SIRPγ mediates TEM of HUVECs under flow. (A) HUVEC monolayers were prepared as described in Figure 2. Isolated CD3⁺ T cells were preincubated with SIRPα (SE7C2), SIRPγ (LSB2.20), or a nonblocking mAb to MHC class I and then cells were drawn across TNF-α–stimulated endothelium. The total number of accumulated T cells was determined 10 minutes after perfusion as described in “Methods.” Data are means (± SEM) of 4 separate experiments. (B) T cells were treated with listed mAb (30 μg/mL; SIRPα, SE7C2; SIRPγ, LSB2.20; nonblocking control mAb MHC class I, W6/32) and then were drawn across TNF-α–stimulated endothelium for 10 minutes. The percentage of T cells that transmigrated the endothelium after 10 minutes was determined as detailed in “Methods.” Data are means ± SEM, n = 4 separate experiments; *P = .001. (C) DIC images of live cells in the TEM assay after 10 minutes were prepared as described in “T-cell locomotion and transmigration under flow.” The polarity of T cells on control (W6/32), SIRPγ (LSB2.20), and SIPRα (SEC7C2) mAbs are shown. show normal, migrated CD3⁺ cells in W6/32-treated monolayers. In contrast, T cells (▶) on SIRPγ-treated T cells exhibit a rounded-up morphology and defects in migratory behavior and show reduced transmigration. (D) The apical migration velocities of T cells on HUVEC monolayers treated with SIRPα (SE7C2), SIRPγ (LSB2.20), or nonblocking control mAb MHC class I mAbs was determined by the Image J software. *P = .01. (E) TNF-α–stimulated HUVEC monolayers were preincubated for 30 minutes with anti-CD47 (B6H12) or SIRPα (SE7C2) mAbs. Medium alone (no additions) or MHC-class I (W6/32) mAb served as controls. The percentage of T cells that transmigrated after 10 minutes was determined as described in “Methods.” Data are means (± SEM) of 4 separate experiments; *P = .001.