PMC

(A) FACS analysis of S1P₁ on lymph node CD4⁺ T cells 4.5 h after in vivo treatment with the indicated amounts of FTY720. (B) Fraction of CD4⁺ T cells remaining in blood (BLD) and lymph (LYM) 4.5 h after treatment with the indicated amounts of FTY720 compared to saline treated controls. (C) FACS analysis of S1P₁ on wild-type littermate control and Edg1^+/- lymph node CD4⁺ T cells. (D) Number of CD4⁺ T cells in blood and lymph of wild-type and Edg1^+/- mice. Data are representative of at least 3 mice for each condition. Bars represent means and dots individual mice.

(A) Egress of PTX or oligomer B treated T cells from lymph nodes of FTY720 treated mice. Splenocytes were treated with PTX or with Oligomer B (Oligo-B) as a control and co-transferred into hosts pre-treated with saline or FTY720 4 h earlier. Three hours after transfer, the mice were treated with integrin-blocking antibodies. The number of remaining cells in lymph nodes 21 h after “entry blockade” was determined. Numbers shown are normalized for the number of input splenocytes. (B and C) PTX restores egress of S1P₁-deficient lymphocytes. (B) Diagram of transfer experiment. (C) Number of transferred Oligomer B or PTX treated Edg1^-/- or Edg1^+/+ T cells in peripheral lymph nodes and blood at 0 h and 27 h of integrin blockade. Numbers shown are normalized for the number of CD62L-high, single-positive input thymocytes. Bars represent mean and points individual animals.

Mice receiving CFSE-labeled OTII T cells were immunized s.c. with OVA in CFA or left unimmunized and draining lymph node cells were analyzed at day 3. (A) Flow cytometric analysis of S1P₁ versus CFSE on OTII T cells. The line indicates the baseline determined by staining cells with a control antibody. (B) Transwell migration assay showing relationship between cell division (Div) number and recovery of S1P responsiveness. OTII T cells that had divided 0-3 times are shown by dashed lines, 4-8 times by thin solid lines and endogenous CD4⁺ T cells by a thick solid line. (C) Flow cytometric analysis of CCR7 versus CFSE on OTII T cells. (D) RT-QPCR analysis of CCR7 on sorted OTII T cells that had divided the indicated number of times based on CFSE dilution. (E) Transwell migration assay showing relationship between cell division number and reduced CCL21 responsiveness. Bars show mean±sd (n=4). (F) Overlay of CFSE profile in lymph node versus blood and lymph, showing enrichment for highly divided cells in circulation. Data are representative of at least 3 experiments.

(A) Flow cytometric analysis of CD45.2⁺ Ccr7^-/- and CD45.1⁺ WT T and B cells in mixed bone marrow chimeras treated for 24 h with saline or FTY720 and with integrin blocking antibodies for 20 h. Numbers indicate percent cells in the indicated gates out of total T or B cells. The means (±SD) of lymphocyte numbers in the samples were as follows: saline lymph=6.3×10³/ul (± 4.5×10³), n=8; FTY720 lymph=7.5×10²/ul (± 9.7×10²), n=9; saline LN=1.3×10⁷ (± 4.0×10⁶), n=8; FTY720 LN=1.6×10⁷ (± 8.1×10⁶), n=9. (B) Frequency of CD45.2⁺ T cells in the peripheral LN, mesenteric LN and lymph of mice that had been reconstituted as in A (Ccr7^-/- mixed chimera) or with a mixture of CD45.2⁺ Ccr7^+/+ and CD45.1⁺ WT bone marrow as a further control (Ccr7^+/+ mixed chimera). (C) Same data as in B plotted as the ratio of each type of T cell in lymph versus peripheral lymph nodes for each animal. Bars in B and C represent mean and points individual animals and the data are pooled from three experiments.

(A) Serial sections of lymph node stained for LYVE-1 (brown) and IgD (blue) or CD11b (blue) showing LYVE-1 expressing structures extending from cortical areas into the macrophage-rich medullary region. (B) Proximity of cortical LYVE-1⁺ structures (blue) to PNAd expressing HEV (brown). (C) LYVE-1⁺ cortical structures (brown) contain B cells (IgD, blue) and T cells (CD3, blue) but are relatively devoid of dendritic cells (CD11c, blue). (D) CCL21 expression (blue) in wild-type lymph nodes with respect to LYVE-1 (brown). (E) Effect of S1P₁-deficiency on T cell appearance in cortical sinusoids. CD45.2⁺ thymocytes from wild-type or S1P₁ deficient fetal liver chimeras were labeled with CFSE and each population was co-transferred with wild-type (CD45.1⁺) thymocytes into B6 (CD45.2⁺) mice. Thirty-six hours after transfer, recipient lymph nodes were sectioned and stained to detect the fetal liver chimera-derived (CSFE⁺) T cells (blue) and the co-transferred wild-type cells (CD45.1, red) and for LYVE-1 (E). Objective magnifications: (A) 5×; (B-D) 10×; (E) 20×. (F and G) Enumeration of (F) Edg1^+/+ and Edg1^-/- cells or (G) Ccr7^+/+ and Ccr7^+/- cells and co-transferred wild-type control T cells inside LYVE-1⁺ sinusoid structures (In) and in a 30 μm thick area surrounding each sinusoid (Out). Data are plotted as ratio of wild-type, Edg1^-/- or Ccr7^+/- cells to co-transferred control cells in each region. Each dot represents data from a single sinusoid cross-section and bars represent the means. (H) Transwell migration of Ccr7^+/+ or Ccr7^-/- CD4⁺ T cells in response to the indicated concentrations of S1P. Cells were added to the upper wells in the absence or presence of 1 μg/ml CCL21 as indicated. Data are representative of three experiments.

(A) Wild-type and Ccr7^-/- spleen cells were co-transferred to wild-type recipients and after 24 h, the mice were treated with integrin-blocking antibodies for 8 h. The data shown indicate the fraction of T cells remaining in recipient mesenteric (m) and peripheral (p) lymph nodes at 8 h compared to 0 hr. (B) Irradiated mice were reconstituted using mixtures of CD45.2⁺ Ccr7^+/+ or Ccr7^-/- bone marrow and CD45.1⁺ WT bone marrow and the representation of CD45.2⁺ cells in peripheral and mesenteric lymph nodes (LN) and in lymph (LYM) was determined. (C) Flow cytometric analysis of S1P₁ on Ccr7^-/- and Ccr7^+/+ CD4 T cells from lymph nodes of mixed-bone marrow chimeras (left panel) and Ccr7^+/- (middle panel) or Ccr7-Tg (right panel) CD4⁺ T cells together with co-transferred control cells. Shaded histograms show staining with control antibody. (D) Flow cytometric data showing CCR7 expression using anti-CCR7 mAb on Ccr7^+/- and littermate control (upper panel) or Ccr7-Tg and littermate control (lower panel) lymph node CD4⁺ T cells. Shaded histograms show staining of Ccr7^-/- cells. (E and F) Transfers and integrin blockade were performed as in A. In E the fraction of cells remaining after 14 h in the indicated lymph nodes was determined. F shows the ratio of transferred Ccr7^+/- and Ccr7^+/+ T cells in peripheral and mesenteric lymph nodes (LN) and in lymph (LYM) at 0 h. (G) Wild-type control and Ccr7-Tg cells were transferred to wild-type recipients and the mice were treated as in A. The data shown indicate the fraction of T cells remaining in the indicated lymph nodes at 12 h compared to 0 h of entry blockade. In A-B and E-G, bars indicate mean and points indicate data from individual mice. (H) Distribution of WT, Ccr7^-/-, Ccr7^+/- and Ccr7-Tg cells within peripheral lymph nodes. CFSE labeled T cells were transferred into wild-type recipients and one day later lymph node sections were stained to detect transferred cells (blue) and LYVE-1⁺ sinusoids (brown). Objective magnification, 5×.