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The Journal of Immunology, 2004, 173: 2222-2226.
Copyright © 2004 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: LFA-1 Integrin-Dependent T Cell Adhesion Is Regulated by Both Ag Specificity and Sensitivity1

Kristen L. Mueller, Mark A. Daniels2, Alicia Felthauser, Charlly Kao, Stephen C. Jameson and Yoji Shimizu3

Department of Laboratory Medicine and Pathology, Center for Immunology, Cancer Center, University of Minnesota Medical School, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Ab stimulation of the TCR rapidly enhances the functional activity of the LFA-1 integrin. Although TCR-mediated changes in LFA-1 activity are thought to promote T cell-APC interactions, the Ag specificity and sensitivity of TCR-mediated triggering of LFA-1 is not clear. We demonstrate that peptide/MHC (pMHC) tetramers rapidly enhance LFA-1-dependent adhesion of OT-I TCR transgenic CD8+ T cells to purified ICAM-1. Inhibition of src family tyrosine kinase or PI3K activity blocked pMHC tetramer- and anti-CD3-stimulated adhesion. These effects are highly specific because partial agonist and antagonist pMHC tetramers are unable to stimulate OT-I T cell adhesion to ICAM-1. The Ag thresholds required for T cell adhesion to ICAM-1 resemble those of early T cell activation events, because optimal LFA-1 activation occurs at tetramer concentrations that fail to induce maximal T cell proliferation. Thus, TCR signaling to LFA-1 is highly Ag specific and sensitive to low concentrations of Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The interaction of the LFA-1 ({alpha}L{beta}2; CD11a/CD18) integrin with its ligand, ICAM-1, is believed to be a critical event in facilitating the sustained interactions between T cells and APCs that are necessary for T cell activation (1, 2, 3, 4). The ability of anti-CD3 Abs to rapidly enhance LFA-1-dependent adhesion of T cells to ICAM-1 (1) is consistent with models where engagement of a small number of TCRs with cognate peptide/MHC (pMHC)4 initiates intracellular signaling events that promote LFA-1-dependent interactions between T cells and APCs that are critical for initiating the full spectrum of signaling pathways necessary for complete T cell activation (4, 5). Although Ag-dependent T cell-APC interactions are largely mediated by rapid increases in LFA-1 activity, the effects of Ag sensitivity and specificity on changes in LFA-1 activity are not known.

In this study, we used specific pMHC tetramers to trigger the activation of purified TCR transgenic T cells. These tetramers are capable of engaging up to three TCRs and have been used to identify Ag-specific T cells (6, 7, 8). At certain concentrations, pMHC tetramers can also induce a variety of T cell responses, including calcium flux, proliferation, cytokine secretion, and differentiation into cytolytic effectors (8, 9, 10). pMHC tetramers have allowed us to investigate changes in LFA-1-dependent adhesion to purified ligand induced by defined antigenic peptides. Using this system, we demonstrate that pMHC tetramers rapidly induce increases in LFA-1-dependent T cell adhesion to ICAM-1 that are both highly sensitive to Ag and display a high degree of Ag specificity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice

OT-I mice (11) were kindly provided by Dr. K. Hogquist (University of Minnesota, Minneapolis, MN). All mice were used between 6 and 12 wk of age. All experimental protocols involving the use of mice were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.

T cell purification

T cells from OT-I mice were isolated by negative selection using MACS (Miltenyi Biotec, Auburn, CA) magnetic separation technology as previously described (12). Cells were >95% CD3+ as assessed by flow cytometric analysis using a PE-conjugated anti-CD3 mAb (eBioscience, San Diego, CA).

Peptides and MHC tetramers

The peptides OVA (SIINFEKL), SIY (SIYRYYGL), A6 (SIYRYAGL), G4 (SIIGFEKL), and E1 (EIINFEKL) have been described previously (13, 14) and were synthesized by Research Genetics (Carlsbad, CA). pMHC tetramers were prepared as previously described (6, 8, 15). Monomeric pMHC complexes were biotinylated using BirA (Avidity, Denver, CO) and purified using fast protein liquid chromatography on a Superdex 200 column (Amersham Biosciences, Piscataway, NJ). Fractions were concentrated and incubated with streptavidin PE (Molecular Probes, Eugene, OR) at an 8:1 molar ratio.

Adhesion assays

Adhesion assays were performed as previously described (16) by assessing adhesion of purified OT-I T cells labeled with calcein acetoxymethyl ester to 96-well microtiter plates (Costar, Cambridge, MA) precoated with murine recombinant ICAM-1/Fc chimera (R&D Systems, Minneapolis, MN) at 0.05–0.3 µg/well. Function-blocking anti-CD11a mAb (clone M17/4; eBioscience) or anti-CD49e (integrin {alpha}5 chain) (clone 5H10-27; BD Pharmingen, San Diego, CA) were added to the appropriate wells at 10 µg/ml. Inhibitors were used at the following concentrations: 30 nM wortmannin (Sigma-Aldrich, St. Louis, MO), 30 µM LY294,002 (Calbiochem, San Diego, CA) and 30 nM PP1 (Calbiochem). Cells were activated for the indicated time periods, and nonadherent cells were removed by washing.

Western blotting

Immunoprecipitation and Western blotting was performed as previously described (16). Briefly, 106 T cells were preincubated on ice for 15 min with anti-CD3 mAb 2C11 (2 µg/ml) or pMHC tetramer (for concentrations, see Figs. 1–4). Cells were rapidly warmed to 37°C for 10 min and lysed by adding an equivalent volume of lysis buffer. Immunoprecipitations were performed by incubating anti-linker for activation of T cells (LAT) Ab (Upstate Biotechnology, Lake Placid, NY)-coated protein A-Sepharose beads with cellular lysates. Samples were separated on 10% precast SDS-polyacrylamide gels (Invitrogen Life Technologies, Carlsbad, CA), transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and immunoblotted with an anti-LAT Ab or the anti-phosphotyrosine mAb 4G10 (both from Upstate Biotechnology) (1/5,000 dilution for anti-LAT; 1/1,000 dilution for 4G10 in PBS/3% milk/0.5% Tween) followed by appropriate dilutions of HRP-conjugated secondary Abs (1/25,00 for anti-LAT or 1/15,000 for 4G10). Membranes were developed by ECL (Pierce, Rockford, IL).



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  		FIGURE 1. pMHC tetramers induce LFA-1-dependent T cell adhesion to ICAM-1. A, Adhesion of OT-I T cells to mouse ICAM-1 was assessed following no stimulation (U), or following stimulation for 10 min at 37°C with 1 µg/ml KbA6, PMA, anti-CD3 mAb, or 1 µg/ml KbOVA. B, Adhesion of purified OT-I T cells to purified ICAM-1 was assessed as in A, but in the presence of 10 µg/ml function-blocking anti-CD11a mAb (Anti-LFA-1) or control anti-CD49e mAb (Control). Results are represented as the mean percentage of adhesion of triplicate wells ± SD.

 


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  		FIGURE 2. LFA-1-dependent T cell adhesion requires a high-affinity peptide ligand. A, Adhesion of OT-I T cells to mouse ICAM-1 was assessed following stimulation for 10 min at 37°C with the indicated concentrations of KbOVA, KbG4, KbE1, or KbSIY. B, Adhesion of OT-I T cells to mouse ICAM-1 was assessed following stimulation with 1 µg/ml KbOVA, KbG4, or KbA6 for 10, 20, 30, or 60 min. Results are represented as the mean percentage of adhesion of triplicate wells ± SD.

 


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  		FIGURE 3. Anti-CD3 mAb and pMHC tetramer-stimulated T cell adhesion to ICAM-1 are regulated by similar mechanisms. Adhesion of OT-I T cells to mouse ICAM-1 was assessed following stimulation for 10 min at 37°C with 1 (A) or 10 (B) µg/ml KbA6, PMA, anti-CD3 mAb, or 1 (A) or 10 (B) µg/ml KbOVA following 30-min preincubation in 10 µM PP1 (A) or 30 nM wortmannin (B) or DMSO (vehicle). Results are represented as the mean percentage of adhesion of triplicate wells ± SD.

 


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  		FIGURE 4. pMHC tetramers stimulate LFA-1-dependent adhesion at low concentrations and display Ag thresholds similar to other early T cell activation events. A, Purified OT-I T cells were left unstimulated (U) or stimulated with the indicated concentrations of KbOVA, 10 µg/ml KbSIY (SIY), or anti-CD3 mAb (CD3) for 5 min, immunoprecipitated with anti-LAT, and immunoblotted with the anti-phosphotyrosine mAb 4G10 (top panel), or an anti-LAT Ab (bottom panel). B, Purified OT-I T cells were stimulated with the indicated concentrations of KbG4 or KbE1, 10 µg/ml KbSIY (SIY) or 10 µg/ml KbOVA (OVA) for 5 min, and analyzed for LAT tyrosine phosphorylation as in A. C, OT-I T cells were stimulated with the indicated concentrations of KbOVA as described in Materials and Methods. Relative activation responses were calculated for OT-I T cell adhesion to ICAM-1, blastogenesis, CD69 expression, and proliferation. Blastogenesis and CD69 expression were assessed by flow cytometry following 3 h of stimulation. Proliferation was assayed by [3H]thymidine incorporation following 60 h of stimulation. Relative activation was calculated as follows: (response following stimulation with indicated concentration of KbOVA – unstimulated response)/(response following stimulation with 10 µg/ml KbOVA – unstimulated response) x 100.

 
Flow cytometry

Increased cell size (blastogenesis) and CD69 expression were assessed by flow cytometry. Purified OT-I T cells were stimulated with pMHC tetramers (see Fig. 4 for concentrations) for 3 h at 37°C in 96-well microtiter plates. Cells were harvested and stained with an allophycocyanin-conjugated anti-CD8 mAb (eBioscience). Blastogenesis was assessed using the forward scatter of CD8+ events. Cells were considered to be blasting if their forward scatter was increased compared with the forward scatter of cells stimulated with 10 µg/ml KbA6 or KbSIY control pMHC tetramers. To assess CD69 expression, cells were stained with anti-CD69-FITC (eBioscience).

Proliferation assays

Purified OT-I T cells were cultured with KbOVA (see Fig. 4 for concentrations) or 10 µg/ml KbA6 or KbSIY for 60 h at 37°C. Cells were pulsed for 8 h with [3H]thymidine and counted on a beta plate reader.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
TCR stimulation by agonist pMHC tetramers induces LFA-1-dependent T cell adhesion to ICAM-1

We used the OT-I TCR transgenic mouse system (11) to determine whether TCR stimulation by pMHC tetramers is capable of enhancing LFA-1 functional activity. T cells from OT-I mice express a transgenic TCR that is H2-Kb restricted and specific for the chicken OVA peptide fragment SIINFEKL (OVA). Incubation of purified OT-I T cells with H2-Kb tetramers presenting OVA (KbOVA) resulted within 10 min in increased adhesion to purified mouse ICAM-1 (Fig. 1A). In contrast, Kb tetramers presenting control peptides SIYRYAGL (KbA6) or SIYRYYGL (KbSIY), which are not recognized by the OT-I TCR, were unable to enhance OT-I T cell adhesion to ICAM-1 above background levels (Fig. 1A and data not shown). This suggests that Ag-specific stimulation of T cells can rapidly induce enhanced LFA-1-dependent adhesion of T cells to ICAM-1. The percentage of adherent T cells following KbOVA stimulation was similar to the percentage of T cells that adhered following stimulation by anti-CD3 mAb or with the phorbol ester, PMA, which induces T cell adhesion by directly activating protein kinase C (1) (Fig. 1A). Enhanced T cell adhesion to ICAM-1 observed following stimulation with KbOVA was mediated by the integrin, LFA-1, because T cell adhesion to ICAM-1 was inhibited by function-blocking Abs against the {alpha}L subunit of LFA-1, CD11a (Fig. 1B). Together, these data demonstrate that Ag stimulation of the TCR, in the form of pMHC tetramers, can induce increases in T cell adhesion to ICAM-1 that are dependent on the integrin, LFA-1. KbOVA was also able to stimulate T cell adhesion to ICAM-2 and VCAM-1 (data not shown), suggesting that KbOVA stimulation enhances the functional activity of multiple integrins expressed on T cells.

LFA-1-dependent T cell adhesion is specific to high-affinity ligands

The ability of pMHC tetramers to enhance LFA-1-dependent T cell adhesion to ICAM-1 allowed us to assess the effects of peptide ligand affinity on TCR-mediated activation of LFA-1. Although partial peptide agonists are capable of stimulating T cell activation in the OT-I system, they do so with slower kinetics and several early activation events are not observed (17). To determine whether altered peptide ligands are capable of enhancing LFA-1 functional activity, we used pMHC tetramers that provide an agonist, partial agonist, antagonist, or nonactivating signal to the OT-I TCR. Of the various peptide ligands examined, only the full agonist ligand, KbOVA, was able to induce dose-dependent increases in OT-I T cell adhesion to ICAM-1 (Fig. 2A). In contrast, the partial agonist KbG4, the antagonist KbE1, and the nonactivating KbSIY peptide ligands resulted in similar levels of T cell adhesion to ICAM-1 that did not increase upon increasing concentrations of pMHC tetramer (Fig. 2A). These results suggest that rapid Ag-induced increases in integrin-mediated adhesion are dependent on engagement of the TCR with a high-affinity peptide ligand. The delayed response of OT-I T cells to partial peptide agonists observed in previous studies (17) may thus be due in part to the inability of these ligands to enhance LFA-1 function, which is critical for optimal interactions of T cells with APCs.

Because G4 peptide stimulates OT-I T cell activation with slower kinetics than OVA peptide (17), we also examined the kinetics of T cell adhesion to ICAM-1 following pMHC tetramer stimulation. T cell adhesion to ICAM-1 peaked at 10 min and steadily declined up to 60 min following stimulation with KbOVA (Fig. 2B). Stimulation with anti-CD3 mAb or PMA showed a similar trend (data not shown). Unlike KbOVA, KbG4 stimulation resulted in T cell adhesion to ICAM-1 similar to background levels at all of the time points tested (Fig. 2B), suggesting that a partial agonist signal is insufficient to stimulate integrin-dependent T cell adhesion.

Ag- and anti-CD3 mAb-stimulated TCR signaling to LFA-1 is blocked by inhibitors of src family tyrosine kinases and PI3K

Both the src family tyrosine kinase, Lck, and the lipid kinase PI3K are involved in regulation of integrin activity mediated by anti-CD3 mAb stimulation of the TCR (18, 19, 20). We used pharmacological inhibitors to investigate whether Lck and PI3K also regulate pMHC-stimulated T cell adhesion to ICAM-1. Incubation of OT-I T cells with the src family tyrosine kinase inhibitor PP1 blocked both KbOVA-induced and anti-CD3 mAb-induced T cell adhesion to ICAM-1 to comparable levels (Fig. 3A). PP1 did not inhibit T cell adhesion induced by PMA, suggesting that the inhibitor does not globally impair LFA-1 function. The PI3K inhibitors wortmannin and LY294,002 also partially inhibited the increased LFA-1 activity stimulated by both anti-CD3 mAb and KbOVA (Fig. 3B and data not shown). Similar to our results with PP1, the degree of inhibition of anti-CD3 mAb-induced adhesion and KbOVA-induced adhesion by the PI3K inhibitors was comparable. In addition, inhibition of adhesion was specific, because PMA-stimulated adhesion was unaffected by wortmannin or LY294,002. Although a previous study using inhibitors suggested differences in the intracellular signaling pathways that regulate integrin activation induced by anti-CD3 Ab vs peptide Ag (21), our results suggest that src family tyrosine kinases and PI3K participate in changes in LFA-1 functional activity induced by both anti-CD3 mAb stimulation and KbOVA stimulation of the TCR.

TCR signaling to LFA-1 is sensitive to low concentrations of Ag

Because Ag-induced changes in LFA-1 avidity are both rapid and specific to high-affinity peptide ligands, LFA-1 may be uniquely suited to promote T cell interactions with APCs upon engagement of a small number of TCRs with cognate pMHC. To determine whether LFA-1 activity is sensitive to low concentrations of Ag, we examined the dose response of KbOVA-induced T cell adhesion to ICAM-1. Adhesion of OT-I T cells to ICAM-1 above the basal levels of adhesion in the presence of control pMHC tetramers could be clearly detected at KbOVA concentrations as low as 0.1 µg/ml, with a response that peaks at 1 µg/ml KbOVA (Fig. 2A). These studies demonstrate that KbOVA doses >0.01 µg/ml induce strong increases in T cell adhesion to ICAM-1.

To compare the Ag sensitivity of increases in LFA-1 activity to other T cell activation events that occur rapidly following ligation of the TCR with Ag, we used KbOVA tetramers to stimulate phosphorylation of the T cell-specific adapter protein LAT. LAT is rapidly phosphorylated on multiple tyrosine residues following T cell activation (22) and is critical for TCR-mediated integrin activation in human T cells (16). Stimulation with 10, 1, or 0.1 µg/ml KbOVA for 5 min resulted in tyrosine phosphorylation of LAT, as detected by anti-phosphotyrosine immunoblotting of anti-LAT immunoprecipitates (Fig. 4A). Because similar KbOVA concentrations were able to stimulate increases in LFA-1-dependent T cell adhesion, these results suggest that the Ag thresholds required for phosphorylation of LAT, an early marker of T cell activation, are similar to the Ag thresholds necessary for induction of LFA-1 activity. We also observed that the altered peptide ligands KbG4 and KbE1 were unable to induce tyrosine phosphorylation of LAT, even at the highest pMHC tetramer concentration tested (Fig. 4B). This is consistent with the inability of KbG4 and KbE1 to induce T cell adhesion to ICAM-1 and suggest that these early T cell activation events require a high degree of Ag specificity.

Because similar pMHC tetramer concentrations are able to stimulate LAT tyrosine phosphorylation and LFA-1-dependent adhesion, we next sought to determine whether the Ag sensitivity and specificity of LFA-1 activity closely resembled the Ag sensitivity and specificity of other well-established T cell activation markers, such as increased cell size (blastogenesis), induction of expression of the CD69 activation Ag, and proliferation. We stimulated purified OT-I T cells with various concentrations of KbOVA, and then assessed blastogenesis by determining the percentage of T cells under each culture condition that exhibited enhanced forward-scatter properties 3 h after stimulation. CD69 expression on activated T cells was also assessed by flow cytometry at the same time point. T cell proliferation was assessed by tritiated thymidine incorporation 60 h after stimulation. To be able to compare these different activation responses, we determined the relative activation response by normalizing the responses observed at various doses of KbOVA to the response observed at the highest dose of KbOVA tested (10 µg/ml), which was given an activation value of 100.

Similar to the results obtained with KbOVA-induced OT-I T cell adhesion to ICAM-1 and tyrosine phosphorylation of LAT, KbOVA-induced blastogenesis was clearly induced at KbOVA concentrations of 0.1 µg/ml or higher (Fig. 4C). Below 0.1 µg/ml KbOVA, much lower levels of blastogenesis were observed. Similar results were obtained when analyzing CD69 expression, although a maximal response occurred with 1 µg/ml KbOVA. The altered peptide ligands KbG4 and KbE1 were unable to induce either blastogenesis or CD69 expression at any of the pMHC tetramer concentrations tested (data not shown). In contrast, much higher amounts of KbOVA were required to induce optimal T cell proliferation. At 0.1 µg/ml KbOVA, proliferation was <20% of the level observed at the highest KbOVA concentration tested, 10 µg/ml. Even at 1 µg/ml KbOVA, which induces optimal levels of T cell adhesion to ICAM-1, blastogenesis, and CD69 expression, proliferation was still <50% of the level observed with 10 µg/ml KbOVA. These results suggest that, similar to other early T cell activation events, Ag-stimulated integrin-dependent T cell adhesion displays a low threshold for Ag.

These data demonstrate that induction of LFA-1-dependent T cell adhesion by Ag requires a high-affinity peptide ligand and is sensitive to low concentrations of Ag. The role of Ag sensitivity and specificity in regulating LFA-1 activity has remained undefined, because previous studies used anti-CD3 mAb to stimulate integrin-dependent adhesion to purified ligands (1). By using pMHC tetramers, we were able to assess Ag-dependent regulation of LFA-1 adhesion to purified ligand for the first time. We demonstrate that only a high-affinity peptide ligand, KbOVA, is sufficient to induce LFA-1-dependent adhesion to ICAM-1 in a short kinetic window. These results are consistent with in vitro conjugate data demonstrating that only OVA peptide, and not G4 or E1 peptide, can induce OT-I T cell conjugation with Ag-pulsed APCs (23). In addition, neither weak agonist nor antagonist peptide ligands are able to stimulate formation of the immunological synapse (23, 24), suggesting that LFA-1 activation by high-affinity peptide ligand is particularly critical to subsequent formation of the immunological synapse.

In addition to showing high specificity for Ag, rapid increases in LFA-1 activity are also highly sensitive to Ag concentration. We demonstrate that the concentrations of Ag that stimulate optimal levels of LFA-1 activity are similar to those that stimulate early T cell activation events such as blastogenesis, CD69 activation, and tyrosine phosphorylation of LAT. However, low concentrations of Ag that stimulated these early T cell activation events were not sufficient to induce optimal T cell proliferation. These results suggest that, although low Ag concentrations can induce signaling cascades that initiate early T cell activation events, higher concentrations of Ag are necessary to fully activate a T cell. Our data are consistent with recent models that propose that initial contact with Ag-pulsed APCs results in the ligation of a small number of TCRs with cognate pMHC, which transduces a signal sufficient enough to increase the avidity of LFA-1 for ICAM-1 (4). This increased adhesiveness between the T cell and APC is critical for further ligation of TCRs with cognate pMHC, resulting in immunological synapse formation and induction of signaling cascades necessary for full T cell activation. Thus, the high Ag specificity and sensitivity of LFA-1 activation promotes productive T cell contact with rare, Ag-expressing APCs.


   Acknowledgments
 
We thank Dr. K. Hogquist for providing the OT-I mice and A. Quale for technical assistance.


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

1 This work was supported by National Institutes of Health Grant AI38474 (to Y.S.), the Harry Kay Chair in Biomedical Research (to Y.S.), and National Institutes of Health Training Grant AI07313 (to K.L.M.). Back

2 Current address: Transplantation and Immunology, University Hospital Basel, Department of Research, Basel, Switzerland. Back

3 Address correspondence and reprint requests to Dr. Yoji Shimizu, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Mayo Mail Code 334/Room 6-112 Basic Sciences and Biomedical Engineering Building, Minneapolis, MN 55455. E-mail address: shimi002{at}umn.edu Back

4 Abbreviations used in this paper: pMHC, peptide/MHC; LAT, linker for activation of T cells. Back

Received for publication April 6, 2004. Accepted for publication June 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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