(Received for publication, April 14, 1995; and in revised form, June 12, 1995)
From the
The B7-related molecules CD80 and CD86 are expressed on
antigen-presenting cells, bind the homologous T cell receptors CD28 and
CTLA-4, and trigger costimulatory signals important for optimal T cell
activation. All four molecules are immunoglobulin superfamily members,
each comprising an extracellular Ig variable-like (IgV) domain, with
CD80 and CD86 containing an additional Ig constant-like (IgC) domain.
Despite limited sequence identity, CD80 and CD86 share similar overall
receptor binding properties and effector functions. We have identified,
by site-directed mutagenesis of soluble forms of CD80 and CD86,
residues in both the IgV and IgC domains that are important for CTLA4Ig
and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11
amino acids that support receptor binding. Many of these residues are
conserved in the B7 family, are hydrophobic, and approximately map to
the GFCC`C`` -sheet face of an IgV fold. Mutagenesis of
corresponding residues in CD86 established that some, but not all, of
these residues also played a role in CD86 receptor binding. In general,
mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby
indicating that both receptors bind to overlapping sites on CD80 and
CD86. Further, mutagenesis of several conserved residues in the ABED
-sheet face of the IgC domain of CD80 completely ablated receptor
binding. Point mutagenesis had a more pronounced effect than complete
truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to
B7 molecules is dependent upon residues in the GFC`C`` face of the IgV
domain and the ABED face of the IgC domain.
Distinct activation signals from antigen-presenting cells are required for T lymphocyte-dependent immune responses (1, 2, 3) . Antigen-specific activation of T cells is mediated by peptide/major histocompatibility complexes on antigen-presenting cells interacting with specific T cell antigen receptors. Binding of CD28 and/or CTLA-4 on T cells to B7-related receptors on antigen-presenting cells provides important antigen-nonspecific costimulatory signals essential for optimum immune responses. Blocking the delivery of these costimulatory signals in vitro can lead to T cell unresponsiveness or anergy(4, 5, 6) . Two B7-related molecules, each having different patterns of expression, have been identified: CD80 (B7-1) (7, 8) and CD86 (B7-2/B70)(9, 10, 11) .
CD80 and CD86 are
members of the immunoglobulin superfamily (IgSF), ()with
their extracellular regions consisting of one amino-terminal Ig
variable-like (IgV) and one membrane proximal Ig constant-like (IgC)
domain. The B7 IgV domains may include some structural features that
depart from currently known Ig folds (12) and share sequence
similarity with three major histocompatibility complex-encoded members
of the IgSF(13) . In contrast, the IgC domains display
significant sequence-structure compatibility with
-microglobulin(12) . B7 molecules are not
disulfide-linked, unlike their coreceptors CTLA-4 and CD28, which are
also IgSF members(14, 15, 16) .
Despite
having only 25% sequence homology, CD80 and CD86 have similar
equilibrium receptor binding properties. Both molecules bind CTLA-4
with high avidity and CD28 with low avidity(17, 18) .
CD80 and CD86 also share T cell costimulatory
properties(9, 10, 11) . However, some kinetic
and molecular properties distinguishing the B7 molecules were observed,
implying that these molecules have distinct interactions with CD28 and
CTLA-4(18) . Little is known of the molecular details of the
interaction between CD80 and CD86 and their counter-receptors. B7
residues critical for interactions with CTLA-4 and CD28 are currently
unknown. We report the results of site-directed mutagenesis of the IgV-
and IgC-like domains in CD80Ig and CD86Ig and the effects that deletion
of the IgC domain of a recombinant CD80Ig has on CTLA4Ig and CD28Ig
binding. We demonstrate that residues in both Ig-like domains are
critical for binding CTLA4Ig and CD28Ig.
CTLA4 mIg
and CD28 mIg were prepared by PCR-amplification of the extracellular
domains of human CTLA-4 and CD28 with primers that allowed fragments to
be digested with HindIII and BclI and ligated in
frame to a mouse Fc IgG2a cDNA fragment (18) encoded in a HindIII/BglII-cut LN expression vector. CD80Ig
and CD86Ig were prepared in transiently transfected COS
cells(17, 18) . CD80VIg, a fusion protein lacking the
IgC-like domain of CD80, was prepared using the PCR. cDNA encoding the
IgV-like domain was amplified using full-length CD80 as template, a
forward primer from the expression vector, and a reverse primer,
CTCACCCTCGGGATCCTTGACTGATAACGTCAC, encoding a BamHI
restriction site and the final six amino acids of exon 3, corresponding
to the carboxyl terminus of the CD80 V domain(19) . This PCR
fragment was digested with HindIII and BamHI and
ligated in frame to a human IgG1 genomic DNA fragment (E
1, (20) ) in the CDM8 expression vector. A control CD80Ig fusion
protein having the same IgG1 Fc region (CD80E
1) was prepared by a
similar method, except the reverse primer used ensured that cDNA
encoding the complete extracellular region of CD80 was PCR-amplified.
In control experiments, CD80E
1 fusion protein had identical
receptor binding properties to CD80Ig. CD80VIg and CD80E
1 were
prepared in transiently transfected COS cells, which were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. Media from transfected COS cells were used as a
source for Ig fusion proteins.
Figure 1:
Sequence
alignment of the extracellular regions of CD80/CD86 family members.
Sequences are aligned from the NH terminus of mature human
CD80 (8) and that predicted for human CD86(10) .
Residues at the end of each row are numbered from
their respective NH
terminus. Tentative
-strand
assignment of residues in IgV/I-set (amino-terminal) and IgC1-set
(carboxyl-terminal) domains are indicated; G-strand assignment in the
IgV domain was possible using only I-set consensus residues. The
relative position of the exon boundary between the two domains is
indicated with an arrowhead. Areas with a dashedunderline highlight positions that could not be assigned
to an IgV domain (structurally ambiguous) based on the absence of IgSF
consensus residues(13) . Darkboxedareas highlight residues common to other IgV/I-like or IgC-like domains; open and shadedboxedareas highlight additional residues completely conserved and
conservatively substituted within the B7 family, respectively. Asterisks highlight human CD80 residues selected for
mutagenesis. Accession numbers of sequences are described
elsewhere(13) .
Site-directed mutants of
soluble Ig fusions of CD80 and CD86 were prepared using PCR
oligonucleotide primer-directed mutagenesis. The concentration of each
mutant Ig fusion protein in serum-free COS cell culture media was
determined by an Ig quantitation assay, and the mutant was tested for
its ability to bind CTLA4Ig and CD28Ig in enzyme immunoassays. The
structural integrity of mutant fusion proteins was assessed by testing
their ability to bind mAbs specific for CD80 (BB-1, IgG; BB-1, IgM;
104; CD80.3), or CD86 (IT2.2) in indirect immunoassays. Failure of mAbs
to bind suggested that either the mutations directly disrupted the mAb
epitope, or that the mutation had compromised the structural integrity
of the molecule. All CD80 IgV domain mutants except V39R, V39A, Y53A,
and D60A, bound at least one of the anti-CD80 mAbs at levels
50-100% of wild-type CD80Ig. Asp-60 is charged and is
predicted to map to a loop region between putative
-strands D and
E (Fig. 1). Thus, global conformational defects caused by
mutagenesis of this residue are unlikely. Since Val-39 and Tyr-53 map
to a structurally ambiguous region (Fig. 1), it is less clear
whether mutation of these residues would affect conformation of the
molecule. All CD86 IgV domain mutants except Y59A bound mAb IT2.2.
Tyr-59, like the equivalent Tyr-53 in CD80, maps to a structurally
ambiguous region, so the effect of its mutation on conformation of the
molecule is difficult to assess. All CD80 and CD86 mutants bound
anti-human Fc antibodies indicating the presence of this domain.
S metabolic labeling of transfected COS cells followed by
protein A purification from conditioned media and autoradiography of
SDS-polyacrylamide gel electrophoresis, showed that all mutant proteins
migrated as M
70,000 proteins (data not
shown).
Figure 2: Representative CD80 IgV domain mutants binding to CTLA4Ig and CD28Ig. Site-directed mutant fusion proteins were produced in transiently transfected COS cells, quantitated, and tested for their ability to bind to CTLA4Ig (A) and CD28Ig (B) immobilized on 96-well plates. Fusion proteins were quantitated as described under ``Materials and Methods.'' Binding data (representative of at least three experiments) are expressed as the average of duplicate determinations and replicates differed from the mean by <10%.
These results led to two conclusions. First, the large number of mutations that disrupted receptor binding suggested the presence of an extensive contact area between CD80 and CTLA4Ig. Second, seven of the residues have hydrophobic side chains, suggesting that hydrophobic interactions are important determinants of CTLA-4/CD80 binding. This is consistent with the role of the hydrophobic MYPPPY region in CTLA-4/CD28 binding to CD80(22) .
However, binding of CD80 and CD86 to
CTLA4Ig is not completely equivalent. CD80 and CD86 bind their
counter-receptors with different kinetics and bind differently to the
CTLA4Ig mutant Y100A(18) . These observations suggest that CD80
and CD86 differ in their interaction with CTLA4Ig. We have identified
three mutations in CD86Ig that had different effects than mutation of
the equivalent residues in CD80Ig. D66A in CD86Ig resulted in 30%
of wild-type binding, but the equivalent mutation D60A in CD80Ig
completely prevented CTLA4Ig binding (Table 1). CD86Ig mutant
H56A had wild-type CTLA4Ig binding activity, while mutagenesis of the
equivalent residue in CD80Ig, W50A, resulted in >10-fold loss of
binding (Table 1). Finally, the CD86Ig mutation G61N bound
CTLA4Ig about one-third of wild-type levels, but substitution of the
equivalent residue in CD80Ig (N55A) increased receptor binding relative
to wild-type CD80Ig (
1.5-fold, Table 1). These molecular
differences may contribute to the different binding properties of
CTLA4Ig for CD80 and CD86.
Figure 3:
Mapping of CD80 site-directed mutants onto
a schematic representation of an IgV-fold. Residues in the CD80
amino-terminal (or V-like) domain whose mutation affects ligand binding (red) are mapped onto approximately corresponding positions of
an IgV-fold. Potential N-linked glycosylation sites are blue. The -strands and termini of the domain are labeled.
Regions that can be assigned to an IgV-fold are shown in yellow; regions where no V-set consensus residues are present
are shown in gray.
Figure 4: CD80 mutagenesis demonstrating that deletion of IgC-like domain diminishes but does not eliminate binding to CTLA4Ig and CD28Ig. Soluble CD80VIg lacking the IgC-like domain was produced and assayed for its ability to bind CTLA4Ig (A) and CD28Ig (B) as described in Fig. 2. Data are expressed as the average of duplicate determinations and differed from the mean by <10%. The data are representative of three separate experiments.
Mutagenesis of hydrophobic residues, Phe-108, Pro-111, and Ile-113
abolished CTLA4Ig binding, while Q157A, D158A, E162A, and L163 bound to
CTLA4Ig at 6-35% relative to wild-type CD80Ig (Fig. 5A and Table 1). Mutagenesis of Ser-156 and
Ser-167 resulted in
2-3-fold increased binding to CTLA4Ig.
Perhaps increased hydrophobicity of this region because of the serine
to alanine mutation enhanced receptor binding. The effects of
mutagenesis on CD28Ig binding were more dramatic. All mutations, except
S156A and S167A, significantly reduced or completely abolished CD28Ig
binding (Fig. 5B and Table 1). While deletion of
the CD80 IgC domain had a greater effect on binding to CTLA4Ig than
CD28Ig, point mutagenesis of residues in the IgC domain had the
opposite effect. Thus, some conserved residues in the IgC domain of
CD80Ig play an important role in CTLA4Ig and CD28Ig binding.
Figure 5: Mutagenesis in CD80 IgC-like domain demonstrates the involvement of specific residues in CTLA4Ig and CD28Ig binding. Site-directed mutant fusion proteins were prepared and assayed for their ability to bind CTLA4Ig (A) and CD28Ig (B) as described in Fig. 2. Data are expressed as the average of duplicate determinations and differed from the mean by <10%. The data are representative of at least four experiments.
Figure 6:
Mapping of CD80 site-directed mutants onto
a schematic representation of the IgC domain. Residues in the C-like
domain of CD80 whose mutation affect ligand binding are mapped onto a
three-dimensional model of the domain (generated on the basis of
sequence structure compatibility with -microglobulin, (12) ). Mutants and potential N-linked glycosylation
sites are color coded according to Fig. 3. With the exception of
Phe-108, all residues are predicted to be accessible on the domain
surface.
We have shown that the IgC domain of CD80 exerts considerable influence on CTLA4Ig and CD28Ig binding. Complete absence of the IgC domain in CD80VIg resulted in >10-fold reduced binding to both CTLA4Ig and CD28Ig. Thus, with human CD80, the presence of both the amino-terminal IgV and membrane proximal IgC domains was essential for full binding to CTLA4Ig/CD28Ig. However, several conserved non-IgSF consensus residues in the IgC domain were identified whose mutagenesis completely abolished CTLA4Ig/CD28Ig binding in our assays. Thus, point mutations in the IgC domain had a greater effect on receptor binding than complete removal of the domain. It is possible that mutagenesis of some residues in the IgC domain led to structural defects affecting the whole extracellular region. More likely, however, this implies that the IgC domain participates in the presentation of the receptor binding site.
Several possible interactions could occur between CD80 and CTLA-4/CD28. Since deletion of the IgC domain and mutagenesis of residues in both CD80 domains affected receptor binding, neither domain can act independently to provide full CTLA4Ig or CD28Ig binding. Instead, the results favor an interaction where both CD80 IgV and IgC domains contribute to binding. Rather than spatial separation of the IgV and IgC domains, the IgC domain may support binding by forming an extensive interface between the domains, similar to that seen with CD4 and tissue factor(31, 32, 33) . This domain interface could support CTLA-4/CD28 binding by, for example, shielding the hydrophobic binding face in the amino-terminal IgV domain from solvent. The consensus hydrophobic residues in the CD80 IgC domain, identified here as critical for receptor binding, may contribute to the formation of the domain interface. Changes in the domain-domain orientation as a consequence of interface mutations could prohibit counter-receptor binding. Alternatively, these hydrophobic residues may be directly associating with CTLA-4 and CD28, particularly via the hydrophobic MYPPPY loop. However, if these and other consensus residues in the IgC domain were directly interacting with CTLA-4 and CD28, they probably do not contribute significantly to the overall binding affinity, as the IgV domain alone can sustain some receptor binding activity.
Evidence for a close association of the IgV and IgC
domains of B7 molecules comes from assignment of putative -strands
in the two domains. Overlapping assignment of residues can be made of
-strand G in the IgV domain with
-strand A in the IgC domain (Fig. 1). An exon boundary, which often defines domain limits,
is found after the first nucleotide of the triplet codon encoding
alanine 106(19) , a residue assigned to
-strand A in the
IgC domain. However, the upstream amino acid valine 104, an IgV-fold G
strand consensus residue, can also be assigned to
-strand A of the
IgC domain. This is analogous to the closely associated D1D2 and D3D4
Ig-like domains seen in the crystal structures of human and rat
CD4(31, 32) .
Both CD80 and CD86 are extensively
glycosylated(11) , but potential N-linked
glycosylation sites in the CD80 IgV and IgC domains are localized to
regions essentially opposite the site of receptor interaction, and are
therefore unlikely to modulate ligand binding. Extensive glycosylation
of CD80 and CD86 may instead be required to aid in solubility since
their extracellular domains probably contain a number of hydrophobic
residues on their surfaces. CTLA-4 and CD28 are also glycosylated.
Removal of N-linked glycosylation sites by mutagenesis of
CTLA4Ig does not alter its ability to bind CD80 or CD86. ()Thus carbohydrate moieties are not likely to play a role
in B7/CTLA-4/CD28 interactions.
Reversed orientation of the CD80 IgV and IgC domains would allow residues that affect receptor binding to align on one face of the molecule. While we have not extensively mutated residues on the opposite faces of these domains, we believe they are unlikely to be involved in receptor binding for two reasons. First, the conserved residues on this face, particularly in the IgV domain, are predominantly IgSF consensus residues; these most likely function to maintain structural stability rather than to mediate receptor binding. Second, bulky carbohydrate moieties that could mask receptor binding sites, map to the opposite faces. The precise nature of the interaction of the single IgSF domain receptors, CTLA-4 and CD28, with multiple domains of B7-related molecules is yet to be determined. However, the present study provides insights into the molecular nature of these interactions and establishes the basis of future structural studies.