FIGURE 1. Defective emigration of S1P₁^-/- mature thymocytes and recirculation of S1P₁^-/- B cells.

Irradiated CD45.1 mice were reconstituted with wild-type (S1P₁^+/+) or knockout (S1P₁^-/-) CD45.2 fetal livers and analysed 6–10 weeks later. a, Flow cytometric analysis of blood from S1P₁^+/+ and S1P₁^-/- chimaeras stained for CD4 or CD8 and CD45.2. Numbers show the per cent total lymphocyte-sized cells. b, c, Number of circulating S1P₁^+/+ and S1P₁^-/- T cells in blood (b) and peripheral lymphoid tissues (c). Bars indicate averages and circles indicate values for individual mice (n = 3). d, Flow cytometric analysis of thymocytes from S1P₁^+/+ and S1P₁^-/- chimaeras stained to detect CD4, CD8 and CD45.2. Profiles shown are gated on CD45.2⁺ donor-derived cells, which comprised >98% of total thymocytes. Numbers shown are per cent of CD45.2⁺ cells. e, Expression of maturation markers l-selectin (CD62L), CD69, integrin 7 and CD24 (heat-stable antigen) on S1P₁^+/+ (thin lines) and S1P₁^-/- (thick lines) CD4 single-positive thymocytes. Similar numbers of total thymocytes were analysed for the wild-type and knockout samples and the higher value of the histograms for S1P₁^-/- samples reflects the greater number of single-positive cells. f, Number of CD4, CD8 double-positive (DP) and l-selectin^lo (immature) or l-selectin^hi (mature) single-positive (SP) thymocytes in S1P₁^+/+ or S1P₁^-/- chimaeras (n = 3). g, h, Numbers of donor-derived CD45.2⁺ S1P₁^+/+ or S1P₁^-/- CD19⁺ B cells in blood and lymph (g) or secondary lymphoid organs (h) of fetal liver chimaeras. Total in h represents the sum of B-cell numbers shown in the indicated tissues (n = 3). i, Bone marrow cells from two femurs and tibias of each fetal liver chimaera were enumerated and stained for CD45.2, B220, IgM and IgD. Bars show number of CD45.2⁺ B220⁺ pro/pre-B cells (Pro/pre; IgM^-, IgD^-), immature B cells (Imm.; IgM⁺, IgD^-) and mature recirculating B cells (Mat.; IgM⁺, IgD⁺) (n = 3). More than 98% of total B220⁺ bone marrow cells were CD45.2⁺ donor-derived cells.