Mel 14

As previously described (6 (link)), 2×10⁶ CFSE labeled CD4 (treated with/without 10 μM S1PR antagonist or 10 μM S1PR4 antagonist) in 30 μl PBS were injected into the footpads that were treated with/without0.5 nM S1PR2 antagonist. For endogenous T cell migration, mice were pretreated with 100 μg anti-CD62L (MEL-14, BioXCell, West Lebanon, NH) i.v., 24 hours later footpads treated with 0.5 nM S1PR2 antagonist, and dLN assessed 16 hours later (29 (link)). For LPS (Sigma-Aldrich, St. Louis, MO) induced inflammation, 1 μg LPS in 20 μl PBS was injected per footpad 2 hours prior to T cell transfer. CFSE-labeled CD4 T cells in 30 μl PBS were mixed with 1 μg mouse control IgG or anti-S1P, or 1 μg rat control IgG or anti-VCAM-1, and injected into the footpads. In the paired analysis for footpad migration, T cells are transferred to both hind footpads of a recipient mouse. T cells on one side are treated with active reagent (e.g., receptor blocker), while T cells on the other side are treated with vehicle or control compound. Each mouse thus acts as its own control. Two or 16 hours after injection, mice were euthanized, the draining popliteal lymph nodes (LN) were harvested, and single cell suspensions or tissue sections prepared for flow cytometric or immunohistochemical analysis, respectively.

Mice were injected i.p. with 600μg IgG, or with 200μg anti-P-selectin (RME-1, Biolegend), 200μg anti-E-selectin (RMP-1, Biolegend) and 200μg anti-L-selectin (Mel-14, BioXcell), or 200μg anti-P-selectin and 200μg anti-PSGL-1 (4RA10) i.p. 0, 3-, and 5-dpi of WT mice with 2 × 10⁶ Cl13. Viral titers in blood were determined at 8- and 36-dpi. To deplete CD4⁺ cells, mice received two 500μg i.p. injections of anti-CD4 antibody (GK1.5, BioXCell) at day -1 and 0 with respect to Cl13 infection. Efficacy of CD4 depletion was confirmed in blood and spleens by flow cytometry.

Dexamethasone (Sigma) was administered to mice fed ad libitum at a dose of 2 mg/kg i.p. every day for 2 weeks. For inducible gene deletion, 75 mg/kg tamoxifen (Sigma) in 90% corn oil: 10% ethanol for 5 consecutive days i.p. Experiments were performed 3 weeks after the final treatment. FTY720 (Sigma) was administered i.p. four times every other day at a dose of 1 mg/kg, with or without 0.5 mg of an anti-CD62L antibody (Mel-14, BioXcell). For AMD3100 (Sigma) experiments, mice were put on DR for 3 weeks then injected with 10 mg/kg subcutaneously for 4 consecutive days. 1 mg/kg BrdU (BD) was administered i.p. every day for 7 days prior to the experiment. Anti-CD4 (GK1.5, BioXcell) and anti-CD8 (2.34, BioXcell) was administered i.p. at a dose of 0.2 mg for three consecutive days prior to infection.

LN egress of T cell was performed as described previously (Brinkman et al., 2016 (link)). CD4⁺ T cells were purified from FoxP3GFP CD45.1 mice with magnetic bead negative selection (Miltenyi, Auburn, CA). 2×10⁷ cells were injected i.v. into tamoxifen treated WT^fl or KO^fl mice (CD45.1⁻). After 18 hours, 100 μg anti-CD62L (MEL-14, BioXCell) or control Rat IgG2a (2A3, BioXCell) was injected i.v. After another 18 hours, LNs were collected for each group and analyzed by flow cytometry for transferred naïve CD4⁺ T cells (CD45.1⁺Foxp3⁻) and tTregs (CD45.1⁺Foxp3⁺). The numbers and ratios of transferred naïve CD4 T cells or tTregs in WT^fl and KO^fl LN were calculated.