Identification, Characterization, and Epitope Mapping of Human Monoclonal Antibody J19 That Specifically Recognizes Activated Integrin α₄β₇

Background: The activation-specific antibodies against integrins are powerful tools in integrin studies.

Results: An activation-specific antibody J19 against α₄β₇ was identified and characterized.

Conclusion: J19 specifically binds to the activated α₄β₇ by recognizing the epitope only exposed in extended conformation.

Significance: J19 is a potentially powerful tool for studying α₄β₇ function and treatment of α₄β₇-related inflammatory diseases.

Abstract

Integrin α₄β₇ is a lymphocyte homing receptor that mediates both rolling and firm adhesion of lymphocytes on vascular endothelium, two of the critical steps in lymphocyte migration and tissue-specific homing. The rolling and firm adhesions of lymphocytes rely on the dynamic shift between the inactive and active states of integrin α₄β₇, which is associated with the conformational rearrangement of integrin molecules. Activation-specific antibodies, which specifically recognize the activated integrins, have been used as powerful tools in integrin studies, whereas there is no well characterized activation-specific antibody to integrin α₄β₇. Here, we report the identification, characterization, and epitope mapping of an activation-specific human mAb J19 against integrin α₄β₇. J19 was discovered by screening a human single-chain variable fragment phage library using an activated α₄β₇ mutant as target. J19 IgG specifically bound to the high affinity α₄β₇ induced by Mn²⁺, DTT, ADP, or CXCL12, but not to the low affinity integrin. Moreover, J19 IgG did not interfere with α₄β₇-MAdCAM-1 interaction. The epitope of J19 IgG was mapped to Ser-331, Ala-332, and Ala-333 of β₇ I domain and a seven-residue segment from 184 to 190 of α₄ β-propeller domain, which are buried in low affinity integrin with bent conformation and only exposed in the high affinity extended conformation. Taken together, J19 is a potentially powerful tool for both studies on α₄β₇ activation mechanism and development of novel therapeutics targeting the activated lymphocyte expressing high affinity α₄β₇.

Introduction

Integrins are a family of α/β heterodimeric adhesion receptors that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions and transmit signals bidirectionally across plasma membrane (1). Because of their unique function of integrating extracellular environment with cytoskeleton, integrins play important roles in adhesion-dependent cellular processes including cell migration, proliferation, survival, and differentiation (1–4). Integrin α₄β₇ is a lymphocyte homing receptor, which can mediate both rolling and firm adhesion of lymphocytes, two of the critical steps in lymphocytes homing to the intestine and gut-associated lymphoid tissues (5, 6). Its ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1),³ is preferentially expressed on high endothelial venules of gut-associated lymphoid organs and on lamina propria venules, helping lymphocyte traffic to mucosal organs (7). The activation of integrin α₄β₇ is a critical step in the progression of inflammatory bowel disease (8, 9). Thus, α₄β₇ is a promising therapeutic target for the treatment of inflammatory bowel disease.

Different from most integrins supporting only firm adhesion of cells upon activation, integrin α₄β₇ can mediate both rolling and firm adhesion of lymphocytes (10, 11). The resting integrin α₄β₇ supports rolling adhesion of lymphocytes via its low affinity interaction with MAdCAM-1. Upon activation, α₄β₇ binds to MAdCAM-1 in high affinity, which results in firm cell adhesion. The transition from rolling adhesion to firm adhesion is regulated by the shift of integrin from low affinity to high affinity state (11, 12). Affinity regulation is associated with the conformational rearrangement of the integrin molecule (13, 14). Previous studies have shown that integrin extracellular domains exist in at least three distinct global conformational states that differ in affinity for ligand: low affinity bent conformation with a closed headpiece, intermediate affinity extended conformation with a closed headpiece, and high affinity extended conformation with an open headpiece (15–19). The equilibrium among these different states is regulated by integrin inside-out signaling and extracellular stimuli, such as divalent cations (20, 21). Compared with the low affinity state in Ca²⁺ + Mg²⁺, removal of Ca²⁺ or addition of Mn²⁺ strikingly increases ligand binding affinity of almost all integrins (11). Electron micrographic studies of integrins α_Vβ₃ and α₅β₁ demonstrate that integrin activation is coupled with the switchblade-like extension of the extracellular domain and a change in angle between the βI and hybrid domains (18, 19). Crystal structures of integrin α_IIbβ₃ headpiece in the high affinity conformation demonstrate that the C-terminal α7-helix of the βI domain moves axially toward the hybrid domain, causing the β hybrid domain to swing outward by 60°, away from the α subunit (15, 22). The conformational rearrangement in the integrin headpiece destabilizes the bent conformation and induces integrin extension in which the headpiece extends and breaks free from an interface with the leg domains that connect it to the plasma membrane. This conversion from the low affinity to the high affinity conformation of integrin can be mimicked by the introduction of glycan wedges into the interface between the hybrid and the I domains of β₇, β₃, and β₁ integrins, which activates integrins by stabilizing the outward swing of the hybrid domain and the high affinity headpiece conformation (12, 23). This wedge mutant integrin therefore can be used as a target for screening activation-specific antibodies that exclusively recognize the activated integrin. Up to date, there is no well characterized activation-specific antibody against integrin α₄β₇. Thus, a well characterized activation-specific antibody to α₄β₇ will be extremely useful for both studies on α₄β₇ activation mechanism and development of drug delivery system targeting the activated lymphocyte for the treatment of inflammatory bowel disease.

In this study, we discovered the activation-specific human mAb J19 against integrin α₄β₇ by screening from the human scFv phage library using the activated wedge mutant α₄β₇ as a target. J19 IgG specifically bound to the α₄β₇ activated by Mn²⁺, DTT, ADP, or CXCL12 but not to the low affinity integrin. Moreover, J19 IgG did not interfere with α₄β₇-MAdCAM-1 interaction, suggesting it was a mAb distinct from “ligand mimetic” group, and we demonstrate that J19 IgG recognizes an activation-dependent epitope on α₄β₇ consisting of residues from both α₄ and β₇ subunits. This epitope is buried in the low affinity integrin with bent conformation and only exposed in the extended conformation induced by integrin activation. These data also provide strong supporting evidence for the conformational rearrangement during integrin α₄β₇ activation.

EXPERIMENTAL PROCEDURES

Cell Lines and mAbs

K562 cells stably expressing human α₄β₇ integrin was cultured in RPMI 1640 medium (Invitrogen) and 10% FBS (Biocherom AG) supplemented with 0.2 mg/ml hygromycin and 0.5 mg/ml G418 (all from Amresco). 293T stable cell lines expressing human integrin α₄β₇ WT or wedge mutant were cultured in DMEM (Invitrogen) with 10% FBS supplemented with hygromycin (0.1 mg/ml).

The following integrin antibodies were used in this study: mAb TS2/16 (Santa Cruz) to human β₁, murine mAb Ber-ACT8 (Santa Cruz) to human α_E, murine mAb HP2/1 (Abcam) to human and rat α₄, and rat mAb FIB27 (BD biosciences) to human and mouse β₇. The murine mAb 9F10 against human α₄ was prepared from hybridoma (Developmental Studies Hybridoma Bank, University of Iowa). The murine mAb Act-1 specific for human α₄β₇ was previously described (24, 25). Human IgG1 control was from Pierce.

Plasmid Construction and Transient Transfection into 293T Cells

J19 IgG expression constructs were built on the backbone of pIRES2-EGFP (Invitrogen). cDNAs encoding human IgG1 light chain and heavy chain constant regions were amplified by RT-PCR from human endothelial cell total mRNA.

cDNAs of human α₄, α_E, β₇, and β₁ subunits were inserted into vector pcDNA3.1/Hygro (−) (Invitrogen), respectively. Chimeric β₁/β₇ subunits were generated by overlap extension PCR (26, 27). Chimeric α₄/α_E subunits were generated by an improved PCR mutagenesis strategy for sequence swapping (28). Chimeras were named according to the origin of their segments. For example, β₇542β₁ indicates that residues 1–542 are from β₇ subunit and residues 543 to the C terminus are from the β₁ subunit. Amino acid sequence numbering was according to the mature α₄ or β₇ sequence. The β₇ site-directed mutations were generated by using QuikChange (Stratagene). All of the constructs were confirmed by DNA sequencing. Transient transfection of 293T cells was performed as described (11).

Protein Purification and Analytical Gel Filtration

The J19 IgG was purified by protein A (Pierce) affinity chromatography. 293T cells were transiently transfected with J19 IgG expressing construct and cultured in DMEM supplemented with 10% “ultralow IgG” fetal bovine serum (Invitrogen).

The soluble integrin α₄β₇ with all ectodomains was purified as previously described (19, 22). Briefly, soluble integrin was purified from culture supernatant of 293T cells stably expressing soluble integrin α₄β₇ ectodomains with C-terminally fused His tag and Strep-tag II using nickel-nitrilotriacetic acid-agarose (Qiagen) followed by Strep-Tactin (IBA) affinity chromatography and gel filtration (Superdex 200; GE Healthcare).

Analytical gel filtrations were performed using precalibrated Superdex 200 on an ÄKTA purifier system running Unicorn 5.11 software at a flow rate of 0.5 ml/min at room temperature (29, 30). The elution profiles were monitored in-line by UV adsorption at 280 nm. Hepes-buffered saline (HBS) buffer (150 mm NaCl, 20 mm Hepes, pH 7.4) containing 1 mm Ca²⁺ and 1 mm Mg²⁺ or 0.5 mm Mn²⁺ was used throughout. 5.2 μg of purified WT or wedge mutant integrin α₄β₇ was loaded.

Selection of Integrin-binding Phages

Human scFv phage library was purchased from Geneservice. Antibody screening was done according to the protocol provided by Geneservice with minor modifications. Soluble integrin α₄β₇ was biotinylated and immobilized to streptavidin-coated Dynabeads (Invitrogen). Phage displaying scFv binding to α₄β₇ was captured by integrin-labeled beads (so called “panning”). In each round of panning, the binders to WT α₄β₇ were depleted first followed by screening the specific scFv fragments against wedge mutant. After three rounds of panning, specific binders were identified by monoclonal phage ELISA.

Flow Cytometry

Immunofluorescence flow cytometry was done as described (31). Before staining with antibody, 2.5 × 10⁵ cells were washed with HBS containing 5 mm EDTA and then resuspended in either HBS containing 1 mm Ca²⁺ + 1 mm Mg²⁺ or activating HBS containing 2 mm Mn²⁺. For activation of α₄β₇ by other stimuli than divalent cations, 2.5 × 10⁵ cells were resuspended and incubated at 37 °C for 15 min in HBS (1 mm Ca²⁺ + 1 mm Mg²⁺) with ADP and DTT at a final concentration of 10 and 500 μm, respectively. Stained cells were then measured using FACSCalibur (BD Biosciences) and analyzed using WinMDI 2.9 software.

Primary splenic lymphocytes (SPLs) were isolated as previously described (32) and suspended in HBS and stimulated by 0.2 μg/ml CXCL12 (R & D Systems) for 5 min at 37 °C. The cells were fixed with an equal volume of 2× formaldehyde (7.4%) before staining with 5 μg/ml J19 IgG.

Fluorescence Microscopy

Immunofluorescence staining assay was performed as reported (33). Cover glasses were coated with 10 μg/ml human MAdCAM-1 fused to the Fc1 and Fc2 regions of human IgG1 (huMAdCAM-1/Fc) in the presence or absence of 2 μg/ml CXCL12. The cells were incubated on the cover glass for 20 min at 37 °C, fixed with 3.7% polyformaldehyde, and blocked by 10% FBS. The cells were then stained with 5 μg/ml J19 IgG or FIB27, followed by Alexa Fluor® 488 goat anti-human IgG (H + L) and Cy3-conjugated goat anti-rat IgG (Invitrogen), respectively.

Flow Chamber Assay

The flow chamber assay was performed as described (11, 12). A polystyrene Petri dish to be used as the lower wall of the chamber was coated with a 5-mm-diameter, 20-μl spot of 10 μg/ml purified huMAdCAM-1/Fc in coating buffer (phosphate-buffered saline, 10 mm NaHCO₃, pH 9.0) for 1 h at 37 °C, followed by 2% BSA in coating buffer for 1 h at 37 °C to block nonspecific binding sites. The cells were washed twice with HBS containing 5 mm EDTA and 0.5% BSA, resuspended at 1 × 10⁷/ml in buffer A (HBS, 0.5% BSA), and kept at room temperature. The cells were diluted to 1 × 10⁶/ml in buffer A containing different divalent cations immediately before infusion in the flow chamber using a Harvard apparatus programmable syringe pump. Then shear stress was increased from 0.3 dyn/cm² up to 16 dyn/cm². The number of cells remaining bound at the end of 1 dyn/cm² was determined.

FRET

For detecting the orientation of integrin ectodomain relative to cell membrane, FRET was measured as described (30, 35). Briefly, 293T cells stably expressing integrin α₄β₇ treated with indicated cations were fixed by 3.7% paraformaldehyde and then stained with 20 μg/ml Alexa Fluor 488-Act-1 Fab, followed by staining with 10 μm FM4–64 FX (Invitrogen).

RESULTS

Generation of High Affinity Integrin α₄β₇ with Glycan Wedge Mutation

Previous studies have shown that the introduction of N-glycan at the integrin βI/hybrid domain interface will activate integrins and stabilize the high affinity conformation (12, 23). To obtain the high affinity human integrin α₄β₇, we introduced an N-glycosylation site at Asn-322 in the α4-β5 loop of β₇ I domain by mutating Gln-324 to Thr as previously described (12). The WT and wedge mutant (Q324T) human α₄β₇ were transiently expressed in 293T cells, and the adhesive behavior in shear flow of those transfectants was characterized by allowing them to adhere to MAdCAM-1 in a parallel wall flow chamber. WT α₄β₇ 293T transfectants behaved as previously described for lymphoid cells expressing α₄β₇ (36). In 1 mm Ca²⁺ + 1 mm Mg²⁺, more than 75% of adherent cells expressing WT α₄β₇ rolled on MAdCAM-1 substrates at the wall shear stress of 1 dyn/cm² (Fig. 1A). In contrast, the cells were firmly adherent in 0.5 mm Mn²⁺ (Fig. 1A). Rolling and firm adhesions represent the low and high affinity interactions of integrin α₄β₇ with MAdCAM-1, respectively. By contrast with the rolling adhesion of WT α₄β₇ 293T transfectants, 293T transfectants expressing α₄β₇ wedge mutant showed significantly increased firmly adherent cells in 1 mm Ca²⁺ + 1 mm Mg²⁺, which is similar to the adhesion behavior of WT α₄β₇ activated by 0.5 mm Mn²⁺ (Fig. 1A). α₄β₇ transfectants treated with the α₄β₇ blocking antibody Act-1 did not accumulate on MAdCAM-1 substrates. Thus, integrin α₄β₇ is constitutively activated by the glycan wedge introduced into the I/hybrid domain interface of β₇ subunit.

FIGURE 1.

The activation and conformational change of α₄β₇ induced by wedge mutation. A, rolling and firm adhesions on MAdCAM-1 substrates of 293T transfectants. The number of rolling and firmly adherent WT and wedge mutant α₄β₇ transfectants was measured in the indicated divalent cations at a wall shear stress of 1 dyn/cm². The error bars are ± S.D. (n = 3). B, purified WT α₄β₇ was analyzed on a Superdex 200 column in HBS, containing 1 mm Ca²⁺ + 1 mm Mg²⁺ (dashed line) or 0.5 mm Mn²⁺ (dotted line) and purified wedge mutant in HBS, containing 1 mm Ca²⁺ + 1 mm Mg²⁺ (solid line). The values next to the peaks indicate elution volumes (in milliliters). mAU, milliabsorbance unit.

Next, we introduced Q324T mutation into the human integrin α₄β₇ soluble construct with all ectodomains. To eliminate the heterogeneity resulting from partial cleavage of α₄ subunit, a previously described Arg-558 to Ala mutation was introduced into α₄ subunit to remove the protease cleavage site (37). Both WT and wedge mutant α₄β₇-soluble proteins were expressed in 293T cells and purified by nickel-nitrilotriacetic acid, Strep-Tactin affinity chromatography, and gel filtration. To study the conformational change of integrin in solution, the isocratic elution profiles of purified WT and wedge mutant α₄β₇ were compared using analytical gel filtration. Previous studies have shown that the shape change of integrin from the low affinity bent conformation to high affinity extended conformation could lead to the increase in hydrodynamic radius of integrin and the decrease of retention volume in gel filtration (19, 30). As expected, the low affinity WT α₄β₇ was eluted at 10.83 ml in 1 mm Ca²⁺ + 1 mm Mg²⁺, whereas the elution volume of high affinity WT α₄β₇ in 0.5 mm Mn²⁺ decreased to 10.67 ml, suggesting the more extended high affinity conformation of α₄β₇ activated by Mn²⁺ (Fig. 1B). Similar to WT α₄β₇ in Mn²⁺, the wedge mutant α₄β₇ showed decreased retention volume (10.72 ml) in Ca²⁺ + Mg²⁺ compared with WT α₄β₇, suggesting the extended conformation of wedge mutant α₄β₇. Thus, the wedge mutant can mimic the high affinity α₄β₇ by stabilizing the extended conformation of integrin.

Identification of Human Antibody to Activated Integrin α₄β₇

To identify an activation-specific antibody against α₄β₇, we performed human single fold scFv phage display library (Tomlinson I + J) selection using the high affinity wedge mutant α₄β₇ as target. The Tomlinson I + J libraries purchased from Geneservice contain over 100 million different human scFv fragments. The library was first depleted by the low affinity WT α₄β₇ soluble protein and then selected against the high affinity wedge mutant α₄β₇. In this way, binders specific for high affinity α₄β₇ were enriched after each round of selection. After three rounds of selections, the remaining binders were validated by monoclonal phage ELISA using purified WT and wedge mutant protein as target, respectively. Among a number of isolates that showed higher binding signals to wedge mutant than to WT α₄β₇ (data not shown), phage clone J19 bound specifically to wedge mutant and high affinity WT α₄β₇ activated by 2 mm Mn²⁺, but not to the low affinity WT α₄β₇ in 1 mm Ca²⁺ + 1 mm Mg²⁺ (Fig. 2A).

FIGURE 2.

The binding specificities of J19 phage and J19 scFv to integrin α₄β₇. A, monoclonal phage ELISAs were carried out with coated soluble WT α₄β₇, wedge mutant α₄β₇, and BSA control in the presence of indicated divalent cations. ELISA signals of J19 phage are shown. The error bars are ± S.D. (n = 3). B, 5 μg/ml J19 scFv or Act-1 binding to inactive (1 mm Ca²⁺ + 1 mm Mg²⁺) or active (2 mm Mn²⁺) α₄β₇ stably expressed on K562 cells were analyzed by flow cytometry. A representative experiment (of three) is shown as a histogram. The numbers within the panels show the specific mean fluorescence intensity of Act-1 and J19 scFv. The results are the means ± S.D. of three independent experiments. Open histogram, control mouse IgG or control scFv; filled histogram, Act-1 or J19 scFv.

To further characterize the J19 scFv phage isolate, the J19 scFv was expressed and purified. The binding of purified J19 scFv to integrin α₄β₇ was analyzed using flow cytometry with K562 cells stably expressing human α₄β₇ (Fig. 2B). Different from the specificity of J19 phage for high affinity α₄β₇, J19 scFv showed similar binding to the low affinity α₄β₇ in 1 mm Ca²⁺ + 1 mm Mg²⁺ and the high affinity α₄β₇ in 2 mm Mn²⁺. The binding of J19 scFv to α₄β₇ was comparable with that of α₄β₇ mAb Act-1 (Fig. 2B). Considering that scFv is of a smaller size than the phage particle, it is tempting to speculate that the binding sites of J19 in low affinity α₄β₇ can be accessed by smaller scFv but not the larger phage particle expressing the same scFv.

J19 IgG Specifically Binds to Activated Integrin α₄β₇

To further investigate the function of J19, we reformatted the J19 scFv to full-length human IgG1. The binding of J19 IgG to integrin α₄β₇ was determined using flow cytometric analysis with 293T cells stably expressing WT or wedge mutant α₄β₇ (Fig. 3A). Comparable with J19 phage, J19 IgG specifically bound to 293T cells expressing wedge mutant α₄β₇ in 1 mm Ca²⁺ + 1 mm Mg²⁺ and WT α₄β₇ 293T transfectants in 2 mm Mn²⁺, but not WT α₄β₇ transfectants in 1 mm Ca²⁺ + 1 mm Mg²⁺, suggesting its specificity for the activated α₄β₇. As control, the binding activity of α₄β₇ mAb Act-1 was shown in parallel. In contrast to J19 IgG, Act-1 showed nonselective binding to both low and high affinity α₄β₇ (Fig. 3A). Both J19 IgG and Act-1 did not bind to mock 293T cells transfected with pcDNA3.1 vector alone (Fig. 3A). Similar results were obtained using K562 cell stably expressing integrin α₄β₇, suggesting that the specificity of J19 IgG for activated integrin α₄β₇ is not cell type-dependent (Fig. 3B). Moreover, J19 IgG bound to Mn²⁺-activated α₄β₇ in a dose-dependent manner (Fig. 3C). From the best fit curve generated with GraphPad Prism 5.01 software, the EC₅₀ of J19 IgG for binding to activated α₄β₇ is 29.96 nm.

FIGURE 3.

J19 IgG specially recognizes the activated integrin α₄β₇. A, 293T cells stably expressing WT or wedge mutant α₄β₇ were stained with human IgG1 control, Act-1, or J19 IgG at 5 μg/ml in the presence of 1 mm Ca²⁺ + 1 mm Mg²⁺ or 2 mm Mn²⁺. Mock represents pcDNA3.1 vector 293T transfectants. B, K562 cells stably expressing α₄β₇ were stained with 5 μg/ml indicated antibodies in the presence of 1 mm Ca²⁺ + 1 mm Mg²⁺ or 2 mm Mn²⁺ and analyzed by flow cytometry. Mock represents K562 cells transfected with pcDNA3.1 vector. C, concentration-dependent binding of J19 IgG and human IgG1 control to K562 transfectants stably expressing WT α₄β₇ in the presence of indicated divalent cations. The cells were incubated with serially diluted J19 IgG or human IgG1 control. The error bars are ± S.D. (n = 3). D, binding of J19 IgG to K562 cells stably expressing α₄β₇ in the presence of DTT (500 μm) or ADP (10 μm) were determined by flow cytometric analysis as described under “Experimental Procedures.” A representative experiment (of three) is shown as a histogram. The numbers within the panels show the specific mean fluorescence intensity of human IgG1, Act-1, or J19 IgG. The results are the means ± S.D. of three independent experiments.

In addition to the strong activation by Mn²⁺, integrin can also be activated by other stimuli like DTT and ADP (38–40). DTT has been shown to activate integrin in a number of systems (38, 39). ADP was reported to induce integrin activation through inside-out signaling by activating PI3K pathway (41). To further study the binding specificity of J19 IgG to α₄β₇ activated by different stimuli, K562 cells stably expressing α₄β₇ were treated with Mn²⁺, DTT, or ADP and then followed by staining with 5 μg/ml J19 IgG. As shown in Fig. 3D, J19 IgG specifically bound to K562 α₄β₇ cells treated with these stimuli. By contrast, J19 IgG did not bind to the same cells without any stimulation (Fig. 3D). These results suggested that epitope recognized by J19 IgG in integrin α₄β₇ was only expressed after integrin activation. Moreover, the binding of J19 IgG to K562 α₄β₇ cells treated with Mn²⁺ is higher in comparison with those stimulated by DTT or ADP. The different expression level of J19 epitope induced by the above stimuli could be due to the different activation states and conformations of integrin α₄β₇.

J19 IgG Recognizes Integrin α₄β₇ Heterodimer from Human, Mouse, and Rat

Because integrin α₄β₇ shares α₄ subunit with integrin α₄β₁ and β₇ subunit with integrin α_Eβ₇, J19 IgG may also bind to α₄β₁ or α_Eβ₇ if it recognizes either subunit of α₄β₇ heterodimer. To evaluate the potential cross-reactivity of J19, its binding to human α₄β₁ and α_Eβ₇ was determined using 293T cells transiently expressing α₄β₁ or α_Eβ₇ (Fig. 4A). The expressions of α₄β₁ and α_Eβ₇ were confirmed by mAbs HP2/1 (α₄ mAb), TS2/16 (β₁ mAb), Ber-ACT8 (α_E mAb), and FIB27 (β₇ mAb). The flow cytometric results showed no J19 IgG binding to α₄β₁ or α_Eβ₇ expressing 293T cells in both Ca²⁺ + Mg²⁺ and Mn²⁺, indicating that J19 IgG does not recognize α₄β₁ or α_Eβ₇ both pre- and postactivation (Fig. 4A). These results demonstrate that neither α₄ nor β₇ subunit alone is sufficient to support J19 binding and that this antibody recognizes α₄β₇ heterodimeric complex.

FIGURE 4.

J19 IgG recognizes α₄β₇ heterodimer and cross-reacted with mouse and rat α₄β₇. A, 293T cells were transfected with human α₄β₁ or α_Eβ₇, and the integrin expression level was determined by indicated antibodies, respectively. The binding of J19 IgG to α₄β₁ or α_Eβ₇ was analyzed by flow cytometry in the presence of 1 mm Ca²⁺ + 1 mm Mg²⁺ or 2 mm Mn²⁺. B, human, mouse, or rat α₄β₇ was transiently expressed in 293T cells, and the integrin expression level was determined by 5 μg/ml indicated antibodies, respectively. Reactivity of J19 IgG with α₄β₇ 293T transfectants was determined by flow cytometry. A representative experiment (of three) is shown as a histogram. The numbers within the panels show the specific mean fluorescence intensity of indicated mAbs. The results are the means ± S.D. of three independent experiments.

We next test the cross-reactivity of J19 IgG with α₄β₇ from other species. The mouse and rat α₄β₇ were transiently expressed in 293T cells, respectively. The expression level of α₄β₇ was determined using mAb FIB27 against human and mouse β₇ and mAb HP2/1 against rat α₄. J19 IgG showed comparable binding to the activated human, mouse, and rat α₄β₇ but not to inactive ones (Fig. 4B).

J19 IgG Specifically Binds to Chemokine-activated Mouse SPLs

Having shown that J19 IgG was specific for the activated α₄β₇ expressed on K562 and 293T cell lines, we next tested whether J19 IgG also bound to the high affinity form of α₄β₇ expressed on primary lymphocytes. The mouse SPLs that highly express α₄β₇ were isolated, and the activation of integrin by CXCL12 stimulation was done as previously described (33, 42). In flow cytometric assay, binding of J19 IgG to SPLs was undetectable before CXCL12 treatment, whereas it significantly increased 5 min after adding 0.2 μg/ml CXCL12 (Fig. 5A). Similar results were obtained by immunostaining with J19 IgG and β₇ mAb FIB27 (Fig. 5B). J19 IgG signal was only observed at the surface of SPLs after α₄β₇ was activated by CXCL12 or Mn²⁺, whereas FIB27 bound to α₄β₇ in an activation-independent manner. Thus, J19 IgG specifically recognizes the activated α₄β₇ of primary lymphocytes.

FIGURE 5.

J19 IgG binds to activated α₄β₇ on primary lymphocytes. A, binding of J19 IgG to mouse SPLs in the presence or absence of 0.2 μg/ml CXCL12 was analyzed by flow cytometry. A representative experiment (of three) is shown as a histogram. The numbers within the panels show the specific mean fluorescence intensity of J19 IgG. The results are the means ± S.D. of three independent experiments. Open histogram, isotype control IgG filled histogram, J19 IgG. B, mouse SPLs were plated on coverslips coated with 10 μg/ml MAdCAM-1 or 10 μg/ml MAdCAM-1 plus 2 μg/ml CXCL12 for 20 min and then stained with 5 μg/ml J19 IgG and FIB27 in the presence of 1 mm Ca²⁺ + 1 mm Mg²⁺ or 2 mm Mn²⁺ as indicated. Bar, 20 μm.

Effect of J19 IgG on α₄β₇-MAdCAM-1 Interaction

To further characterize J19 IgG, we studied the effect of this antibody on cell adhesion mediated by α₄β₇-MAdCAM-1 interaction in shear flow using K562 cells stably expressing human α₄β₇. The cells were preincubated with J19 IgG or α₄β₇ blocking mAb Act-1 and then infused into a parallel wall flow chamber with MAdCAM-1 immobilized on the lower wall. 20 μg/ml Act-1 almost completely abolished the cell adhesion at the wall shear stress of 1 dyn/cm², whereas J19 IgG showed no effect on cell adhesion even at much higher concentration of 100 μg/ml (Fig. 6). Thus, J19 IgG does not affect the interaction between integrin α₄β₇ and MAdCAM-1, suggesting that it is distinct from the ligand-mimetic mAb.

FIGURE 6.

J19 IgG has no effect on α₄β₇-MAdCAM-1 interaction. K562 cells stably expressing human α₄β₇ were incubated with 100 μg/ml J19 IgG or 20 μg/ml mAb Act-1 in the presence of 1 mm Ca²⁺ + 1 mm Mg²⁺ or 2 mm Mn²⁺ for 15 min at 37 °C, followed by flow chamber assay as described under “Experimental Procedures.” The number of rolling and firmly adherent cells was measured under the wall shear stress of 1 dyn/cm². The error bars are ± S.D. (n = 3).

Epitope Mapping of J19 IgG

Because of the lack of cross-reactivity with α₄β₁ by J19 IgG and the high homology between β₁ and β₇ subunits, we constructed a panel of β₁/β₇ chimeras to locate the epitope of J19 IgG in β₇ subunit. A schematic of the constructed chimeras is shown in Fig. 7A. These chimeras were all transiently co-expressed with human α₄ subunit in 293T cells, the expression level of which was confirmed by immunostaining with 9F10 against α₄. Flow cytometric analysis of these 293T transfectants showed that the β₇ segment 331–348 located in βI domain was absolutely required for the binding of J19 IgG to α₄β₇. All of the chimeras in which segment 331–348 was of β₁ origin failed to be stained with 5 μg/ml J19 IgG, whereas all other chimeras in which this segment was of β₇ origin were stained, as well as WT β₇ (Fig. 7A). Within region 331–348, seven amino acids differ between human β₁ and β₇. Thereafter, we substituted the seven β₇ residues with the corresponding β₁ residues. Mutations of S331E, A332E, and A333F decreased ∼30–50% of J19 IgG binding in comparison with WT α₄β₇. By contrast, mutations of the other four residues, L334Q, Q338K, S341K/K342N, almost had no effect on the recognition of α₄β₇ by J19 IgG. Moreover, the β₇ triple mutation S331E/A332E/A333F completely abolished the recognition of α₄β₇ by J19 IgG (Fig. 7B). These results strongly suggest that residues Ser-331, Ala-332, and Ala-333 in β₇ I domain represent a direct binding site for J19 IgG.

FIGURE 7.

Epitope mapping of J19 IgG. A, mapping of J19 IgG epitope with β₇ chimeras. mAb reactivity was determined with chimeric human β₁/β₇ subunits co-expressed with human α₄ in 293T cells in the presence of 2 mm Mn²⁺ by using flow cytometry. J19 IgG recognition was measured as specific mean fluorescence intensity and quantitated as a percentage of total α₄β₇ expression defined by staining with mAb 9F10 to α₄. B, fine mapping of J19 IgG epitope in β₇ subunit. Human WT β₇ or mutant β₇ containing multiple or single β₇ to β₁ amino acid substitutions was co-expressed with human α₄ subunit in 293T cells. J19 IgG recognition was quantified as in A. C, mapping of J19 IgG epitope with α₄ chimeras. The human α₄/α_E chimeras were co-expressed with human β₇ in 293T cells. The transfectants were stained with J19 IgG or mAb FIB27 to β₇, followed by flow cytometry in the presence of 2 mm Mn²⁺. J19 IgG recognition was quantified as in Fig. 7A. The error bars are ± S.D. (n = 3).

The J19 IgG binding site in human α₄ subunit was mapped by using α₄/α_E chimeras because α₄ and α_E share the same β₇ subunit. Considering β-propeller domain in α subunit is close to the above mapped J19 epitope in βI domain, we first swapped β-propeller domain of α₄ and α_E subunits, whereas the swap of β-propeller domain of α₄ and α_E subunits resulted in no expression of both α₄β₇ and α_Eβ₇ chimeric integrins. The abnormal expression of α₄/α_E chimeras is possibly due to the difference in structure between αI domain-less integrin α₄ and αI domain-containing integrin α_E. Thus, based on J19 binding sites in β₇ subunit and the crystal structures of integrin α_IIbβ₃ and α_Vβ₃, several segments in α₄ β-propeller domain close to the epitope in β₇ subunit were substituted with corresponding α_E sequences, respectively. These chimeric cDNAs were all cloned into pcDNA 3.1 expression vectors and transiently co-expressed with human β₇ subunit in 293T cells, then followed by immunostaining with 5 μg/ml J19 IgG. The expression of these chimeras was confirmed by immunostaining with mAb FIB27 against β₇. Swapping of α₄ segments 211–216 and 240–246 with those of α_E had no effect on the binding of J19 IgG, whereas swapping of the α₄ 184–190 segment completely abolished J19 IgG binding to chimera α₄184α_E190α₄ (Fig. 7C). These results demonstrate that the epitope of J19 IgG in α₄ subunit locates in a seven-residue segment from 184 to 190 in β-propeller domain. However, J19 did not bind to chimeric α_E (α_E184α₄190α_E) when swapping the 184–190 segment of α₄ into α_E subunit (Fig. 7C), which might be due to the interference of J19 binding by the αI domain on the top of the β-propeller domain in α_E subunit.

The Epitope Exposure of J19 IgG Is Coupled with Extension of Integrin α₄β₇ Ectodomain

The headpiece of integrin folds over its legs and faces down toward the membrane in the low affinity bend conformation and extends upward in a switchblade-like opening upon activation (2, 43). We next studied the relationship between J19 IgG epitope exposure and the conformational rearrangement of integrin α₄β₇ during its activation. To assess the orientation of integrin α₄β₇ ectodomain relative to the cell membrane using a FRET system, α₄β₇ was labeled with Alexa Fluor 488-Act-1 Fab fragment as donor, which binds to the top of α₄β₇ βI domain (44). The outer face of plasma membrane was labeled with FM4–64 FX (FM) as acceptor (30, 35). Compared with the inactive α₄β₇ in 1 mm Ca²⁺ + 1 mm Mg²⁺, the FRET efficiency of 293T transfectants bearing activated α₄β₇ in 2 mm Mn²⁺ was significantly decreased from 22 to 6%, indicating the extension of the α₄β₇ ectodomain (Fig. 8A). In parallel, immunostaining results showed that J19 IgG bound to activated α₄β₇ in 2 mm Mn²⁺ but not to inactive α₄β₇ in 1 mm Ca²⁺ + 1 mm Mg²⁺, suggesting that the epitope of J19 IgG was only exposed in the activated α₄β₇ with extended conformation in 2 mm Mn²⁺ (Fig. 8B). These data strongly indicate that the J19 IgG epitope is exposed when the integrin head domain moves away from the cell membrane in the extended conformation.

FIGURE 8.

The expression of J19 IgG epitope is associated with the conformational change of integrin α₄β₇ upon activation. A, FRET between integrin α₄β₇ βI domain and the plasma membrane. The error bars are ± S.D. (n = 10). B, the expression of J19 IgG epitope was measured as specific mean fluorescence intensity and quantitated as a percentage of total α₄β₇ expression defined by staining with mAb Act-1. 293T cells stably expressing α₄β₇ were stained with 5 μg/ml J19 IgG and Act-1 in the presence of 1 mm Ca²⁺ + 1 mm Mg²⁺ or 2 mm Mn²⁺ as indicated and followed by flow cytometry. The error bars are ± S.D. (n = 3).

DISCUSSION

In this study, we screened and characterized an activation-specific human monoclonal antibody to integrin α₄β₇ by panning a scFv-displaying phagemid library using an active form of α₄β₇ (wedge mutant) as target. The subtractive selection was performed by depleting the library on the isolated, inactive WT α₄β₇ protein first and then panning against the constitutively activated α₄β₇ wedge mutant. The specific scFv clone J19 was isolated from the library and reformatted to full-length human IgG1. The J19 IgG specifically binds to integrin α₄β₇ activated by different stimuli, other than the inactive α₄β₇. The binding epitope of J19 IgG was mapped to two small segments located in α₄ β-propeller domain and β₇ I domain, respectively (supplemental Fig. S1). The seven-residue segment from 184 to 190 in α₄ subunit locates between β-strands 2 and 3 in β-sheet 3 of the β-propeller domain. The other segment consists of residues Ser-331, Ala-332, and Ala-333 located in a turn between β-strand 5 and α-helix 5 at the top of βI domain. In the low affinity bend conformation, the integrin β-propeller domain and the βI domain sit on their leg domains and face the cell membrane, leading to the epitope buried in the inactive integrin. Upon activation, integrin converted from the bent to extended conformation with either closed headpiece (intermediate affinity state) or open headpiece (high affinity state), which led to the exposure of J19 epitope (supplemental Fig. S1). Thus, J19 IgG is an activation-dependent mAb, which recognizes an epitope expressed only in integrin α₄β₇ with extended conformation.

The high affinity wedge mutant α₄β₇ used for J19 selection contains a mutation-introduced N-glycan at the integrin β₇ I/hybrid domain interface and mimics the swing-out of hybrid domain in β₇ subunit, which is predicted to activate integrins and stabilize the high affinity conformation. In our study, we showed that J19 IgG bound both wedge mutant and WT integrin α₄β₇ activated by physiological agonist, such as CXCL12. Thus, the epitope recognized by J19 IgG is expressed in both high affinity wedge mutant and the physiologically activated WT integrin α₄β₇. These results strongly suggest that the hybrid domain swing-out is the key conformational rearrangement during integrin activation and can induce a high affinity conformation mimicking the physiological activation of integrin α₄β₇.

Different from the J19 phage and J19 IgG, which specifically recognize the activated integrin α₄β₇, J19 scFv showed similar binding to both inactive and activated α₄β₇. The loss of specificity to activated α₄β₇ could be due to the smaller size of scFv compared with phage particle and full-length IgG1. The epitope of J19 in the inactive α₄β₇ with bend conformation is not accessible to large size J19 phage or IgG, whereas the space is large enough for smaller scFv to access the epitope. This result also provides supporting evidence for the induction of J19 epitope expression by the conformational rearrangements of integrin.

In αI domain-containing integrins, the αI domain is inserted into a loop between β-sheets 2 and 3 at the top of the β-propeller domain (45, 46). As part of the J19 epitope, the seven-residue segment from 184 to 190 locates in β-sheet 3 of the β-propeller domain, which will be masked by the αI domain in the α_E subunit (supplemental Fig. S2). Thus, J19 IgG cannot bind to integrin α_Eβ₇ even after replacement of the seven-residue segment in α_E subunit with that from α₄ (Fig. 7C).

Interestingly, the binding of J19 IgG to α₄β₇ could be enhanced by a number of stimuli, including Mn²⁺, DTT, and ADP, but to different levels. The binding of J19 IgG showed stronger binding to α₄β₇ expressing cells treated with Mn²⁺ than the same cells stimulated with DTT or ADP, suggesting that integrin activated by different stimuli could have different conformations and expression of J19 epitope. The different expression levels of J19 epitope were consistent with the detection of increased affinity for ligand after those stimulations, as measured by a sensitive flow chamber assay (34, 35, 47). Thus, J19 IgG could serve as a reporter for the activation extent of α₄β₇.

In addition to serving as a research tool for in vitro and in vivo studies of integrin activation, the J19 antibody also represents a therapeutic candidate for treatment of inflammatory bowel disease. J19 IgG selectively targets the activated lymphocytes and leaves the inactive ones intact, which may result in fewer potential side effects.