37±2.84); compared to the difference in total distance, the difference in beeline distance was smaller with Treg covering 88.8 μm±9.51 and non-Treg covering 49.24 μm±5.25, indicating that Treg exhibited a higher rate of direction changes

during laminin-specific 2D migration compared to non-Treg. To analyze T-cell diapedesis, we used freshly isolated, primary CNS endothelium as an in vitro model of the blood–brain barrier (BBB) cultured in a transwell migration assay. Naïve, lymph node-derived CD4+ T cells were applied on the luminal side of the cultured murine brain microvascular endothelial cell (MBMEC) layer and were collected from the three compartments after 18 h as delineated in Fig. 1C (upper chamber, MBMEC layer and lower chamber) to check whether Treg accumulated among CD4+ T cells. Fig. 1B depicts a representative population of CD4+

T cells PKC412 supplier incubated for 18 h to serve as a reference. Between 4.8 and 6.3% Treg were Lapatinib clinical trial found in all experiments (n=5, data not shown). When no attracting stimuli was added to the medium, CD4+ T cells showed very low migration (data not shown) so we used FBS, which is known to contain low concentrations of different cytokines as a chemoattractant agent. Eighteen hours after application of the CD4+ T cells to an FBS gradient, Treg accumulated to 20.7% of the entire CD4+ T-cell population within the MBMEC fraction (n=5, 15.1–29.8%). In the basolateral compartment, Treg enriched to 10.8% of total CD4+ T cells (n=5, 8.4–20.2%) (Fig. 1D). As CCR6 is expressed on both T-cell subsets (Supporting Information Fig. 1D), we tested whether CCL20 (the CCR6 ligand) contributes to the preferential migration of Treg in the MBMEC layer. Although enrichment of Treg within the MBMEC layer was nearly completely abrogated (5.7–6.7%), the accumulation of Treg in the lower chamber was threefold enhanced by addition of CCL20 from 10.8 to 34.1% of migrated cells (Fig. 1E). Activation of the MBMEC layer 24 h before starting the

migration assay with murine TNF-α and IFN-γ revealed a similar Treg accumulation as under non-inflammatory conditions while, as expected, the total counts of migrated cells from the lower chamber increased under inflammatory conditions (n=3, data not shown). To verify our findings in vivo, we Docetaxel examined naïve C57BL/6 mice for ratios of Treg versus non-Treg in the CNS, spleen, lymph nodes and peripheral blood by flow cytometry after animal perfusion with PBS (Fig. 1F). We were able to isolate approximately 2×104–1×105 leukocytes with a Percoll density gradient from the CNS of healthy mice. Strikingly, Treg were present to a significantly higher extent in the CNS compared to the three other examined organs (mean±SE blood: 4.5±0.5, lymph nodes: 10.6±0.9, spleen: 12.1±1, CNS: 19.55±1.4, n=5). Taken together, murine Treg showed higher expression of surface markers indicative for activation, adhesion and migration, and exhibited higher motility in 2D migration on a laminin substrate.