Lymphocyte adhesion molecules play a critical role in the complex orchestration of immune system function. The ability to modulate interactions among these molecules holds considerable potential for regulation of immune system participation in allograft rejection and autoimmune diseases. The present work focuses on the interaction of lymphocyte function associated antigen-1 ([LFA-1*] reviewed in  and one of its ligands, intercellular adhesion molecule-1 ([ICAM-1] reviewed in ). The interaction of these molecules is important for several T cell functions, including surveillance and migration, T and B cell contact, and cytotoxic T cell function. Several in vivo studies suggest the therapeutic utility of blocking the LFA-1/ICAM-1 interaction. A combination of antibodies to the LFA-1 α-subunit and to ICAM-1 successfully blocked rejection of cardiac allografts in allogeneic mice (3). In addition, allograft acceptance occurred in mice after treatment with ICAM-1 antisense oligonucleotides (4). Effective treatments of renal (5), heart (6), and thyroid (7) allografts, bone marrow transplants (8), diabetes models (9), and rheumatoid arthritis (10, 11) have been accomplished using antibodies to either ICAM-1 or LFA-1 or both.
The integrin LFA-1 is a heterodimer composed of a unique α-subunit (CD11a) and a β-subunit (CD18) common to the β2 subfamily. LFA-1 mediates intercellular adhesion by interacting with ICAM-1, -2, or -3. ICAM-1 exists as a monomer or a noncovalent dimer and interacts with LFA-1, MAC-1 (CD11b/CD18), p150/95 (CD11c/CD18), and CD43 (sialophorin). Contact sites between LFA-1 and ICAM-1 have been described in the first immunoglobulin-like domain of ICAM-1 (12), as well as the I-domain and domains V and VI of the LFA-1 α-subunit (13-15). Amino acids D134 and S136 in the LFA-1 β-subunit also are critical for binding (16). In addition to antibodies, some peptides derived from LFA-1 (14) and from ICAM-1 have been shown to inhibit the LFA-1/ICAM-1 interaction (17-19). Similar peptide inhibition studies have been performed using other adhesion systems, including ICAM-2 (20, 21), gpIIbIIIa (22, 23), and CD28 (24). An important step in the development of therapeutic agents that target the LFA-1/ICAM-1 system is the development of small, nonimmunogenic peptides that can block adhesion. We have begun to investigate short-chain amino acid peptides derived from either LFA-1 or ICAM-1 for their ability to inhibit T cell adhesion and modulate immune function.
Perhaps most important, we have tested these peptides in the mixed lymphocyte reaction (MLR). The MLR is a reasonably valid model for bone marrow transplantation because, as with both graft versus host and host versus graft responses, an MLR is mediated by MHC-responsive T cell subsets that react with lymphocytes that they regard as foreign.
We have generated peptides derived from LFA-1 and ICAM-1 that modulate the interaction of these two molecules, and an evaluation of the activity of five of these peptides is presented. The peptides IB and IE, derived from ICAM-1, as well as the LFA-1-derived peptides LAB (α-subunit), LAB2 (α-subunit), and LBE (β-subunit), inhibited LFA-1- /ICAM-1-dependent homotypic adhesion, were specific for their target proteins, and did not function as agonists to stimulate T cells. In addition to inhibition of binding, we present the first evidence that peptides derived from LFA-1 or ICAM-1 inhibit biological function of allogeneic T cells in a one-way mixed lymphocyte reaction.
MATERIALS AND METHODS
Antibodies and reagents. The anti-CD18 (TS1/184.108.40.206; cat# HB-203) and anti-CD3 (OKT3; cat# CRL-8001) hybridomas were purchased from American Type Culture Collection (ATCC; Bethesda, MD). Antibodies were purified from cell culture supernates using protein G Sepharose (Pharmacia, Piscataway, NJ) and used at 5 μg/ml, unless otherwise indicated. ICAM-1 cDNA was generously provided by Dr. D. Simmons (ICRF, Nuffield, England). PDB (phorbol-12,13-dibutyrate) was obtained from Sigma (St. Louis, MO) and was used at a concentration of 10−8 M.
Cell purification and culture. Fresh human tonsil T cells were isolated, as previously described (25). Typically, >98% of the purified cells were positive for the T cell surface marker CD3, as assessed by flow cytometric method. Tonsil T cells and Molt-3, a leukemia-derived T cell line (ATCC; cat# CRL-1552), were cultured in RPMI 1640 (Mediatech, Herndon, VA) containing 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 50 U/ml each of penicillin and streptomycin (Life Technologies, Grand Island, NY), and 20 mM glutamine (Life Technologies). COS-7, an SV40-transformed African green monkey kidney cell line (ATCC; cat# CRL-1651), was cultured in Dulbecco's modified Eagle's medium (Life Technologies) containing 10% fetal bovine serum, penicillin, streptomycin, and glutamine.
Peptides. Peptides were either synthesized on site using standard solid phase protocols employing t-boc amino acid chemistry or purchased from La Jolla Cancer Research Foundation (La Jolla, CA). Peptides were purified and purity was verified by high-performance liquid chromatography using a C18 column. All peptides were analyzed by fast atom bombardment mass spectrometric method and were confirmed by nuclear magnetic resonance. Before use, peptides were resuspended in culture medium at a concentration of 5 mM.
Homotypic adhesion assay. Freshly purified, rested or PDB-stimulated tonsil T cells were incubated at 37° in a 96-well tissue culture plate (Falcon, Franklin Lakes, NJ) for 6 hr in the presence of peptides. For analysis, wells were photographed randomly at 40× magnification, and photos were assessed for index of clumping relative to PDB-treated positive control samples. In a scoring system adapted from other investigators (26, 27), PDB-stimulated samples were arbitrarily assigned a clumping index of 10, and nonstimulated samples an index of 1. Test samples were ranked from 1 to 10, on the basis of degree of clumping relative to PDB- and non-treated samples. Results were verified by blind, independent ratings performed by a second observer. Clumping index for each peptide was roughly quantified by determining the mean clumping index per experiment ± SE. All experiments were performed at least three times, with similar results.
Cell viability and cell number determination. Tonsil T cells (4×105 cells/well) were incubated alone or with 500 μM peptide in a 96-well plate for 72 hr. Samples were mixed, and an aliquot was removed from each well for counting. Aliquots were mixed 1:1 with a 0.4% trypan blue solution (Sigma), and samples were counted at 100× magnification. Total number of cells and number of trypan blue positive cells were recorded for each well, and values were calculated from mean of triplicate samples ± SE. The percentage of viability for each well was determined by total number of cells minus number of trypan blue-containing cells, divided by total number of cells.
COS-7 cell transfection and heterotypic adhesion assay. COS-7 cells were not transfected or were transfected with 10 μg of ICAM-1 cDNA using calcium phosphate (28), and were seeded in a 24-well plate at 2×104/well. After 48 hr, cells were assessed for ICAM-1 expression using flow cytometry (not shown). For the adhesion assay, cells were pretreated with 250 μM peptides for 30 min at 37°. Molt-3 cells were then resuspended at 4×106 cells/ml in RPMI, treated with PDB (10−8 M) for 30 min at 37°, then added to nontransfected or transfected COS-7 cells for 60 min at 37°. Unattached cells were removed with five PBS washes, and photographs were taken of each sample at 200× magnification.
Proliferation assay. Antibodies (0.2 μg/well) were attached to plastic by incubation in a 96-well tissue culture plate for 1 hr at 37°, and the wells were washed three times with PBS. Tonsil T cells (2×106 cells/ml) in the absence or presence of peptides were added to each well. After 72 hr, 3H-thymidine was added to each well (1 μCi/well, 67 Ci/mmol; Amersham, Arlington Heights, IL) for 6 hr, samples were harvested using a PHD Cell Harvester (Cambridge Technology, Inc., Watertown, MA), and incorporated 3H was assessed using a liquid scintillation counter (Packard Instrument Company, Inc., Downers Grove, IL).
One-way mixed lymphocyte reaction. One-way MLR was performed, as described by others (29). Responders were human tonsil T cells suspended at 4×106 cell/ml in culture medium. Stimulator cells were human mononuclear cells isolated from first Ficoll separation of peripheral blood (PBMC) suspended at 4×106 cells/ml in culture medium. Stimulator cells were inactivated before use by 5000 rad of ionizing irradiation from a γ-137Cs source. 100 μl of each cell suspension were combined in each well of a 96-well plate in the absence or presence of peptides. Proliferation was assessed after 72 hr using 3H-thymidine, as described above.
Homotypic adhesion assay. Homotypic adhesion between two T cells from the same donor is a reasonable model for adhesion events that take place during certain normal immune functions. Assessment of the ability of the peptides to inhibit homotypic adhesion was made using an LFA-1/ICAM-dependent homotypic adhesion system and fresh human tonsil T cells. Purified T cells were stimulated with PDB in the absence or presence of antibody, and photographed at 6 hr (Figure 1). Panel A shows nontreated tonsil T cells, which did not homotypically adhere. Tonsil T cells stimulated with PDB formed large clumps, as shown in Panel B. In Panel C, anti-CD18 inhibited homotypic adhesion of T cells. Results were quantified as described in Materials and Methods and presented in Panel D. Relative to the PDB-induced control, anti-CD18 inhibited adhesion by 80%. This indicated that the homotypic adhesion was guided, in the greatest part, by LFA-1 interactions. Control antibodies (anti-CD11b, anti-CD11c, IgG2a isotype control) had no effect (not shown).
LFA-1- and ICAM-1-derived peptides inhibited homotypic adhesion. Peptides derived from ICAM-1 or LFA-1 (see Table 1) were tested in the homotypic adhesion assay for the ability to inhibit LFA-1/ICAM-dependent adhesion. IB and IE are from adjacent segments of the first immunoglobulin-like domain of ICAM-1 (see also 17, 18). LAB is homologous to a region in the I-domain of the LFA-1 α subunit (CD11a). LAB2 is from the divalent cation binding region of the LFA-1 α subunit. LAB2-L is a fragment of LAB2, and BAL2-L is the reverse of this sequence. LBE is from the β subunit of LFA-1 (CD18), and EBL is the reverse of this sequence. EBL-L and EBL-R are fragments of EBL. Results from a representative experiment are shown in Figure 2. The control monoclonal antibody (mAb) against CD18 inhibited adhesion by 80%. The ICAM-1-derived peptide IB inhibited adhesion by 44%. IE, from an adjacent segment of ICAM-1, inhibited adhesion by 37%. The LFA-1-derived peptides LAB, LAB2, and LBE inhibited by 23%, 48%, and 27%, respectively. EBL was used as a control and did not inhibit adhesion (0%). Thus, in this experiment, the selected peptides inhibited homotypic adhesion one third to one half as well as did the antibody.
Inhibition of homotypic adhesion was concentration-dependent. Peptides were tested at varying concentrations ranging from 1 μM to 500 μM (Fig. 3). IB, LAB, and LBE all significantly inhibited adhesion (P<0.05 by student's t test) at concentrations as low as 10 μM, with maximal inhibition occurring at 250 μM for LAB and LBE, and by 100 μM for IB. IE significantly inhibited adhesion by 100 μM, with the maximum inhibition at 250 μM. LAB2 significantly inhibited adhesion by 250 μM, with the maximum decrease occurring at 500 μM. In addition, all five of the test peptides exhibited dose-dependent inhibition. Whereas these concentrations are relatively high, they fall within the range of concentrations used by others for peptides derived from LFA-1α (14) and ICAM-1 (18). Effective concentrations as low as 24 and 42 μM have been observed, in some cases, using peptides derived from ICAM-1 (17) and ICAM-2 (21), respectively. EBL did not inhibit homotypic adhesion at any dose.
Peptides did not alter viability or cell number. Tonsil T cells were incubated with 500 μM of each peptide for 3 days, then the number of viable cells and the number of total cells were counted using a hemacytometer (Fig. 4). None of the peptides significantly altered the percentage of viable cells or the total cell number. This suggests that the peptides are not generally toxic to the cells, and, therefore, inhibition of homotypic adhesion occurs by binding interference rather than alteration of membrane structure.
T cell adhesion to ICAM-1-transfected COS-7 cells was inhibited by LBE. We periodically verified our results using a more defined, heterotypic adhesion system that is dependent solely on the interaction of LFA-1 with ICAM-1. A representative example is shown in Figure 5. COS-7 cells transiently transfected with ICAM-1 cDNA were used as a target for adhesion by the Molt-3 T cell line. Routinely, between 20% and 30% of the transfected COS-7 cell population expressed ICAM-1 at 48 hr, as assessed by flow cytometric analysis (not shown). For Figure 5, nontransfected or ICAM-1-transfected COS-7 cells were incubated with PDB-stimulated Molt-3 T cells in the absence or presence of LBE or EBL, then rinsed to remove nonbound cells. Three random fields of each well were photographed at 200× magnification. T cells effectively bound in patches to the transfected COS-7 cells, but did not bind to the nontransfected cells. All patches of bound T cells were in a single layer, suggesting that homotypic adhesion of T cells did not artificially increase the number of T cells bound. LBE completely inhibited T cell binding to ICAM-1-transfected cells; that is, no bound cells were ever observed in the LBE-treated samples. In contrast, the reverse-synthesized EBL had no effect on T cell binding (similar patches of T cells were observed in the transfected and transfected plus EBL samples). LFA-1-derived peptides LAB and LAB2 had similar inhibitory effects to LBE in this assay (not shown). Thus, inhibition in a defined ICAM-1-specific adhesion system was also specific to the peptide sequence and supported results obtained from the homotypic adhesion system. In contrast, the ICAM-1-derived peptides IB and IE had little effect on the ICAM-1-specific adhesion event in the heterotypic adhesion assay (not shown). This may be a result of binding to other ICAM-1 ligands, such as CD43.
Peptides did not stimulate T cell proliferation. Using antibody to cross-link LFA-1 on the cell surface provides a second signal for T cell proliferation (30-35), and recent evidence from our lab and others has suggested that cross-linking of ICAM-1 may also induce internal signaling of cells (25, 36-40). In theory, to perform in vivo in a manner analogous to that of antibodies used by other investigators, candidate peptides not only should inhibit a second signal, but also should not provide a second signal themselves. Thus, if peptides described herein or their derivatives are to have utility as therapeutic agents, the potential ability of the peptides to serve as agonists must be determined. We tested the effects of our peptides on T cell proliferation stimulated by anti-CD3. Antibodies to CD3 (OKT3) were coated on a 96-well tissue culture plate. As a positive control, anti-CD3 was co-coated with anti-CD11a (mAb38). Tonsil T cells in the absence or presence of peptides (see Fig 4 legend) were added to each well and incubated for 72 hr. The degree of proliferation was determined using the standard 3H-thymidine incorporation assay, as described in Materials and Methods. Costimulation with anti-CD3 plus positive-control anti-CD11a induced proliferation (Fig. 6). In contrast, peptides used alone or peptides in combination with anti-CD3 did not stimulate T cell proliferation. In fact, cells treated with any of the peptides routinely showed decreases in proliferative response. In the example presented, LBE and LAB2 exhibited 50% and 41% decreases compared with anti-CD3 stimulation alone. Finally, the peptides had no effect on proliferation induced by anti-CD3 plus anti-CD11a (not shown).
Inhibition of one-way mixed lymphocyte reaction. Mixed lymphocyte reactions (MLR) provide an in vitro system that mimics the allogeneic response observed during allograft transplant rejection and, to some extent, mimics the graft versus host response evident in bone marrow transplantation. Thus, MLR may provide a more useful evaluation of peptide efficacy than models relying only on intercellular adhesion, because the MLR mimics adhesion-related biological events that are similar to those that occur in vivo. For MLR, human tonsil T cells (responders) were combined with irradiated PBMC (stimulators) from mismatched human donors. Peptides were evaluated at 250, 150, and 50 μM, and antibodies against LFA-1 or ICAM-1 were used as controls. Proliferation of responder cells was assessed after 72 hr by 3H-thymidine incorporation. Similar to the homotypic adhesion system, all of the test peptides inhibited the MLR (Fig. 7). With the exception of LBE and LAB, all peptide inhibitions were dose-dependent in this representative experiment. At concentrations of 250 μM, IB and IE inhibited proliferation by 34% and 24%, relative to the untreated mixed lymphocyte sample. LAB and LBE inhibited by 28% and 20%, whereas LAB2 inhibited in this MLR by 70%. With respect to the antibody controls, anti-ICAM-1 (CD54) inhibited the MLR by 59% and anti-LFA-1 (CD11a) inhibited by 19%. Thus, the level of inhibition observed using the peptides was comparable to that seen with anti-LFA-1 Ab, and roughly half as effective as anti-ICAM-1 used at 120 nM.
To control for nonspecific peptide activity in the MLR, we also tested reverse sequences of some of the peptides. Figure 8 is an example. We tested fragments of one of the parent peptides, LAB2, as well as a control reverse peptide of the same fragment. The 10 amino acid LAB2 NH-terminal fragment, LAB2-L, inhibited the MLR by 26%. BAL2-L, the reverse sequence of the same fragment, did not inhibit the MLR. Reverse sequences of EBL (EBL-L and EBL-R) also did not inhibit the MLR, further demonstrating that inhibition of the MLR by the peptides is sequence-specific.
Interaction of the adhesion molecules LFA-1 and ICAM-1 is important for many immune system functions, and modulation of LFA-1/ICAM-1 interactions is being examined for controlling immune responses in various clinical situations. Although some peptides have been used in vitro to inhibit LFA-1/ICAM-1 interactions, it was imperative to test efficacy in an assay that measures biological function. Thus, after establishing efficacy in a simple adhesion assay, we tested the ability of peptides to inhibit the allogeneic response characteristic of allograft rejection or graft versus host disease.
Here, short peptides derived from LFA-1 or ICAM-1 interfered with these two adhesion molecules and modulated the biological effects of their interaction in the MLR, a model for bone marrow transplantation. IB and IE, derived from the first domain of ICAM-1, span residues Q1-C21 and D26-K50 of the primary amino acid sequence and overlap with peptides studied by other groups (17, 18). Recent work has demonstrated that E34 of ICAM-1 interacts with the I-domain of LFA-1 (41). Our results with these peptides agree with that previous work and serve as a good control for the other peptide studies undertaken here. Previous reports using LFA-1- or ICAM-1-derived peptides have demonstrated physical inhibition of protein binding without demonstrating functionality in a biological system. We extend the previous observations concerning inhibition of protein binding and demonstrate lack of toxicity or stimulation of cells by the peptides. We also demonstrate inhibition of function in a more biological assay, the MLR.
LAB, from residues I237-I261 of the α-subunit of LFA-1, was derived from the 190-amino acid I-domain that is necessary for ICAM-1 binding. This region was selected for study because of its structural similarity to the functional A-domain of von Willebrand factor (42). Since we began this study, the functional importance of this region for ligand binding has been demonstrated by others, using binding assays with the purified I-domain and using mutational analysis (13, 15, 43, 44). More specifically, D137 and D239 of the I-domain were shown by mutation to be essential for binding to ICAM-1; M140, G146, T243, and S245 were also shown to be required. It is noteworthy that residues K127 and N129 of the I-domain have been identified as necessary for ICAM-3 binding (45), suggesting that the ICAM-1 and ICAM-3 contact domains in LFA-1 may differ. Because LAB overlaps with several of the key amino acids for ICAM-1 binding, it is perhaps not surprising that LAB inhibited LFA-1/ICAM-1-dependent adhesion and biological activity.
LAB2 (G441-G466) is a peptide from the divalent cation-binding region of the LFA-1 α-subunit, which encompasses residues 422-616 (1). This domain is essential for stabilization of the α/β heterodimer. Thus, inhibition by LAB2 may involve interference with cation binding or direct disruption of dimerization, or this region may represent another contact domain for ICAM-1. Other work has shown that domains V and VI, in which LAB2 is found, contain an ICAM-1 binding site, and peptides derived from this region are capable of inhibiting LFA-1/ICAM-1 adhesion (14).
Until recently, the role of the β-subunit of LFA-1 in ligand binding has not been known. The area surrounding LBE was chosen because of its similarity to the βIIIa domain of the platelet integrin gpIIbIIIa, which binds to the RGD peptide (46, 47). While this work was in progress, site-directed mutagenesis studies using recombinant αLβ2 (CD11a/CD18) or αMβ2 (CD11b/CD18) demonstrated that D134 and S136 are important for binding to purified ICAM-1 or iC3b, respectively (16). LBE, derived from β-subunit residues D134-Q159, encompasses both of these amino acids, and inhibited homotypic adhesion and MLR in our system.
As reflected in the in vitro experiments, the single peptides do not inhibit as well as the antibodies, because they have a smaller molecular weight (less steric hindrance). In addition, the peptides likely have a lower affinity for the target protein than the monoclonal antibodies. In future experiments, we expect that combinations of stabilized peptides will be able to immunosuppress as well as the antibodies.
Both LFA-1 and ICAM-1 induce intracellular signaling in T cells in response to binding events. Cross-linking of LFA-1 α-subunit initiates inositol phosphate hydrolysis and increased free intracellular Ca2+ concentrations (34), as well as phosphorylation on tyrosine of a number of other proteins, including phospholipase Cγ and p130cas(30-35). ICAM-1 stimulation alters gene expression (38) and Ca2+ mobilization (40) in B cells, and initiates tyrosine phosphorylation in brain endothelial cells (37) and oxidative burst (39) in neutrophils, respectively. Our group previously reported a transient tyrosine phosphorylation and inactivation of cdc2 kinase in response to cross-linking of ICAM-1 in T cells (25). For these reasons, we investigated the possibility that the peptides could provide a means of T cell co-stimulation. A mAb to the LFA-1 α-subunit induced proliferation of fresh human tonsil T cells in combination with anti-CD3, but did not induce proliferation when used alone. None of the peptides used alone, or in combination with CD3 stimulation, induced an enhanced T cell proliferation. It was noteworthy that LAB2 and LBE slightly inhibited anti-CD3-induced proliferation.
Thus, we have examined five different peptides: two from ICAM-1, and three from LFA-1. These peptides were capable of inhibiting LFA-1/ICAM-1-dependent adhesion, supporting the supposition that these regions are involved in contact between the two cell surface proteins, or, in the case of LAB2, dimerization of the LFA-1 heteroduplex. Our observations that these peptides inhibit MLR without inducing co-activation of T cells suggest that they or their derivatives have potential for therapeutic intervention of LFA-1/ICAM-1 adhesion events in vivo. The work presented here has enabled us to identify peptides that may form parent compounds for useful therapeutic agents. We are currently developing protease-resistant synthetic derivatives of these peptides that may be more potent in vivo. Preliminary testing of derivatives of the peptides used in the present work indicates that such peptides may be active in vivo.
Acknowledgments. We are grateful to Dr. D. Simmons for the generous gift of ICAM-1 cDNA, Michael Lemon and Judith de Champlain for assistance with cesium source, and Drs. S. Segebrecht, R. Dinsdale, and L. Reusner of Lawrence Otolaryngology Associates and Drs. L. Price and S. Mersmann of Lawrence Memorial Hospital for assistance with tonsil acquisition.
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* Abbreviations used: LFA, leukocyte function-associated antigen; ICAM, intercellular adhesion molecule; MLR, mixed-lymphocyte reaction; PBMC, peripheral blood mononuclear cells; PDB, phorbol-12,13-dibutyrate.