Nrp-1 is expressed in immature thymic and peripheral iNKT cells in naive mice. (A) Nrp-1 expression in thymic iNKT cells. Thymocytes from 7-week-old mice were enriched in iNKT cells by depletion of CD8⁺ cells, and the resulting cell suspensions were stained for FACS analysis of Nrp-1 expression in thymic iNKT-cell stages. iNKT cells were defined as CD3^int CD1d Tet⁺ (left dot plot) and stage I (CD44^low NK1.1⁻), stage II (CD44^highNK1.1⁻), and stage III (CD44^high NK1.1⁺) iNKT cells were gated as shown on the middle dot plot. The right histogram plot is representative of Nrp-1 expression in stage I (full black histogram), stage II (thick black line), and stage III (dotted line) iNKT cells overlaid on control isotype staining of total iNKT cells (shaded gray histogram). (B) Representative FACS analysis of Nrp-1 expression in splenic TCRβ⁺ CD1d Tet⁺ iNKT cells and TCRβ⁺ CD1d Tet⁻ conventional T cells of 8-week-old naive mice. Note the subset of iNKT cells expressing Nrp-1 at the same levels as Treg cells (CD4⁺ Nrp-1⁺ conventional T cells). (C) Intranuclear staining for the proliferation-associated marker Ki67 in splenic Nrp-1⁻ (shaded gray) and Nrp-1⁺ (thick black line) iNKT cells overlaid on control isotype staining (dotted line) of total iNKT cells. The percentage of Ki67⁺ cells in both subsets is indicated (n = 5, ***P < .001 by paired Student t test). (D) Representative histogram overlays showing CD69 and NK1.1 expression in splenic conventional T cells (dotted line), Nrp-1⁻ (shaded gray), and Nrp-1⁺ (thick black line) iNKT cells. (E) Relative proportions of Nrp-1⁺ iNKT cells in spleen, liver, thymus, and pLN iNKT cells of 8-week-old naive mice (n = 4).