Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling

Abstract

T cell receptor (TCR) microclusters form within seconds of T cell contact with supported planar bilayers containing intercellular adhesion molecule-1 and agonist major histocompatibility complex (MHC)-peptide complexes, and elevation of cytoplasmic Ca2+ is observed within seconds of the first detectable microclusters. At 0-30 s after contact, TCR microclusters are colocalized with activated forms of Lck, ZAP-70, and the linker for activation of T cells. By 2 min, activated kinases are reduced in the older central microclusters, but are abundant in younger peripheral microclusters. By 5 min, TCR in the central supramolecular activation cluster have reduced activated kinases, whereas faint peripheral TCR microclusters efficiently generated activated Lck and ZAP-70. TCR microcluster formation is resistant to inhibition by Src family kinase inhibitor PP2, but is abrogated by actin polymerization inhibitor latrunculin A. We propose that Src kinase-independent formation of TCR microclusters in response to agonist MHC-peptide provides an actin-dependent scaffold for signal amplification.

Figure 1.

Dynamic of TCR microclusters during formation of a mature IS. (A) A series of images of Alexa546-H57 Fab–labeled AND TCR Tg T cell blasts interacting with planar bilayer containing ICAM-1 and agonist MHCp at times indicated. Time is relative to first detected contact area. This sequence is representative of three experiments with at least 20 cells observed in each. (B) Alexa546-H57 Fab and Fluo-LOJO dye–labeled AND TCR Tg T cell blasts interacting with planar bilayers containing ICAM-1 and agonist MHCp at early times. TIRFM of Alexa546 anti-TCR to visualize microcluster formation (top row) and wide field image of Fluo-LOJO fluorescence 4 μm above the contact plane indicates relative cytoplasmic Ca²⁺ (bottom row). The TIRFM image was acquired 1 s before the Fluo-LOJO image. Representative of three experiments.

Figure 2.

Relationship of pLck³⁹⁴, pZAP-70³¹⁹, and pLAT¹⁹¹ to TCR microclusters during IS formation. Alexa546-H57 Fab–labeled AND TCR Tg T cells were incubated on bilayers containing ICAM-1 and MHCp. At different times after cell injection, the cells were fixed to yield contacts that formed between 0–0.5 min, 1–2 min, or 4–5 min. Cell were permeabilized and stained with antibodies to pLck³⁹⁴ (A), pZAP³¹⁹ (B), and pLAT¹⁹¹ (C) followed by Alexa488-conjugated secondary antibodies. TCR- (red in overlay) and phosphoprotein-specific antibodies (green in overlay) were imaged by wide-field fluorescence microscopy. (D) Colocalization was quantified based on the Improvision colocalization algorithm (classifier threshold 1.5×). Microclusters from at least 10 contact areas were included for each data point.

Figure 2.

Relationship of pLck³⁹⁴, pZAP-70³¹⁹, and pLAT¹⁹¹ to TCR microclusters during IS formation. Alexa546-H57 Fab–labeled AND TCR Tg T cells were incubated on bilayers containing ICAM-1 and MHCp. At different times after cell injection, the cells were fixed to yield contacts that formed between 0–0.5 min, 1–2 min, or 4–5 min. Cell were permeabilized and stained with antibodies to pLck³⁹⁴ (A), pZAP³¹⁹ (B), and pLAT¹⁹¹ (C) followed by Alexa488-conjugated secondary antibodies. TCR- (red in overlay) and phosphoprotein-specific antibodies (green in overlay) were imaged by wide-field fluorescence microscopy. (D) Colocalization was quantified based on the Improvision colocalization algorithm (classifier threshold 1.5×). Microclusters from at least 10 contact areas were included for each data point.

Figure 3.

Colocalization of pLck³⁹⁴ and pZAP³¹⁹ with peripheral TCR microclusters in the IS. Cells were prepared exactly as for 4–5-min time point in Fig. 2, and then TCR (red in overlay) and the indicated phosphoprotein (green in overlay) were imaged by TIRFM for cells stained with pLck³⁹⁴ (A) and pZAP³¹⁹ (B). Examples of locations where peripheral TCR clusters colocalize with active kinases are indicated with arrowheads; positions where the large central TCR clusters contained active kinases are indicted by an arrow. The dividing line between central and peripheral structures used for calculation of the signaling efficiency is indicated in the overlay by the dotted white line. Representative of three experiments.

Figure 4.

Effect of inhibition of Src family kinases and actin polymerization on TCR microclusters. Alexa546-H57 Fab–labeled AND TCR Tg T cells were incubated on bilayers containing ICAM-1 and MHCp after treatment by relevant drug vehicles, 10 μM PP2 or 1 μM latrunculin A. These concentrations inhibit Src family kinases and actin polymerization, respectively, >90% in intact cells. In each case, cells were treated with the drugs for 1 h before introduction to the planar bilayers. (A) TIRFM time course of TCR clustering (top) in the presence of inhibitory concentration of PP2. Activity of PP2 was verified by lack of robust Ca²⁺ elevation (bottom). (B) Reduction of pLck³⁹⁴ in TCR microclusters in the presence of 100 μM PP2 versus DMSO vehicle at 5 min. (wide-field images) Similar results were obtained by TIRFM. (C) Wide-field image of unlabeled AND T cell blasts on bilayers containing Oregon Green I-E^k (green in overlay) and Cy5 ICAM-1 (red in overlay) after cell treatment with vehicle, 10 μM PP2 or 1 μM latrunculin A, for 1 h before exposure to the bilayer for 1 h. The IRM image shows the contact area in darker gray. Individual fluorescence images are as indicated. The percent adhesion is indicted in the column adjacent to the images. Representative of three experiments each.