T-cell chemotaxis assays

T-cell chemotaxis was measured as previously described [9, 20]. Human PB T-cells acclimated to 37°C were suspended in prewarmed RPMI (37°C) with 0.5% bovine serum albumin (BSA; Sigma-Aldrich; St. Louis, MO). Costar 24-well transwell plates with 6.5 mm diameter inserts with 5.0 μm pores (Sigma-Aldrich, St. Louis, MO) were prepared by placing 650 μl of prewarmed RPMI with 0.5% BSA that contained 0, 1200 ng/mL rhCCL21 (R&D Systems, Minneapolis, MN) or 1200ng/mL rhCCL21 pretreated for 1 hour with sample clones #7–39 in the bottom well and allowing plates to acclimate at 37°C for half an hour prior to chemotaxis assay. Cells were suspended at 300,000 cells/100 μl prewarmed RPMI with 0.5% BSA and loaded to the top chamber of the transwell assay. Transwell plates were placed in a 37°C incubator (95% humidity, 5% CO2) for 4 hours. Percent migration was determined using flow cytometry (counts determined by running samples for the same amount of time at the same speed) with background migration (cells that migrated toward media alone; always <4%) subtracted from total migrated cells.