Crystal structure of the complex betweenprogrammed death-1 (PD-1) and its ligand PD-L2Eszter Lazar-Molnar*†, Qingrong Yan†‡, Erhu Cao†‡, Udupi Ramagopal§, Stanley G. Nathenson*‡¶, and Steven C. Almo§¶�

Departments of *Microbiology and Immunology, ‡Cell Biology, §Biochemistry, and �Physiology and Biophysics, Albert Einstein College of Medicine,1300 Morris Park Avenue, Bronx, NY 10461

Contributed by Stanley G. Nathenson, May 7, 2008 (sent for review April 18, 2008)

Programmed death-1 (PD-1) is a member of the CD28/B7 super-family that delivers negative signals upon interaction with its twoligands, PD-L1 or PD-L2. The high-resolution crystal structure of thecomplex formed by the complete ectodomains of murine PD-1 andPD-L2 revealed a 1:1 receptor:ligand stoichiometry and displayed abinding interface and overall molecular organization distinct fromthat observed in the CTLA-4/B7 inhibitory complexes. Furthermore,our structure also provides insights into the association betweenPD-1 and PD-L1 and highlights differences in the interfaces formedby the two PD-1 ligands (PD-Ls) Mutagenesis studies confirmed thedetails of the proposed PD-1/PD-L binding interfaces and allowedfor the design of a mutant PD-1 receptor with enhanced affinity.These studies define spatial and organizational constraints thatcontrol the localization and signaling of PD-1/PD-L complexeswithin the immunological synapse and provide a basis for manip-ulating the PD-1 pathways for immunotherapy.

costimulation � coinhibition � inhibitory receptor � T cell activation

T cell activation requires a primary antigen-specific signal thatresults from the engagement of the T cell receptor (TCR)

with antigenic peptide presented in the context of the majorhistocompatibility complex (MHC). The strength, duration, andcourse of this response are modulated by antigen-independentsignals provided by a number of distinct costimulatory mole-cules, including members of the CD28/B7 family. Upon bindingits ligands, B7–1 and B7–2, the constitutively expressed CD28delivers stimulatory signals for T cell proliferation, expansion,and differentiation. In contrast, CTLA4 (�30% sequence iden-tity with CD28) expressed on activated T cells delivers negativesignals upon binding to the same B7 ligands (1). The balancebetween costimulatory and coinhibitory signals is crucial formaximizing immune responses while maintaining immunologicaltolerance. Both CTLA-4 and CD28 consist of a single immuno-globulin variable (IgV) ectodomain linked to a cytoplasmic tailcontaining tyrosine-based signaling motifs (1). The IgV domaincontains a proline-rich motif (MYPPPY) that is responsible forligand binding. In vivo both CD28 and CTLA4 exist as covalenthomodimers because of an interchain disulfide formed by aconserved cysteine residue in the linker region connecting theIgV domain to the transmembrane segment. The in vivo oligo-meric states of the ligands are less characterized, but recentstudies demonstrated that B7–1 has high propensity to oligomer-ize on the cell surface (2). The oligomeric and polyvalentfeatures of these receptors and ligands may contribute to theformation and localization of multicomponent signaling assem-blies at the immunological synapse.

Programmed death-1 (PD-1) is a member of the CD28/B7family that plays an important role in negatively regulatingimmune responses (1). In contrast to other receptors in thisfamily, upon activation, PD-1 expression is induced not only onT cells but also on B cells and myeloid cells (3). Concomitantwith TCR or BCR cross-linking, engagement of PD-1 by itsligands, PD-L1 (4) or PD-L2 (5), induces inhibitory signalsthrough the recruitment of phosphatases, such as SHP-2, to theimmunoreceptor tyrosin-based switch motif (ITSM) of the cy-

toplasmic tail of PD-1, resulting in dephosphorylation of effectormolecules involved in downstream TCR or BCR signaling (5, 6).PD-1 signaling plays an important role in inducing and main-taining peripheral tolerance. PD-1 ligands (PD-Ls) on antigen-presenting cells have been shown to inhibit autoreactive T cellsand induce peripheral tolerance, whereas those on parenchymalcells prevent tissue destruction by suppressing effector T cells tomaintain tolerance (7). The inhibitory role of PD-1 is highlightedby the phenotype of PD-1 deficient mice, which develop variousautoimmune diseases, depending on the genetic background (8,9). In humans, single-nucleotide polymorphisms of the PD-1gene may be linked to various autoimmune diseases, such asrheumatoid arthritis, SLE, and diabetes, and the PD-1/PD-Lpathway is crucial in establishing fetomaternal tolerance andmaintaining the integrity of immuno-privileged sites (7). ThePD-1/PD-L pathway is frequently exploited as a target forimmune evasion by tumor cells (10) and by a wide range ofpathogens (11).

Human and mouse PD-1 share �60% amino acid identity,whereas the extracellular IgV domain shows only 21% and 16%sequence identity with CD28 and CTLA4, respectively. Consis-tent with this modest sequence similarity, PD-1 exhibits impor-tant differences relative to the other CD28 family members,including the lack of the proline-rich ligand recognition loop andthe absence of the cysteine residue responsible for disulfide bondformation (12). Like other B7 homologs, PD-L ectodomainsconsist of a membrane distal IgV and a membrane proximal IgCdomain. PD-L1 and PD-L2 share 34% identity with each otherand �20% identity with B7–1 and B7–2. The PD-Ls differ intheir patterns of expression and affinity for PD-1. PD-L2 exhibits3-fold higher affinity for PD-1 (13) and is restricted to activateddendritic cells and macrophages. In contrast, PD-L1 is consti-tutively expressed and up-regulated on antigen presenting cells(APCs), and T cells and a variety of nonhematopoietic cell types(14).

The roles that PD-L1 and PD-L2 play in T cell activation arediverse and both stimulatory and inhibitory functions for theseligands are reported in ref. 15. It was also recently demonstratedthat B7–1 can bind T cell-associated PD-L1, resulting in theinhibition of T cell proliferation and cytokine production (16).This finding highlights the complexity of T cell costimulation,because competitive binding interactions between multiple mol-

Author contributions: E.L.-M., S.G.N., and S.C.A. designed research; E.L.-M., Q.Y., and E.C.performed research; E.C. contributed new reagents/analytic tools; E.L.-M., Q.Y., U.R., andS.C.A. analyzed data; and E.L.-M., Q.Y., S.G.N., and S.A. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The atomic coordinates and reflections have been deposited in the ProteinData Bank, www.pdb.org (PDB ID codes 3BP5 and 3BOV).

See Commentary on page 10275.

†E.L.-M., Q.Y., and E.C. contributed equally to this work.

¶To whom correspondence may be addressed. E-mail: nathenso@aecom.yu.edu oralmo@aecom.yu.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0804453105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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ecules provide mechanisms for linking the PD-1, CTLA-4, andCD28 pathways.

We report the 1.8-Å-resolution structure of the murine PD-1/PD-L2 complex, which reveals a binding interface differentfrom that observed in the CTLA-4/B7–1 and CTLA-4/B7–2complexes. We also report the 1.77-Å-resolution structure of theisolated PD-L2 IgV domain. The structural and organizationalfeatures of the PD-1/PD-L2 complex provide important mech-anistic constraints that must be accommodated in models de-scribing signaling associated with the PD-1/PD-L2 and PD-1/PD-L1 complexes. Mutagenesis studies confirm the details of thebinding interfaces between PD-1 and the PD-Ls, and suggest thebasis for designing novel tools for immunotherapy.

ResultsOverall Structure of PD-1/PD-L2 Complex. The structure of thePD-1/PD-L2 complex reveals an assembly with 1:1 receptor–ligand stoichiometry and a binding interface formed by the front�-sheets of both the PD-1 and PD-L2 IgV domains (Fig. 1A).Residues from the GFCC� strands and CC�, CC�, and FG loopsof PD-1 contribute to the binding interface and pack against theAGFC strands and the FG loop of the PD-L2 IgV domain,burying a total surface area of 1,915 Å2. Eighteen potentialhydrogen bonds are formed by 11 residues contributed by the C,C�, and G-strands and the CC� and FG loops in PD-1 and 11residues from the A, C, F, and G strands and the AA� and FG

loops in PD-L2 [supporting information (SI) Table S1 and Fig.S1]. A small hydrophobic core, formed by residues from the C,F, and G strands and the FG loop in PD-1 and residues from theF and G strands and the FG loop in PD-L2, also contributes tothe binding interface and buries a surface area of 570 Å2.Additional hydrophobic contacts are formed between residues inthe C� strand and the C�D loop of PD-1 and the A strand inPD-L2 (Fig. 1B and Fig. S1).

Most of the residues in the binding interface are conserved (12of 16 residues from PD-1 and 10 of 14 residues from PD-L2 areidentical) between human and mouse sequences (Figs. 2 and 3).Six of the 14 residues involved in binding PD-1 are fullyconserved between the two ligands in all species examined, andtwo additional residues are very similar between the PD-L2 andPD-L1 sequences (E versus D or Q versus E) (Fig. 3). Thisconservation suggests that both PD-L1 and PD-L2 may formsimilar assemblies with PD-1. Importantly, the positions of allpredicted glycosylation sites in PD-1 and the PD-Ls are consis-tent with the observed mode of interaction between PD-1 andPD-L2 and that proposed for the PD-1/PD-L1 complex (Figs. 2and 3).

The organization of the PD-1/PD-L2 complex differs consid-erably from that observed in the CTLA-4/B7–1 and CTLA-4/B7–2 structures, in which the CTLA-4 and the B-7 IgV domainscross at �90° as opposed to 60° in the PD-1/PD-L2 complex. Theoverall difference in organization is highlighted by direct super-imposition of CTLA-4 and PD-1 in the two complexes (Fig. 4).The PD-1/PD-L2 interface is formed by residues distributed overthe front sheets and associated loops of both molecules, with theapproximate center of the interface defined by the interaction ofW110 and Y112 in the G-strand of PD-L2 and with a concavesurface on the front face of PD-1 formed by the C, F, and Gstrands (Fig. 5A). In contrast, the majority of the CTLA-4/B7interfaces arise from contributions of the invariant FG loop(MYPPPY) in CTLA-4, which packs against a concave surfaceformed by the C,C� strands and the CC� and C�D loops on thefront sheet of the B7 molecules. These different binding inter-actions result in a relatively compact PD-1/PD-L2 complex withan end-to-end distance that spans �76 Å, compared with theCTLA-4/B7 complexes that span �100 Å. Notably, the linkerregions connecting the ectodomains and transmembrane seg-ments are longer for PD-1 (20 residues) and PD-L2 (11 residues)than those present in CTLA-4 (6 residues) and B7–1 (9 residues),and could easily allow the PD-1/PD-L2 complex to span an

Fig. 1. Structure of the PD-1/PD-L2 complex. (A) Overall structure of thePD-1/PD-L2 complex. Green, PD-1; cyan, PD-L2. The strands of PD-1 and PD-L2are labeled in red and blue, respectively. (B) Surface representation of PD-1/PD-L2 binding interface. Red, hydrophilic residues in the binding interface;yellow, hydrophobic residues in the binding interface. PD-L2 is in the sameorientation as in A; PD-1 is rotated 180° about a vertical axis to reveal thebinding surface.

Fig. 2. Alignment of the PD-1 ectodomains. The � strands in mouse PD-1 aredenoted with arrow segments above the sequence. Red shading, conservedresidues; red labeling, residues with similar properties; green triangles, resi-dues bearing potential N-glycans; green asterisks, residues that contribute tobinding to PD-L2.

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end-to-end distance comparable with the linear dimensions ofother pairs of signaling molecules within the immunologicalsynapse (17). Furthermore, based on the structures of thePD-1/PD-L2 complex and the isolated PD-1 and PD-L2 IgVdomains, complex formation results in very modest binding-induced structural alterations, which seem insufficient to directlytransduce signals across the plasma membrane (see SI Resultsand Fig. S2).

PD-L2 Ectodomain Structure. The PD-L2 monomer exhibits arod-like shape, with two Ig domains (IgV and IgC domain)joined by a short linker region (Fig. S3). The PD-L2 IgV domainexhibits classic two-layer �-sandwich topology, with front andback sheets composed of the A�GFCC� and ABED strands,respectively. This IgV domain possesses the hallmark intersheetdisulfide linking the B and F strands that is a characteristicfeature of many IgV domains, including all known PD-L1 andPD-L2 sequences. In the mouse PD-L2 structure, there is oneadditional disulfide bond linking the F strand and the BC loop,which is not present in human PD-L2 or other B7 family ligands.The PD-L2 IgV shares considerable structural similarity with theB7–1 and B7–2 IgV domains (RMSD of 1.59 Å and 2.33 Å).Notably, the PD-L2 IgV domain has a much shorter C� strandthan B7–1 and B7–2, and the C� strand, which is typical ofconventional IgV domains, is missing in the PD-L2 IgV domain.In addition, the A strand present in PD-L2 IgV domain is absentin the B7–1 and B7–2 IgV domains (Fig. S4).

The front and back sheets of the PD-L2 IgC domain arecomposed of the GFC and ABED strands, respectively. Before

the current PD-L2 structure, human B7–1 was the only memberof the B7 family whose full ectodomain structure was available(18, 19), and the IgC domains of these proteins superimpose withan RMSD of 1.55 Å (Fig. S3).

The overall rod-like architecture of PD-L2 is stabilized byspecific interactions between the IgV and IgC domains. The IgVdomain ends at A121 and is followed by a five residue stretch(122SYMRI126) that links the two Ig domains. The interdomaininterface is stabilized by a series of hydrophilic and hydrophobicinteractions: K120 from the end of the G strand of the IgVdomain forms an ionic interaction with E201 from the G strandof the IgC domain; the side chain of S122 and main chain of A121from the linker form potential hydrogen bonds with the sidechain of E201 and main chain of Y148, respectively; V94 andV119 from the IgV domain and Y148 from the IgC domaincontribute hydrophobic interactions (Fig. S3B). These residuesare identical between all PD-L2 species, and five of sevenresidues are conserved between PD-L2 and PD-L1, suggestingthat all PD ligand ectodomains adopt an approximately similarorganization (Fig. 3).

Despite the absence of sequence conservation, the interdo-main linkers of PD-L2 and B7–1 are similar in length (five versussix residues) and share similar main chain conformations (Fig.S3C). The PD-L2 IgV-IgC interface buries 689 Å2 of surfacearea, which is comparable with the 670 Å2 of buried surface areain B7–1. Superimposition of the N-terminal IgV domains ofPD-L2 and B7–1 results in a significant deviation of the mem-brane-proximal IgC domains, which can be described as arotation of �30° around the long axis of the molecule (Fig. S3C).

Confirmation of the PD-1/PD-L Binding Interfaces: PD-L2 mutants.Our structure predicts that PD-1 binds on the front face ofPD-L2, making the greatest number of contacts with the G

Fig. 3. Alignment of the extracellular domains of PD-L1 and PD-L2. The �

strands in mouse PD-L2 are denoted with arrows above the sequence. Redshading, conserved residues; red labeling, residues with similar properties;triangles, residues bearing potential N-glycans; pink, residues conserved be-tween PD-Ls; green, residues conserved for PD-L2 only; green asterisks, resi-dues that contribute to receptor binding of PD-L2; filled circles, residuesforming the interdomain hydrogen bonds between PD-L2 IgV and IgCdomains.

Fig. 4. Comparison of the PD-1/PD-L2 and the CTLA-4/B7–1 complexes.(Upper Left) IgV domains of PD-1 (green) and PD-L2 (cyan) in the PD-1/PD-L2complex. (Upper Right) IgV domains of CTLA-4 (blue) and B7–1 (magenta) inthe CTLA-4/B7–1 complex. (Lower) Overlay of the entire PD-1/PD-L2 andCTLA-4/B7–1 complexes by superimposition of PD-1 and CTLA-4.

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strand, and additional contacts with the C and F strands. On thisbasis, PD-L2 mutants (E28A in the AA� loop and W110A,�W110, D111A, Y112A, K113A, and Y114A in the G strand)were generated to map the receptor-binding surface of PD-L2(Fig. 5B). Several mutants, such as D111A and K113A, withinthe proposed PD-1/PD-L2 interface abolished binding to PD-1-Ig (Fig. 5C). Mutation or deletion of W110, which resides in themiddle of the interface, results in significantly reduced binding,and the Y114A mutation earlier also diminishes binding to PD-1.In addition, mutagenesis data have shown that the R101S,L103A and I105A mutations in the F strand of PD-L2 resultedin decreased affinity for PD-1 (20), consistent with the crystal-lographically observed binding interface.

Our data highlight the importance of the G strand of PD-L2in contacting PD-1 (Figs. 3 and 5A). Of particular note is W110,located in the core of the binding interface, which forms thelargest numbers of contacts with different residues from PD-1.Indeed, deletion of this residue or substitution with Ala reduces

binding to PD-1 to �20% and 40% of the wild type, respectively.W110 is conserved in all known PD-L2 sequences, but issubstituted with alanine in all PD-L1 sequences (Fig. 3). Resi-dues D111 through K113 are conserved in all known PD-Lsequences, suggesting that they are important for receptorrecognition by both PD-L1 and PD-L2, consistent with idea thatPD-L1 and PD-L2 form similar assemblies with PD-1. Thissimilarity is further supported by earlier data showing that theI117A and K127A mutations in PD-L1, corresponding to PD-L2residues L103 and K113 (F and G strands, respectively), result indecreased binding to PD-1. Notably, mutations of E60A andC115A in PD-L1 corresponding to Q60 (C strand) and R101 (Fstrand) in PD-L2 show �120% wild-type binding to PD-1,suggesting that these residues are also within the binding inter-face in the PD-1/PD-L1 complex (20).

The unique presence of W110 in PD-L2 and its important rolein binding to PD-1 suggests that this residue might be a deter-minant for the higher affinity that PD-L2 exhibits toward PD-1(Fig. S5). However, other differences exist, including a 14-residue insertion in PD-L1 after the C� strand, which could alsomodulate receptor–ligand affinity in the PD-1/PD-L1 complex(Fig. 3). Nine of these residues are conserved in all known PD-L1sequences, and it was reported that mutations in this segmentaffect binding of PD-L1 to PD-1 (20).

PD-1 Mutants. Our previous mutagenesis studies (12) provided agross description of the PD-1 residues that contribute to thebinding interface. Based on the current structure, additionalPD-1 mutants were examined by SPR and flow cytometry (Fig.5D). Mutants such as K45A (C� strand), I93A (F strand), I101Aand E103A (G strand) exhibited unmeasurable or extremelyweak binding to both PD-L1 and PD-L2, suggesting that the sidechains of these residues are involved in recognition of bothligands (Fig. 5E). Notably, the A99L mutation of PD-1 results inhigher binding not only to PD-L1 (12), but also to PD-L2,consistent with an �2-fold lower Kd for PD-L1 and an �3-foldlower Kd to PD-L2 (Fig. S5).

Our structure-based mutagenesis studies also allowed forepitope mapping of several monoclonal antibodies to PD-1.Notably, clone J43 showed reduced binding to the PD-1 mutantsP97A, K98A, and A99L, suggesting that it recognizes an epitopeinvolving residues 97PKA99 on the FG loop. Consistent with thecurrent structure, this antibody has been reported to block thebinding of both PD-L1 and PD-L2 Ig to PD-1-transfected cellsand has been used extensively in in vivo models of immunologicalrelevance (21). Other monoclonal antibodies to PD-1, such asRMP1–14 (blocking) and RMP1–30 (nonblocking), showedbinding to all of the mutants tested, suggesting that they recog-nize different epitopes that are not within the ligand bindinginterface (data not shown).

In total, these mutagenesis experiments validate the crystal-lographically observed PD-1/PD-L2 interface and strongly sup-port the similarity of the assemblies formed by PD-1 with bothPD-L1 and PD-L2. These studies have also unexpectedly iden-tified a mutant PD-1 receptor with novel biochemical propertiesthat exhibits high affinity binding.

Interaction Between PD-1 and PD-Ls Is Sufficient to Drive TheirEnrichment at a ‘‘Pseudosynapse.’’ To further study the interactionbetween PD-1 and its ligands in the context of a cell–cellinterface, an artificial pseudosynapse system was exploited. Bothreceptor and ligand were expressed on the cell membrane aseither C-terminal CFP or YFP fusion proteins. Cells expressingeither receptor or ligand were mixed, and the localization of theintercellular receptor–ligand complex was examined by confocalmicroscopy. A pattern of localization at the cell–cell interfacethat is characterized by increased intensity for both fluorophoresis consistent with the accumulation of both receptor and the

Fig. 5. Mapping the binding interface between PD-1 and PD-L2. (A) The coreof the binding interface: electron density of residues W110 through Y114 fromthe G strand of PD-L2 contacting the front concave surface of PD-1 formed bythe C, F, and G strands. (B) Receptor binding interface of PD-L2. Mutation ofthose residues shown in red results in significantly reduced or no binding.Other residues predicted to form the PD-L2 binding site based on the structureof the complex are shown in magenta. Mutation of those residues shown ingray had little or no effect on binding to PD-1. (C) Binding of cell surfaceexpressed PD-L2 mutants (DW110 indicates deletion of residue W110) to PD-1Ig (0.5 and 5 �g/ml), detected by flow cytometry. Data are shown as percent-ages of MFI values of wild-type PD-L2 binding to PD-1. (D) Ligand bindingsurface of PD-1. Mutation of those residues shown in red results in significantlyreduced or no binding to either of the ligands. Mutation of those residuesshown in blue decreases binding to PD-L1 only, but not to PD-L2. Mutation ofA99 (green) increases affinity for both ligands. Other residues contributing tothe ligand-binding site based on the structure of the complex are shown inmagenta. Mutation of those residues shown in gray had little or no significanteffect on binding to PD-L2. (E) Binding of PD-1 mutants to PD-L1 (blue) andPD-L2 (purple) Ig. Data are shown as percentages of MFI values of wild-typePD-1 binding to either PD-L1 or PDL-L2.

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ligand at the pseudosynapse (Fig. 6 A and B). Our data show thatnonimmune CHO cells expressing PD-1 can form stable conju-gates with cells expressing either PD-L1 or PD-L2. These resultssuggest that the diffusive movement of costimulatory moleculesdriven by their mutual affinity could make important mecha-nistic contributions to the initiation and maturation of synapseformation.

DiscussionHigh-resolution crystallographic analysis revealed that the PD-1/PD-L2 complex is distinct from the CTLA-4/B7 inhibitorycomplexes in both overall organization and the detailed atomicinteractions responsible for binding and specificity. Mutagenesisstudies validated the basic features of this model and, in com-bination with primary sequence considerations, indicate thatPD-L1 and PD-L2 form similar complexes with PD-1. Thesestructural models provide several constraints that must beaccommodated by any detailed mechanistic model describingsignaling through the PD-1 pathway and the integration of thesesignals into the overall immune response.

Primary sequence considerations, including the lack of aproline-rich ligand recognition loop and the absence of a cys-teine residue in the linker segment connecting the IgV andtransmembrane domains, identify PD-1 as a unique member ofthe CD28/B7 family. In CTLA-4, the MYPPPY sequence motifin the FG loop contributes a large fraction of the contactsresponsible for binding the B7 ligands. Within this motif, thethree consecutive prolines adopt an unusual high-energy cis-trans-cis backbone conformation that provides the geometriccomplementarity required for efficient recognition of B7–1 andB7–2 (19, 22). It is notable that this same detailed FG loopconformation is present in the unliganded CTLA-4, indicatingthat CTLA-4 is poised in a preformed productive binding modebefore ligand engagement (23). CD28 contains this same FGloop motif that is critical for B7 binding and the CD28 structurealso exhibits the unique cis-trans-cis backbone conformation(24), suggesting that CTLA-4 and CD28 bind the B7 ligands with

similar geometries. In contrast to these receptors, the FG loopof PD-1 possesses only a single proline and only the base of theloop contacts PD-L2. In addition, the apex of the FG loop, whichcorresponds to the highly rigid MYPPPY loop in CTLA-4 andCD28, displays considerable disorder in both the bound andunbound forms of PD-1 and makes no contacts, probablybecause of the absence of the C� strand and the shortness of theC� strand in PD-L2. Furthermore, our structural and mutagen-esis results demonstrate that the residues involved in ligandrecognition are more fully distributed over the front sheet ofPD-1 than in CTLA-4 and other family members.

These differences in the binding interface result in a consid-erable difference in the overall organizations of the PD-1/PD-L2and CTLA-4/B7 complexes. In particular, the PD-1/PD-L2 as-sembly is significantly more compact than the CTLA-4/B7complexes, displaying end-to-end distances that span �76 and100 Å, respectively. The dimensions of receptor–ligand com-plexes provide a convenient mechanism for the sorting andlocalization of signaling molecules within the immunologicalsynapse, with small signaling molecules going to the central zoneand larger adhesion molecules residing in the peripheral zone.Notably, the apparently small size of the PD-1/PD-L complexesis potentially compensated by the long linker segment connect-ing the PD-1 IgV domain to the transmembrane segment,allowing for appropriate colocalization with the pMHC/TCRcomplex and other costimulatory receptor/ligand pairs (Fig. 6C).

Another important feature of PD-1 is its oligomeric state.Both CTLA-4 and CD28 exist as disulfide-linked homodimers.In addition, B7–1 exhibits a considerable propensity to formnoncovalent oligomers in solution and on the plasma membrane(2, 18). The higher order oligomeric states of these receptors andligands afford the opportunity to assemble multicomponentsignaling complexes with specific stoichiometries and geome-tries. For example, the crystal structures of the CTLA-4/B7ectodomain complexes (19, 22) revealed that both receptor andligand form unusual dimer interfaces that place their respectivebinding sites distal to the dimer interface, resulting in receptorsand ligands that are bivalent. The bivalency of both CTLA-4 andthe B7s supports the formation of a periodic, alternating ar-rangement of CTLA-4 and B7 homodimers, characterized by an�100-Å repeat between receptors that extends throughout thecrystal. This periodic network provides a model for the assemblyof these molecules at the T cell-APC interface and offers amechanism for the localized enrichment of signaling moleculesat the central zone of the immunological synapse. Although theorganization of these assemblies are driven by interactionsinvolving the ectodomains, these same constraints are imposedon the noncovalently associated cytoplasmic signaling and scaf-folding proteins that are responsible for propagating and am-plifying extracellular cues. In contrast to CTLA-4 and CD28,which are disulfide-linked homodimers, PD-1 lacks the extra-cellular equivalent of Cys-122 in CTLA-4 and, consequently,exists as a monomer in solution, in the crystalline state, and onthe cell surface (12).

The monomeric state of PD-1 precludes the types of multi-valent assemblies that may be associated with the localizationand assembly of CTLA-4-containing complexes. Instead, ourstructural and cell-based data suggest that simple diffusiveprocesses allow for the engagement of PD-1 and the PD-Ls ininteracting cells and for the subsequent enrichment of PD-1/PD-L complexes at the central zone of the immunologicalsynapse. Our demonstration of PD-1/PD-L complex formationin pseudoconjugates, which occurs in the absence of primary orcostimulatory signals, supports an important role for diffusionwithin the plasma membrane for recognition, engagement, andsynapse development. These mechanistic considerations areconsistent with the recent report that the degree of enrichment

Fig. 6. PD-1/PD-L interaction at the cell–cell interface. Noncovalent inter-actions between PD-1 and PD-Ls are sufficient to drive their enrichment at apseudosynapse. (A and B) PD-1 and PD-L1 (A) or PD-L2 (B) expressed in CHOcells are recruited to the cell–cell contact area and form conjugates that areanalogous to the immunological synapse. (Left) PD-1-CFP-expressing cells inblue. (Center) PD-L1-YFP or PD-L2-YFP-expressing cells in yellow. (Right) Over-lay of the CFP and YFP images. (C) Model of the PD-1/PD-L2 complex in theimmunological synapse. A number of receptor–ligand assemblies have dimen-sions that are compatible with colocalization to the central zone of theimmunological synapse.

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of PD-1 at the immunological synapse depends on the affinityand availability of its ligands (25).

Finally, our mutagenesis studies identified a high affinityA99L mutant PD-1 receptor with twofold and threefold en-hanced affinities for PD-L1 and PD-L2, respectively. It is notablethat Belatacept, a modified CTLA-4-Ig containing two pointmutations, possesses only a modest twofold increase in affinityfor the B7 ligands relative to the wild type but remarkablyexhibits a 10-fold enhancement in biological potency (26). Inclinical trials Belatacept has demonstrated equivalent efficacywith fewer side effects than existing immunosuppresants forrenal transplantation (27). By analogy, soluble (Ig-fusion) formsof the high affinity A99L PD-1 receptor could represent asuperior reagent for the therapeutic modulation of the PD-/PD-L pathways.

In summary, our data demonstrate a unique 1:1 PD-1/PD-L2assembly that exhibits distinct structural and organizationalfeatures compared with the CTLA-4/B7 inhibitory complexes.Structure-based mutagenesis studies have confirmed the uniqueligand binding interfaces proposed for the PD-1/PD-L com-plexes and resulted in the generation of a high-affinity mutantwith potential therapeutic value.

Experimental ProceduresExpression, Crystallization, Data Collection, and Structure Determination. Allmaterials for crystallization were expressed in Escherichia coli and refoldedfrom inclusion bodies. Crystals of murine PD-1 in complex with PD-L2 or PD-L2IgV alone were obtained by sitting drop vapor diffusion at 293K. All structureswere solved by molecular replacement and refined by standard methods,resulting in Rwork/Rfree values of 18.8%/22.6% and 19.0%/22.3% for the PD-1/PD-L2 complex and the isolated PD-L2 IgV structures, respectively (Table S2).

Details of expression, purification and structure determination are provided inSI Experimental Procedures.

Mutagenesis of PD-1 and PD-L2 and Transfection into HEK293T Cells. Mutants ofPD-1 for bacterial expression were designed by PCR-based mutagenesis, ex-pressed in E. coli, and refolded as described in ref. 12. For mammalian cellsurface expression, the PD-1 and PD-L2 mutants were transfected intoHEK293T cells. Details are provided in SI Experimental Procedures.

Binding Assays. SPR experiments were performed with a Biacore X opticalbiosensor at 25°C, using immobilized murine PD-L1-Ig or PD-L2-Ig fusionproteins. For flow cytometry, 293T cells were transiently transfected with PD-1or PD-L2 and, as appropriate, incubated with PD-1, PD-L1, or PD-L2 Ig fusionproteins. Details are provided in SI Experimental Procedures.

Pseudoconjugate Assays. PD-1, PD-L1, and PD-L2 were expressed as C-terminalfusion proteins of CFP or YFP. CHO cells were transiently transfected withPD-1-CFP, PD-L1-YFP, or PD-L2-YFP, using FuGENE (Roche). Conjugates wereprepared by incubating cells expressing PD-1-CFP with cells expressing PD-L1-YFP/PD-L2-YFP and analyzed by using laser-scanning confocal microscopy.Details are provided in the SI Experimental Procedures.

Note. While this manuscript was under review, the crystal structure of thecomplex between human PD-L1 and mouse PD-1 was reported (28). Thestructure-based alignment of the PD-L IgV domains (Fig. 2D) was manuallyedited based on the reported PD-L1 structure.

ACKNOWLEDGMENTS. We thank the staff of the X29 beam lines at theNational Synchrotron Light Source, R. Toro for assistance with the crystalliza-tion robot, and X. Zang for critical reading of the manuscript. This work wassupported by National Institute of Health Grant AI07289 (to S.G.N. and S.C.A.);a postdoctoral fellowship from Cancer Research Institute (to E.L.-M.); andAlbert Einstein Cancer Center Grant P30CA013330; and the Flow Cytometry,the Structural Biology Core Facilities, and the Analytical Imaging Facility.

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10488 � www.pnas.org�cgi�doi�10.1073�pnas.0804453105 Lazar-Molnar et al.

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