Data Supplement

Files in this Data Supplement:

• Video 1 (MOV, 7.02 MB)
  - Intravital imaging of transferred wild type and Gnai2-/- CD4 T cells in the inguinal lymph node. Ten million CMTMR-labeled Gnai2-/- CD4 T cells and the same number of CMFDA-labeled wild-type CD4 T cells were intravenously transferred to recipient mice. Twenty-four hours after cell transfer, inguinal lymph nodes were imaged intravitally using multiphoton microscope immediately following injection of mixture of red and green fluorescent dextrans (Gnai2-/- CD4 T cells-red, wild-type T cells-green). A 750 um x 750 um image was collected every second at 1 um intervals to a depth of 266 um. The movie was made using the orthoslicer feature of Imaris software (frame rate is 10 frames/sec). The most superficial slice is first seen and the movie proceeds to the last image acquired at 266 um. The bloods vessels are outlined by fluorescent dextrans.
• Video 2 (MOV, 3.37 MB)
  - Wild-type and Gnai2-/- CD4 T cell movement in inguinal lymph node. Ten million CMFDA-labeled Gnai2-/- CD4 T cells (green) and the same number of CMTMR-labeled wild-type CD4 T cells (red) were intravenously transferred to recipient mice. Twenty-four hours after cell transfer, inguinal lymph node was imaged intravitally using multiphoton microscope. Images are a rendition of 11 optical sections spaced 3 um apart in z-stack (total 30 um) and was collected every 30 sec over a period of 10 min at a depth of 100 um. Sequences of image stacks were transformed into volume-rendered four-dimensional movies using Imaris software. Gnai2-/- CD4 T cell have accumulated along the edge of blood vessels, which can be discerned by the absence of labeled cells with the exception of occasional fast moving cells within them. Frame rate is 10 frames/sec.
• Video 3 (MOV, 4.64 MB)
  - Visualization of wild-type and Gnai2-/- CD4 T cell movement in inguinal lymph node at higher magnification. Ten million CMFDA-labeled Gnai2-/- CD4 T cells (green) and the same number of CMTMR-labeled wild-type CD4 T cells (red) were intravenously transferred to recipient mice. Twenty-four hours after cell transfer, inguinal lymph node was imaged intravitally using multiphoton microscope. Images are a rendition of 9 optical sections spaced 3 um apart in z-stack (total 24 um) and was collected every 30 sec over a period of 15 min at a depth of 100 um in the region of HEVs. Sequences of image stacks were transformed into volume-rendered four-dimensional movies using Imaris software. Images were electronically zoomed 2x. The movie plays at a frame rate of 10 frames/sec. Time is shown in minutes and seconds.
• Video 4 (MOV, 9.47 MB)
  - Wild-type CD4 T cell tracks in the inguinal lymph node. Imaging data was used for semi-automated tracking of cell motility in three dimensions by using the parameters of autoregressive motion as an algorithm. Wild-type CD4 T cells are visualized as green balls and their tracks colored corresponding to the advancing time shown in the lower right corner of the movie. Frame rate is 15 frames/sec.
• Video 5 (MOV, 3.11 MB)
  - Gnai2-/- CD4 T cell tracks in the inguinal lymph node. Imaging data was used for semi-automated tracking of cell motility in three dimensions by using the parameters of autoregressive motion as an algorithm. Gnai2-/- CD4 T cells are visualized as red balls and their tracks colored corresponding to the time shown in the right lower corner of the movie. Frame rate is 15 frames/sec.
• Video 6 (MOV, 2.67 MB)
  - Visualization of wild-type and Gnai2-/- CD4 T cell movement in the T cell cortex of the inguinal lymph node. Ten million CMTMR-labeled Gnai2-/- CD4 T cells (red) and the same number of CMFDA-labeled wild-type CD4 T cells (green) were intravenously transferred to recipient mice. Twenty-four hours after cell transfer, inguinal lymph node was imaged intravitally using multiphoton microscope. Images are a rendition of 5 optical sections spaced 3 um apart in z-stack (total 12 um) and was collected every 30 sec over a period of 15 min. Sequences of image stacks were transformed into volume-rendered four-dimensional movies using Imaris software. Images were electronically zoomed 4x. Movie at played at a frame rate of 10 frames/sec.