Mark de Boern, Paul Parreno, Jeffrey Dove", Ferry Ossendorp", Gerda van der HorstO and John Reeder" Department of Immunology", Cetus Corp., Emeryville, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology., University of Amsterdam, Division of Immunologyo, The Netherlands Cancer Institute, Amsterdam
Functional characterization of an anti-B7 mAb
Eur. J. Immunol. 1992. 22: 3071-3075
Functional characterization of a novel anti-B7 monoclonal antibody For optimal activation of T cells, binding of their Tcell receptor to antigenic peptides in the context of major histocompatibility complex molecules on antigen-presenting cells (APC) is not sufficient. Accessory signals, provided by accessory cells, are needed to induce proliferation and clonal expansion of normal Tcells. It has been shown previously that the B7 molecule, present on the cell surface of activated APC, can provide the second signal by binding to the CD28 molecule onTcells. Here we describe a novel anti-B7 (mAb), B7-24.This mAb binds to a functionally important epitope of the B7 molecule. Fab fragments of B7-24 can almost completely block anti-CD3-induced, B7-dependent T cell proliferation when tested in a model system where purified T cells are co-cultured with 3T6 cells expressing the human FcyRII and human B7, in the presence of anti-CD3 mAb. In contrast, mAb B7-24 is not able t o inhibit T cell proliferation in primary mixed lymphocyte reactions where purified T cells are co-cultured with Epstein-Barr virus-transformed B cells. These findings suggest that other cell surface molecules allow for maximal proliferation of T cells in mixed lymphocyte reactions, even when the interaction between B7 and CD28 is blocked by B7-24.
1 Introduction Activation of T cells is the result of ligand-receptor interactions. Under physiological conditions the TcR/CD3 complex binds to antigenic peptides presented by MHC molecules of APC. The TcR/CD3 complex has two functions in antigen-induced activation: a recognition function in which a spccific antigen is recognized in the context of the appropriate MHC molecule, and a signaling function in which the recognition event is transmitted across the plasma membranc [ 1, 21. Studies on activation of T cells have been facilitated by the availability of mAb reactive with the TcR or CD3 antigens. These mAb can function as polyclonal activators of T cells, resulting in expression of high-affinity IL-2 receptors, secretion of lymphokines including TL-2 for autocrine growth, and proliferation [3-61. However, when freshly isolated resting Tcells are used, stimulation with mAb to theTcR/CD3 complex is dependent on accessory signals . Apart from theTcR/CD3 complex, an increasing number of accessory molecules on the surface of Tcells have been shown to play a role in Tcell activation. Thc function of these molecules can involve adhesion or signaling. These two functions are not always mutually exclusive. For imtance, thc CD2 molecule on Tcells can bind to CD58 (LFA-3) on APC [XI, but it has also been shown that binding of specific mAb to CD2 can augment signaling via the TcR/CD3 complex [9, 101. Other ligand pairs that are involved in Tcell activation are CDlldCD18 (LFA-1) and CD54 (ICAM-I), CD28 or CTLA-4 and B7, and CD29/CD49d (VLA-4) and VCAM-1 [ll-151.
[I 104401 ~
Correspondence: Mark dc Boer. Innogenetics, Industriepark Zwijnaarde 7. box 4. B-9052 Ghent, Belgium (3 VCH Veilagsgcsellachaft mbH, D-6940 Weinhelm, 1992
Interestingly, one of these accessory molecules, CD28, regulates a signal transduction pathway distinct from that stimulated by the TcR/CD3 complex [16-171. CD28 is a homodimeric transmembrane glycoprotein with an apparent molecular mass of 44 kDa and is a member of the immunoglobulin superfamily. The CD28 molecule is expressed on approximately 95% of the CD4+ Tcells and 50% of the CD8+ Tcells. Co-stimulation of Tcclls with mAb to the TcR/CD3 complex and CD28 results in greatly enhanced activation [ 17-19]. This effect apparently involves stabilization of mRNA for several lymphokines, including IL-2, resulting in a greatly enhanced production of these lymphokines [la, 191. Furthermore, a CD28responsive element has been demonstrated in the enhancer of the IL-2 gene, suggesting that there is also regulation at the transcriptional level [20, 211. Recently, it was described that the B cell activation marker B7 can bind to CD28 . B7 is a monomeric transmembrane glycoprotein with an apparent molecular mass of 45-65 kDa and is, like CD28, a member of the immunoglobulin superfamily . Although it was initially reported that the expression of B7 is restricted to Bcells , the B7 molecule was later described as also being expressed on monocytes activated with IFN-y . Moreover, B7-expressing Chinese hamster ovary (CHO) cells are able to synergize with TcR stimulation, resulting in IL-2 secretion and proliferation by T cells [24,25]. However, B7 cannot only bind to CD28, but also to a recombinant fusion protein of the CTLA-4 molecule . CTLA-4 is also a member of the immunoglobulin superfamily and the cytoplasmic regions of CTLA-4 and CD28 show significant homology, but in the absence of specific mAb, it remains unknown whether CTLA-4 is expressed on the cell surface of Tcells. Here we report on the characterization of a new mAb to human B7, B7-24. This mAb binds to a functional epitope on the B7 molecule involved in the interaction with the CD28 or CTLA-4 molecule on T cells and can completely block B7-mediated Tcell proliferation. However, mAb B7-24 is not able to inhibit T cell proliferation in mixed lymphocyte reactions. The implications of these findings are discussed.
0014-2980/92/12 12-3071$3.50 .25/0
M. de Boer, P. Parren, J. Dove et al.
Eur. J. Immunol. 1992. 22: 3071-3075
2 Materials and methods
2.5 Generation of B7-expressing 3T6-FcyRII cells
2.1 Cells and cell lines
Human B7 was cloned by PCR . For the generation of stable cell lines expressing B7, the B7 cDNA was cloned in the Bam HI-Eco RI site of the vector pCDNAl (Invitrogen, San Diego, CA). Subsequently, the fragment was transferred to the vector pCDNA1-NEO as a Bam HIXho I fragment. Plasmid DNA was prepared using the QIAGEN system (QIAGEN, Studio City, CA). For transfection, at day 0,3T6-FcyRII cells (0.8 x lo6)were plated in a 100-mm tissue culture disk. On day 1, the cells were transfected with 10 pg plasmid DNA employing the calcium phosphate method . On day 2, the transfection medium was replaced with fresh complete DME/HAM-F10 medium, and on day 3 the medium was replaced with DME/HAM-F10 medium containing 400 pglml G418. The cells were given fresh medium every 3-4 days. After 10-14 days, colonies of viable cells were isolated, expanded, and tested for cell surface expression of B7.
Peripheral blood mononuclear cells were isolated from heparinized blood by Ficoll-Hypaque (Sigma, St. Louis, MO) density centrifugation. T cells were enriched by depleting monocytes and B cells using Lymphokwik (One Lambda, Los Angeles, CA) The EBV-transformed B cell line ARC was obtained from the ATCC (Rockville, MD). The mouse fibroblast cell line 3T6 expressing human FcyRIIa high responder allele (CD32, 3T6-FcyRII cells) was kindly provided by Dr. J.van de Winkel (University Hospital, Utrecht,The Netherlands).This human Fc receptor interacts efficiently with mouse IgGl mAb [27, 281.
2.2 Culture media Purified T cells and EBV-transformed B cells were cultured in Iscove’s modification of Dulbecco’s modified Eagle’s medium supplemented with streptomycin (200 pg/ml), penicillin (200 U/ml) and 10% heat-inactivated fetal bovine serum (complete IMDM). 3T6-FcyRII cells were cultured in medium consisting of 50% Dulbecco’s modified Eagle’s medium and 50% HAM-F10 medium, supplemented with aminopterin (0.2 pg/ml), thymidine (5 pg/ml), xanthine (10 pg/ml), hypoxanthine (15 pg/ml), mycophenolic acid (20 pg/ml), deoxycytidine (2.3 pg/ml) and 10% heat-inactivated fetal bovine serum (complete DME/HAM-F10). 3T6-FcyRIIIK7 cells were cultured in complete DME/HAM-F10 medium containing 400 pg/ml G418 (Gibco, Grand Island, NY).
Proliferation of purified T cells in the presence of transfected 3T6 fibroblasts was measured by [3H]thymidine incorporation. Briefly, 4 x lo4T cells were cultured with lo4 irradiated (2500 rad) 3T6-FcyRII or 3T6-FcyRIIIB7 cells in 96-well flat-bottom tissue culture plates in 200 pVwell complete IMDM with or without anti-CD3 mAb CLBT3/4.1 (1 pg/ml). During the last 16 h of a 72-h culture period, the cells were pulsed with 1 pCi/well [3H]thymidine. Proliferation of T cells is expressed as the mean cpm of triplicate wells. SD were always smaller than 10%.
2.3 Monoclonal antibodies
2.7 Mixed lymphocyte cultures
mAb B7-24 (IgG2,,x) was obtained from a fusion with splenocytes from a mouse immunized with Sf9 insect cells expressing the human B7 molecule  and was used as purified antibody. Anti-human IL-6 mAb CLB-IL6/8 (IgG1,x)  was a gift of Dr. L. Aarden (Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). Anti-CD28 mAb CLBCD28/1 (IgC1,x) was used as purified antibody and was a gift of Dr. R. van Lier (Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam,The Netherlands). AntLCD3 mAb CLB-T3/4.1 (IgG1,x) was used as diluted tissue culture supernatant.
Proliferation of purified T cells was measured in mixed lymphocyte cultures using the EBV-transformed B cell line ARC as stimulator cells. Fifty thousand Tcells were cultured with 5 x lo4irradiated (6500 rad) stimulator cells in 96-well round-bottom tissue culture plates (Corning, Corning, NY) in 200 pVwell complete IMDM medium. During the last 16 h of a 72-h culture period, the cells were pulsed with 1 pCi/well [3H]thymidine. Proliferation of Tcells is expressed as the mean cpm of triplicate wells. SD were always smaller than 10%.
2.6 Proliferation of T cells
2.8 Radiolabeling, immunoprecipitation and gel electrophoresis 2.4 Fluorescence cell staining ARC cells (lo6 sample) were incubated for 15 rnin at 4°C with 50 pl Fab fragments of mAb B7-24 or mAb CLBIL-6/8 (10 pg/ml in PBS-BSA). Subsequently, 50 pl of biotinylated mAb B7-24 (1 pglml in PBS-BSA) was added and the mixture was incubated for an additional 15 min at 4°C. After three washes with PBS-BSA, cells were incubated in 100 p1 streptavidine-PE (Becton Dickinson, San Jose, CA) for 15 min at 4°C. After three washes with PBS-BSA and one wash with PBS, the cells were resuspcnded in 0.5 ml PBS and fixed by adding 0.5 ml of 2% paraformaldehyde in PBS. Analyses were performed with a FACScan V (Becton Dickinson).
For cell surface iodination, 50 x 106 cells of the EBVtransformed B cell line JY were washed and resuspended in PBS and labeled with lmCi Na1251(Amersham Co.) using lactoperoxidase as a catalyst. After labeling, cells were lysed in immunoprecipitation buffer (IPB), consisting of 0.01 M triethanolamine-HCL, pH 7.8, 0.15 M NaCl, 5 mM EDTA, 1 mM PMSF, 0.02 mg/ml ovomucoid trypsin inhibitor, 1 mM sodium p-tosyl-L-lysine chloromethyl ketone, 0.02 mg/ml leupeptin and 1% Nonidet P-40 (NP40). Nuclear debris was removed by centrifugation for 15 min at 13000 X g. Lysates were centrifuged for 30 rnin at 100000 x g and precleared by three incubations of 1h with 30 pl of a 10% V/V suspension of protein A-CL 4B Sepharose beads
Functional characterization of an anti-B7 mAb
Eur. J. Immunol. 1992. 22: 3071-3075
(Pharmacia, Uppsala, Sweden) coated with normal mouse serum immunoglobulin. Specific precipitation was carried out for 2 h with purified anti-B7 mAb B7-24, coupled non-covalently to protein A beads. Beads were washed with IPB and resuspended in SDS sample buffer. For SDSPAGE, 10%-15% polyacrylamide gradient gels were used, according to a modification of the Laemmli procedure. Samples were analyzed either under reducing conditions (5% 2-ME in SDS sample buffer) or nonreducing conditions (1 mM iodoacetamide in SDS sample buffer). Autoradiography took place at - 70 "C, using Kodak XAR-5 film in combination with intensifier screens.
3 Results To study the role of the B7 molecule in accessory celldependent T cell proliferation, we generated anti-B7 mAb. The anti-B7 mAb B7-24 was obtained from a fusion with splenocytes from a mouse immunized with Sf9 insect cells expressing the human B7 molecule .We have previously demonstrated the specificityof mAb B7-24 in a competition experiment in which soluble B7, but not soluble CD40,was able to block the binding of mAb B7-24 to EBVtransformed B cells expressing B7. Here we show that mAb B7-24 only binds to a single molecule on the cell surface of EBV-transformed B cells. Fig. 1 shows that mAb B7-24 is able to precipitate a single molecule with an estimated M, of 45-65 kDa under both reducing and nonreducing conditions. This finding is in good agreement with what has been reported for two other anti-B7 mAb [34, 331. To find out whether B7-24 binds to a functional domain of the B7 molecule involved in the interaction with its ligand CD28 on T cells, we tested whether B7-24 could block B7-mediated T cell proliferation. These experiments were performed by assessing anti-CD3-induced T cell proliferation in the preA
Figure 2. Proliferation of purified T cells stimulated with 3T6FcyRII cells in the absence (gray bars) or presence (open bars) of anti-(CD3) mAb CLB-T3/4.1. Proliferation was measured as described in Sect. 2.6 and represents the mean of triplicate wells. The additions to the culture medium were none, anti-(CD28) mAb CLB-CD28/1 (1 pg/ml), or recombinant human IL-2 (100 U/ml) .
sence of mouse 3T6 cells transfected with human FcyRII (3T6-FcyRII cells). In this system, accessory cell-depleted peripheral blood T cells are activated by FcyRJI-bound anti-CD3 mAb CLB-T3/4.1 . Fig. 2 shows that this induction of T cell proliferation is absolutely dependent on a second signal. A second signal could be provided by addition of antLCD28 mAb CLB-CD28/1 or by addition of recombinant human IL-2. The absolute requirement for a second signal indicated that this system could be very useful as a model to assess the role of various accessory molecules, present on APC, in the activation of Tcells. Therefore, we decided to generate 3T6 cells co-expressing human B7 and the FcyRII (3T6-FcyRIIIB7 cells). Fig. 3 shows that in the presence of anti-CD3 mAb CLB-T3/4.I, purified T cells could be stimulated by 3T6-FcyRIIIB7cells. In contrast, control 3T6-FcyRII cells lacking B7 expression were not able to induce proliferation of purified T cells, even in the presence of anti-CD3 mAb. In this svstem we tested whether mAb B7-24 interacts with a functional
F i g u r ~1. Immunoprecipitation with anti-B7 mAb B7-24 of cellsurface 12sI-labeledEBV-transformed B cells JY. The immunoprecipitated material was subjected to SDS-PAGE and run under nonreducing conditions (lanes A and B) and reducing conditions (lanes C and D). Lanes A and C: immunoprecipitation with B7-24; lancs B and D: protein A preclear.
- 68 - 29 - 18 - 14
Figure 3. Proliferation of purified Tcells co-cultured with 3T6FcyRII cells in the presence (open bars) or in the absence (gray bars) of anti-(CD3) mAb CLB-T3/4.1, or co-cultured with 3T6FcyRIUB7 cells in the presence (hatched bars) or absence (black bars) of anti-(CD3) mAb CLB-T3/4.1. Proliferation was measured as described in Sect. 2.6 and represents the mean of triplicate wells. The data is representative for five experiments using Tcells from different donors.
Eur. J. Immunol. 1992. 22: 3071-3075
M. de Boer, I? Parren, J. Dove ct al.
Table 1. Inhibition of anti-CD3-induced proliferation of purified T cells by antik(B7) Fab fragments
Concentration of Rib fragments (PgW
3T6-FcyRII/B7-induced proliferation") of Tcells i n presence of anti-B7 anti-IL-6
5 0.5 0.05 None
2 343 5 701 18056 13594
5 0.5 0.05 None
24 853 30 789
IS 635 10926 I1511 26 562 29 683 29 822
a) Proliferation of purified Tcells was measured as described in Sect. 2.6 and is expressed as the mean cpm of triplicate wells. The data is representative for three experiments using Tcells from different donors.
epitope on B7. Table 1 shows that Fab fragments of mAb B7-24 could almost completely inhibit anti-CD3-induced activation of purified T cells co-cultured with 3T6FcyRIIIB7 cells. Fab fragments of control mAb CLB1L6/X were without effect. This demonstrates that mAb B7-24 indeed binds to a functional epitope on the B7 molecule. Ncxt we studied whether mAb B7-24 could block the activation ot T cells in a mixed lymphocyte reaction using EBV-transformed B cells as stimulator cells. In this experiment B7-24 Fab concentrations that completely blocked B7-mediated T cell proliferation, as tested in Table 1, had no inhibitory effect on the mixed lymphocyte response (results not shown). Furthermore, Table 2 shows that concentrations of up to 100 Kg/ml of intact mAb B7-24, like intact mAb to CD28, had no inhibitory effect on the mixed lymphocyte response using EBV-transformed B cells expressing B7 as stimulator cells. I n this experiment anti-CD3 m Ab almost completely blocked the activation of the Tcells.
rahle 2. Lack of inhibition of mixed lymphocyte reaction by intact mAb B7-24
C'onccntrat ion ol mAh
(pg/nil) 100 -1
II 3.7 None
MLR inciuccd proliferation") o f accessory cell-depleted Tcells Anti-B7 Anti-CD25 Anti-CD3 19 500 19090 19 820 22 130 18410
20 4-10 19360 21 270 21 130 18410
2 110 3 940 -1350 3 270 15410
Proliferation of purified T cells was measured as described in Sect. 2.6 and is expressed as the mean cpm of triplicate wells. The data is representative for three experiments using Tcells from different donors.
4 Discussion Tcells are activated after binding of their TcR to antigenic peptides in the context of MHC class I1 molecules on APC. This signaling through the TcR/CD3 complex alone, however, is not enough for complete activation of Tcells. Accessory signals, which may be provided by accessory cells, are needed to induce proliferation of Tcells. It has been suggested that only activated APC are able to provide these co-stimulatory signals , and that direct cell-cell contact is required [35, 36l.Thislead to the postulation that upon TcR-antigen/MHC class I1 interaction, B7 on APC can provide the co-stimulatory signal . This hypothesis is supported by the findings that B7 is not expressed on resting B cells [34, 371, but can be induced by stimulating B cells with anti-immunoglobulin antibodies  or crosslinking of MHC class I1 molecules . Likewise, B7 was not found on unstimulated monocytes, but could be induced by IFN-y . To study the function of the B7 molecule on APCS in T cell activation, we wanted to use a simple model system.We hve recently demonstrated that highly purified T cells can be activated by the anti-CD3 monoclonal antibody CLBT3/4.1 when presented on the cell surface of mouse 3T6 cells expressing the human FcyRII . Induction of T cell proliferation in this system is, however, absolutely dependent on a second signal. This absolute requirement for a second signal makes this system very useful as a model to assess the individual role of various accessory molecules normally present on APC. Here, we demonstrate that 3T6 cells co-expressing the human B7 molecule and the FcyRII are very efficient in eliciting anti-CD3-induced T cell proliferation. This is in agreement with what has been shown in other systems [24,25] and demonstrates that the B7 molecule might have an important role in accessory cell-dependent T cell proliferation. To further address the importance of the B7 molecule in accessory cell-dependent T cell mitogenesis, we wanted to generate mAb to functional epitopes on the B7 molecule. We have demonstrated that B7-24 is an anti-B7 mAb that binds to an epitope of the B7 molecule which is important for its biological activity.We have shown that Fab fragments of mAb B7-24 bind to a functionally important epitope of the B7 molecule, since these Fab fragments almost completely blocked anti-CD3-induced, B7-dependent T cell proliferation. Several lines of evidence have indicated that the B7/CD28 interaction is important for allogeneic T cell activation. Koulova et al.  have shown that the anti-B7 mAb BB-1 and Fab fragments of the anti-CD28 mAb 9.3, can almost completely block mixed lymphocyte reactions. Furthermore, the B7-negative B cell line Ramos is a weak inducer of T cell proliferation in mixed lymphocyte reactions, whereas transfection of this cell line with the B7 cDNA completely restores the capacity of this cell line to induce allogeneic T cell proliferation . It was therefore surprising to find that B7-24 had no inhibitory effect on T cell proliferation in primary mixed lymphocyte reactions with EBV-transformed B cells.This discrepancy may be due to differences in the experimental conditions used.The fact that we did not see inhibition with intact anti-CD28 mAb 15E8 is not inconsistent with the findings of Koulova et al. ,since we known that intact mAb 15E8 can stimulate Tcells (see Fig. 2). The inhibition of the mixed lymphocyte
Eur. J. Immunol. 1992. 22: 3071-3075
response by the anti-CD3 mAb demonstrates that the lack of inhibition with anti-B7 mAb B7-24 is due to the unique properties of the mAb. It is possible that mAb BB-1,which is of the IgM isotype, blocks more membrane molecules besides B7. These molecules might exist in a complex with B7 and it could be possible that cell-cell interactions involving these unknown molecules may allow for maximal T cell proliferation even when the interaction between B7 and CD28 or CTLA-4 is blocked by mAb B7-24. Experiments are in progress to investigate these possibilities, and thc outcome of these studies will tell us more about the physiological role of the B7 molecule in Tcell activation. We would like to thank Drs. Hyc>Yeorzg Min and Lucien Aarden f o r rrilicully reudirig the manuscript and f o r helpful discussions during the course of this work. Received March 12, 1992; in final revised form August 17, 1992.
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Functional characterization of an anti-B7 mAb
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