Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: T cell receptor triggering and effector responses are abrogated by pMHC elongation in a murine system. A , schematic representation of native and elongated versions of D b SCT containing the indicated membrane-proximal IgSF spacers, tethered to the plasma membrane by the native H-2D b stalk and transmembrane segments. B , surface expression levels of SCT constructs in TAP2-deficient CHO cells. C , primed transgenic T cells expressing the F5 TCR (10 4 T cells/well) were incubated with varying numbers of SCT-expressing CHO cells (denoted as APC/F5 ratio). Culture supernatants were assayed for IL-2 by ELISA after 24 h of incubation. A representative plot of three independent experiments is shown. The error bars represent ± S.D. D , F5/APC conjugates were incubated for 2 min at 37 °C before fixing and stained for phosphotyrosine and H-2D b (SCTs). The conjugates were imaged by confocal immunofluorescence microscopy (Fig. S1A ). Untransfected CHO cells were used to determine basal phosphorylation levels. Phosphotyrosine accumulation at the T cell interface was quantified as the ratio of interface/noninterface fluorescence ( black bars ). At least 25 conjugates were randomly chosen for analysis. Accumulation of SCTs was measured using a similar approach ( supplemental Fig. S1 B ). As a control, conjugates of B3Z hybridomas expressing the noncognate OT1 TCR and CHO cells expressing D b SCT were used. Accumulation of SCT at the interface was quantified for 19–25 randomly chosen conjugates for each SCT ( purple bars ). The error bars represent S.E. The dashed line represents enrichment ratio of 1 (no enrichment) E , fully differentiated F5 CTL were assessed for sensitivity to elongated pMHC expressed at high levels (as in B ) on CHO cells. Cytotoxic responses of F5 CTL to SCTs was tested in a 6-h 51 Cr release assay using CHO cells expressing native and maximally elongated (SCT-CD4) SCTs as targets. The data are representative of two independent experiments. The error bars represent ± S.D. F , cytotoxic responses by F5 CTL to low levels (∼50-fold lower than cells shown in B ) of SCT expression ( inset ). The data are representative of two independent experiments. G , cytolytic activity of F5 CTL and IL-2 release by F5 RAG −/− T cells in response to high levels of SCT expression (as in B ) in the presence of 0–10 μ m PP2 was measured as in C and E . The data are expressed as the percentages of reduction in response relative to responses in the absence of PP2. The concentration of PP2 that gave 50% inhibition (IC 50 ) was derived from error-weighted sigmoidal fitting of the data. The error bars represent ± S.D.

Article Snippet: For IL-2 measurements, culture supernatants and mouse recombinant IL-2 standards (Sigma) were incubated in microtitre plates coated with a capture anti-mouse IL-2 antibody (BD Pharmingen) for 2 h at 37 °C, washed, and incubated for another 1 h with a biotinylated detection anti-mouse IL-2 antibody (BD Pharmingen).

Techniques: Expressing, Construct, Transgenic Assay, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Microscopy, Fluorescence, Release Assay, Activity Assay, Concentration Assay, Inhibition, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: Elongation of a human pMHC (gagSLY/HLA-A2) abrogates activation of G10 T cells expressing cognate TCR. A , schematic representation of single-chain constructs of native and elongated single-chain versions of HLA-A2/gag and corresponding coreceptor binding mutants ( yellow stars ). Constructs were stably expressed in TAP2-deficient CHO cells and sorted for comparable expression for use as surrogate antigen-presenting cells ( supplemental Fig. S3 A ). Also shown are soluble forms of native and elongated constructs comprising the extracellular portion followed by a C-terminal biotin acceptor site ( red dot ) and His 6 tag ( green line ). B , activation of primed G10 T cells was assessed by incubating 10 4 G10 cells/well with varying numbers of SCT-expressing APC (denoted as APC/G10 ratio) and IFNγ release measured by ELISA of culture supernatants after 8 h incubation. A representative plot of three independent experiments is shown. C , the human T cell clone SLY10 was used to assess responses to SCTs with mutated coreceptor-binding sites. Varying numbers of SCT-expressing CHO cells were incubated with 10 4 SLY10 cells/well, and MIP1b release was measured by ELISA of culture supernatants after 24 h of incubation. D , coreceptor binding to native and elongated SCTs was assessed by surface plasmon resonance (BIAcore). Monomeric biotinylated SCTs were immobilized by coupling to streptavidin conjugated CM5 flow cells (∼500 reference units). Binding curves were obtained by injecting CD8αα at a range of concentrations (0.9–154 μ m ) over flow cell surfaces immobilized with native or elongated SCTs. Immobilized biotinylated polyclonal anti-hamster IgG antibody was used as a control surface. The experiments were performed at 25 °C at a flow rate of 5 μl/min. Plateau response units were plotted against CD8αα concentration, and K D values were obtained by nonlinear curve fitting. The data are representative of two independent experiments. E , binding of native and elongated SCTs to G10 TCR was compared by surface plasmon resonance as described for D . The binding curves were obtained by injecting G10 TCR (0.1–7 μ m ) over immobilized SCTs, HLA-A2/NY-ESO, in addition to an irrelevant antibody-bound control surface.

Article Snippet: For IL-2 measurements, culture supernatants and mouse recombinant IL-2 standards (Sigma) were incubated in microtitre plates coated with a capture anti-mouse IL-2 antibody (BD Pharmingen) for 2 h at 37 °C, washed, and incubated for another 1 h with a biotinylated detection anti-mouse IL-2 antibody (BD Pharmingen).

Techniques: Activation Assay, Expressing, Construct, Binding Assay, Stable Transfection, Enzyme-linked Immunosorbent Assay, Incubation, SPR Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: TCR·CD3 triggering but not clustering is diminished by pMHC elongation. A , clean poly- l -lysine coated coverslips were incubated at 37 °C for 2 h with 50 μg/ml streptavidin/PBS. Biotinylated SCTs were immobilized on coverslips at comparable densities (Fig. S3 ). G10 cells were placed on SCT-coated coverslips and incubated for 1 min at 37 °C. The cells were fixed and permeabilized before serial staining CD3, Zap70, and CD45. The images were acquired using uniform settings between experiments. The cells adhered poorly on uncoated coverslips; therefore coverslips coated with biotinylated anti-HLA antibody was used as a control surface (without cognate TCR ligands). The scale bar represents 4 μm. B–D , the mean intensities for CD3, Zap70, and CD45 accumulation at the T cell interface was quantified. For a better appreciation of regions of TCR clustering pseudocolor scaled images of the CD3 distribution are shown (CD3 Scale) in A and E . The regions of CD3 clustering were arbitrarily defined by thresholding at two times the mean CD3 fluorescence intensity at the T cell interface (corresponding to the yellow regions in the pseudocolored scale images) and expressed as percentages of total CD3 interface fluorescence (Fig. S4 ). E , G10 cells were incubated with 10 μ m PP2 for 30 min prior to placing on to SCT-coated coverslips. Preparation and labeling were performed as described above. The scale bar represents 4 μm. The horizontal black bars represent the means. Statistical significance was determined by analysis of variance with correction for multiple comparisons. ns , p > 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: For IL-2 measurements, culture supernatants and mouse recombinant IL-2 standards (Sigma) were incubated in microtitre plates coated with a capture anti-mouse IL-2 antibody (BD Pharmingen) for 2 h at 37 °C, washed, and incubated for another 1 h with a biotinylated detection anti-mouse IL-2 antibody (BD Pharmingen).

Techniques: Incubation, Staining, Fluorescence, Labeling