3-dimensional quantitative immunofluorescence (3DQIF) of lymphocyte homing in the absence of ICAM-1, ICAM-2 and VCAM-1 during lymphocyte trafficking. (A) Representative 3D reconstructions of WT, ICAM-1/2^−/−, and ICAM-1/2^−/− + anti–VCAM-1 mAb (MK2.7) PLNs after adoptive transfer of untreated lymphocytes (green), or treated with blocking anti–α4-integrin (PS/2, blue) or anti–LFA-1 (FD441.8, red). The MECA79⁺ HEV network is shown in brown. One square line corresponds to 50 μm. (B) Absolute cell counts per mm³ in control and ICAM-1/2^−/− PLNs in the presence of blocking mAbs. In some experiments, WT or ICAM-1/2^−/− mice were pretreated with anti–VCAM-1 (MK2.7). Pooled from 5 experiments with 15 (ICAM-1/2^−/−) and 16 (WT) independent scans. (C) Intravascular frequency of adoptively transferred untreated lymphocytes in WT and ICAM-1/2^−/− PLNs (right bar), and FD441.8-treated lymphocytes in WT PLNs (middle bar). (D) Perivascular frequency of adoptively transferred untreated lymphocytes in WT and ICAM-1/2^−/− PLNs (right bar), and FD441.8-treated lymphocytes in WT PLNs (middle bar). Total cell counts in panels C and D were normalized to 100%. Data in panels C and D are pooled from 5 independent experiments. Bars represent mean plus minus SEM. n.s. indicates not significant. *P < .05; **P < .001; ***P < .0001.