Supplementary Figure 6: FRCs promote B cell migration.

(a) Ccl19-Cre x iDTR mice were injected with CFSE-labeled, CD45.1⁺ congenic B cells 24 h after DTxn administration. Homing of B cells to lymph nodes was evaluated 90 min or 24 h later. Data are representative of three independent experiments (mean+/-sd, n=3 mice/group per experiment). (b) Lymph nodes from Ccl19-Cre x iDTR mice were collected, lysed in trizol and analyzed for the presence of Cxcl12 transcript. Lines indicate mean. Data are normalized to cyclophillin. Each data point represents one lymph node from individual mice (n=4-10 mice/group from 2-3 separate experiments). (c) Purified FRCs were cultured in a 24 well plate. CFSE-labeled B cells were seeded on top of a transwell insert and allowed to migrate towards the FRCs for 2 h. After that time, cells were recovered and B cells enumerated by flow cytometry. The figure shows three combined experiments (mean+/-sd, n= 3 wells/condition per experiment). (d) WT mice received one single dose of CD62L-specific antibody i.p. and B cell numbers were determined in lymph nodes at different time points. The figure shows two combined experiments (mean+/-sd, n=3 mice/group per experiment). (e) Ccl19-Cre x iDTR mice were injected with 1 mg/kg FTY720 i.p. and, 4 h later, with DTxn. B cell numbers were determined 24 or 72 h after DTxn administration. For analysis at 72 h, mice received an additional dose of FTY720. The figure shows two combined experiments (mean+/- sd, n= 3-4 mice/group per experiment). ^NS non significant, *p<0.05, **p<0.01, ***p<0.001

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