Nrp-1 expression is induced on iNKT cells after TCR activation. (A) Mice were injected IP with α-GalCer or control diluent, and CD69 and Nrp-1 expression on gated TCRβ⁺ CD1d Tet⁺ spleen iNKT cells was analyzed after 3 and 7 days. Representative data of 1 mouse of 2 per time point in 1 of 2 experiments are shown. (B) FACS-sorted spleen Nrp-1⁻ iNKT cells were cultivated in lymphocyte culture medium alone (Control) or activated with anti-CD3ϵ and anti-CD28, and Nrp-1 expression on activated iNKT cells was analyzed by FACS at 24 and 48 hours. Representative data of 1 of 3 experiments are shown. (C) Analysis of Nrp-1 expression on CD1d Tet⁺ cells after in vitro culture of CFSE-labeled spleen cells in lymphocyte culture medium alone (Control) or supplemented with IL-7, IL-15, or α-GalCer for 72 hours. These data are representative of 2 distinct experiments performed in duplicate wells.