PMC

Th1 (A) and Th2 (B) cells were injected onto supported planar bilayers containing ICAM-1-Cy5 (red) and peptide-loaded I-E^k-488 (green). Wide-field fluorescence microscopy was used to image T-cell:APC contacts. One set of ICAM-1-Cy5/I-Ek-488 images was obtained every minute. The time indicated is relative to the initial detection of T cell-bilayer contact. MHC/TCR clusters did not coalesce into a cSMAC in most Th2 IS. Data are from one representative experiment of two with n=14 for Th1 cells and n=83 for Th2 cells. Scale bars represent 5 μm.

Th1 (A) and Th2 (B) cells were incubated with MCC:GFP fibroblasts for 30 minutes. The conjugates were then fixed, permeabilized and stained with a biotinylated antibody to phosphotyrosine followed by Cy-5-conjugated streptavidin (red). Following deconvolution, 3-D reconstructions of the T cell:APC interfaces were made. (C) A greater percentage of the MHC/TCR at the T cell-APC interface is colocalized with pTyr in Th2 cells. Colocalization analysis was performed as in . The difference between Th1 and Th2 cells is significant (P = 0.01; Student’s unpaired t test) Data are representative of three independent experiments with n=32 for Th1 cells and n=28 for Th2 cells. Scale bars represent 5 μm.

For these experiments, 10 or 1 μM MCC was used during the peptide-loading step instead of 100 μM MCC, as in the preceding figures. Th1 (A and B) or Th2 (C and D) cells were incubated on supported planar bilayers containing ICAM-1-Cy5 (red) and I-E^k-488 (green) loaded with 10 μM MCC (A and C) or 1 μM MCC (B and D) for 15 minutes prior to imaging. Images were then captured between 15 and 35 minutes. Cells marked with an asterisk were scored as multifocal for MHC/TCR. Data are from one representative experiment of three. Scale bars represent 5 μm.

Th1 and Th2 cells were incubated on supported planar bilayers containing ICAM-1-Cy5 (red) and peptide-loaded I-E^k-488 (green) for 20 minutes prior to imaging. Images were then captured between 20 and 35 minutes. (A) Th1 cells consistently formed IS with a compact, monofocal accumulation of MHC/TCR. (B) The majority of Th2 cells formed multifocal IS. (C) Th2 cells were imaged over time, 20 min after IS formation. Th1 (D) and Th2 (E) cells were allowed to interact with MCC:GFP fibroblast APC for 30 minutes. Conjugates were fixed, permeabilized and stained with an antibody to LFA-1 followed by a Cy-5-conjugated secondary antibody. Following deconvolution, 3-D reconstructions of T-cell:APC interfaces were made. En face views from the 3D reconstructions of T cell:APC interfaces are shown. Data are representative of two experiments with n=20 for Th1 cells and n=19 for Th2 cells. Scale bars represent 5 μm.

Th1 (A) and Th2 (B) cells were incubated with MCC:GFP fibroblast APC for 10 minutes, fixed, permeabilized and stained with an antibody to CD45 followed by a Texas Red-conjugated secondary antibody (red). 3-D reconstructions of T-cell:APC interfaces were made after deconvolution. (C) Conjugates were scored for the exclusion of CD45 from the center of the T cell-APC interface. (D) Although CD45 is not excluded from the center of the T cell-APC interface in Th2 cells, there is not a significant difference in the colocalization between MHC/TCR and CD45 in Th1 and Th2 cells. Colocalization analysis was conducted with Imaris software from Bitplane using the thresholding technique. The percentage of the total volume of MHC/TCR at the T cell-APC interface that was colocalized with CD45 is shown. The difference between Th1 and Th2 cells was not statistically significant (P=0.3; Student’s t Test). Data shown are representative of three independent experiments with n=32 for Th1 cells and n=33 for Th2 cells. Scale bars represent 5 μm.