Immunoprecipitation and immunoblotting
The following antibodies were used in these experiments: rabbit antiserum to ZAP-701; 4G10, a mouse mAb to phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY); biotinylated TCRb chain mAb H57-597 (PharMingen, San Diego, CA); anti-human pan TCRab mAb (Endogen, Woburn, MA); rabbit antiserum to z1; C-14 and C16, rabbit polyclonal antibodies to extracellular signal-regulated kinase (ERK)-1 and –2, respectively (Santa Cruz Biotech., Inc., Santa Cruz, CA); rabbit polyclonal antibodies to SHP-1 (Upstate Biotechnology, Inc., Lake Placid, NY); a mouse mAb to SHP-1 (Transduction Laboratories, Lexington, KY); mouse mAb 3A5 to Lck (Santa Cruz Biotech., Inc., Santa Cruz, CA); mouse mAb and rabbit polyclonal antibodies to HA (Berkeley Antibody Company); and peroxidase-linked goat antibodies to mouse and rabbit Ig (Bio-Rad Laboratories, Richmond, CA). The following antibodies were used as controls for non-specific binding in immunoprecipitations: biotinylated hamster anti-mouse CD80 (PharMingen, San Diego, CA); mouse IgG (Zymed Lab., San Francisco, CA); rabbit polyclonal antibodies to CD3e (Dako, A/S, Denmark); rabbit IgG (Jackson ImmunoResearch). ZAP-70, MAPK, SHP-1, and TCRs were immunoprecipitated by incubation of lysates with optimized amounts of polyclonal antisera or mAb on ice for 4 hr, collected using ImmunoPure immobilized Streptavidin (Pierce, Rockford, IL) (biotinylated hamster Ab), Pansorbin (Calbiochem, La Jolla, CA) (rabbit Ab), or goat anti-mouse IgG Dynabeads (Dynal, Oslo, Norway) (mouse Ab) and analyzed by SDS-PAGE and immunoblotting performed as previously described2. Quantitative data were obtained from multiple film exposures using a Kodak ImageStation 440CF and Kodak Digital Sciences 1D software.
Enzyme
assays
MAPK activity in 5C.C7 T cells was analyzed by flow cytometry after cell fixation with 4% paraformaldehyde in PBS and permeabilization with buffer containing 0.1% saponin, 0.1% BSA, and 0.01 M HEPES, followed by staining with anti-phospho-MAPK mAb (Cell Signaling, Beverly, MA), biotinylated goat anti-mouse antibody (Jackson ImmunoResearch), and PE Streptavidin (PharMingen, San Diego, CA). For measurement of MAPK activity in human 2G4 T cells, immunoprecipitated MAPK samples were incubated with 30 µl of 25 mM HEPES (pH 7.5), containing 10 mM magnesium acetate, 50 µM ATP, 5 µCi of [g-32P]ATP (specific activity 3000 Ci/mM) (NEN, Life Science Products, Boston, MA) and 1 µg of MAPK substrate PHAS-I (Stratagene, La Jolla, CA) for 15 min at 30oC. The MAPK assay with immunoprecipitated TCRs was done in the presence of 0.3 µM protein kinase C (PKC) inhibitor Bisindolylmaleimide I (Calbiochem, La Jolla, CA). Activity of isolated PKC under these conditions was almost completely inhibited (data not shown). Phosphorylated PHAS-I was separated on Pierce phosphocellulose units (Pierce, Rockford, IL), washed twice with 0.75% H3PO4 and incorporated activity measured by liquid scintillation counting. For the protein kinase assay, immunoprecipitated samples were incubated with 50 µl of 25 mM HEPES (pH 7.2), containing 3 mM MnCl2, 0.1% Brij 96, and 5 µCi of [g-32P]ATP (specific activity 3000 Ci/mM) for 15 min at 25˚C. After washing with lysis buffer, proteins were eluted with SDS-PAGE sample buffer and subjected to SDS-PAGE and autoradiography. For the protein phosphatase assay, immunoprecipitates were incubated in 50 µl of an assay buffer containing 100 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 10 mM p-nitrophenyl phosphate (Sigma, St. Louis, MO) at 30˚C for 1 h while shaking. Reactions were stopped by addition of 950 µl of 0.1 M NaOH, the immunoprecipitates were separated by brief centrifugation, and phosphatase activity was assessed by measuring the absorbance at 405 nm of the supernatant. For both human and mouse cells, the duration of MAPK activity was considered to be the interval between the initial peak and the point when the MAPK activity level declined to 50% of this initial peak.
Immunofluorescence imaging
For immunofluorescence staining, T cells were first fixed in 4% paraformaldehyde in PBS for 5 min at 37˚C and incubated with biotinylated mAb H57-597 (PharMingen, San Diego, CA) directed against the mouse TCRb -chain followed by Streptavidin-FITC (Southern Biotechnology). After cell permeabilization with buffer containing 0.1% saponin, 0.1% BSA, 0.01 M HEPES in PBS, SHP-1 was visualized by rabbit antibody to SHP-1 and goat anti-rabbit IgG conjugated to Texas Red (Jackson ImmunoResearch). Images were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Exon, PA USA) using a 100x oil immersion objective (NA 1.4), at a zoom setting of 2.8. Fluorochromes were excited using an argon laser at 488 nm for FITC and a krypton laser at 568 nm for Texas Red. Detector slits were configured to minimize any cross-talk between the channels. Z stacks were collected using 0.203 µm optical sections. DIC (differential interference contrast) images were collected using the transmitted light detector. Images were processed using the Leica TCS-NT/SP software (version 1.6.551) with Imaris 3.0.4 (Bitplane AG, Zurich, Switzerland) and co-localization software (Bitplane AG, Zurich, Switzerland).
1. Burkhardt, A. L. et al. Temporal regulation of non-transmembrane protein tyrosine kinase enzyme activity following T cell antigen receptor engagement. J. Biol. Chem. 269, 23642-7 (1994).
2. Hemmer, B., Stefanova, I., Vergelli, M., Germain, R. N. & Martin, R. Relationships among T cell receptor ligand potency, thresholds for effector function, and the quality of early signaling events in human T cells. J. Immunol. 160, 5807-5814 (1998).
Web Figure
Legends
Web Figure 1. T cell exposure to antagonist leads to SHP-1 recruitment to the TCR complex and functional inhibition
(a-b) Inhibition of single-cell IL-2 responses of 5C.C7 T cells stimulated with the indicated concentrations of agonist peptide PCC in the presence of a fixed concentration (100 mM) of antagonist peptide102G. Data are reported as the percentages of T cells showing intracellular staining for IL-2 above background and as the percentages of response inhibition . (c) The pattern of tyrosine phosphorylation in 5C.C7 T cells stimulated with agonist peptide PCC or with antagonist peptide 102G. T cells were stimulated with APC previously pulsed with PCC or 102G peptide. Tyrosine phosphorylation of ZAP-70 and the z-chain in anti-ZAP-70 immunoprecipitated complexes was visualized by anti-phosphotyrosine immunoblotting. Control lanes marked as (-) involved proteins from T cells exposed to APC with no peptide added. (d,e) Peripheral lymph node T cells from 5C.C7 TCR transgenic RAG–2-/- mice were incubated with APC previously pulsed with the indicated peptide. Control T cells were exposed to APC that had not been exposed to peptide. After 1, 5, and 10 minutes of cell contact, the cells were lysed and (d) ZAP-70 or (e) TCR-associated proteins were immunoprecipitated, then analyzed by anti-phosphotyrosine, anti-ZAP-70, or anti-SHP-1 and anti-z immunoblotting. The amount of the SHP-1 relative to SHP-1 detected in control TCR immunoprecipitates after 1 min. of T-APC contact is indicated below the lanes of the SHP-1 immunoblot.
Web Figure 3. Delayed and transient nature of ERK activation in T cells exposed to partial agonists (a) Summary of MAPK activity kinetics in 2G4 human CD8+ T cells stimulated with APC previously pulsed with Tax or Y5A peptide. The left panel displays the time to the first peak of MAPK activity; the right panel displays the duration of MAPK activity, defined as in Methods. (b) Summary of MAPK activity kinetics in 5C.C7 mouse CD4+ T cells stimulated for varying times with APC prepulsed with MCC or 102S peptide. After stimulation, the cells were fixed and analyzed by flow cytometry using phospho-MAPK antibodies. The left panel displays the time to the first peak of MAPK activity; the right panel displays the duration of MAPK activity, defined as in Methods. Paired t-test P values are indicated in each panel.
