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Mutational analysis and an alternatively spliced product of B7 defines its CD28/CTLA4-binding site on immunoglobulin C-like domain.

Costimulatory molecules B7 and B7-2 interact with T cell surface receptors CD28/CTLA4 and deliver a costimulatory signal essential for T cell growth. However, the structure basis of this interaction is not known. B7 and B7-2 are members of immunoglobulin (Ig) superfamily and their extracellular portion consists of an IgV- and IgC-like domain. Here we report that a naturally occurring, alternatively spliced form of B7 reveals that exon 3-encoded IgC domain is essential for CD28/CTLA4 binding. Mutational analysis of B7 demonstrates a critical role of several amino acids around loops between strands B and C and D and E, for binding CTLA4/CD28. These amino acids are clustered to form a single binding site centered at 201Y. A comparison of the effects of mutations on the binding of CD28 and CTLA4 reveals that CD28 and CTLA4 binds to the same site on B7. These results have important implications on the role of CTLA4 and CD28 in T cell costimulation. The structure of the CD28/CTLA4-binding site also provides valuable information for immune intervention targeted at the B7/B7-2-CD28/CTLA4 interactions.

ctivation of T cells requires two types of biological signals: signal I is delivered by interaction between the TCR and MHC-peptide complex, whereas signal 2 is called the costimulatory signal (1)(2)(3)(4)(5)(6). Recent studies from various laboratories indicate that signal I determines the specificity of T cell activation, whereas signal 2 determines the fate of T cells. T cells that receive both signals 1 and 2 clonally expand and differentiate into effector cells. In contrast, T cells that receive signal 1 alone are either functionally inactivated (anergized) and/or undergo programmed cell death (7)(8)(9)(10)(11).
B7 and B7-2 are members of the Ig super family (15)(16)(17)31). The extracellular portion consists of an IgV-like domain and an IgC-like domain. Murine B7 gene contains 5 exons encoding, respectively, signal peptide, IgV-like domain, IgClike domain, transmembrane domain, and cytoplasmic domain. B7 and B7-2 bind to CD28 (32) and, with a higher affinity, to a less abundant receptor CTLA4 (33). It has been suggested that CD28 and CTL4 may transduce qualitatively different signals when engaged by B7 and/or B7-2 (34). As a first step to understand the mechanism ofT cell costimulalion mediated by B7/B7-2-CD28/CTLA4 interaction, we set out to determine the structural basis of these interactions. Here we report the B7-binding site for CD28 and CTLA4 as defined by a naturally occurring, alternatively spliced B7 and by site-directed mutagenesis.

Experimental Animals, Cell Lines, and mAbs
Male CBA/CaJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) as donors of lymphocytes. B cell lymphoma CRCS-2, provided by Dr. Thorbecke (New York University Medical Center) (35), CH27, and M12 were cultured in KPMI medium containing 5% of FCS. Anti-B7 mAb 16.10A (36) was kindly provided by Dr. H. Reiser (Harvard Medical School, Boston, MA), mAb 7A5 was described fully (18).

Construction of the Fusion Proteins CD28Ig and CTLA4Ig
Fusion proteins CTLA4Ig and CD28Ig, comprised, respectively, the extracellular domains of murine either CTLA4 or CD28 and Fc portion of murine IgG2a, were generated according to a described procedure (33). Briefly, DNA encoding the extracellular region of murine CTLA-4 was amplified by PCR using the synthetic oligonucleotide CACAAGCTTGCCATGGCTTGTCTT-GGACTC as forward primer, ~TCCTGATCAAGGTCAGA-ATCCGGGCATGGTTC as reverse primer, and cDNA from Con A-activated murine spleen T cells as template. The product of this PCR reaction was digested with HindlII and BclI and ligated together with a BdI/XbaI cleaved DNA fragment encoding the hinge, CH2 and CH3 domains of murine Ig Cy2a. The latter fragment was amplified from cDNA prepared from a murine IgG2a hybridoma using the oligonucleotide GACCTGATCAGCGAGCCCAGA-GGGCCCACA as forward primer, and GACCAGTCTAGATTT-ACCCGGAGTCCGGGAGAA as reverse primer. Ligation products were cloned into the CDM8 mammalian expression plasmid (Invitrogen, San Diego, CA) and the construction was verified by DNA sequence analysis. The encoded molecule comprises residues 1-161 of murine CTLA-4 fused to the hinge region of murine heavy chain C3~2a. Similarly, the extracellular domain of CD28 (AA 1-150) was amplified from CD28 cDNA (37) kindly provided by Dr. J. Allison (University of California, Berkeley, CA), using CCCAAA-GCTTCAATGACACTCAGGCTGCTG as forward primer and TTGTGGGCCCTCTGGGCTCGAGCTTAGGAGATGACTG as reverse primer. The PCR product was digested with ApaI. The IgG2a fragment was prepared by digesting CTLA4Ig construct with ApaI and XbaI. The CD28 fragment and the Ig fragment were ligated into pCDM8 vector digested with HindlII plus XbaI. The final construct was verified by DNA sequencing. The fusion proteins were prepared from CHO cells stably transfected with either CD28Ig or CTLA4Ig construct. The CHO cell supernatants were concentrated 10 times and used for this study. The supernatant contains 10 times the excess of fusion proteins required for saturating binding against wild-type B7 in flow cytometry.
Construction of B7-HSA, B7IgV-HSA, and B7IgC-HSA All fusion proteins were generated by three piece ligation including HindIII plus XhoI-digested B7-fragments, XhoI + XbaI-digested HSA fragment and HindlII + XbaI-digested pCDM8 vector. The B7 and HSA fragments are generated as follows: B7. The extracdlular portion orB7, including the signal peptide, IgV and IgC domain, were amplified by PCR, using GCTCGA-AGCTTATGGCTTGCAATTGTCAG as forward primer, and GTC-AGCC~GAGTTTTI~CCAGGTGAAGTC as reverse primer. pCDM8-B7, which contains the entire reading frame of B7, was used as template. The product of this reaction encodes M1-K236 which is the entire extracellular portion of B7.
B7IgV. pCDM8-B7 was used as template and the GCTCGA-AGCTTATGGCTTGCAATTGTCAG as the forward primer and GGGCTCGAGGTCTGCAGATGGGTTTCC as reverse primer. The product of this reaction encodes M1-D159, which contains the signal peptide, B7IgV domain plus 15 AA from B7IgC domain as spacer.
B7-IgC. First, the signal sequence of B7 (B7SP) was amplified with GCTCGAAGCTTATGGCT'[GCAATTGTCAG as a forward primer and GAAGTCAGCATCTGAAGACACTTGTGAAAGACG as a reverse primer, and exon 3 sequence of B7 (B7E3) was amplified with GTGTCTTCAGATGCTGACTTCTCTACCCCCAAC as a forward primer and GTCAGCCATCTCGAGTTTTTCCCAGGT-GAAGTC as a reverse primer. A mixture of B7SP and B7E3 fragments were used as template, GCTCGAAGCTTATGGCTT-GCAATTGTCAG as a forward primer and GTCAGCCATCTC-GAGTTTTTCCCAGGTGAAGTC as a reverse primer. The final PCR product encodes the signal peptide and exon 3 encoded B7 IgC domain (M1-D37 + A143-K236).
lISA. The HSA fragment that contains all HSA protein sequence except signal peptide was amplified using GAAAAACTC-GAGATGAACCAAACATCTGTTGCA as forward primer and CACAAGTAAGGTTCCTI~ACAAAG as rewet~ primer, pCDM8-HSA (13) was used as template. The final PCR product encodes LE as linker ptus extraceUular portion and GPI-signal motif of HSA sequence, N27-stop codon.
All PCR. products were verified by DNA sequencing.
Analysis of B7 mRNA by PCR mRNA were prepared from 5 x 106 of either CRCS, M12.4.1, CH27, or total spleen cells by the fast-tract poly(A)* RNA preparation kit (Clontech, Palo Alto, CA). First strand cDNA were prepared using 50 ng of hexamer random primers, 1 t~g mRNA, 200 U Maloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg, MD). Similar results were obtained when poly dT or B7 reverse primer were used to prepared the first strand cDNA. The B7 or GAPDH DNA were amplified from the cDNA by PCR reaction as has been described. The primers used were: B7 forward primer GAAGCTA_TGC~TTGCAATTGTCAG, B7 reverse primer AGAAGAACTAAAGGAAGACGGTCT, GAPDH forward primer ATGGTGAAGGTCGGTGTGAACGGATTTGGC, and GAPDH reverse primer CATCGAAGGTGGAAGAGTGGGAGTTGCTGT. The RT-PCR products were detected by Southern blot using 32p_ labeled murine B7 or GAPDH cDNA as probe.

Cloning of the RT-PCR Products
Two forms of the B7 RT-PCK products were purified from agarose gels and cloned into the pCDM8 vector. The DNA inserts were sequenced and confirmed to contain the fixll-length B7 open reading frame sequence or that of an alternatively spliced B7.

Site-directed Mutagenesis
Site-directed mutagenesis was carried out based on PCR. Briefly, oligonucleotide primers between 25 and 40 bp tong were synthesized to contain the desired mutations. These primers were used to generate B7-fragments that were then used to replace the wildtype fragment to generate the B7 mutants. The DNA sequences of the regions that contain the mutation and the junction of ligations were verified by DNA sequencing. The mutations are listed in Table 1.

T Cell Proliferation
CD4 T cells were purified from CBA/CaJ mouse spleen cells by two rounds of treatment with complement plus an antibody cocktail, that consists of: anti-B220 mAb KA3-3/6.1, anti-HSA mAb Jlld (38), anti-CD8 mAb M31(39), anti-Mac-1 mAb M1/70.15: 11.5H (40), and anti-FcR mAb 2.4G2 (41), all hybridoma supernarants mixed in an equal proportion. CD4 T cells were stimulated with anti-CD3 mAb 2Cl1.145 (1:40 supernatants) (42). COS cells transfected with either Fc'yRIIB2 (FcR), or FcR plus B7 or B7 mutants were treated with mitomycin C (100/zg/ml, 37~ for 1 h), and used as accessory cells. After The data shown are MF of transfected COS cells after being stained with either anti-B7 mAb 10.16A.1 or chimeric molecules CD28Ig or CTLA4Lg, with that of controls (staining in the absence of first step-reagents) substracted. Numbers in the parentheses are % wild-type B7 binding after normalizing the cell surface expression of B7 mutants as described in experimental procedure. Each staining has been repeated for at least three times.  Guo et al.
goat anti-mouse IgG, goat anti-hamster IgG, and mouse anti-rat IgG. All of these second step reagents give essentially identical background staining in the absence of primary antibody, therefore only one control is shown in each experiment. Details of flow cytometry have been described (44). Data shown in Table 1 are mean fluorescence (MF) ~ obtained by flow cytometry with that of the controls (in the absence of first step reagents) subtracted.
1 Abbreviation used in this paper: MF, mean fluorescence.

Results
A Naturally Occurrin,r Alternatively Spliced B7 Reveals a Critical Role of BlIgC Domain in Binding CD28 and CTLA4, In the process of analyzing the B7 expression in a number of different cell lines by PCR, we have observed two major forms of B7 cDNA: a longer form of 930 bp and a shorter form of '~,650 bp ( Fig. 1 a). Cloning and c D N A sequencing revealed that the larger band is B7, whereas the shorter band is a truncated B7 with a deletion from base 428 to base 699 (number begins at the start codon), which is the entire sequence of exon 3 (Fig. 1 b). Thus this shorter form of B7 is a product from an alternative splicing of B7. As the only extracellular portion of the protein encoded by this alternatively spliced B7 gene is the IgV domain, we call it B7IgV. To test whether BIlgV contains CD28/CTLA4-binding sites,

1348
CD28/CTLA4 Binding Site on B7 IgC-like Domain we generated fusion proteins that consist of murine IgG2a Fc portion and extraceUular domains of either murine CD28 (CD28Ig) or routine CTLA4 (CTLA4Ig). We then used these fusion proteins and a panel of anti-B7 mAbs to monitor the cell surface expression of B7 and B7IgV on COS cells transiently transfected with these genes. Whereas the B7 binds anti-B7 mAbs ( Fig. 1 c and data not shown) CD28Ig and CTLA4Ig, B7IgV binds anti-B7 mAbs but not CTLA4Ig and CD28Ig (Fig. I c). The lack of binding to CTLA4 was not due to poor cell surface expression of the B7IgV, because CTLA4Ig binding to wild-type B7 is better than the anti-B7 mAbs used. Thus the structure encoded by exon 3, which is the B7IgC domain, is involved in binding CD28/CTLA4. Consistent with this notion, a mutation of 165C>G that destroys the disulfate chain in the IgC domain, eliminates B7 binding to CD28Ig and reduces its binding to CTLA4Ig by 10-fold ( Fig. 1 d, Table 1).

Site-directed Mutagenesis Defines the Roles of the Conserved
Residues within the B7IgC Domains for CD28/CTLA 4 Binding.
So far, four molecules that bind CD28/CTLA4 have been identified, namely human B7, human B7-2, murine B7, and murine B7-2 (15-17, 26, 31). As these four molecules crossreact with both human and murine CD28/CTLA4, we reasoned that the structure involved in binding CD28/CTLA4 should be conserved among these four molecules. Previous analysis of B7 sequence homology (15-17, 26, 31) revealed that 17 of the 98 amino acids in the IgC domain are conserved. Among them, two are cysteines at positions 165 and 219. All I1 conserved amino acids between two cysteines are clustered at or near two loops, if we use the structure of IgC-domain as a reference (Table 1 legend). One is the loop between strands B and C (loop B-C), and the other between strand D and E (loop D-E). This analysis suggests that these two regions are involved in binding to CD28/CTLA4. To further define the CD28/CTLA4-binding sites, we generated a series of B7 mutants by site-directed mutagenesis and tested their binding to CD28Ig and CTLA4Ig. Binding to anti-B7 mAbs was used as an indicator for cell surface expression of B7 mutants. As shown in Table I Fig. 3 and Table 1, mutation 194Q>L195D>E reduces CD28 binding by 10-fold and CTLA4Ig binding by fivefold. An additional mutation in this region 194Q>L195D>E196P>H, or 194Q>L195D> E196P>K eliminates CD28-binding site and reduces CTLA-4Ig binding by 10-fold. The most drastic effect is caused by mutation 200L>V201Y>D, that leads to elimination of binding to both CTLA4Ig and CD28Ig. This result demonstrates that one or both of these amino acids is essential for binding CD28/CTLA4Ig. To test this, we replaced, one by one, three amino acids 199E, 200L, and 201Y with A and tested the effect of these mutations on B7 binding to CD28 and CTLA4. As shown in Fig. 4, replacement of either 199E or 200L by A does not significantly reduce the binding of B7 to CD28 and CTLA4, suggesting that these two amino acids are not critical for CD28/CTLA4Ig binding. In contrast, mutation 201Y>A eliminated the binding of both CD28 and CTLA4, suggesting that the 201Y plays a critical role in binding CD28 and CTLA4.
The results of the site-directed mutagenesis reveal that all mutations that affect CD28Ig binding affect CTLA4Ig binding. Interestingly, the effects of mutations on CD28 and CTLA4 binding follow a grossly similar hierarchy (Table 1)

Binding to CD28/CTLA4 Is Essential for tke Costimulatory Activity of BT.
To determine whether binding to CD28/ CTLA4 was necessary for the costimulatory activity of B7, we compared the costimulatory activity of wild-type B7 that binds CD28/CTLA4 with the B7 mutants that have lost the ability to bind CD28/CTLA4. As shown in Fig. 6 a,  poorly (Fig. 1), this lack of costimulation could be attributed to poor cell surface expression of B7IgV. To overcome this problem, we generated two fusion proteins. One consists of the extracellular domain of B7 and that of the HSA, the other is made of HSA and B7 fragments AA 1-159 (that consists of B7IgV domain plus 15 amino acids from IgC domain as spacer between B7IgV and the HSA). This truncated molecule does not contain the structures necessary for CD28/ CTLA4 binding. Again, B7-HSA but not B7IgV-HSA costimulates T cell proliferation (Fig. 6 b). These results demonstrate that the B7IgV domain does not costimulate T cell proliferation. COS cells transfected with HSA failed to costimulate T cell proliferation as opposed to HSA-transfected CHO cells (13,45), presumably because of differential glycosylation of HSA (6). As measured by anti-HSA antibody M1/69, the amount of fusion proteins expressed on the cell surface is comparable to B7-HSA and B7IgV transfected COS cells, although the anti-B7 mAb 7A5 binds B7-HSA fusion protein *10-fold better than the B7IgV-HSA (Fig. 6 c). The HSA portion does not interfere with the conformation of wild-type B7 and B7IgV as their binding to a panel of three anti-B7 mAbs as well as CTLA4/CD28 is similar to that of B7 and B7IgV ( Fig. 1 and Fig. 6 (Fig. 7). Thus binding to CD28/CTLA4 is critical for the costimulatory activity of B7, consistent with a previous study that showed that the Fab fragment of anti-CD28 blocks the costimulatory activity of B7 (26).

Discussion
We have reported here that a naturally occurring, alternatively spliced B7 that lacks IgC domain has lost the CD28/CTLA4Ig-binding sites. Site-directed mutagenesis based on homology analysis revealed several conserved amino acids within the IgC-like domains are involved in binding CD28 and CTLA4. To determine whether these amino acids can be clustered in the three-dimensional structure, we have mapped the residues of our mutagenesis study to an available Fab C-domain structure (3FAB, reference 46). There are a few x-ray protein structures available from the Brookhaven Protein Data Bank that contain IgC-like domains. Supposition of these structures shows that the loops B-C and D-E and these four strands from all known Fab IgC domains align very well structurally (data not shown). Residues which upon mutation significantly reduce CD28/CTLA4 binding around loops B-C and D-E form a localized region towards one end of the molecule even though they come from different locations of the sequence. This indicates that B7 has a localized CD28/CTLA4-binding site (Fig. 8). Although the backbone of 201Y is on the other side of the molecule from this region, its side chain is located approximately at the center of this region, sandwiched between loop B-C and loop D-E, consistent with our results that mutation of this residue has the most drastic effect on CD28/CTLA4 binding. This tyrosine is conserved among human B7, B7-2, and murine B7, and in murine B7-2, it has been conservatively replaced by phenylalanine.
Whereas it is formally possible that mutations of the amino acids in B-C and D-E loops affect the CD28/CTLA4 binding by causing a general distortion of the three-dimensional structure of B7, the fact that several mutations in such a localized area selectively affect CD28/CTLA4 binding indicates that this is highly unlikely. Using three anti-B7 mAbs (7A5, 3A12, and 10.16A.1, which bind three independent epitopes in B7IgV domain as determined by cross-blocking studies, data not shown) we found that the conformation of B7 is not significantly affected by the mutations we generated, although some mutations have a minor effect on the strength of 7A5 binding (data not shown). It is of interest to note that 172P and 174P are conserved among a large number of IgC-like domains, thus raising a possibility that these two amino acids are important for the overall structure of Ig. However, it is equally possible that the CD28/CTLA4-binding site evolves from a conserved structure of IgC-like domain.
An unresolved question is the contribution of the B7IgV domain in CD28/CTLA4 binding. Several lines of evidence can be interpreted in light of the possibility that B7IgV may be involved in binding CTLA4/CD28. First, our present study reveals that several anti-B7 mAbs (18,36) that blocked CTLA4Ig binding and B7-mediated T cell costimulation bind to the IgV domain. It should be emphasized that because the amino acids involved in binding CD28/CTLA4 defined by the site-directed mutagenesis are located in an area close to the IgV domains, the effect of several anti-IgV antibodies can be explained by steric hindrance. Second, the IgC domains have not been shown to bind CD28/CTLA4 in the absence of IgV domain. Third, Inobe et al. (47) reported that a stable cell line thought to be transfected with the B7IgV domain can bind CD28 and CTLA4. However, as no characterization of the stable transfectant has been done to verify the gene product, it is hard to reconcile the results with our study.
An important conclusion that can be derived from the results of our mutational analysis is that CD28 and CTLA4 bind to the same site on the B7IgC domain. Thus, all mutations that affect CD28 binding affect CTLA4 binding. More strikingly, the effects of mutations on CD28 and CTLA4 binding follow a grossly similar hierarchy (Table 1), although CTLA4Ig binding is generally more resistant to mutations, consistent with an earlier finding that CTLA4 has a ~17-fold higher affinity for B7 (33). This conclusion has an important implication on the mechanism of costimulation by B7. As CD28 and CTLA4 have different cytoplasmic domains, they may transduce qualitatively different signals (34). As CD28 and CTLA4 do not form heterodimers (33), and because they are located at distinct patches on the T cell surface as detected by confocal microscopy (34), the simplest mechanism that allows direct interaction of CD28omediated signaling machinery with that mediated by CTLA4 would be a crosslinking of the two molecules by B7 or B7-2. By necessity, this model implies that CTLA4 and CD28 have distinct binding sites on B7 to allow cross-linking by a single B7 molecule. Our results that CTLA4 and CD28 bind B7 at the same sites do not support this model. We favor an alternative hypothesis that CTLA4 and CD28 bind different B7 molecules and transduce distinct biological signals. The facts that CTLA4 is expressed after T cell activation and that CTLA4 has a higher affinity for B7 or B7-2 suggests that CD28-B7/ B7-2 interaction is likely to dominate at the induction phase of immune responses, whereas CTLA4-B7/B7-2 interaction is likely to dominate effector phase of immune responses. Our previous study (30) demonstrated that B7 can play an important role at both the induction and effector phases of T cell responses, thus raising an interesting possibility that CD28-B7 interaction may deliver a signal necessary for induction of T cell responses, whereas CTLA4-B7 interaction may deliver a signal for effector phase of T cell responses. Experiments are underway to test this hypothesis.
Whereas it is known that antibody stimulation through CD28 transduces the costimulatory signal for T cells, and that B7 binds CD28 and delivers the costimulatory activity, it is still formally possible that B7 costimulate T cells via other yet unidentified receptors on T cells. Our results demonstrate that B7 mutants that lose their binding to CD28 and CTLA4 also lose their costimulatory activity. These results demonstrate that B7 costimulate T cells by binding CD28/CTLA4.
We thank Drs. V. Nussenzweig and S. Vukmanovic for reviewing this paper, Dr. J. Hess for help with flow cytometry, and J. Orlin for help in preparation of the manuscript.