Cxcl9

For migration studies, 5 × 10⁵ purified CD4⁺ T cells in complete RPMI 1640 medium were added to the top chambers of a 96 well Transwell plate with a pore size of 5 μm (Corning Costar, Acton, MA). Mouse recombinant chemokines CXCL9, CXCL10, CXCL11, and CCL21 (Peprotech, Rocky Hill, NJ) were diluted in complete RPMI 1640 medium and added to the bottom chamber of the Transwell plate to a final concentration of 200 μM for CXCL9 and CXCL11 and 100 μM for CXCL10 and CCL21. As a control, complete RPMI 1640 medium alone was added to the bottom chamber. After incubation for 4 h at 37°C in 5% CO₂, the cells were collected from the bottom chamber and counted in a hemocytometer. The chemotaxis index was calculated by dividing the number of cells that migrated in response to chemokines by the number of cells in the bottom chamber containing medium alone.
To identify the population of cells that had migrated to each chemokine, cells were collected from individual bottom chambers and surface stained with FITC-conjugated CD4 mAb followed by intracellular staining with APC-conjugated Foxp3 mAb as previously described. Cells were analyzed by FACS and the percentage of CD4⁺Foxp3⁺ Tregs in the migrated cell population was determined.

Skin T_RM in VacV-immunized hosts were activated in situ with 50μg transdermal injection of indicated peptides at the immunization site as described [34 (link)]. Single cells suspension from the ears is obtained 6 hours later and stained for surface and intracellular antigens/cytokines in the absence of BFA. When stated, 3μg of CXCL9 and CXCL10 (Peprotech) was added to the site of peptide injection as previously described [64 (link)].

We assessed the influence of chemokines on cell migration of MSCs using the CytoSelect^TM 96-well cell migration assay (Cell Biolabs), according to the manufacturer’s instructions^{44 (link)–46 (link)}. Feeder tray wells were filled with 150 µl of medium in the presence of seven chemokines (CCL19, CCL21, CCL25, CXCL9, CXCL10, CXCL11, and CXCL12; Peprotech; all chemokines were used at a concentration of 100 ng/ml). Migration assays were run for 8 h at 37 °C in a 5% CO₂ incubator. The cells were treated with a fluorescent dye (CyQuant^®) and measured at 480 nm using a SpectraMax M2 Plate Reader (Molecular Devices).