The Nonintegrin Laminin Binding Protein (p67 LBP) Is Expressed on a Subset of Activated Human T Lymphocytes and, Together with the Integrin Very Late Activation Antigen-6, Mediates Avid Cellular Adherence to Laminin

Cell lines

The Jurkat T leukemia lines B2.7 and D1.1 have been described previously (33). Briefly, Jurkat B2.7 is a CD4⁺ human T leukemia line. D1.1 is a Jurkat subclone that lacks surface CD4 expression, constitutively expresses surface CD40 ligand, and is capable of providing contact-dependent T cell help to B cells (34). Cells were maintained in IMDM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Summit Biotechnologies, Fort Collins, CO), 5 U/ml penicillin, and 5 μg/ml streptomycin (Sigma, St. Louis, MO) in a 5% CO₂ incubator at 37°C.

Flow cytometry

Cells analyzed by flow cytometry were pelleted at 300 × g for 5 min at 4°C, and resuspended at 2 × 10⁶ cells/ml in DMEM (Life Technologies) supplemented with 1% FBS and 20 μg/ml heat-aggregated human IgG (International Enzymes, Fallbrook, CA). Cells were aliquoted at 50 μl/well into a polystyrene 96-well plate (Becton Dickinson, San Jose, CA), and the appropriate mAb was added to a final concentration of 2–5 μg/ml. Cells were incubated at 4°C for 30 min, pelleted at 800 × g for 5 min at 4°C, and washed with 200 μl DMEM/1% FBS. Cells stained with fluorochrome-conjugated primary mAbs were resuspended in 100 μl PBS containing 1% formaldehyde. Cells stained with nonconjugated primary mAbs were resuspended in 50 μl fluorochrome-conjugated goat anti-mouse (GAM) IgG/IgM (Jackson ImmunoResearch, West Grove, PA) at 1.5 μg/ml in DMEM/1% FBS and incubated at 4°C for 15 min, washed, and resuspended in 100 μl PBS containing 1% formaldehyde. In some experiments, fluorochrome-conjugated GAM IgG (γ-chain-specific) and anti-mouse IgM (μ-chain-specific) were used in dual color staining to distinguish mAb MLuC5 (mouse IgM) from any of several unconjugated anti-integrin mAbs (all mouse IgGs).

The following mAbs directed against human cell surface Ags were used in flow cytometry: anti-p67 LBP mAb MLuC5, nonconjugated (NeoMarkers/Lab Vision, Fremont, CA); anti-CD3 mAb HIT3A, R-PE-conjugated (PharMingen, San Diego, CA); anti-CD4 mAb RPA-T4, R-PE-conjugated (PharMingen); anti-CD8 mAb RPA-T8, R-PE-conjugated (PharMingen); anti-CD45RO mAb UCHL1, R-PE-conjugated (PharMingen); anti-integrin α₁ (CD49a) mAb HP2B6, unconjugated (Beckman Coulter, Fullerton, CA); anti-integrin α₂ (CD49b) mAb 12F1-H6, R-PE-conjugated (PharMingen); anti-integrin α₃ (CD49c) mAb C3 II.1, unconjugated (PharMingen); anti-integrin α₆ (CD49f) mAb GoH3, R-PE-conjugated (PharMingen); anti-integrin β₁ (CD29) mAb 4B4, PE-conjugated (Beckman Coulter); anti-integrin α_L (CD11a) mAb B-B15, PE-conjugated (Biosource International, Camarillo, CA); anti-CD69 mAb L78, R-PE-conjugated (Becton Dickinson); anti-IL-2 receptor α-chain (CD25) mAb 143-13, R-PE-conjugated (Biosource International); anti-transferrin receptor (CD71) mAb YDJ.1.2.2, R-PE-conjugated (Beckman Coulter); anti-CD26 mAb M-A261, R-PE-conjugated (PharMingen); and anti-CD44 mAb A3D8, R-PE-conjugated (Sigma). The unconjugated mAbs TS2/7 (anti-integrin α₁) and P1H5 (anti-integrin α₂) were the gift of Dr. Eugene Marcantonio (Columbia University, New York, NY).

Flow cytometry was performed on a FACScan (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson), except in experiments involving cell sorting by cytometry. All cell sorting was performed on a FACStar sorter (Becton Dickinson). Cells were prepared for sorting as described above, except that all solutions were presterilized by filtration through 0.2-μ pore cellulose acetate filters (Nalge Nunc International, Rochester, NY), and cells were resuspended in ice-cold IMDM/10% FBS after the final wash step, instead of PBS/1% formaldehyde.

Differential display and cloning of p67 LBP

Oligo(dT)-primed cDNA sets derived from the Jurkat cell lines B2.7 and D1.1 were screened for messages differentially expressed in the D1.1 subclone, using the method of differential display RT-PCR (DDRT-PCR) (35, 36). Briefly, mRNA was isolated from each line using the FastTrack System (Invitrogen, Carlsbad, CA) and reverse transcribed using oligo(dT) priming and Superscript II reverse transcriptase (Life Technologies). A set of 23 pairs of arbitrary decanucleotides (Operon Technologies, Alameda, CA) was used to screen the cDNA sets by PCR at low annealing temperature (42°C) designed to amplify DNA fragments of 150–500 bp in length. This amplification was performed in the presence of [α-³²P]dCTP (NEN Life Sciences, Boston, MA), and products derived from B2.7 and D1.1 were compared by PAGE followed by autoradiography, on 10% polyacrylamide gels (Bio-Rad, Hercules, CA). All reactions were performed in duplicate with independently isolated mRNA preparations, and PCR products reproducibly amplified from D1.1 cDNA, but not B2.7 cDNA, were excised from the gel, eluted in TE buffer (10 mM Tris (pH 8.0), 1 mM EDTA), and reamplified using the same primer pair. These putatively differentially expressed cDNA fragments were cloned using the TA Cloning System (Invitrogen) and partially sequenced using an automated ABI 373 Sequencer (Perkin-Elmer, Norwalk, CT). Fragment-specific cDNA primers were designed based on cloned fragment sequence, and PCR used to confirm the differential expression of the represented messages by RT-PCR. Finally, the cDNAs amplified using the fragment-specific primers were radiolabeled with [α-³²P]dCTP (NEN Life Sciences) by random hexanucleotide priming using the NEBlot system (New England Biolabs, Beverly, MA) and used to probe a D1.1 cDNA library consisting of 2 × 10⁶ independent cDNA inserts cloned directionally into the expression vector pCDNAI (Invitrogen).

A 180-bp PCR product from DDRT-PCR was used to screen 180,000 colonies from the D1.1 cDNA library, yielding two independent clones (no. 74 and no.79) that were further characterized. Both showed >98% identity to the published sequence of the p67 laminin receptor (NCBI BLAST Search: www.ncbi.nlm.nih.gov). Clone no. 74 was incomplete, lacking 10 base pairs of 5′ coding sequence; clone no. 79 represented the complete coding sequence, as well as 27 bp of 5′ untranslated sequence. Clone no. 79 was excised from the pCDNAI vector by restriction endonuclease digestion, and ligated into the vector pCEP4 (Invitrogen), yielding pCEP-p37 LBPP. This vector confers stable episomal replication in eukaryotic cells selectable on the basis of hygromycin resistance. Transcription of the insert is initiated within the vector-encoded CMV immediate-early promoter.

Transfection of Jurkat tumor cells

The plasmid pCEP-p37 LBPP was transfected into Escherichia coli strain XL1 Blue MRF′ (Stratagene, La Jolla, CA) by electroporation using a Bio-Rad Gene Pulser under the following conditions: 1.8 kV, 200 Ω, 25 μF, 0.1 cm gap (Bio-Rad). A single colony was amplified by growth overnight at 37°C in 1 L Luria Broth supplemented with 50 μg/ml ampicillin. Plasmid DNA was purified from this culture by a standard alkaline lysis procedure, followed by two cycles of CsCl density centrifugation (37).

Before electroporation, Jurkat B2.7 cells were grown in IMDM/10% FBS to a density of 1 × 10⁶ cells/ml, collected by centrifugation at 300 × g for 5 min, resuspended in IMDM/10% FBS at a density of 8 × 10⁶ cells/ml, and divided into 0.5-ml aliquots in sterile polypropylene tubes. Purified plasmid DNA was precipitated in ethanol, washed once with 70% ethanol, and dissolved in sterile water at a concentration of 1 μg/ml. A total of 10 μg of plasmid DNA in sterile water was added to each aliquot of cells, the cells and DNA were gently mixed by stirring with a pipet tip, and the mixture was transferred to a sterile electroporation cuvette (Bio-Rad; 0.4 cm gap). The DNA was transfected into the B2.7 cells by electroporation using a Bio-Rad Gene Pulser (280 V, 975 μF), after which transfectants were cultured in IMDM/10% FBS for 48 h before drug selection in IMDM/10% FBS containing 800 μg/ml hygromycin B (Boehringer Mannheim, Indianapolis, IN). Finally, the hygromycin-resistant transfectants were cloned by flow cytometric sorting into 96-well plates on the basis of staining with mAb MLuC5 on a FACStar cell sorter (Becton Dickinson), as described above.

PCR analysis for vector-encoded p37 LBPP expression

Jurkat B2.7 transfectant clones expressing the pCEP-p37 LBPP cDNA were grown to a density of 1 × 10⁶ cells/ml in IMDM/10% FBS/800 μg/ml hygromycin, and 1 × 10⁷ cells were collected by centrifugation at 800 × g, washed once with PBS, and pelleted. Total RNA was isolated from each cell pellet using the RNEasy RNA Isolation System (Qiagen, Valencia, CA). A total of 5 μg of total RNA was reverse transcribed into cDNA using a dT₁₅ primer and SuperScript II reverse transcriptase (Life Technologies) in a 20-μl reaction. A total of 1 μl of the resulting single-stranded cDNA product was subjected to PCR amplification using Taq DNA polymerase (Fisher, Pittsburgh, PA) in a forty cycle reaction using a forward primer specific for the 5′ untranslated region of the expression vector pCEP4 (5′-AACCGTCAGATCTCTAGAAGCTGGG-3′), and a reverse primer specific for the p37 LBPP cDNA (5′-AATTCCTCCTTGGTCACTGCCTTC-3′). Cycle parameters included denaturation at 94°C (0.5 min), annealing at 56°C (1 min), and chain extension at 72°C (0.5 min). PCR amplification products were separated by gel electrophoresis on a 1% agarose gel, and visualized by staining in ethidium bromide (0.3 μg/ml). The primers resulted in the generation of an 800-bp cDNA fragment in clones expressing the p37 LBPP cDNA transcribed from the CMV immediate/early promoter in pCEP4.

Substrate-specific cell adhesion assay

Cell adhesion to laminin was assayed by a method modified from St. John (38). Briefly, 96-well polystyrene tissue culture plates (Becton Dickinson) were precoated with human laminin (Life Technologies) at 5 μg/ml in PBS, 50 μl/well, in a humidified environment at room temperature overnight (control wells received PBS alone). At the end of this period, all wells were washed three times with PBS (400 μl/well) and blocked with 3% BSA in PBS, 200 μl/well, at 37°C for 3 h. Finally, wells were washed with PBS, and the plates were stored inverted and sealed at 4°C for up to 48 h before use. In other assays, plates were precoated with human fibronectin (Life Technologies), VCAM-1 (R&D Systems, Minneapolis, MN), or collagen IV (Life Technologies) by an identical procedure.

Test cells were removed from culture, washed once with IMDM/10% FBS, and resuspended in the same media supplemented with 90 μCi/ml ⁵¹Cr sodium chromate (NEN Life Sciences) at a cell density of 20 × 10⁶ cells/ml. The cells were incubated in ⁵¹Cr-loading media at 37°C with agitation for 90 min, then washed three times with 30-fold excess IMDM/10% FBS. ⁵¹Cr-loaded cells were plated at a density of 100,000 cells/well in 400 μl of the same media and allowed to adhere for 2 h in a humidified incubator at 5% CO₂ and 37°C. Following adhesion, the test plate was submerged in 1.5 L PBS at room temperature in a vessel large enough to accommodate the entire plate. The plate, entirely beneath the PBS surface, was then inverted and fixed in position using a burette stand and clamp. The PBS bath was oscillated relative to the plate for 20 min on a rotary shaker at 40 cycles/min, applying a mild shear force to all wells simultaneously. Following this shear wash, the plate, still beneath the liquid surface, was reinverted (to the upright position) and removed from the PBS bath. Half of each well volume (200 μl) was removed by pipet, and cells were lysed by the addition of 200 μl 20% SDS (final concentration 10% SDS). To determine cpm/cell, 100,000 ⁵¹Cr-labeled cells from each test sample were incubated in parallel with the test plate, washed, pelleted, and lysed in 400 μl 10% SDS. All cell lysates (400 μl) were transferred to counting vials and analyzed using a Clinigamma 1272 gamma counter (EG&G Wallac, Gaithersburg, MD). All conditions were assayed in qua-druplicate. The laminin-specific adhesion was calculated as the difference in average cpm per well between the laminin-coated and noncoated wells, and expressed as number of cells, based on the measured cpm/cell for each clone tested. The results presented in Fig. 7⇓ derive from a representative experiment. Similar results were found in at least four independent experiments for each cell line tested.

Isolation and activation of normal human PBL

Peripheral blood specimens from normal healthy human volunteers were collected in heparinized tubes by venipuncture. PBL were isolated by ficoll density centrifugation (Sigma) and subsequently enriched to >90% CD3⁺ T cells by rosetting with SRBC (Colorado Serum, Denver, CO). After isolation of erythrocyte rosetting (E+) lymphocytes on a second ficoll density gradient, SRBC were lysed by agitation at room temperature for 3 min in a solution of 0.5 M NH₄Cl, 33 mM KHCO₃, 0.4 mM Na₂EDTA. E+ lymphocytes were then washed three times in DMEM (Life Technologies) supplemented with 1% FBS (Summit Biotechnologies) before transfer to IMDM/10% FBS for culture. T cell stimulation was performed by incubating the cells for 6 h at 37°C in IMDM/10% FBS in the presence of 10 ng/ml phorbol dibutyrate (PDB) and 600 ng/ml ionomycin (Sigma). Following activation, cells were washed once with IMDM/10% FBS and maintained in culture at 37°C for up to 10 days.

Differential expression of p67 LBP in Jurkat T leukemia subclones

To isolate genes expressed in activated T cells, DDRT-PCR (35, 36) was used to compare two subclones of the Jurkat T leukemia line. The Jurkat subclone D1.1 is a model for activated T lymphocytes, whereas the Jurkat B2.7 subclone is a model for resting T cells (33). Using DDRT-PCR, a cDNA fragment of 180 bp was reproducibly amplified from D1.1 mRNA, but not from B2.7 mRNA. This 180-bp PCR product was radiolabeled and used to screen a D1.1 cDNA library. Two independent cDNA clones were identified, both with >98% identity with the cDNA encoding the 37-kDa polypeptide precursor (p37 LBPP) to p67 LBP (16). Together, these data suggested that p37 LBPP is differentially expressed by Jurkat D1.1; however, due to the presence of p37 LBPP-hybridizing transcripts in B2.7 (presumably from pseudogenes (17)), the differential expression of productive LBPP transcripts was difficult to substantiate by Northern or RT-PCR analysis.

To determine whether the expression of p37 LBPP mRNA by Jurkat D1.1 is associated with differential surface expression of p67 LBP, the Jurkat clones were examined by flow cytometry using the anti-p67 LBP mAb, MLuC5 (39). Consistent with the DDRT-PCR results, the D1.1 subclone expresses high level surface p67 LBP, whereas B2.7 does not express detectable p67 LBP (Fig. 1⇓). Together, these data corroborate the DDRT-PCR result and confirm that p67 LBP is differentially expressed by Jurkat D1.1, a model for activated T cells.

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FIGURE 1.

Differential surface expression of p67 LBP on Jurkat D1.1, but not B2.7. Shown are FACS histograms of Jurkat B2.7 (left column) and D1.1 (right column) depicting surface expression of the following proteins: top row, p67 LBP (mAb MLuC5); second row, integrin α₁ (CD49a, mAb TS2/7); third row, integrin α₂ (CD49b, mAb P1H5); fourth row, α₃ (CD49c, mAb C3 II.1); fifth row, integrin α₆ (CD49f, mAb GoH3). In each panel, staining with the indicated mAb is depicted by a solid line in comparison with an isotype control (broken line).

Expression of p67 LBP on activated, normal T cells

To determine the significance of the selective expression of p67 LBP in the Jurkat model of activated T cells, the next series of experiments examined the expression of p67 LBP on normal human peripheral blood T cells either resting (freshly isolated) or 36 h after activation with PDB and ionomycin. T cells were analyzed by dual channel flow cytometry for p67 LBP expression relative to the expression of CD3, CD4, CD8, and CD45RO. High level surface expression of p67 LBP was detected on a subset (10–15%) of activated T cells, but only on very few resting cells (Fig. 2⇓). In fact, the rare freshly isolated T cells (1–2%) that stain with MLuC5 appear to represent a small population of circulating, recently activated T cells because these cells are CD69⁺ and CD71⁺ (data not shown). The subset of PDB/ionomycin-activated T lymphocytes that express p67 LBP are predominantly CD4⁺, CD8⁻, and CD45 RO⁺ (Fig. 2⇓). Therefore, these data suggest that a subset of activated, but not resting, memory T cells expresses p67 LBP.

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FIGURE 2.

Expression of p67 LBP on activated human peripheral blood T cells. Shown are dual-color FACS contour plots of resting (left column) and activated (right column) peripheral blood human T cells depicting p67 LBP expression (mAb MLuC5) on the x-axis and CD3 (mAb HIT3A), CD4 (mAb RPA-T4), CD8 (mAb RPA-T8), and CD45 RO (mAb UCHL1), as indicated on the y-axis. The percentage of total T cells represented in selected quadrants is indicated within each quadrant. T cells were activated by PDB (10 ng/ml) and ionomycin (600 ng/ml), as described in Materials and Methods.

The next experiments studied the time course of p67 LBP expression by T cells after activation. Expression of p67 LBP is detectable within 6 h after activation and peaks between 18 and 36 h after activation (Fig. 3⇓). Thereafter, p67 LBP expression declines gradually over 5 days (Fig. 3⇓) and returns to baseline by 10 days (data not shown). To further define the T cell population expressing p67 LBP, 36 h-activated T cells were FACS sorted for MLuC5⁺ phenotype and analyzed by dual channel flow cytometry for a variety of other T cell surface molecules (Fig. 4⇓). The p67 LBP⁺ subset is uniformly CD45 RO⁺ (Fig. 4⇓), confirming the results of the studies on unsorted cells (Fig. 2⇑). The p67 LBP⁺ subset is composed of mature, single-positive T cells, 85% CD4⁺8⁻ and 15% CD4⁻8⁺ (Fig. 4⇓). The p67 LBP⁺ T cells express the IL-2 receptor p55 polypeptide (CD25) and CD69, consistent with their activated state. The p67 LBP⁺ subset also expresses CD11A, CD26, and CD44 at high levels (Fig. 4⇓), a phenotype that has been associated with T cell migratory potential, in vitro (8). Taken together, these data indicate that a subset of activated memory T cells, including both CD4⁺ and CD8⁺ cells, expresses p67 LBP for 7–10 days after activation.

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FIGURE 3.

Time course of p67 LBP expression on activated peripheral blood T cells. Shown are dual-color FACS contour plots of human peripheral blood T cells depicting p67 LBP expression (mAb MLuC5) on the x-axis, and CD3 expression (mAb HIT3A) on the y-axis, at the indicated time points after activation with PDB/Ionomycin: 6 h (A), 24 h (B), 48 h (C), and 5 days (D). The percentage of T cells expressing p67 LBP is indicated in the upper right quadrant of each panel.

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FIGURE 4.

Analysis of p67 LBP-expressing T cells for expression of other T cell surface proteins. Shown are single-color FACS histograms of the MLuC5⁺ (sorted) fraction of activated human peripheral blood T cells depicting staining for various T cell markers, as indicated in each panel (solid line) relative to an isotype control (broken line). Top row: CD3 (mAb HIT3A), CD4 (mAb RPA-T4), CD8 (mAb RPA-T8); middle row: CD45 RO (mAb UCHL1), CD25 (mAb 143–13), CD69 (mAb L78); and bottom row: CD11a (mAb), CD44 (mAb A3D8), CD26 (mAb M-A261).

The FACS studies also revealed that p67 LBP⁺ T cells express higher levels of both α₆ (CD49f) and β₁ (CD29), relative to the p67 LBP⁻ T cell subset (Fig. 5⇓). The α₆ and β₁ integrin chains pair in VLA-6, the integrin laminin-specific receptor. High VLA-6 expression is consistent with the memory phenotype of these cells (9), and is of interest since, in tumor cells, p67 LBP and α₆ associate, are coordinately expressed, and α₆ is thought to play a role in p67 LBP expression (15, 22). In addition, these studies revealed that the p67 LBP⁺ T cell subset also expresses higher levels of surface α₁, α₂, and α₃ than the p67 LBP⁻ T cell subset (Fig. 5⇓), suggesting that these cells may interact with laminin through a number of different VLA receptors.

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FIGURE 5.

Analysis of p67 LBP-expressing T cells for expression of integrin laminin receptors VLA-1, VLA-2, VLA-3, and VLA-6. Shown are FACS histograms of the MLuC5⁺ (solid bold lines) and MLuC5⁻ (solid light lines) fractions of activated human peripheral blood T cells depicting the expression of the polypeptides that compose the surface proteins VLA-1, -2, -3, and -6. Top row: integrin chains α₁ (CD49a, mAb HP2B6), α₂ (CD49b, mAb 12F1-H6), α₃ (CD49c, mAb C3 II.1); bottom row: α₆ (CD49f, mAb GoH3), β₁ (CD29, mAb 4B4). In each panel, staining for the indicated integrin chain is compared with that of an irrelevant isotype control mAb (broken line).

Expression of p37 LBPP cDNA in Jurkat B2.7

To determine whether the differential expression of p67 LBP on D1.1 is due solely to differential expression of p37 LBPP mRNA, the next series of experiments examined whether transfection of p37 LBPP cDNA confers p67 LBP expression on the p67 LBP⁻ Jurkat subclone B2.7. The B2.7 line was electroporated with p37 LBPP cDNA in an expression vector (pCEP4; Invitrogen), and stable transfectants were selected on the basis of hygromycin resistance. Drug-resistant clones were sorted by flow cytometry for MLuC5⁺ phenotype, and vector-encoded p37 LBPP expression was verified in the sorted clones by RT-PCR. Transfectant clones were analyzed by FACS for surface expression of p67 LBP, as well as α₁, α₂, α₃, and α₆ (Table I⇓). All clones expressed high levels of β₁ (data not shown). B2.7 transfectants expressing pCEP4-p37 LBPP express high level surface p67 LBP (Table I⇓; Fig. 6⇓, clones 42 and 48). In contrast, control B2.7 transfectants expressing a CD8 α-chain cDNA in pCEP4 (B2.7/CD8) do not express p67 LBP (Table I⇓; Fig. 6⇓). Together, these data show that expression of a single cDNA encoding p37 LBPP confers high level p67 LBP surface expression on B2.7 cells.

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FIGURE 6.

Surface expression of p67 LBP on representative clones of Jurkat B2.7 stably expressing the p37 LBPP cDNA driven by an exogenous promoter. Shown are FACS histograms of two representative Jurkat B2.7 transfectants expressing vector-encoded p37 LBPP (B2.7/p37 LBPP, clones 42 and 48), in comparison with a B2.7 transfectant expressing vector-encoded CD8α-chain as a control (B2.7/CD8). Clones were examined for the following surface proteins: top row, p67 LBP (mAb MLuC5); second row, α₁ (CD49a, mAb TS2/7); third row, α₂ (CD49b, mAb P1H5); fourth row, α₃ (CD49c, mAb C3 II.1); fifth row, α₆ (CD49f, mAb GoH3). In each panel, staining with the indicated mAb (solid line) is compared with that of an isotype control mAb (broken line).

Laminin-specific adherence of p67 LBP⁺ Jurkat B2.7 transfectants

To determine whether the presence of p67 LBP on the surface of the Jurkat clones is associated with increased cellular adherence to laminin, chromium-labeled D1.1, B2.7, or B2.7 transfectant clones were allowed to interact with immobilized laminin (or BSA in control wells) for 2 h at 37°C, before nonadherent cells were removed and adherent cells were lysed in 10% SDS and quantified by gamma counting. Each Jurkat clone was studied in at least four independent assays, and a representative experiment is shown in Fig. 7⇓A. As a positive control for laminin-specific adherence, D1.1, which expresses high levels of both p67 LBP and VLA-6 (Fig. 1⇑), adheres avidly to the laminin-coated surface (Fig. 7⇓A). As negative controls, the parental B2.7 clone (Fig. 1⇑) and a B2.7 transfectant expressing the CD8α polypeptide (B2.7/CD8) (Fig. 6⇑) show no significant laminin-specific adherence. B2.7 transfectants that express high p67 LBP display significant laminin binding (Fig. 7⇓A, clones 18, 28, and 42). These data indicate that p67 LBP expression confers laminin-specific adherence on B2.7 cells.

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FIGURE 7.

Laminin-specific adherence of Jurkat B2.7, D1.1, and transfectants of Jurkat B2.7. A, The laminin-specific binding of Jurkat B2.7, D1.1, and selected transfectants of B2.7. Cells were labeled with ⁵¹Cr and allowed to adhere for 2 h to polystyrene wells coated either with laminin or BSA (control). Following adherence, nonadherent cells were removed, and the cpm remaining in each well obtained. The cpm/cell was determined separately by counting 100,000 labeled cells. Laminin-specific adherence refers to the difference in average number of cells remaining between the laminin-coated and control wells. In all experiments, a given clone was assayed in quadruplicate wells, and each clone was assayed in at least four independent experiments. The results shown derive from one experiment, and are typical of performance over multiple trials. Horizontal bars depict laminin-specific adherence (in number of cells) for parental B2.7 and D1.1 (light grey bars), the B2.7 transfectant expressing pCEP-CD8 (B2.7/CD8, light stippled bar), and B2.7 transfectants expressing pCEP-p37 LBPP (B2.7/p37 LBPP, dark stippled bars). The phenotypes of all clones with respect to p67 LBP and α₆ are shown in the table to the left of the bar plot. B, The fibronectin-, VCAM-1-, and collagen IV-specific binding of Jurkat B2.7, D1.1, and selected transfectants of B2.7. The assay was performed exactly as for laminin-specific adherence, except that the indicated ECM protein was substituted for laminin. Horizontal bars depict fibronectin adherence (striped), VCAM-1 adherence (light grey), and collagen IV adherence (dark grey). The phenotypes of all clones with respect to p67 LBP and α₆ are shown in the table to the left of the bar plot.

Since B2.7 transfectants expressing p67 LBP display varying levels of laminin-specific adherence (Fig. 6⇑), these lines were studied for their expression of α₆ (CD49f) by flow cytometry (Fig. 6⇑; Table I⇑). Clones 18, 28, and 42 are representative of transfectants with low α₆ and high p67 LBP (α₆^lowp67 LBP^high) (Table I⇑; Fig. 7⇑A). Clones 29, 34, and 48 are representative of transfectants with high α₆ and high p67 LBP (α₆^highp67 LBP^high) (Table I⇑; Fig. 7⇑A). The α₆^lowp67 LBP^high and α₆^highp67 LBP^high groups of clones had similar levels of α₁, α₂, and α₃, although α₂ expression was slightly increased in the latter group (Fig. 6⇑; Table I⇑). Clones with the phenotype α₆^lowp67 LBP^high show moderate laminin-specific adherence (Fig. 7⇑A). In contrast, B2.7 transfectants with the phenotype α₆^highp67 LBP^high show avid laminin-specific adherence (Fig. 7⇑A). Interestingly, B2.7 and B2.7/CD8, which express surface α₆ in the absence of p67 LBP, do not exhibit laminin-specific adherence. Taken together, these data indicate that p67 LBP expression on B2.7 transfectants confers increased cellular laminin adherence; however, avid laminin binding requires high level expression of both p67 LBP and α₆. Since Jurkat B2.7 is β₁⁺ (data not shown) and α₆ is a component of VLA-6 (α₆β₁), these data suggest that p67 LBP and VLA-6 function together to mediate high-avidity laminin adherence.

We next sought to determine whether the adherence of the Jurkat transfectants was specific to laminin relative to other β₁-integrin substrates, such as VCAM-1 and fibronectin, or to the basement membrane matrix protein collagen IV. Similar to the case for laminin, the parental B2.7 clone and the B2.7/CD8 transfectant do not adhere to collagen IV (Fig. 7⇑B). In contrast, all Jurkat clones show significant baseline adherence to VCAM-1 and to fibronectin (Fig. 7⇑B). Although p67 LBP expression in the B2.7/p37 LBPP transfectants is associated with increased laminin adherence (Fig. 7⇑A), p67 LBP expression does not induce collagen IV adherence. In addition, expression of p67 LBP is not associated with increased adherence to either fibronectin or to VCAM-1. Therefore, p67 LBP expression is associated with increased cellular adherence that is restricted to laminin, consistent with the previously reported specificity of p67 LBP for this β₁-integrin substrate.