Figure 1
Figure 1. AKAP450 is required for the correct configuration and activation of LFA-1 at the IS. (A) J77 cells expressing GFP or C-term AKAP450-GFP were incubated with SEE-pulsed Raji B cells. Conjugates were stained with antibodies to LFA-1 (blue) and CD3ζ (red). Confocal z slices and corresponding 3-dimensional reconstruction of SMAC area (right) are shown. Histograms show the fluorescence intensity profiles of staining for CD3 (red) and LFA-1 (blue) along the diagonal black lines in the DIC images. Graph represents quantification of immune synapse with LFA-1 ring showing correct architecture. Data are the arithmetic mean ± SD of correct LFA-1 segregation. *P < .05 compared with J77 cells transfected with GFP (Student t test). (B) T lymphoblasts expressing GFP or C-term AKAP450-GFP were incubated with SEE-pulsed Raji B cells. Conjugates were stained with antibodies to LFA-1 (red). Confocal z slices and corresponding 3-dimensional reconstruction of SMAC area (right) are shown. Scale bars represent 10 μm. Graph represents quantification of immune synapse with LFA-1 ring showing correct architecture. Data are the arithmetic mean ± SD of correct LFA-1 segregation. *P < .05 compared with J77 cells transfected with GFP (Student t test). (C) Cell conjugates were formed between CH7 cells expressing GFP or C-term AKAP450-GFP and HA-stimulated HOM-2 APCs. Cells were stained with the antibodies to active conformation of LFA-1 (red), m24 (top panel), and KIM127 (bottom panel). A confocal z section of conjugates formed and processed as in panel A is shown (3-dimensional). Quantification of the active LFA-1 accumulation (m24, top panel; KIM127, bottom panel) at the IS. A total of 50 conjugates were counted for each condition. *P < .01 compared with CH7C17 cells transfected with GFP control (Mann-Whitney test). (A-C) Arrowheads in DIC images indicate the cell-cell junction selected for 3-dimensional projection. Yellow arrows indicate the positions of immune synapses; blue arrow, the position of the mislocalized MTOC in cells overexpressing C-term AKAP450-GFP. Asterisks in DIC images identify CMAC-loaded Raji APCs. Scale bars represent 10 μm.

AKAP450 is required for the correct configuration and activation of LFA-1 at the IS. (A) J77 cells expressing GFP or C-term AKAP450-GFP were incubated with SEE-pulsed Raji B cells. Conjugates were stained with antibodies to LFA-1 (blue) and CD3ζ (red). Confocal z slices and corresponding 3-dimensional reconstruction of SMAC area (right) are shown. Histograms show the fluorescence intensity profiles of staining for CD3 (red) and LFA-1 (blue) along the diagonal black lines in the DIC images. Graph represents quantification of immune synapse with LFA-1 ring showing correct architecture. Data are the arithmetic mean ± SD of correct LFA-1 segregation. *P < .05 compared with J77 cells transfected with GFP (Student t test). (B) T lymphoblasts expressing GFP or C-term AKAP450-GFP were incubated with SEE-pulsed Raji B cells. Conjugates were stained with antibodies to LFA-1 (red). Confocal z slices and corresponding 3-dimensional reconstruction of SMAC area (right) are shown. Scale bars represent 10 μm. Graph represents quantification of immune synapse with LFA-1 ring showing correct architecture. Data are the arithmetic mean ± SD of correct LFA-1 segregation. *P < .05 compared with J77 cells transfected with GFP (Student t test). (C) Cell conjugates were formed between CH7 cells expressing GFP or C-term AKAP450-GFP and HA-stimulated HOM-2 APCs. Cells were stained with the antibodies to active conformation of LFA-1 (red), m24 (top panel), and KIM127 (bottom panel). A confocal z section of conjugates formed and processed as in panel A is shown (3-dimensional). Quantification of the active LFA-1 accumulation (m24, top panel; KIM127, bottom panel) at the IS. A total of 50 conjugates were counted for each condition. *P < .01 compared with CH7C17 cells transfected with GFP control (Mann-Whitney test). (A-C) Arrowheads in DIC images indicate the cell-cell junction selected for 3-dimensional projection. Yellow arrows indicate the positions of immune synapses; blue arrow, the position of the mislocalized MTOC in cells overexpressing C-term AKAP450-GFP. Asterisks in DIC images identify CMAC-loaded Raji APCs. Scale bars represent 10 μm.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal