©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Monoclonal Antibodies That Block the Activity of Leukocyte Function-associated Antigen 1 Recognize Three Discrete Epitopes in the Inserted Domain of CD11a (*)

(Received for publication, July 25, 1994; and in revised form, November 7, 1994)

Mark Champe (1) Bradley W. McIntyre (2) Phillip W. Berman (1)(§)

From the  (1)Department of Immunology, Genentech, Inc., South San Francisco, California 94080 and the (2)Department of Immunology, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The epitopes recognized by eight independently isolated monoclonal antibodies to the alpha chain of human and murine leukocyte function-associated antigen 1 (LFA-1), all able to inhibit receptor function, were identified. Initial localization of epitopes was accomplished using chimeric proteins constructed by splicing fragments of cDNAs encoding the alpha subunit of LFA-1 (CD11a) and the alpha subunit of the closely related leukocyte integrin, Mac-1 (CD11b). Antibody binding to CD11a/CD11b chimeras, expressed in the 293 human kidney cell line, demonstrated that the epitopes recognized by six monoclonal antibodies to human CD11a were located in a 200-amino acid sequence found in all beta(2)-integrin alpha subunits, termed the inserted (I) domain. Three distinct epitopes within the I domain (IdeA, IdeB, and IdeC) were identified using a series of mutants in which sequences from murine CD11a were substituted into human CD11a. A series of mutants incorporating single amino acid substitutions was used to identify individual amino acids essential for antibody binding. The location of these residues accounts for the binding specificity of LFA-1-blocking antibodies and identifies particular conserved sequences (residues 126-150) in the I domain of CD11a and homologous sequences in other beta(2)-integrin alpha subunits that may be important for ligand binding.


INTRODUCTION

Integrins are a large family of homologous, heterodimeric cell surface receptors that mediate cell to cell and cell to substratum adhesion(1, 2) . All integrins possess alpha(1),beta(1) subunit structure where the alpha chains and the beta chains associate through noncovalent interactions. Integrin families are classified on the basis of their beta subunits (3) , which typically form heterodimers with any of several different alpha subunits. In the immune system, integrins serve as accessory molecules that facilitate intercellular interactions required for antigen recognition, cellular activation, and leukocyte trafficking, as well as effector functions such as cytotoxic killing, phagocytosis, and antibody-dependent cytotoxicity(4, 5, 6) . The beta(2) family of leukocyte integrins consists of three homologous receptors, LFA-1 (^1)(CD11a/CD18), Mac-1 (CD11b/CD18), and p150,95 (CD11c/CD18), each consisting of a unique alpha chain (CD11a, CD11b, and CD11c, respectively) binding to a common beta chain (CD18). The distribution of the beta(2)-integrins is tissue-specific, with LFA-1 present on all leukocytes and Mac-1 and p150,95 occurring primarily on macrophages, neutrophils, and other myeloid cells. Like other integrins, the members of the beta(2) family are promiscuous and interact with several different ligands or contrareceptors. LFA-1 is known to bind to the three structurally related immunoglobulin superfamily members termed intercellular adhesion molecule 1 (ICAM-1)(7, 8, 9, 10, 11) , intercellular adhesion molecule 2 (ICAM-2)(12, 13) , and intercellular adhesion molecule 3 (ICAM-3)(14) . Mac-1 is known to bind several diverse and unrelated ligands including the C3Bi component of complement(15) , ICAM-1(16, 17) , fibrinogen (18) , factor X(19) , and an unidentified ligand on neutrophils that mediates homotypic aggregation(20) . The functional activity of beta(2)-integrins has been investigated through the use of monoclonal antibodies (mAbs) to LFA-1, Mac-1, and p150,95 (21) that are able to block adhesive interactions that mediate leukocyte function. These studies have shown that mAbs to either the alpha or beta chains can be potent inhibitors of leukocyte function(15, 22, 23, 24, 25) . Additional insight into the function of beta(2)-integrins has come through the analysis of a rare genetic disease, leukocyte adhesion deficiency, which results from the inability to synthesize functional CD18(26) . Individuals with this disease are unable to express LFA-1, Mac-1, or p150,95 and, as a consequence, are severely immunocompromised and exhibit multiple defects in lymphocyte and granulocyte function (27, 28) .

Little is known of the molecular basis of ligand recognition for the beta(2) family of integrins. Like other integrins, the alpha chains in the beta(2) family possess multiple divalent cation binding sites (29, 30, 31) and require Ca, Mg, or Mn to function(32, 33) . The ligands for beta(2)-integrins are unique in that none are known to possess the tripeptide receptor recognition sequence, RGD, that is common in many of the ligands for other integrin families (e.g. beta(1) and beta(3) family)(1, 2, 3, 34) . Besides the difference in ligand specificity, the beta(2) family of integrins is unusual in that the alpha subunits are larger (molecular size, 150-170 kDa) than most other integrins by virtue of a unique insertion of approximately 200 amino acids, termed the inserted domain (I domain). The I domain contains sequences homologous with the type A domains of von Willebrand factor, the repeats of cartilage matrix-binding protein, and complement factor B and is located approximately 150 amino acids from the NH(2) terminus(29, 31, 35) . Recent studies (20) have used antibodies to localize the binding sites for C3Bi, ICAM-1, fibrinogen, and an as yet unidentified ligand responsible for homotypic aggregation of neutrophils to the I domain of the CD11b/CD18 heterodimer (Mac-1). However, the regions within the I domain responsible for ligand binding have not yet been described.

In the present study, we have localized the epitopes recognized by eight mAbs to human and mouse CD11a that are all known to inhibit LFA-1 function. Mutagenesis to replace I domain sequences of human CD11a with those of murine CD11a permitted the identification of three discrete sequences in the I domain of CD11a that are recognized by antibodies able to block the binding of LFA-1 to ICAM-1.


MATERIALS AND METHODS

Antibodies

A panel of mAbs to CD11a that are known to block LFA-1 function was obtained from various sources. The mAb MHM.24, known to block a mixed lymphocyte reaction, cytotoxic T lymphocyte (CTL) lysis of target cells, and antigen-induced T cell proliferation(23, 36) , was kindly provided by Dr. James Hildreth (Johns Hopkins University, Baltimore, MD). The mAb CLB-LFA-1/2, known to block antigen-induced proliferation of T cell clones and to augment proliferation induced by immobilized CD3 antibodies(37) , was kindly provided by R. A. W. Van Lier (Amsterdam Blood Transfusion Center). mAb 25.3, known to inhibit CTL killing and T cell-dependent antibody production(38, 39) , was purchased from Amac, Inc. (Westbrook, ME). Three mAbs to human CD11a antibodies, 3D6, 32E6, and 50G1, are described for the first time in this study. Both the 32E6 and 50G1 mAbs were able to block mixed lymphocyte reactions and LFA-1-dependent lymphocyte binding to keratinocytes, whereas the 3D6 mAb failed to show inhibitory activity in either assay. (^2)The mouse mAb to human CD11a, TS-1/22(40) , and the rat mAb to murine CD11a, M17(22) , both able to block CTL-mediated killing (22, 40) were obtained from the ATCC. Another rat mAb to murine CD11a that blocks CTL activity, I21/7(41) , was purchased from Endogen, Inc. (Boston, MA). Polyclonal rabbit antisera to recombinant soluble Mac-1 were prepared in New Zealand White rabbits by immunization with recombinant soluble Mac-1 (43) as described previously(42) .

Mutagenesis and Expression of the cDNAs Encoding CD11a and CD11b

A cDNA used for the expression of CD11b was isolated as described previously(43) . Standard techniques (44) were employed to clone a cDNA encoding approximately 75% of CD11a from a [lamda] gt-10 cDNA library prepared from oligo(dT)-primed peripheral blood mononuclear cell mRNA. Synthetic DNA primers synthesized on the basis of published sequence data for CD11a (31) were used to clone cDNA fragments encoding the 5` end of CD11a from a random primed U937 cell cDNA library using the polymerase chain reaction(45) . The oligonucleotide sequence of the CD11a fragments was determined after subcloning into the bacteriophage M13 (46) using the method of Sanger et al.(47) . Full-length CD11a cDNA was contained on a 4.1-kilobase pair fragment beginning at the first EcoO109I site in the 5`-untranslated region and extending to a unique XbaI site in the 3`-untranslated region of the CD11a gene and encoded a protein of 1145 amino acids. Chimeric CD11a/CD11b cDNAs were constructed using standard techniques.

Expression and Immunoprecipitation of CD11a and CD11b Variants in 293 Cells

Chimeric transcription units for the expression of cDNAs encoding CD11a and CD11b were created by cloning the integrin genes into the RK 5 and RK 7 expression plasmids (48) as described previously(43) . The resulting plasmids were transfected into the 293 human kidney adenocarcinoma cell line (49) using a CaPO(4)-precipitated plasmid DNA(50) . 2-3 days after transfection, the cells were metabolically labeled in methionine-free Dulbecco's modified Eagle's medium with 100 µCi/ml [S]methionine for 4-5 h. The medium was removed, and the cells were lysed in 1 ml of lysis buffer (1% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 2 mM CaCl(2), and 2 mM MgCl(2)) per 10-cm dish. Cell debris and nuclei were pelleted, and the supernatant was used for subsequent immunoprecipitations. Antibodies (1-2 µg/immunoprecipitation) were incubated with 100 µl of labeled lysate and 2 µg of rabbit anti-mouse IgG (Cappel, Inc., West Chester, PA) for 2 h at room temperature. To reduce nonspecific binding, the protein A-Sepharose CL-4B beads were preincubated with unlabeled lysate of 293 cells for 30 min prior to immunoprecipitation. The antibody-antigen complexes were precipitated using 2 mg of protein A-Sepharose CL-4B beads (Pharmacia Biotech Inc.) per reaction for 15 min at room temperature with gentle agitation. After adsorption, the beads were washed two times in wash buffer (0.5% Nonidet P-40, 0.01% SDS, 400 mM NaCl, 10 mM Tris-HCl, pH 7.5, 2 mM CaCl(2), and 2 mM MgCl(2)) and resuspended in 30 µl of 2 times concentrated SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer(51) , boiled for 3 min, and centrifuged briefly. The clarified sample was then resolved on 6% SDS-PAGE gels (51) and visualized by autoradiography.


RESULTS

Localization of mAb Binding Sites Using CD11a/CD11b Chimeras

A panel of six mAbs to human CD11a known to inhibit LFA-1 function (TS-1/22, MHM.24, 25.3, CLB-LFA-1/2, 32E6, and 50G1) and one mAb known to bind LFA-1 without blocking function (3D6) were assembled. The ability of these antibodies to immunoprecipitate full-length recombinant CD11a from detergent lysates of transiently transfected 293 cells was evaluated. It was found (Fig. 1, A and B, CD11a) that all of the mAbs were able to immunoprecipitate a 160-165-kDa protein corresponding to the intracellular (i.e. high mannose) form of recombinant CD11a in the absence of the beta subunit of LFA-1, CD18. In control studies (data not shown), no differences were seen in mAb binding to the high mannose form of intracellular CD11a and the sialic acid containing the extracellular form of CD11a produced by co-transfection of CD11a with CD18. The specificity of these antibodies for CD11a was investigated in control experiments (Fig. 1, A and B, CD11b and Mock) in which the CD11a mAbs failed to specifically immunoprecipitate proteins from mock transfected 293 cells or cells transfected with full-length recombinant CD11b as described previously(43) . These results demonstrated that the epitopes recognized by the mAbs to LFA-1 did not depend on CD18 and were not present on the closely related integrin, CD11b. Although several of the mouse antibodies to human CD11a (25.3, MHM.24, and 32E6) reacted with CD11a on Western blots (data not shown), we cannot be sure that they recognized conformation-independent epitopes. Based on the lack of cross-reactivity between CD11a and CD11b, we reasoned that a series of intermolecular chimeras produced by splicing complementary fragments of CD11a and CD11b cDNAs might encode unique molecules in which the secondary and tertiary structures of CD11a and CD11b portions would be preserved and the epitopes recognized by the CD11a and CD11b mAbs could be localized.


Figure 1: Immunoprecipitation of recombinant human CD11a, CD11b, and CD11a/CD11b chimeras with monoclonal antibodies to human CD11a. The human kidney adenocarcinoma cell line 293 was transfected with expression plasmids containing genes encoding full-length human CD11a, full-length human CD11b, or chimeric genes produced from the ligation of complementary regions of human CD11a and CD11b genes (LM2, LMN, LM3, and ML9). The chimeric genes, but not the native genes, were mutated so as to delete sequences encoding the transmembrane domains and cytoplasmic tails. A schematic diagram of these constructions is shown in Fig. 2. Murine monoclonal antibodies to human CD11a CLB-LFA-1/2, MHM24, and 50G1, (A) and 32E6, 3D6, 25.3, and TS-1/22 (B) or a rabbit polyconal antisera to human CD11b (A) were incubated with detergent lysates of metabolically labeled [S]methionine-transfected 293 cells and immunoprecipitated as described previously(43) . The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography as described previously(43) .




Figure 2: Diagram of CD11a/CD11b chimeric proteins. Intermolecular chimeras between CD11a and CD11b were produced by ligation of complementary sequences of cDNAs encoding CD11a and CD11b. Regions selected for chimerization exhibited a high degree of sequence homology. For all but one of the chimeras, site-directed mutagenesis was used to introduce common restriction sites at the chimeric junctions without changing the predicted amino acid sequence. To construct the LM2 and ML9 plasmids, SacII sites were introduced to join the fragments of CD11a and CD11b. In the LMN plasmid, NheI sites were introduced into CD11a and CD11b to join the complementary fragments. To construct the LM3 plasmid, naturally occurring Sse8387I sites were used to splice the complementary CD11a and CD11b fragments. Sequences from CD11a are shown as open bars; sequences from CD11b are shown as dark bars. The locations of the I domain, the transmembrane domain, and the intracellular domain are also indicated. Amino acid numbering for CD11a and CD11b and the resulting chimeras is indicated.



The sequences of CD11a and CD11b were examined, and regions of high homology were selected as locations for chimerization. A schematic diagram of the four chimeras that were constructed is shown in Fig. 2. The chimeric genes, mutagenized to remove the transmembrane domains and cytoplasmic tails, were all cloned into the pRK expression vector and transiently expressed in 293 cells as described previously(43) . It was found that all of the chimeric proteins, but not CD11a, could be immunoprecipitated from lysates of transfected cells with polyclonal rabbit sera to recombinant soluble Mac-1 (Fig. 1A). When the binding of the CD11a mAbs to the panel of CD11a/CD11b chimeras was evaluated, it was found that none of the antibodies known to block LFA-1 function bound to the ML9 construction containing residues 1-340 of CD11b fused to amino acids 332-1063 of CD11a (Fig. 2). However, the 3D6 mAb, known to bind to CD11a without blocking function, was able to immunoprecipitate this protein (Fig. 1, A and B, ML9). These results suggested that the epitopes recognized by the LFA-1-blocking mAbs were located within the first 340 residues of CD11a. The mAbs were then tested for binding to the LM3 protein that contained the first 25 amino acids of CD11a fused to residues 62-1092 of CD11b (Fig. 2). In contrast to the ML9 protein, neither 3D6 nor any of the LFA-1-blocking mAbs were able to immunoprecipitate this protein (Fig. 1, A and B, LM3). Thus the amino-terminal sequence of CD11a did not appear to contain epitopes recognized by any of the mAbs. When mAb binding to the LM2 protein was examined, all six of the CD11a-blocking antibodies were able to immunoprecipitate the protein (Fig. 1, A and B, LM2) that consisted of amino acids 1-331 of CD11a fused to amino acids 341-1092 of CD11b (Fig. 2). Similarly, all six of the CD11a-blocking mAbs bound to the LMN protein (Fig. 1, A and B, LMN) that consisted of residues 1-278 of CD11a fused to residues 289-1092 of CD11b (Fig. 2). These studies localized the epitopes recognized by the six LFA-1-blocking mAbs to an amino-terminal fragment of CD11a spanning residues 26-278 that contains the I domain but excludes the divalent cation-binding sites (Fig. 2).

Localization of I Domain Sequences Essential for the Binding of mAbs to Human CD11a

Because pilot studies demonstrated that none of the mAbs to human CD11a cross-reacted with murine CD11a, we reasoned that sequences in the I domain that are important for mAb binding might be localized by replacing sequences from human CD11a with those from murine CD11a. Comparison of the sequences of human CD11a with murine CD11a (Fig. 3) revealed a high degree of homology in the I domain, with a limited number of sequence differences between the two proteins. A series of full-length CD11a replacement mutants was constructed (H/M 48-54) that incorporated the most radical amino acid differences between human and murine CD11a (Fig. 3). In control experiments (Fig. 4A), it was found that the nonblocking CD11a mAb 3D6 immunoprecipitated all of the proteins with I domain mutations. When mAbs known to block CD11a function were examined (Fig. 4), all were able to immunoprecipitate the H/M 48 protein (mutated at residues 182 and 184), the H/M 49 protein (mutated at residues 189 and 191), the H/M 50 protein (mutated at residue 210), and the H/M 51 protein (mutated at residues 216, 217, and 218). Together, these results demonstrated that multiple mutations at various locations in the I domain could be induced without disrupting the overall conformation of the molecule (Fig. 4, A and B, H/M 48-51). In contrast, mAbs TS-1/22 and 25.3 were unable to bind to the H/M 53 protein, whereas this protein was readily immunprecipitated by the MHM.24, CLB-LFA-1/2, 32E6, or 50G1 mAbs (Fig. 4). Thus, replacement of human CD11a residues Ile and Asn with residues from murine CD11a (the Met and Lys, respectively) specifically inhibited the binding of two of the six CD11a-blocking antibodies (Fig. 4B, H/M 53). Replacement of human CD11a residues Gln, Pro, Asp, and Gln with murine CD11a residues Asp, Arg, Lys, and Glu, respectively (H/M 52), preserved the 25.3, TS1/22, and MHM.24 mAbs but abolished the binding of the CLB-LFA-1/2, 32E6, and 50G1 mAbs (Fig. 4, A and B, H/M 54). Finally, replacement of human CD11a residues Lys, His, Lys, His, and Leu with the murine CD11a residues Gly, Ser, Gln, Pro, and Phe, respectively (H/M 54), preserved the binding of the 25.3 and TS-1/22 mAbs but prevented the binding of mAbs MHM.24, CLB-LFA-1/2, 32E6, and 50G1 (Fig. 4, A and B, H/M 54). Thus, this study suggested that the I domain contains three distinct epitopes recognized by mAbs that block CD11a function. Based on these results we have termed the epitope recognized by the 25.3 and TS-1/22 mAbs (involving residues Ile and Asn) as I domain epitope A (IdeA), the epitope recognized by mAbs 32E6, CLB-LFA-1/2, and 50G1 (involving residues 143, 144, 145, and 148) as I domain epitope B (IdeB), and the epitope recognized by MHM.24 as well as 32E6, CLB-LFA-1/2, and 50G1 (involving residues 197, 198, 200, 201, and 203) as I domain epitope C (IdeC).


Figure 3: Alignment of I domain sequences from human and murine CD11a and description of human to mouse mutants. The specific amino acid changes incorporated into each member of the H/M mutant series are indicated. The boxes above the alignment identify the three epitopes defined by the H/M mutants.




Figure 4: Immunoprecipitation of human CD11a variants incorporating selected sequences from murine CD11a. Human kidney adenocarcinoma cell line 293 was transfected with expression plasmids containing genes encoding full-length human CD11a or full-length human CD11a mutated to contain short stretches of murine CD11a sequence. Murine monoclonal antibodies to human CD11a 3D6, 32E6, CLB-LFA-1/2, and 50G1 (A) and 25.3, TS-1/22, and MHM24 (B) or rat monoclonal antibodies to murine CD11a M17 and I27/1 (B) were incubated with detergent lysates of metabolically labeled [S]methionine-transfected 293 cells and immunoprecipitated as described previously(43) . The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography as described previously(43) .



Localization of Epitopes Recognized by Rat mAbs to Murine CD11a

Two independently isolated rat mAbs to murine CD11a (M17 and I21/7) were tested for the ability to bind to the H/M series of replacement mutants. These studies were conducted to determine whether introducing the murine sequences into human CD11a might endow the human CD11a mutants with the capacity to bind the antibodies specific for murine CD11a. It was found that the rat mAbs were unable to bind to the H/M 48, H/M 49, H/M 50, H/M 51, H/M 52, and H/M 54 mutants (Fig. 4B). However, both M17 and I21/7 were able to bind to the H/M 53 mutant. These results demonstrated that the same mutations that destroyed the TS-1/22 and 25.3 mAb binding sites on human CD11a created binding sites recognized by the rat mAbs to murine CD11a. Thus, epitopes recognized by M17 and I21/7 were located in a region corresponding to IdeA and depended on Met and Lys of the H/M 53 chimera, whereas binding by mAbs 25.3 and TS-1/22 to H/M 53 depended on Ile and Asn at these positions.

I Domain Residues Essential for Binding mAbs to Human and Murine CD11a

A series of full-length human CD11a variants incorporating single amino acid substitutions were constructed in order to further localize the residues of human CD11a important for the binding of mAbs to LFA-1 (Fig. 5). The residues selected for mutagenesis were those in IdeA, IdeB, and IdeC that were identified using the H/M mutants (Fig. 3). The first region examined was IdeB in which the mutations in H/M 52 prevented the binding of mAbs 32E6, 50G1, and CLB LFA-1/2. It was found that replacement of Gln with Ala (Fig. 5, Q143A) diminished the binding of mAbs 32E6, 50G1, and CLB-LFA-1/2 but had no effect on the binding of 3D6 or MHM.24. Replacement of human CD11a residue Pro with Arg (as occurs in murine CD11a) completely blocked the binding of 32E6, 50G1, and CLB-LFA-1/2 without affecting the binding of the 3D6 mAb (Fig. 5, P144R). The importance of Pro is highlighted by the observation that replacement of the adjacent residues Asp with Lys (Fig. 5, D145K), Glu with Lys (Fig. 5, E146K), and Gln with Glu (Fig. 5, Q148E) all failed to have any impact on the binding of mAbs 32E6, 50G1, and CLB-LFA-1/2.


Figure 5: Localization of residues in I domain epitopes B and C using human CD11a variants incorporating single amino acid substitutions. Human kidney adenocarcinoma cell line 293 was transfected with expression plasmids containing genes encoding full-length human CD11a or full-length human CD11a with single amino acid substitutions as indicated. Murine monoclonal antibodies to human CD11a 3D6, 32E6, 50Gl, CLB-LFA-1/2, and MHM24 were incubated with detergent lysates of metabolically labeled [S]methionine-transfected 293 cells and immunoprecipitated as described previously(43) . The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography as described previously(43) .



We next explored the importance of residues 198-200 in IdeC, which were found to be important for the binding of mAbs 32E6, 50G1, CLB-LFA-1/2, and MHM.24 (Fig. 4). It was found that replacement of Lys with Asp completely inhibited the binding of all four mAbs but did not affect the binding of 3D6 (Fig. 5, K197D). Replacement of the adjacent His with Ala, had no effect on the binding of the four CD11a-blocking mAbs or 3D6 (Fig. 5, H198A). Replacement of the neighboring residue, Lys, with Asp (Fig. 5, K200D) diminished the binding of MHM.24 without affecting the binding of any of the other mAbs. Replacement of His with Ala (Fig. 5, H201A) completely inhibited the binding of 32E6, diminished the binding of MHM.24, and had little effect on the binding of the 50G1 and CLB-LFA-1/2 mAbs.

Finally, the mutations identified in IdeA that prevented the binding of 25.3 and TS1/22 and conferred binding of rat mAbs to murine CD11a (M17 and 121/7 mAbs) were investigated. Replacement of Ile with Met or Asp (Fig. 6, I126M and I126D) failed to prevent the binding of mAbs 25.3 and TS1/22. Similarly, replacement of Asn with Lys also failed to affect the binding of these antibodies. Thus, whereas the H/M 53 double mutant that incorporated both of these substitutions blocked the binding of both antibodies (Fig. 4B, H/M 53), replacement of either residue alone had no effect on antibody binding. To determine if other nearby residues might affect antibody binding, Lys was replaced with Ala (Fig. 6, K127A) and Gly was replaced with Ala (Fig. 6, G128A). It was found that mutagenesis of position 127 reduced the binding of both mAbs TS1/22 and 25.3, whereas mutagenesis at position 128 had no effect. When the rat mAbs to murine CD11a (M17 and I21/7) were tested against this series of variants, no binding could be detected (Fig. 6). Thus, like the mAbs to human CD11a, the mAbs to murine CD11a required the double mutation at residues 126 and 129 for binding (Fig. 4B, H/M 53).


Figure 6: Localization of residues in I domain epitope A using human CD11a variants incorporating single amino acid substitutions. Human kidney adenocarcinoma cell line 293 was transfected with expression plasmids containing genes encoding full-length human CD11a or full-length human CD11a with single amino acid substitutions as indicated. Murine monoclonal antibodies 3D6, 25.3, and TS 1/22 were incubated with detergent lysates of metabolically labeled [S]methionine-transfected 293 cells and immunoprecipitated as described previously. The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography as described previously.




DISCUSSION

The location and structure of ligand binding sites on integrin alpha and beta subunits have been the subject of intense speculation. The discovery that all three beta(2)-integrin alpha chains possess an I domain suggested that this family of integrins evolved a unique strategy for ligand binding. Data supporting the role of the I domain in ligand binding by the closely related integrins, Mac-1 and p150,95, were provided in experiments (20, 21) in which the binding properties of intermolecular chimeras between CD11b and CD11c were analyzed. Direct data demonstrating that the I domain of CD11a contains the ligand binding was provided in studies utilizing a bivalent chimeric protein consisting of the CD11a I domain fused to the Fc fragment of immunoglobulin G(52) . In these studies, binding of CD11a I domain/Fc chimeras to purified recombinant soluble ICAM-1 could be detected by enzyme-linked immunosorbent assay. Neither of these studies, however, provided any indication as to which part of the 200-amino acid I domain might actually be involved in ligand binding. Data suggesting that the ICAM-1 binding site of CD11a might involve residues outside the I domain was provided in other studies (53) in which fragments of CD11a lacking the I domain expressed in an in vitro translation system could be precipitated using a chimeric ICAM-1 variant that contained the first three domains of ICAM-1 fused to the Fc fragment of immunoglobulin G(1).

The present study provides evidence that the I domain of CD11a, like that of CD11b(20) , contains sequences that are important for ligand binding. Using reciprocal intermolecular chimeras of CD11a and CD11b, it was possible to localize the epitopes recognized by six receptor-blocking mAbs to human LFA-1 and two blocking mAbs to murine CD11a to sequences within the NH(2)-terminal region of the CD11a I domain. A series of novel CD11a mutants constructed by replacement of human CD11a sequences with sequences from murine CD11a further localized the epitopes recognized by all eight blocking mAbs to three distinct epitopes within the I domain. Proteins incorporating amino acid substitutions at residues 126-129 (H/M 53) completely inhibited the binding of the TS-1/22 and 25.3 mAbs without affecting the binding of the other CD11a mAbs. This epitope (IdeA) falls immediately NH(2)-terminal to the sequence that previously was considered the start of the I domain(30) . In other studies, we found that the same mutations that blocked the binding of the TS-1/22 and 25.3 mAbs to human CD11a allowed for the binding of the rat mAbs to murine CD11a (M17 and I21/7). The possibility that these antibodies actually bind to the ICAM-1 binding site on LFA-1 is supported by the experiments of Lollo et al.(54) , who have shown that M17 competes with soluble recombinant murine ICAM-1 for binding to LFA-1.

A second epitope (IdeB) important for the binding of CD11a-blocking mAbs was identified by mutations in the region of 143-149 (H/M 52). This epitope is located 12 amino acids away from the H/M 53 site and does not appear to contain residues important for the binding of TS-1/22 or 25.3. Interestingly, the sequence located between these two epitopes (i.e. residues 130-143) is highly conserved among the beta(2)-integrins and among the domains of the other proteins (i.e. cartilage matrix protein, von Willebrand factor, and factor B) that share homology with the beta(2)-integrin I domains (Fig. 7). In contrast, the sequences of the two mAb recognition sites themselves (126-129 and 143-149) differ between the three beta(2)-integrin alpha chains (CD11a, CD11b, and CD11c) and exhibit no homology with the domains of the other proteins (i.e. von Willebrand factor, cartilage matrix protein, and factor B) that share homology with the I domain. Thus, these results suggest that these two classes of antibodies may inhibit LFA-1 binding by disrupting the function of highly conserved structural elements important for ligand binding. The fact that the residues important for mAb binding are located in sequences that vary among the beta(2)-integrin alpha subunits accounts for their LFA-1 binding specificity.


Figure 7: Alignment of I domain sequence homologies. The amino acid sequences of the NH(2)-terminal portion of the I domains of human CD11a, mouse CD11a, human CD11b, and human CD11c aligned with each other and with three regions of human von Willebrand factor A domains (VWFA) and human cartilage matrix protein (CMP). Boxed regions represent regions of sequence homology. Shaded regions represent nonhomologous regions corresponding to the epitopes identified in this study. The locations of I domain epitopes IdeA, IdeB, and IdeC are indicated above the alignment.



The third epitope (IdeC) recognized by CD11a-blocking mAbs was localized to residues 197-203 (H/M 54). Of the four mAbs that bound to this site, two distinct specificities could be identified: mAbs that depended on epitopes at residues 143-149 and 197-203 (CLB-LFA-1/2, 32E6, and 50G1) and mAbs (e.g. MHM.24) whose binding depended only on residues 193-207. The sequence at 193-207, like the other two epitopes, is unique to CD11a but lies between two conserved sequences (PXXLL and TXTXXA(I/L)) that are found in all the beta(2)-integrin alpha chains and are homologous to sequences in von Willebrand factor A repeats, cartilage matrix protein, and factor B (Fig. 7). These results suggest that the mAb MHM.24 might inhibit LFA-1 function by interacting with a sequence that is distinct from the epitopes recognized by the other LFA-1-blocking mAbs.

Although all of the mAbs used in this study have been shown to inhibit functional responses in vitro (e.g. CTL killing and mixed lymphocyte reactions), the possibility exists that the mechanism of action may differ between antibodies to different epitopes. Several different mechanisms have been discussed in accounting for the activities of integrin-blocking mAbs. In principle, the simplest mechanism whereby antibodies can block adhesive interactions is by competitive binding to the ligand binding site. Studies by Lollo et al.(54) suggest that this is the case for the M17 mAb. Because mAbs 25.3 and TS-1/22 appear to bind to the same epitope as M17, they would also be expected to inhibit LFA-1 function competitively binding to the ligand (ICAM-1) binding site. Another mechanism by which antibodies are thought to modulate LFA-1 function is stabilization of ``activated'' or ``unactivated'' conformations. Dransfield et al.(32) have described an antibody that is thought to inhibit LFA-1 function by preventing ``de-adhesion,'' thus locking LFA-1 and Mac-1 into a permanently activated state. Finally, antibody binding could inhibit function by altering interactions with divalent cations mediating the transition between activated and unactivated conformations. Several studies have described cation-dependent epitopes recognized by antibodies that can activate or inhibit LFA-1 function(32, 33) . This type of mechanism might account for the activity of the CLB-LFA-1/2 mAb that binds to the IdeB site and is known to activate T cells in T cell receptor co-stimulation studies and to inhibit adhesion required for cytotoxic T cell-mediated lympholysis in studies with human T cell clones(37, 38) . Michishita et al.(55) have described a novel divalent cation binding site in the I domain of CD11b that is essential for ligand binding. Mutagenesis experiments identified two sites of CD11b (Asp/Ser and Asp) that were critical for cation binding and receptor function but did not affect the binding of several mAbs known to block CD11b function. In addition, a third residue, Pro, was identified that appeared to shift the optimal cation concentration required for ligand binding but did not inhibit the binding of CD11b mAbs known to inhibit receptor function. These residues are conserved among all three beta(2)-integrin alpha chains and correspond to residues Asp, Ser, Asp, and Pro of CD11a. The observations that Pro was located just 5 residues away from Lys (critical for the binding of mAb MHM.24) and that Asp and Ser were located in the conserved region between the epitopes IdeA and IdeB (critical for the binding of the 25.3, TS-1/22, M17, I21/7, 32E6, 50G1, and CLB-LFA-1/2 mAbs) support our hypothesis that the conserved regions between residues between IdeA and IdeB, as well as the regions flanking IdeC, are important for CD11a function. These data are consistent with the possibility that the CD11a-blocking mAbs that we have studied may inhibit LFA-1 by affecting the conformation or the activity of this novel cation binding site. This model is also consistent with a recent report (53) suggesting that ICAM-1 can bind to CD11a variants that lack the I domain.

Whether or not the I domain sequences that we have identified contain the LFA-1 ligand (ICAM-1) binding site remains to be established. Because murine LFA-1 does not bind human ICAM-1(56) , analysis of the H/M mutants in functional assays provides a means to test the role of these sequences in ICAM-1 binding. Studies utilizing the mutants described in this paper in cell-based adhesion assays are in progress to determine which of the mutations that inhibit antibody binding also inhibit ligand binding. In this regard it will be interesting to test the inhibitory activity of synthetic peptides corresponding to the I domain sequences identified by the epitope-mapping studies described herein. Studies of this type should help to determine whether the I domain participates in receptor function by providing contact residues that interact with ligand or by providing structural elements necessary to maintain the conformation of a ligand binding site in a remote part of the molecule. An interesting possibility is that the I domain represents an ion-sensitive regulatory domain that has been incorporated into the prototypic integrin structure to govern the transition between activated and unactivated conformations.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Immunology, Genentech, Inc., 460 Pt. San Bruno Blvd., South San Francisco, CA 94080. Tel.: 415-225-1570; Fax: 415-225-8221.

(^1)
The abbreviations used are: LFA-1, leukocyte function-associated antigen 1; I domain, inserted domain; ICAM, intercellular adhesion molecule; mAb, monoclonal antibody; CTL, cytotoxic T lymphocyte; PAGE, polyacrylamide gel electrophoresis; H/M mutant, human to mouse mutant; IdeA, I domain epitope A; IdeB, I domain epitope B; IdeC, I domain epitope C.

(^2)
P. Jardieu, manuscript in preparation.


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