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. S3A). 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 His₆ tag (green line). B, activation of primed G10 T cells was assessed by incubating 10⁴ 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 10⁴ 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 μm) 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 μm) over immobilized SCTs, HLA-A2/NY-ESO, in addition to an irrelevant antibody-bound control surface.

Small pMHC dimensions allow efficient segregation from CD45 following TCR engagement. A, experiments were performed as in Fig. 3. The data were acquired as 8-bit 1024 × 1024 pixel images. B, background subtracted images acquired in the CD3 and CD45 channels were analyzed for colocalization by determining Manders colocalization coefficients using ImageJ. The mean proportion of CD3 colocalized with CD45 expressed as a percentage (Manders M1 coefficient) was calculated from 20 interfaces chosen at random for each coated surface. Manders M2 coefficient (proportion of CD45 colocalized with CD3) is 100% for all. The horizontal black bars represent the means. Statistical significance was determined by analysis of variance with correction for multiple comparisons. *, p < 0.05; ***, p < 0.001. The scale bar represents 4 μm.

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^bSCT 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 (10⁴ 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. S1B). As a control, conjugates of B3Z hybridomas expressing the noncognate OT1 TCR and CHO cells expressing D^bSCT 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 ⁵¹Cr 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 μm 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 (IC₅₀) was derived from error-weighted sigmoidal fitting of the data. The error bars represent ± S.D.

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 μm 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.