FIGURE 2. Transferred S1P₁^-/- T and B cells accumulate in secondary lymphoid organs and fail to exit.

a, b, CD45.2⁺ thymocytes from S1P₁^+/+ or S1P₁^-/- fetal liver chimaeras were co-transferred with approximately equal numbers of CMTMR-labelled CD45.1⁺ wild-type thymocytes into CD45.1 B6 mice. After 24 h, blood, lymph and peripheral lymphoid tissues were examined for the presence of transferred cells by flow cytometry. Frequency of transferred S1P₁^+/+ or S1P₁^-/- CD4 single-positive (a) and CD8 single-positive (b) T cells in the blood, secondary lymphoid organs and lymph of recipients 24 h after transfer, as a per cent of the CMTMR-labelled co-transferred control cells (see Methods). LNs, lymph nodes. c, Top panel: spleen sections from CD45.1 B6 mice 24 h after receiving CD45.2⁺ S1P₁^+/+ or S1P₁^-/- thymocytes as described in a, stained to detect transferred CD45.2⁺ T cells (blue) and endogenous B220⁺ B cells (brown). Bottom panel: spleen sections from Igh^b mice 24 h after receiving Igh^a+ spleen and lymph node cells from S1P₁^+/+ or S1P₁^-/- mice, stained to detect transferred IgM^a+ IgD^a+ B cells (blue) and endogenous B220⁺ B cells (brown). Objective magnification 5. d, CD45.2⁺ S1P₁^+/+ or S1P₁^-/- B cells were co-transferred with equal numbers of CMTMR-labelled CD45.1⁺ wild-type B cells into CD45.1 B6 mice. Frequency of transferred CD19⁺ B cells in blood, secondary lymphoid tissues and lymph of the recipients 40 h after the transfer was determined as for T cells in a, b. For transfer experiments, the average and individual values for 3–4 recipients per group is shown. The data are representative of three experiments using chimaeras made from at least two different S1P₁^+/+ and S1P₁^-/- fetal livers.