3/T-cell-receptor complexes (3-10). The mb-i products are called a chains, and the B29 products (3 chains (9). Characteristic of the sIg-mediated signal transduction pathways is that the initial activation cascades result in different biological effects, depending on the B-cell maturation stage and the sIg isotypes that are crosslinked (11-13). These complicated effects of sIg crosslinkage are not yet fully explained since, with the exception of variation in molecular mass of the mb-i products due to differential glycosylation (5, 14), the components of the B-cell antigen receptor complex appear identical regardless of the B-cell maturation stage and the sIg isotype (1, 2, 6, 7). In the present experiments, IgM-associated molecules on human B cells were isolated and used to produce monoclonal antibodies (mAbs) against exposed determinants on the human Ig-associated molecules. These mAbs were employed to investigate the antigen receptor units on B cells at various stages of maturation and to begin to explore their therapeutic potential.- Here we describe the distribution and biochemical features of the Ig-associated molecules, using two mAbs that appear to recognize extracellular epitopes of the human B29 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: sIg, surface immunoglobulin; mAb, monoclonal antibody; TdT, terminal deoxynucleotidyltransferase; NP-40, Nonidet P-40.

8522

Proc. Natl. Acad. Sci. USA 89 (1992)

Immunology: Nakamura et al. rescein-labeled goat anti-human ,u antibody and peridinin chlorophyll protein-labeled anti-CD19 mAb. For intracytoplasmic staining, unstained cells or cells stained with a rhodamine-labeled goat anti-human ,u antibody were centrifuged onto glass slides, fixed with 5% acetic acid/95% ethanol, and then stained with appropriate antibody preparations. For nuclear staining of terminal deoxynucleotidyltransferase (TdT), cells fixed in 100% methanol were incubated with rabbit anti-TdT antibody (Supertechs, Bethesda, MD) and then with fluorescein-labeled goat anti-rabbit Ig antibody. Counterstains were with biotinylated CB3-1 or SA-DA4-4 antibodies, followed by rhodamine-conjugated streptavidin. Modulation Assay. Blood mononuclear cells were cultured with CB3-1, CB3-2, or mixture of goat anti-human K and anti-human A antibodies (50 u.g/ml) for 16 hr at 37°C. Cells were then washed and stained with CB3-1 or CB3-2 plus fluorescein-labeled goat anti-mouse Ig antibody, fluoresceinlabeled anti-CD20 mAb or fluorescein-labeled goat antihuman heavy chain antibodies. After incubation with normal mouse serum, the B cells were counterstained with phycoerythrin-labeled anti-CD19 mAb and the mean fluorescence intensity was determined for B-cell expression of each antigen with and without prior antibody modulation. Immunoprecipitation. Cells surface labeled with Na125I (Amersham) were lysed in either 1% NP-40 or 1% digitonin lysis buffer containing 150 mM NaCl, 50 mM Tris HCl (pH 7.4), 5 mM EDTA, 0.05% sodium azide, 1 mM diisopropyl fluorophosphate, leupeptin (10 Mg/ml), pepstatin A (1 ,ug/ ml), chymostatin (2 pg/ml), and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were first cleared with Sepharose 4B coupled with an isotype-matched antibody of irrelevant specificity and then were incubated with Sepharose 4B coupled with specific antibodies. Bound molecules were dissociated with Laemmli's buffer, resolved by SDS/l0o PAGE, and identified by autoradiography. N-Glycanase (Genzyme) treatment of eluted proteins was done according to manufacturer's instructions. Immunoblotting. Digitonin lysates of Ramos, 697, and MOLT4 cells were incubated with Sepharose 4B coupled with SA-DA4-4, and bound molecules were eluted with 1% NP-40 lysis buffer. Eluted molecules were resolved by SDS/ PAGE and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated with CB3-1 and CB3-2 and then with 187-1 labeled with 125I.

RESULTS CB3-1 and -2 mAbs Identify an Exposed Epitope on sIgAssociated Molecules. Of the antibody products from >500 hybridomas, 37 were reactive with the sIgM complex from Daudi B cells but were nonreactive with secreted IgM molecules. Two of these hybridomas were selected because of their reactivity with viable Ramos B cells and 697 pre-B cells, subcloned by limiting dilution, and designated CB3-1 and -2. Both mAbs were determined to be of mouse ylK

isotype. Modulation studies revealed that incubation of normal B cells with CB3-1 or CB3-2 led to down-modulation in expression of the corresponding cell surface antigen and that the sIg molecules were comodulated (Table 1). Conversely, anti-Ig antibodies modulated both the sIg and CB3-1 antigens, whereas cell surface CD20 expression was unaffected by pretreatment with any of these antibodies. These results indicated the physical association of sIg molecules with the CB3 antigens on normal B cells. When cell surface proteins from Ramos B cells were lysed with NP-40 detergent, the CB3-1 and -2 immunoprecipitates migrated on gel electrophoresis as a single broad band at 82-95 kDa (Fig. lc). Under reducing conditions, three mol-

8523

Table 1. Comodulation of slg and the CB3-1/CB3-2 antigen

% of control MFI Antibody CB3-1 CB3-2 treatment sIg CD20 5.5 ND 87.1 Anti-K,+A 6.2 17.7 6.4 ND 89.9 CB3-1 CB3-2 26.2 ND 14.0 94.0 Peripheral blood lymphocytes were cultured for 16 hr with goat anti-K+A, CB3-1, or CB3-2 antibodies, and percentage of the control mean fluorescence intensity (MFI) was calculated for the indicated antigens on CD19+ B cells. ND, not done.

ecules of 45, 40, and 37 kDa (p45, p40, and p37 proteins) were resolved from the CB3-1 and -2 precipitates (Fig. la), whereas an anti-human A antibody precipitated only the Au heavy chains and A light chains under the same reducing conditions. In contrast, when the cells were lysed with a milder detergent, digitonin, to avoid dissociation of noncovalently linked molecules, the anti-human AL, CB3-1, and CB3-2 antibodies all precipitated the A heavy and A light chains in addition to p45, p40, and p37 (Fig. lb). The p40 band was sometimes obscured by diffuse migration of overlapping p45 and p37 bands. These results indicate that both CB3 antibodies recognize covalently linked dimers consisting of p45, p40, and p37 molecules that are noncovalently associated with sIgM molecules on B cells. To determine which of the associated proteins were recognized by the CB3-1 and -2 mAbs, we performed Western blot analysis of Ramos B-cell and 697 pre-B-cell lysates together with MOLT4 T-cell lysates as a control. Both mAbs reacted with a prominent 37-kDa protein of Ramos B cells and a 34-kDa protein of 697 pre-B cells (Fig. 2; see also Fig. Sc, lane 2) but were unreactive with MOLT4 T-cell constituents. A faint 34-kDa band was also recognized by both mAbs on Ramos cells. Since the p45 and p40 IgM-associated molecules have been identified as the human mb-l-encoded a chains (18), these results suggest that the CB3 antibodies recognize human (-chain equivalents which exhibit size heterogeneity in representative pre-B and B cells. Surface Expression of the CB3 Antigen During B-Cell Differentiation. Competition immunofluorescence assays revealed that binding of the CB3-1 antibody to Ramos cells was a. NP-40 1 2 3 4

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8524

Proc. Natl. Acad Sci. USA 89 (1992)

Immunology: Nakamura et al. a. CB3-1

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FIG. 2. Western blot analysis of CB3-1 and -2 antigens expressed by pre-B and IgM+ B cell lines. Ig-associated molecules purified from Ramos (lanes 1), 697 (lanes 2), and MOLT4 cells (lane 3) were resolved by SDS/PAGE under reducing conditions, transferred onto a nitrocellulose membrane, and incubated with CB3-1 (a) or CB3-2 (b) mAb.

inhibited equally well by the CB3-1 and -2 antibodies and vice versa (data not shown), suggesting that both antibodies recognized the same or contiguous epitopes. Since both mAbs perfomed identically in all assays, only the results obtained with the CB3-1 antibody are shown in the following experiments. The CB3 mAbs were reactive by surface immunofluorescence with all of the sIg+ cell lines tested, but with none of the sIg- cell lines examined, regardless of their lineage (data not shown). Expression of the cell surface CB3 antigen on pre-B and B cell lines paralleled sIg expression (Fig. 3). This was true for both sIgM+ (Ramos) and sIgG1+ (IM9) cell lines. When peripheral blood cells were examined, the antigen was found only on the CD19+ B cells, all of which were CB3+ (Fig. 4a). In contrast, terminally differentiated myeloma cells did not express the CB3 antigen (Fig. 4b). When bone marrow lymphocytes were examined, only 40%o of the CD19+ B-lineage cells expressed the CB3 antigen on their surface (Fig. 4c). Among the CD19+ population, all of the CB3+ cells expressed ,u heavy chains but the CB3cells did not (Fig. 4d). Moreover, the intensity of CB3 antigen expression correlated in linear fashion with that of the ,u heavy chains, as has been shown for the B29 gene product in murine B cell lines (19). Surface expression of the CB3

FIG. 4. Cell surface expression of the CB3-i antigen during B-cell differentiation. (a) Mononuclear cells from normal peripheral blood (peripheral blood lymphocytes, PBL) were differentially stained with the CB3-1 and anti-CD19 mAbs. (b) Bone marrow mononuclear cells from a myeloma patient were stained with CB3-1 and anti-CD38 antibodies. Large cells corresponding to myeloma plasma cells were gated on the basis of light-scatter characteristics for analysis of their CB3-1 and CD38 expression. (c and d) Mononuclear cells from afetal bone marrow sample were differentially stained with CB3-1, goat anti-human 1t antibodies, and anti-CD19 mAb. Lymphoid cells were gated for immunofluorescence analysis of expression of the CB3-1 antigen and CD19 (c). The CD19+ B-lineage cells were gated for analysis of the expression of the CB3-1 antigen and surface ,u (d).

antigen thus begins with surface ,u heavy-chain expression, continues throughout B-cell maturation and isotype switch-

ing, and terminates with plasma-cell differentiation. Cytoplasmic Expression of the CB3 Antigen During B-Cell Differentiation. Next, we examined the cytoplasmic expression of the CB3 antigen in pro-B (,- TdT+), pre-B (,u + TdT-), and B cells (sIgM+) in four fetal bone marrow samples. More than 80o of the u- TdT+ pro-B cells contained detectable amounts of the CB3 antigen in their cytoplasm. In keeping with this observation, 26% of the cytoplasmic CB3+ lymphocytes in fetal bone marrow were ,u-, whereas all of the ,u+ pre-B and B cells contained the CB3 antigen. Interestingly, while most myeloma plasma cells did not appear to express the antigen, CB3 immunofluorescence could be visualized in the Golgi region of a subpopulation of myeloma cells. We conclude that expression of the CB3 antigen begins with 207 697 Ramos IM9 nuclear TdT expression in human pro-B cells and persists until the onset of terminal plasma-cell differentiation. Molecular Heterogeneity of Ig-Associated Molecules During CD B-Cell Differentiation. When normal B cells were lysed in the relatively mild digitonin detergent, both the anti-light chain surface ° ~~~~~~~~~~~~Ig mAbs and CB3 mAbs precipitated A and 8 heavy chains, light chains, and the Ig-associated molecules (Fig. 5b, lanes 3 and 6), indicating that sIgM and sIgD were physically associated log(fluorescence) with the CB3 antigen complex in normal B cells. The 'y heavy chains could not be identified unambiguously in digitonin FIG. 3. Cell surface expression of the CB3-1 antigen and slg on pre-B and B cell lines. The 207 (p-) and 697 (A+) pre-B cells and the lysates because of the overlapping p45 band. Another band Ramos [IgM(A)+] and IM9 [IgGl(K)+] B cells were incubated with between the 8 and y heavy chains, the identity of which is purified CB3-1 and anti-human ,A mAb SA-DA4-4 (207, 697, and uncertain (marked X in Fig. 5a, lane 6), was observed in Ramos) or anti-human K mAb 187-1 (IM9), followed by biotinylated The X protein was prominant in both anti-light B-cell lysates. goat anti-mouse Ig antibody and phycoerythrin-conjugated streptachain and CB3 immunoprecipitates, but not in anti-heavy vidin. Isotype-matched control antibodies served as negative conchain immunoprecipitates. trols (dotted lines).

Immunology: Nakamura et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

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Interestingly, differences were evident in the characteristics of molecules associated with the tL and 8 chains. First, a relative paucity of sIgD-associated molecules was observed in comparison with the relative abundance of sIgMassociated molecules (Fig. 5b, lanes 4 and 5). This relative deficit was especially notable for the p45 a-chain component, instead of which a faint protein band of 53 kDa was found to be associated with sIgD (Fig. 5b, lane 5, arrow). In addition, the sIgM-associated p37 (3-chain protein identified by the CB3 antibodies (Fig. 2) was slightly larger than the comparable sIgD-associated band, which was -36 kDa (Fig. 5b, lanes 4 and 5). From NP-40 lysates of peripheral B cells, anti-light chain mAbs precipitated heavy chains together with light chains, whereas the CB3 antibodies precipitated broad p45 and p37 bands (Fig. 5a, lanes 3 and 6). Unexpectedly, under these relatively harsh detergent conditions some 1L heavy chains were also observed in the CB3 immunoprecipitates (Fig. 5a, lane 6). The relative intensities of the heavy-chain bands in anti-light chain immunoprecipitates were 8 > IL (Fig. 5a, lane 3), whereas the relative intensities in the CB3-1 immunoprecipitates were IL >> 8 (Fig. 5a, lane 6), indicating considerable variability in the strength of association between the different sIg isotypes and the a and 8 chains. The CB3 immunoprecipitates from NP-40 and digitonin lysates of the 697 pre-B cell line also differed in several respects (Fig. 5 c and d). First, in comparison with the anti-P antibody, the CB3 mAbs were relatively inefficient in precipitation of the solubilized receptor units present in digitonin lysates. Second, the p34 j-chain candidate identified by the CB3 mAbs in Western blots of pre-B cells (Fig. 2) was especially prominent in NP-40 lysates. In addition, the p40 and p45 a-chain species noted in B cells were largely replaced by 37- and 48-kDa proteins in the 697 pre-B cells. These differences, which were also observed in other pre-B (Nalm6, SMS-SB, BLIN-1) and B (Daudi) cell lines, reveal an interesting pattern of molecular heterogeneity in the slgassociated a and 8 chains during the progressive differentiation of pre-B cells to isotype-switched B cells. The apparent

molecular mass of a and chains from both pre-B and B cells reduced to -28 kDa following removal of N-linked oligosaccharides by N-Glycanase treatment.

was

DISCUSSION Several lines of evidence indicate that the CB3-i and -2 antibodies recognize an external epitope on one of the Ig-associated molecules. First, from NP-40 detergent lysates of B cells, the CB3 antibodies precipitated a complex of proteins distinct from the Ig heavy and light chains, whereas from B-cell lysates obtained with a milder detergent, digitonin, these antibodies precipitated the same non-Ig complex together with Ig heavy and light chains, although neither antibody reacted with secreted IgM in a sensitive immunoenzymatic assay. Of the three prominent disulfide-linked proteins, p45, p40, and p37, that were noncovalently associated with membrane-bound IgM on the B-cell lines, the CB3 antibodies recognized p37 together with a minor p34 molecular species. Since mAbs raised against peptides of the mb-l-encoded a chains recognize the p45 and p40 molecules (18), our data suggest that the CB3 antibodies recognize the (3chain products of the human B29 gene equivalent. The identification and characterization of the B29 gene counterpart in humans will allow formal testing of this interpretation. Transcriptional activity of the mouse B29 (3-chain gene and ofboth mouse and human mb-i a-chain genes can be detected in pro-B cells, in which Ig heavy-chain genes are in germ-line configuration, and in all types of B cells, but not in terminally differentiated plasma cells (1, 2, 17). In accordance with these results, we observed that most TdT1 pro-B cells and all of the IL + pre-B cells expressed the CB3 antigen in their cytoplasm, whereas most myeloma plasma cells did not. Since nuclear TdT is expressed before B cells productively rearrange their Ig heavy-chain genes (20), this result indicates the onset of (3-chain expression at roughly the same stage in differentiation. However, surface expression of the CB3 (chain antigen was restricted to lymphocytes that expressed either the

8526

Proc. Nad. Acad. Sci. USA 89 (1992)

Immunology: Nakamura et al.

,u heavy chain/surrogate light chain complex or conventional sIg isotypes. An interesting pattern of molecular heterogeneity of the human Ig-associated molecules emerged in our studies of pre-B and B cells. (i) The data indicate size variations in both types of Ig-associated molecules as a function of maturational stage. While the putative a- and (3-chain species associated with the p/surrogate light chain receptors on pre-B cell lines were largely of 48 and 34 kDa, respectively, those associated with IgM on B cells were primarily 45 and 37 kDa in apparent molecular mass. (ii) Size differences in both types of Igassociated chains were observed for the sIgM and sIgD receptors on normal B cells. While a- and (3-chain species of 53 and 36 kDa were associated with IgD, proteins of 45 and 37 kDa were associated with IgM receptors. Differently glycosylated forms apparently account for this size heterogeneity, since the same protein core size (-28 kDa) was observed for a and ( chains from both pre-B and B cells. These results complement and extend data obtained in studies of mice, where only the mb-i a-chain gene products associated with IgM and IgD differ in apparent size due to differential glycosylation (5, 21). (iii) We observed an apparent variation between sIgM and sIgD in the strength of their association with their respective a- and (-chain components on normal B cells. In the digitonin lysates, the CB3 antibodies precipitated ji and 8 heavy chains together with a and 83 chains. The CB3 antibodies also coprecipitated ,u, but not 8, heavy chains from NP-40 lysates. This is in spite of the fact that analysis of sIg isotype distribution revealed higher surface levels of 8 than ,u heavy chains on the normal B-cell population. This relatively weak association of both the a and ,B chains with sIgD on human B cells complements the results of experiments in mice, where gene transfection studies have indicated that sIgD expression may occur without either a or P chains (7, 22). In the latter case, however, the IgD that reached the surface of cells lacking in a and ( chains proved to be attached via glycosyl-phosphatidylinositol linkage rather than via the usual transmembrane domain (23). In humans, the transmembrane form of sIgD appears to predominate, as its expression is resistant to treatment with phosphatidylinositol-specific phospholipase C (data not shown) and both a and P chains are abundant in B cells bearing sIgD and sIgM. Crosslinkage of sIgM molecules on mature B cells induces hydrolysis of phosphatidylinositol bisphosphate leading to cell proliferation (24, 25). Signaling through sIgM on immature B cells instead induces growth inhibition through activation of a protein-tyrosine kinase pathway (26-28), even though these cells have an intact phosphatidylinositol pathway that can be otherwise activated (12). Signaling through IgM and IgD receptors on the same B cell may also result in different biological effects (12, 13). Therefore, it is attractive to speculate that the heterogeneity of the sIg-associated molecules that we have observed may be an important variable in signal transduction via the different slg receptor units expressed as a function of pre-B- and B-cell maturation. The CB3 mAbs provide another surface marker for B cells and should be helpful in further delineation of the signal transduction pathways activated via antibody receptors during different stages in B-cell differentiation. The CB3 antibodies could also be of therapeutic value, since they react with all sIg+ B cells and can down-modulate antibody receptors in a fashion comparable to the modulation of T-cell

receptors by anti-CD3 antibodies, which have proven valuable for the diagnostic and therapeutic manipulation of T cells

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