NKT cells traffic to GVHD priming sites and target organs but do not cause GVHD. (A) NKT cells were sorted to high purity (> 95%) from C57BL6 donors (H-2^b) by first gating TCRβ⁺DX5⁺ then CD4⁺ cells; analysis of sorted cells indicates a population that is approximately 50% positive for PBS-57–loaded CD1d (National Institutes of Health). (B) 5.5 × 10⁵luc⁺NKT cells transferred into lethally irradiated allogeneic BALB/c (H-2^d) mice caused no significant alteration in weight profile compared with mice receiving T cell–depleted C57BL/6 bone marrow (TCD-BM) only. Solid line (●) indicates TCD-BM; dashed line (◇) indicates 550K luc⁺NKT. Bars indicate means ± SE (n = 3). (C) BLI indicated that NKT cells first appear in the spleen and lymph nodes, followed by migration to the skin and other GVHD target organs. Total photons emitted peaked near day 25 after transplantation, followed by a steady decline. Results shown are from a representative mouse of 3 from 1 of 4 independent experiments. Ventral and lateral images of the same mouse are shown with the day after transplant indicated. (D) Ex vivo imaging of internal organs indicates NKT cells trafficking to the spleen and mesenteric lymph nodes at day 11 and infiltration into the gastrointestinal tract, pancreas, and liver tissue by day 37. Images from mice that did not receive any luc⁺NKT cells are shown as a control.