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The Leukocyte Function-Associated Antigen-1 (LFA-1)-Binding Site on ICAM-3 Comprises Residues on Both Faces of the First Immunoglobulin Domain This information is current as of September 28, 2021. Elaine D. Bell, Andrew P. May and David L. Simmons J Immunol 1998; 161:1363-1370; ; http://www.jimmunol.org/content/161/3/1363 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Leukocyte Function-Associated Antigen-1 (LFA-1)-Binding Site on ICAM-3 Comprises Residues on Both Faces of the First Immunoglobulin Domain1

Elaine D. Bell,2* Andrew P. May,† and David L. Simmons3*

ICAM-3 (CD50), a member of the Ig superfamily, is a major ligand for the leukocyte integrin LFA-1 (CD11a/CD18). This interaction represents one of several Ig superfamily/integrin ligand-receptor pairs that have been described to date. ICAM-3 is highly expressed on resting leukocytes and on APCs. In addition to an adhesive function, ICAM-3 can act as a signal-transducing molecule on T cells, providing a costimulatory signal for cell proliferation. Eighteen point mutations in ICAM-3 were generated, and residues important for binding of functional blocking Abs were identiﬁed. Mutation of seven of the residues reduced or abrogated adhesion to LFA-1, including three residues that are located on strand A of the ABED face of domain 1. In contrast, Downloaded from extensive mutagenesis analysis of ICAM-1 has shown that only residues on the GFC face interact with LFA-1. Our results provide evidence for a more extensive binding interface between ICAM-3 and LFA-1 than has previously been described. ICAM-3 appears to be unique among the ICAMs in utilizing residues on both faces of domain 1 for interaction with its ligand LFA-1. The Journal of Immunology, 1998, 161: 1363–1370.

he three intercellular adhesion molecules ICAM-1, LFA-1-binding site to be mapped to the GFC face of domain 1. http://www.jimmunol.org/ ICAM-2, and ICAM-3 are members of the Ig superfamily The two residues homologous to E34 and Q73 in ICAM-1 have T (IgSF)4 composed of five, two, and five Ig domains, re- also been defined as key residues for LFA-1 binding to ICAM-3. ␤ spectively (1–8, 11, 12). All three are counterreceptors for the 2 A large panel of anti-ICAM-3 Abs were generated, of which five ␣ ␤ integrin LFA-1 (CD18/CD11a, L 2). In addition, ICAM-3 has Abs were able to block ICAM-3/LFA-1 interactions and were recently been shown to interact with a newly identified member of functionally effective in inducing homotypic T cell aggregation, ␤ ␣ ␤ the 2 family, CD18/CD11d ( d 2) (9, 10). induction of T cell proliferation, IL-2 production, and expression ICAM-3 appears to be the dominant ligand for LFA-1 during the of cell surface activation markers on T cells (16). The five blocking initiation of the immune response (13), given that ICAM-1 and Abs define three overlapping epitopes in domain 1, none of which ICAM-2 either are not expressed or are expressed at very low was affected by our previous panel of mutations (20). by guest on September 28, 2021 levels on resting leukocytes and APCs (14, 15). In addition to an In recent years, several studies on integrin/ligand interactions adhesive function, ICAM-3 can provide a costimulatory signal for have shown that many ligands use short sequences as recognition T cell proliferation and IL-2 production (16, 17). Cross-linking of motifs for integrin binding (21). RGD, the prototypic peptide mo- ICAM-3 induces changes in calcium flux and tyrosine phosphor- tif, provides adhesive activity for various extracellular matrix com- ylation in a T cell line (18). A consequence of this ICAM-3 sig- ponents including fibronectin, fibrinogen, vitronectin, and von ␤ ␤ naling is increased adhesion mediated by 1 and 2 integrins, a Willebrand’s factor. Other studies on VCAM-1 and the ICAMs process referred to as crosstalk or adhesion amplification (14, 19). have identified a second linear motif with the following consensus The LFA-1-binding site on ICAM-3 has been mapped to domain sequence: L/I-D/E-S/T/V-P/S (22). The solution of the VCAM-1 1 (20), an Ig I-set structure consisting of seven ␤ strands arranged D1-2 crystal structure (23) has revealed that like RGD in fibronec- on two surfaces, an ABED face and a GFC face. Site-directed tin (24), the VCAM-1 IDSP motif is located in a prominently mutagenesis in conjunction with modeling studies permitted the exposed loop, which presumably favors interaction with integrin. In contrast, the recently solved crystal structure of ICAM-2 shows that the critical glutamic acid residue at position 37 is located in a *ICRF Cell Adhesion Laboratory, Imperial Cancer Research Fund Laboratories, Uni- ␤ versity of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Head- strand and is surrounded by a relatively flat surface which may 2ϩ ington, Oxford, OX3 9DS, U.K., and †Laboratory of Molecular Biophysics and Ox- complement the relatively flat surface surrounding the Mg -bind- ford Centre for Molecular Sciences, Department of Biochemistry, University of ing site in the I domains of Mac-1 and LFA-1 (25). It has been Oxford, Oxford, OX1 3UQ, U.K. proposed that these short linear sequences are essential common Received for publication June 18, 1997. Accepted for publication March 30, 1998. components of IgSF/integrin interactions (26). Indeed, the QIDS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance region in domain 1 of VCAM-1 can be replaced with the similar with 18 U.S.C. Section 1734 solely to indicate this fact. ICAM-1 sequence GIET without affecting interaction with VLA-4, 1 This work was supported by the Imperial Cancer Research Fund (E.D.B. and D.L.S.) indicating that this region is part of a general integrin binding and a BBSRC CASE award with the Yamanouchi Research Institute (A.P.M.). motif (27). Despite having common structural binding motifs, in- 2 Current address: The Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, tegrins clearly have discrete receptor specificities presumably de- British Columbia, V5Z 1L3 Canada. termined by residues without the common motifs. The identifica- 3 Address correspondence and reprint requests to Dr. David L. Simmons, Cell Ad- tion of such “specificity” residues will permit the development of hesion Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, U.K. E-mail address: [email protected] novel therapeutics targeted to particular interactions. 4 Abbreviations used in this paper: IgSF, Ig superfamily; ICRF, Imperial Cancer A second common theme that has emerged from structural stud- Research Fund. ies of IgSF proteins is that only residues located on the GFC face

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 1364 LFA-1 BINDING SITE ON ICAM-3 of the Ig fold contribute to ligand binding. This holds true for the (Sigma, Poole, Dorset, U.K.) in bicarbonate buffer (pH 9.6), blocked for ␣ ␤ 2 h at room temperature with 0.4% BSA (fraction V, Sigma) in PBS, and interaction of VCAM-1 with the integrin 4 1, and also for the binding of the adhesive proteins CD2 (29), E cadherin (28), and then coated with chimeric Fc proteins by addition of neat tissue culture supernatant to the wells for at least2hatroom temperature. Before the members of the sialoadhesin family (30, 31) to their respective adhesion assay, HSB2 or KL/4 cells were labeled with the fluorescent dye ligands. Notably, mutational analysis of most of the ABED face BCECF-AM (10 ␮g/107 cells, Molecular Probes, Eugene, OR) for 30 min residues in domain 1 of ICAM-1 has shown that none of these at 37°C and washed twice in assay buffer (DMEM, 10 mM HEPES buffer, residues participates in binding to LFA-1 (32, 33). Interestingly, a 0.2% BSA). LFA-1 is not constitutively avid for ICAM-3 but requires activation to mediate stable adhesion. Therefore, HSB2 cells were stimu- recent study has shown that residues N23 and S25, which comprise 2ϩ lated by addition of either Mn ions (0.5 mM MnCl2) or KIM185 Ab at a potential N-linked glycosylation site located on the ABED face 5to10␮g/ml, or KIM185 Ab at 5 to 10 ␮g/ml plus 50 ng/ml PMA (Sigma) of ICAM-3, contribute to the interaction with LFA-1 (34). for 10 min at room temperature before plating, and KL/4 cells were sim- In this study, we set out to define the epitopes of the functional ilarly stimulated by addition of KIM127 Ab at 5 to 10 ␮g/ml. Labeled, ϫ 5 anti-ICAM-3 blocking Abs and hence delineate additional ICAM- stimulated cells were added to wells at 2 10 cells/well in a volume of 50 ␮l and allowed to adhere for 35 min at 37°C. Plates were washed with 3/LFA-1 contact sites. A panel of 18 single point mutations were prewarmed assay buffer until the cells in the negative control wells were generated, targeting 7 residues in domain 1 and 3 residues in do- sufficiently removed as judged by visual inspection. Typically two to three main 2 of ICAM-3. This panel of mutations has enabled us to washes were required. The percentage of input cells bound (percentage of identify residues that contribute to the epitopes of the five func- cell adhesion) was quantified using a Cytofluor II fluorescent plate reader (Millipore, Watford, U.K.) by comparing the total input fluorescence and tional blocking anti-ICAM-3 Abs. Mutation of seven of the resi- the fluorescence of bound cells after washing. dues reduced or abrogated adhesion to LFA-1, including three res- Downloaded from idues located on strand A of the ABED face of domain 1. Mutation Enzyme-linked immunosorbent assay of ICAM-3 Fc proteins of residues in domain 2 of ICAM-3 had some effect on the inter- Immulon 3 96-well flat-bottom plates were precoated overnight at 4°C with action with LFA-1. Fc-specific goat anti-human IgG in bicarbonate buffer (pH 9.6), blocked for 2 h at room temperature with 2% BSA in PBS, and then coated with Fc proteins by addition of neat tissue culture supernatant to the wells for at Materials and Methods least2hatroom temperature. Primary Ab were added to saturation as Monoclonal antibodies and cell culture either neat tissue culture supernatant, or at 5 to 10 ␮g/ml for purified Ab, http://www.jimmunol.org/ or at 1:500 dilution for the Abs from the workshop panel, with the excep- The mAbs used in this study were as follows. The anti-ICAM-3 Abs tion of AO19 (BU 68) which was added at 1:100 dilution. Bound Ab were CH3.1, CH3.2, CH3.3, CAL3.4, CAL3.10, CAL3.38, and CAL3.41 were detected using peroxidase-conjugated sheep anti-mouse Ig (1:1000 dilu- generated in this laboratory as described previously (16). The anti-ICAM-3 tion, Amersham Life Sciences, Amersham, Buckinghamshire, U.K.) and Abs BY44 and CG106 were provided by Dr. D. Mason (Nuffield Depart- visualized with o-phenylenediamine dihydrochloride (Sigma). Each layer ment of Pathology, John Radcliffe Hospital, Oxford, U.K.). The anti- was incubated at room temperature for 30 min, followed by three washes ICAM-3 Ab KS128 was purchased from Dako, High Wycombe, Bucking- with 0.25% BSA in PBS. The absorbance was measured at 450 nm. hamshire, U.K. Anti-CD18 Abs KIM185 and KIM127 (36) were provided by Dr. M. Robinson (Celltech, Slough, U.K.), and anti-CD11a Ab 38 (35) Modeling of ICAM-3 was obtained from Dr. N. Hogg (Imperial Cancer Research Fund (ICRF), London, U.K.). Six Ab that were submitted to the CD50 (ICAM-3) panel A molecular model of the N-terminal domain of ICAM-3 was produced, of the Sixth International Workshop on Human Leukocyte Differentiation based on the structure of ICAM-2 domain 1 (25). The sequence of ICAM-3 by guest on September 28, 2021 Antigens were also used in this study: AO19 (BU68); AO41 (186–269); was substituted into the ICAM-2 structure using the program MUTATE (R. AO64 (B-N2); AO69 (B-P12); AO70 (B-R1); and AO85 (AZN-ICM3.1). Esnouf, unpublished data). No insertions or deletions were required, and in Ab AO76 (B-S9), an anti-ICAM-2 Ab that cross-reacts with domain 2 of all regions the ICAM-2 main chain conformation was retained. The figure ICAM-3, and the CD50 reference Ab HP2/19 (Ref. 25 in panel) were also was produced using the program MOLSCRIPT (42). included. KL/4 cells (K562 cells stably transfected with CD11a and CD18) (36) Results were a generous gift from Dr. M. Robinson (Celltech, Slough, U.K.) and Mutagenesis strategy were maintained in RPMI 1640 medium, 10% FCS, and 1 mg/ml G418 (Life Technologies, Paisley, U.K.). COS-1 cells were provided by the cell Previous work has established that ICAM-3 domains 1 and 2 con- bank (ICRF, Clare Hall, U.K.) and grown in DMEM supplemented with tain the LFA-1 binding site (7). Using a nested set of ICAM-3 Fc 10% FCS, 2 mM glutamine, and penicillin-streptomycin. domain deletion chimeras, it was shown that binding was not sig- Generation of ICAM-3 mutants nificantly increased by the presence of domains 3 to 5 of ICAM-3 (20). In this latter study, 9 residues in domain 1 (6 on the GFC face ICAM-3(D1-2)Fc and CD31(D1-2)Fc consisting of the first two extracellular domains fused to human IgG1-Fc fragment have previously been and 3 on the ABED face) and 3 residues in domain 2 were selected described (7, 37). Single point mutations were introduced into ICAM- for mutational analysis. The domain 1 residues E37, L66, S68, and 3(D1-2)Fc using a two-step PCR strategy (38, 39) with common forward Q75 were found to contribute significantly to LFA-1 binding. amplification (5Ј-ATAT AAGCTT ATG GTA CCA TCC GTG TTG TGG However, the five functional blocking Abs were able to bind to all Ј Ј CCC-3 ) and reverse amplification primers (5 -ATCT AGATCT ACT of the conformationally intact domain 1 mutants, suggesting that TACCTGT GCG CGG GGG GGT CAC GGG CAG-3Ј) in addition to sequence-specific mutagenic primers (a list of primers is available on re- further sites of interaction between LFA-1 and ICAM-3 remain to quest). All PCR amplifications were performed using Pwo DNA polymer- be defined. ase (Boehringer Mannheim, Mannheim, Germany) with ICAM-3(D1-5)Fc In this study, we have used a two-step PCR strategy to mutate in pcDNA3neo as template. A maximum of 10 cycles in the first round and the 7 previously unmutated charged residues in domain 1 (4 on the 15 cycles in the second round of PCR were performed to reduce the rate of generating unwanted mutations. PCR products were digested with HindIII GFC face and 3 on the ABED face) as well as a further three and BglII and cloned into the HindIII/BamHI-digested Fc expression vector residues in the E-F loop of domain 2. Figure 1 shows an alignment of pIG1 (7, 40). All mutants were verified by sequencing of the amplified region. the primary amino acid sequences of ICAM-1, ICAM-2, and ICAM-3 Recombinant chimeric Fc plasmids were transiently expressed in COS and indicates the location of residues targeted for mutagenesis. cells and tissue culture supernatants containing secreted protein harvested Mutant ICAM-3D1-2Fc proteins were produced as tissue cul- after 7 to 10 days. Recombinant protein production was initially assessed by ELISA using the Ab CH3.1 which bound well to all the mutants. ture supernatants from COS cells and were used undiluted in adhesion assays and ELISAs. To demonstrate that application of neat Adhesion assays Fc supernatants produced saturating conditions for Ab and cell Immulon 3 96-well flat-bottom plates (Dynatech, Chantilly, VA) were pre- binding assays, a titration ELISA was performed (Fig. 2). The coated overnight at 4°C with 1 ␮g/well Fc-specific goat anti-human IgG primary Ab chosen for this ELISA was CH3.2, which binds well The Journal of Immunology 1365 Downloaded from

FIGURE 1. Sequence alignment of ICAM-1, ICAM-2, and ICAM-3.

Sequence alignment of the two N-terminal domains of ICAM-1, ICAM-2, http://www.jimmunol.org/ and ICAM-3. The predicted ␤ strands of the Ig domains are underlined and labeled according to the method of Casanovas et al. (25), as deﬁned for ICAM-2. Asterisks denote those residues in ICAM-3 that were targeted for site-directed mutagenesis. Potential N-linked glycosylation sites are num- bered (-1- etc.) above the alignment. to all the ICAM-3 mutants with the exception of R6A and R6E. The results indicate that use of neat Fc supernatants produced sat- by guest on September 28, 2021 urating conditions for functional assays. Thus, differences observed in LFA-1-mediated cell binding to the mutants cannot be attributed to differences in the amount of mutant Fc protein coated on the plates.

Assessment of structural integrity of ICAM-3 mutants and identification of blocking Ab epitopes by ELISA The structural integrity of the ICAM-3 Fc mutant proteins was assessed by ELISA profile using a panel of 13 Abs that map to domain 1 and 5 Abs which map to domain 2 of ICAM-3 (Table I). Mutation of the domain 2 residue D154 to both alanine and lysine appears to cause a severe disruption of the domain 2 structure as FIGURE 2. Titration of mutant ICAM-3 Fc proteins to demonstrate that indicated by lack of binding of all five Abs that map to domain 2. ELISA and adhesion assays were performed under saturating conditions. These mutants were excluded from functional assays. The remain- Mutant ICAM-3 Fc proteins were produced as supernatants from COS cells. Various dilutions were applied to plates precoated with Fc-specific ing three mutations in domain 2 appeared to have little or no effect goat anti-human IgG and blocked with BSA. Bound protein was quanti- on the overall structure of domain 2. The majority of the domain tated by ELISA with the primary Ab CH3.2 applied at 1/5 dilution of tissue 1 Abs recognize the domain 1 mutants, the main exception being culture supernatant. Binding to wild-type ICAM-3D1-2Fc was used to nor- mutation of those residues that contribute to the epitopes of block- malize the data, giving 100% binding by definition. Data are representative ing and partial blocking Abs. In particular, residues E32 and K33 of two independent experiments. The results are expressed as means of seem to be crucial components of the blocking Ab epitopes, with three wells. The SDs were consistently within 10% of mean values and are residue K42 also making a significant contribution. The binding of not shown for clarity. A, Data for mutants in domain 1 of ICAM-3 that the domain 1 Ab CH3.2 is significantly reduced by mutation of R6 localize to the ABED face; B, Data for mutants in domain 1 of ICAM-3 that to R6E. localize to the GFC face. C, Data for mutants in domain 2 of ICAM-3.

Effect of ICAM-3 mutants on interaction with LFA-1 To investigate the effect of the mutations on LFA-1 binding, ad- R64A, and R64E also resulted in a signiﬁcant reduction in cell hesion assays were performed using two different cell lines that adhesion ( p Ͻ 0.01 compared with wild type in each case). These express LFA-1. Figure 3 shows that HSB2 cells suboptimally ac- residues are all located on the GFC face of domain 1. Interestingly, ␤ tivated with the anti- 2-activating Ab KIM185 were unable to bind LFA-1-mediated cell binding to mutants E2K, R6E, E8A, and to the mutant K42D that maps to the D strand of domain 1 in a E8K, located on the ABED face of domain 1, was also signiﬁ- region close to the LETS motif. The mutants E32A, K33A, K33D, cantly reduced ( p Ͻ 0.02, p Ͻ 0.05, p Ͻ 0.01, and p Ͻ 0.01, 1366 LFA-1 BINDING SITE ON ICAM-3

Table I. Assessment of structural integrity of ICAM-3 mutants and identiﬁcation of blocking Ab epitopes by ELISAa

Domain 1 Abs Domain 2 Abs

B PB B B B B B PB AO41 PB AO85 Location of B CG CAL CAL CAL HP AO19 186– AO69 AZN CAL AO64 AO70 AO76 Mutation Residue CH3.1 CH3.2 CH3.3 BY44 106 3.10 3.38 3.41 2/19 BU68 269 B-P12 ICM3.1 KS128 3.4 BN-2 B-R1 B-S9

D154A D2 ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ND ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩ/Ϫϩ/ϪϪϪϪ D154K D2 ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ND ϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϪϪϪϪϪ H155A D2 ϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩ H155D D2 ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ G156K D2 ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ E2A D1-ABE ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩ E2K D1-ABE ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ R6A D1-ABE ND ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ND ϩϩϩ ND R6E D1-ABE ND Ϫ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ND ϩϩϩ ND E8A D1-ABE ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩ ϩ/Ϫϩ E8K D1-ABE ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩ/Ϫϩ/ϪϪ E32A D1-CFG ϩϩϩ ϩϩ ϩϩϩ Ϫ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩ/ϪϩϩϩϪϩ/Ϫ ϩϩ ϩ ϩϩ ϩϩ ϩϩ E32K D1-CFG ϩϩϩ ϩϩϩ ϩϩϩ Ϫ ϩ/Ϫ ϩϩϩ ϩϩϩ ϩϩϩ Ϫ ϩ/Ϫ Ϫ Ϫ Ϫ ϩϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ K33A D1-CFG ϩϩϩ ϩϩϩ ϩϩϩ Ϫ ϩ/Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ ϩϩϩ Ϫ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ Downloaded from K33D D1-CFG ϩϩϩϩϩϩϩϩϩϪϪϪϪϪϪϪϪϪϪϩϩϩϩϩϩϩϩϩϩϩϩϩϩ K42D D1-CFG ϩ ϩϩϩϩϩ/ϪϪϩϩϩϩϩϩϩϩ/Ϫ ϩϩ ϩϩϩ Ϫ ϩ ϩϩ ϩϩϩ ϩ/Ϫϩ R64A D1-CFG ND ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ND ϩϩϩ ND R64E D1-CFG ND ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ND ϩϩϩ ND

P12A D1-ABE ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩ/ϪϪ ND

F21A D1-ABE ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩϩ ϩϩ ϩϩ ND http://www.jimmunol.org/ D27A D1-ABE ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ND E43A D1-CFG ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ND L66K D1-CFG ϩϩϩ ϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩϩ ND S68K D1-CFG ϩϩϩ Ϫ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ND Q75A D1-CFG ϩϩϩ ϩ/Ϫ ϩϩϩ ϩϩϩ Ϫ ϩϩϩ ϩϩϩ ND Q75H D1-CFG ϩϩϩ ϩ/Ϫ ϩ ϩϩϩ Ϫ ϩϩϩ ϩϩϩ ND E143A D2 ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩϩ

a A panel of 18 Abs, 13 with epitopes in domain 1 and 5 with epitopes in domain 2, was used in an ELISA to assess the structural integrity of the mutant ICAM-3 Fc proteins. Background values for binding to a CD31D1-2Fc construct were subtracted and Ab binding activity was expressed as a percentage of binding to the wild-type ICAM-3D1-2Fc ϩϩϩ Ͼ ϩϩ ϩ ϩ Ϫ Ϫ Ͻ

protein. The results were tabulated as follows: , 80% binding; , 60 to 80% binding; , 40 to 60% binding; / , 20 to 40% binding; and , 20% binding. by guest on September 28, 2021 Comparable results were obtained on sometimes two but mostly three separate occasions. Data presented in the upper part of the table summarizes the results obtained using the complete panel of mAbs against all the newly generated ICAM-3 mutants. Data presented in the lower part of the table summarizes the results obtained from mutants generated previously in this laboratory but assessed with the newly available panel of mAbs from the Sixth CD Antigen Workshop. B, blocking Ab; PB, partial blocking Ab. respectively). The domain 2 mutants H155A and H155D had a ulated with KIM127, the cells bound to the panel of mutants in a significant effect on binding of HSB2 cells, with H155A resulting pattern similar to that observed for adhesion of activated HSB2 in decreased adhesion compared with wild type ( p Ͻ 0.01) and cells; mutant K42D resulted in abrogation of adhesion, while mu- H155D showing enhanced adhesion compared with wild type tants K33D and R64E profoundly reduced adhesion compared ( p Ͻ 0.01). On the whole, residues mutated to opposite charges with the wild-type control (Fig. 5). had greater effects on cell adhesion than those mutated to alanine, To map the location of the mutated residues, a model of domain the exceptions being E32A and E32K. 1 of ICAM-3 was generated based on the crystal structure of To determine the effect of the ICAM-3 mutants under optimal ICAM-2 (Fig. 6). The side chains of residues that, when mutated, binding conditions, HSB2 cells were maximally activated by stim- resulted in reduced binding or no binding to LFA-1 positive cells ulation with KIM185 and the protein kinase C agonist PMA (Fig. are illustrated on the model. In this model, the critical glutamic 4). Under these conditions, the K42D mutant remained unable to acid residue at position 37 is located at the end of a ␤ strand and bind HSB2 cells. The other domain 1 mutants produced a pattern not in a loop region, and is surrounded by a relatively flat surface. of binding similar to that observed under suboptimal activating Interestingly, residues identified in this study as being important conditions. Maximal stimulation of HSB2 cells did not result in for the interaction of ICAM-3 with LFA-1, are located on both increased binding to E32A, K33A, K33D, or R64E. Although in- faces of domain 1. creased cell binding to E32K and R64A was observed, the adhesion was still significantly reduced compared with the wild-type control ( p Ͻ 0.01 in each case). The domain 1 mutants located on Discussion the ABED face produced a pattern of binding similar to that ob- In this study we have used mutant ICAM-3Fc proteins to build on served for suboptimal activation of the cells. For the domain 2 previous work and further investigate the interaction between mutants, adhesion to H155A remained significantly reduced ( p Ͻ ICAM-3 and LFA-1. We have generated 18 single point mutants in 0.01), although binding to H155D was not significantly different an ICAM-3D1-2Fc backbone and used this panel of reagents to compared with the wild-type control. define residues that are crucial components of blocking Ab The effect of the mutations on LFA-1 binding were then exam- epitopes and residues that are components of the ICAM-3/LFA-1 ined in an alternative cell context, because it is known that cell interface. background plays an important role in determining the ability of The panel of ICAM-3 mutants was assessed by ELISA using a LFA-1 to recognize its ligands (43). When KL/4 cells were stim- series of 18 Abs, 13 of which map to domain 1 and 5 that map to The Journal of Immunology 1367 Downloaded from http://www.jimmunol.org/

FIGURE 3. Adhesion of KIM185-stimulated HSB2 cells to the panel of FIGURE 4. Adhesion of KIM185/PMA-stimulated HSB2 cells to the ICAM-3 mutants. HSB2 cells were stimulated with the activating Ab KIM panel of ICAM-3 mutants. HSB2 cells were stimulated with the activating by guest on September 28, 2021 185 at 10 ␮g/ml for 10 min at room temperature before addition to wells Ab KIM 185 at 10 ␮g/ml and the phorbol ester PMA at 10 ng/ml for 10 coated with ICAM-3 mutants, ICAM-3D1-2Fc wild type or the negative min at room temperature before addition to wells coated with ICAM-3 control protein CD31D1-2Fc. The percentage cell adhesion (percentage of mutants, ICAM-3D1-2Fc wild type or the negative control protein input cells bound after two washes) was calculated, and results are ex- CD31D1-2Fc. The percentage cell adhesion (percentage of input cells pressed as mean Ϯ SD (n ϭ 3). Results are representative of three inde- bound after two washes) was calculated, and results are expressed as pendent experiments. *, p Ͻ 0.01; **, p Ͻ 0.02; and ***, p Ͻ 0.05 com- mean Ϯ SD (n ϭ 3). Results are representative of three independent ex- pared with the wild-type control, as determined by Student’s t test. A, Data periments. *, p Ͻ 0.01; **, p Ͻ 0.02; and ***, p Ͻ 0.05 compared with the for mutants of residues H155, G156, E2, E8, E32, K33, and K42; B, Data wild-type control, as determined by Student’s t test. A, Data for mutants of for mutants of residues R6 and R64. residues H155, G156, E2, E8, E32, K33, and K42; B, Data for mutants of residues R6 and R64. domain 2 of ICAM-3. Residues E32 and K33 were identified as crucial components of the epitopes of the 10 blocking and partial We have investigated the role of 9 residues in both domain 1 and blocking Abs, with residue K42 also making a significant contri- domain 2 of ICAM-3 in the interaction with LFA-1. We generated bution. Residues Q75 and S68 are also important for binding of the a panel of 18 single point mutations, which produced a similar Abs AO19 and AO85, which are both partial blocking Abs. Sim- pattern of cell adhesion when analyzed using two different LFA- ilarly, Sadhu et al. (44) have defined the epitopes of six other 1-positive cell types and various activation regimens. A lysine res- blocking Abs. Residues E32, K33, and E37 were identified as be- idue at position 42, which bound the nonblocking mAbs as wild ing important for Ab binding, while mutation of Y70 and Q75 also type, was identified as a key residue for LFA-1 binding. In an had some effect. Our model of ICAM-3 based on the ICAM-2 alternative assay system, where COS cells were transfected with crystal structure indicates that these residues cluster in two distinct mutant ICAM-3 constructs and assayed for adhesion to purified locations on the GFC face of domain 1: the B-C and F-G loops and LFA-1, the mutant K42A displayed wild-type activity and bound adjacent residues located at the ends of ␤ strands in the top half of to a panel of Abs as wild type (34). In the structure of ICAM-2, the domain 1; and the C-D loop and adjacent residues in the lower half side chain of K42 forms a hydrogen bond with the main chain of domain 1. Interestingly, anti-ICAM-1 Abs that block the inter- carbonyl of L36 (25). In our model of ICAM-3, K42 adopts a action with LFA-1 also have epitopes that map to these regions on similar position, and it is possible that this interaction may be the domain 1 structure (33, 45). The anti-ICAM-1 Abs RR1, important in stabilizing the conformation of the C-D loop region. WEHI-CAM-1, 7.5C2, 8.4A6, and R6.5D6 map to the F-G loop at In previous studies, functional analysis of the equivalent residue in the top of domain 1, and the epitopes of the Abs 84H10, BBIG-11, ICAM-1 (K39) has yielded conflicting results. The triple mutant 1B9, and 4D3 include the residues K29, L30, K39, K40, K41, and K39KE/ERQ was found to bind as wild type to LFA-1 (32), Y66 (at equivalent positions to residues E32, K33, K42, none, E43, whereas the single point mutants K39A, K39E, and K39M resulted and S68, respectively, in ICAM-3). in little or no adhesion to LFA-1 (33). 1368 LFA-1 BINDING SITE ON ICAM-3 Downloaded from http://www.jimmunol.org/

FIGURE 5. Adhesion of KIM127-stimulated KL/4 cells to the panel of by guest on September 28, 2021 ICAM-3 mutants. KL/4 cells were stimulated with the activating Ab KIM127 at 10 ␮g/ml for 10 min at room temperature before addition to wells coated with ICAM-3 mutants, ICAM-3D1-2Fc wild type or the negative control protein CD31D1-2Fc. The percentage cell adhesion (percentage of input cells bound after two washes) was calculated, and results are expressed as mean Ϯ SD (n ϭ 3). Results are representative of three independent experiments. *, p Ͻ 0.01; **, p Ͻ 0.02; and ***, p Ͻ 0.05 compared with the wild-type control, as determined by Student’s t test. A, Data for mutants of residues H155, G156, E2, E8, E32, K33, and K42; B, Data for mutants of residues R6 and R64. FIGURE 6. Proposed model of domain 1 of ICAM-3 based on the crys- We have identified mutants of several residues that result in tal structure of ICAM-2. A side-on view of the domain is shown, with the intermediate effects on LFA-1-mediated adhesion. Mutants of GFC face on the left-hand side and the ABED face on the right-hand side. The side chains of residues that, when mutated, resulted in no binding three residues on the GFC face of domain 1, E32, K33, and R64, (K42) or reduced binding (E2, R6, E8, E32, K33, and R64) to LFA-1 are significantly decreased LFA-1 adhesion compared with wild type illustrated. Residue E37, which has been defined as a key residue for even under optimal activation conditions. Residues E32 and K33 ICAM-3 binding to LFA-1, is also shown. map to the B-C loop and the C strand, respectively, while R64 lies on the F strand in the lower region of domain 1. Using a similar system, Sadhu et al. (44) found that the double mutant E32K/AS In addition to residues E32, K33, and R64 on the GFC face of resulted in an approximately sixfold decrease in binding to LFA-1, domain 1, we have also identified mutants of ABED face residues in complete agreement with our data for the single mutants. In an that have statistically significant effects on LFA-1 adhesion. The alternative assay system, where COS cells were transfected with mutants E2K, R6E, E8A, and E8K reduced adhesion compared mutant ICAM-3 constructs and assessed for binding to purified with wild-type ICAM-3 (with p Ͻ 0.05 or less), although in gen- LFA-1, Klickstein et al. (34) found that the mutant E32A behaved eral the magnitude of the reduction in adhesion was less than that as wild type. Interestingly, residues in ICAM-1 at positions equiv- observed for GFC face mutants. In a previous study, LFA-1-trans- alent to E32 and K33 (K29 and L30, respectively) have been mu- fected COS cells that were suboptimally activated with PMA were tated (K29A, K29M, K29Q, and L30A) and found to bind to observed to bind to the mutants P12A and F21A at ϳ50% of LFA-1 equally as well as the wild type construct (33). The residue wild-type levels (20). When the LFA-1-positive COS cells were ␤ R64 has not previously been targeted for mutation and appears to maximally activated using the anti- 2-activating mAb KIM185 in have an effect on LFA-1 adhesion by virtue of its location in the conjunction with PMA, these two mutants were able to bind at main LETS motif region at the bottom of the F strand. 80% of wild type. In a recent study, the mutants N23 and S25, The Journal of Immunology 1369 which both disrupt a potential N-linked glycosylation site on the 4. Staunton, D. E., M. L. Dustin, and T. A. Springer. 1989. Functional cloning of ABED face, were found to profoundly disrupt binding to LFA-1 ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature 339: 361. (24 and 19% binding compared with wild type at 100%) (34). The 5. de Fougerolles, A. R., S. A. Stacker, R. Schwartin, and T. A. Springer. 1991. two residues at positions equivalent to R6 and E8 in ICAM-3 have Characterisation of ICAM-2 and evidence for a third counter-receptor for LFA-1. been analyzed in ICAM-1 (residues S3 and S5, respectively) and J. Exp. Med. 174:253. 6. de Fougerolles, A. R., L. B. Klickstein, and T. A. Springer. 1993. Cloning and found to bind to LFA-1 as wild type (33). Residues in ICAM-1 at expression of intercellular adhesion molecule 3 reveals strong homology to other positions equivalent to P12 and F21 in ICAM-3 have not been immunoglobulin family counter-receptors for lymphocyte function-associated antigen 1. J. Exp. Med. 177:1187. targeted for mutation in any studies of ICAM-1 to date. The res- 7. Fawcett, J., C. L. L. Holness, L. A. Needham, H. Turley, K. C. Gatter, idue in ICAM-1 that aligns with N23 is T20, which was changed D. Y. Mason, and D. L. Simmons. 1992. Molecular cloning of ICAM-3, a third to alanine in the mutant T20CS/ACT and had no significant effect ligand for LFA-1, constitutively expressed on resting leukocytes. Nature 360: 481. on binding to LFA-1 (32). Mutation of S22 to alanine in ICAM-1, 8. Vazeux, R., P. A. Hoffman, K. A. Tomita, E. S. Dickinson, R. L. Jasman, T. St. which corresponds to S25 of ICAM-3, similarly had no effect on John, and W. M. Gallatin. 1992. Cloning and characterisation of a new intercel- adhesion to LFA-1 (33). Taken together, our data and the data lular adhesion molecule ICAM-R. Nature 360:485. 9. van der Vieren, M., H. L. Trong, C. L. Wood, P. T. Moore, T. St. John, from these recent studies seem to indicate that in contrast to ␣ ␤ D. E. Staunton, and W. M. Gallatin. 1995. A novel leukointegrin, d 2, binds ICAM-1, ABED face residues on domain 1 of ICAM-3 may con- preferentially to ICAM-3. Immunity 3:683. tribute to the interaction with LFA-1. 10. Danilenko, D. M., P. V. Rossitto, M. van der Vieren, H. L. Trong, and ␣ ␤ P. F. Moore. 1995. A novel canine leukointegrin, d 2, is expressed by specific Domain 2 mutants were found to have some effect on adhesion macrophage subpopulations in tissues and a minor CD8ϩ lymphocyte subpopu- to LFA-1. Whereas non-domain 2 mutants generally resulted in lation in peripheral blood. J. Immunol. 155:35. Downloaded from statistically significant decreases in adhesion in all three assay sys- 11. Simmons, D. L. 1995. The role of ICAM expression in immunity and disease. Cancer Surv. 24:141. tems, the data for the domain 2 mutants were somewhat variable in 12. de Fougerolles, A. R., and T. A. Springer. 1992. Intercellular adhesion molecule terms of statistical significance. It is therefore difficult to conclude 3, a third adhesion counter-receptor for lymphocyte function-associated molecule whether these domain 2 residues play a major role in the interac- 1 on resting lymphocytes. J. Exp. Med. 175:185. 13. Starling, G. C., A. D. McLellan, W. Egner, R. V. Sorg, J. Fawcett, tion with LFA-1, although it would appear possible that they may D. L. Simmons, and D. N. J. Hart. 1995. Intercellular adhesion molecule 3 is the make some contribution to the interaction. predominant ligand for leukocyte function antigen 1 on human blood dendritic In this study, we have provided evidence for a more extensive cells. Eur. J. Immunol. 25:2528. http://www.jimmunol.org/ 14. Campanero, M. R., M. A. del Pozo, A. G. Arroyo, P. Sanchez-Mateos, binding interface between ICAM-3 and LFA-1 than has previously T. Hernandez-Caselles, A. Craig, R. Pulido, and F. Sanchez-Madrid. 1993. been described. Notably, residues on the ABED face of ICAM-3 ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion domain 1, as well as on the GFC face, appear to contribute to the pathway. J. Cell Biol. 123:1007. 15. Hernandez-Caselles, T., G. Rubio, M. R. Campanero, M. A. del Pozo, M. Muro, interaction with LFA-1, although GFC face residues predominate. F. Sanchez-Madrid, and P. Aparicio. 1993. ICAM-3, the third LFA-1 counter- We have also compared the residues in ICAM-3 and ICAM-1 that receptor, is a costimulatory molecule for both resting and activated T lympho- mediate interaction with LFA-1 and show that although some res- cytes. Eur. J. Immunol. 23:2799. 16. Bossy, D., C. D. Buckley, C. L. Holness, A. J. Littler, N. Murray, I. Collins, and idues, such as E37 in ICAM-3 and E34 in ICAM-1, are identical D. L. Simmons. 1995. Epitope mapping and functional properties of anti- intercellular adhesion molecule-3 (CD50) monoclonal antibodies. Eur. J. Immu-

and located at equivalent positions in both molecules, other resi- by guest on September 28, 2021 nol. 25:459. dues and their locations are very different. These findings comple- 17. de Fougerolles, A. R., X. Qin, and T. A. Springer. 1994. Characterisation of the ment several studies that show that LFA-1 can bind selectively to function of intercellular adhesion molecule (ICAM)-3 and comparison with its ligands ICAM-1 and ICAM-3 and that there are distinct but ICAM-1 and ICAM-2 in immune responses. J. Exp. Med. 179:619. 18. Juan, M., O. Vinas, M. R. Pino-Otin, L. Places, E. Martinez-Caceres, overlapping binding sites in the I domain of LFA-1 for ICAM-1 J. J. Barcelo, A. Miralles, R. Vilella, M. A. S. de la Fuente, J. Vives, J. Yague, and ICAM-3 (35, 36, 46–48). It has recently been shown that prior and A. Gaya. 1994. CD50 (intercellular adhesion molecule 3) stimulation induces exposure to ICAM-1 increases the binding of LFA-1 to ICAM-3 calcium mobilisation and tyrosine phosphorylation through p59fyn and p56lck in Jurkat T cell line. J. Exp. Med. 179:1747. (43), which further suggests that the binding sites on LFA-1 for 19. Cid, M. C., J. Esparza, M. Juan, A. Miralles, J. Ordi, R. Vilella, these two ligands are distinct. In this article, we show that ICAM-3 A. Urbano-Marquez, A. Gaya, J. Vives, and J. Yague. 1994. Signaling through CD50 (ICAM-3) stimulates T lymphocyte binding to human umbilical vein en- is clearly very different from ICAM-1 in utilizing residues on both ␤ ␤ dothelial cells and extracellular matrix proteins via an increase in 1 and 2 faces of domain 1 to interact with LFA-1, which may reflect a integrin function. Eur. J. Immunol. 24:1377. distinct stoichiometry of interaction. Although this study extends 20. Holness, C. L., P. A. Bates, A. J. Littler, C. D. Buckley, A. McDowall, D. Bossy, our knowledge of the interaction face between ICAM-3 and N. Hogg, and D. L. Simmons. 1995. Analysis of the binding site on intercellular adhesion molecule 3 for the leukocyte integrin lymphocyte function-associated LFA-1, cocrystallization studies will be required to determine the antigen 1. J. Biol. Chem. 270:877. precise interactions between LFA-1 and its ICAM ligands. 21. Yamada, K. M. 1991. Adhesive recognition sequences. J. Biol. Chem. 266:12809. 22. Newham, P., and M. J. Humphries. 1996. Integrin adhesion receptors: structure, function and implications for biomedicine. Mol. Med. Today 2:304. Acknowledgments 23. Jones, E. Y., K. Harlos, M. J. Bottomley, R. C. Robinson, P. C. Driscoll, R. M. Edwards, J. M. Clements, T. J. Dudgeon, and D. I. Stuart. 1995. Crystal We thank Dr. D. Mason (Nuffield Department of Pathology, John Radcliffe structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at Hospital, Oxford, U.K.) for the BY44 and CG106 Abs, and in particular 1.8 A resolution. Nature 373:539. Dr. M. K. Robinson (Celltech, Slough, U.K.) for the gift of KL/4 cells and 24. Main, A. L., T. S. Harvey, M. Baron, J. Boyd, and I. D. Campbell. 1992. The KIM Abs. We also thank Justin Newton and Dr. Chris Buckley for critical three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions. Cell 71:671. reading of the manuscript. 25. Casanovas, J. M., T. A. Springer, J. Liu, S. C. Harrison, and J. Wang. 1997. Crystal structure of ICAM-2 reveals a distinctive integrin recognition surface. Nature 387:312. References 26. Vonderheide, R. H., T. F. Tedder, T. A. Springer, and D. E. Staunton. 1994. Residues within a conserved amino acid motif of domains 1 and 4 of VCAM-1 1. Rothlein, R., M. L. Dustin, S. D. Marlin, and T. A. Springer. 1986. A human are required for binding to VLA-4. J. Cell Biol. 125:215. intercellular adhesion molecule distinct from LFA-1. J. Immunol. 137:1270. 27. Osborn, L., C. Vassallo, B. G. Browning, R. Tizard, D. O. Haskard, 2. Simmons, D. L., M. W. Makgoba, and B. Seed. 1988. ICAM-1, an adhesion C. D. Benjamin, I. Dougas, and T. Kirchhausen. 1994. Arrangement of domains, ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM. and amino acid residues required for binding of vascular cell adhesion molecule-1 ␣ ␤ Nature 331:624. to it counter-receptor VLA-4 ( 4 1). J. Cell Biol. 124:601. 3. Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin, and T. A. Springer. 28. Overduin, M., T. S. Harvey, S. Bagby, K. I. Tong, P. Yau, M. Takeichi, and 1988. Primary structure of ICAM-1 demonstrates interaction between members M. Ikura. 1995. Solution structure of the epithelial cadherin domain responsible of the immunoglobulin and integrin supergene families. Cell 52:925. for selective cell adhesion. Science 267:386. 1370 LFA-1 BINDING SITE ON ICAM-3

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