Teff cell entry into BM of unmanipulated mice is chemokine receptor-dependent. B6 mice (A; supplemental Video 1) or B6.ubGFP→ B6 BM chimeric mice (B; supplemental Video 2) were IV injected with untreated (red) or PTX-treated (blue) OT-1 effectors. Three hours later, mice were injected with dextran-FITC (A) or Qdot655 (B) to visualize blood vessels and then underwent 2PIM imaging of the calvarium. Representative images (A-B). Arrows highlight extravascular OT-1 cells adjacent to blood vessels. Scale bar represents 50 μm. PTX-treated OT-1 cells were excluded from LN but not spleen (C). PTX treatment reduced the E/I ratio of OT-1 effectors, the absolute number of extravascular OT-1 effectors (D), and the distance extravascular OT-1 cells traveled from the nearest vessel (E). (F) Rapidly moving (streaming) intravascular OT-1 cells (supplemental Video 3) were more numerous and more frequent among PTX-treated cells. Number of streaming cells per frame (left panel); the percentages of PTX– and PTX+ cells that were streaming (middle panel); the numbers of streaming PTX+ and PTX– cells per image frame (right panel). (D-E) Data from 3 nonchimeric and 3 chimeric mice with a total of 12 imaged volumes. (F) Data from 2 mice with 2 imaged volumes. Significance was determined by a 2-tailed Student t test. ∗P < .001; ∗∗P < .0001. ns, not significant.
