Characterization of Treg cells and CCR2-Treg cells. (a) Expression of CCR2 gene in Treg cells and CCR2-Treg cells. Quantitative real-time polymerase chain reaction analysis was performed on total RNA prepared from Treg and CCR2-Treg cells. Results are calculated as a ratio of CCR2 expression to the expression of hypoxanthine phosphoribosyl transferase 1 (HPRT1). (b) Chemotactic activity of Treg and CCR2-Treg cells to monocyte chemoattractant protein-1 (MCP-1). Aliquots (100 μl) of cells (5 × 106 per milliliter) were added to the upper chambers, and MCP-1 was added to the lower wells at various concentrations. After 2 hours, cell migration was quantified by counting cells in each lower chamber and cells adhering to the bottom part of the filter. Each assay was performed in triplicate. (c) Suppressive function of Treg and CCR2-Treg cells to alloantigen. One hundred thousand freshly isolated CD3+ T cells from MRL/+ mice were stimulated with 1 × 105 irradiated allogeneic splenocytes from DBA1 mice. Graded numbers of the Treg and CCR2-Treg cells were added to the cultures. Wells were pulsed with [3H]thymidine (3H-TdR) for the last 18 hours of the 4-day culture. Control (no addition of regulatory T cells) is 16,429 ± 5,160 cpm. CCR2-Treg cell, CD4+CD25+Foxp3+ CCR2-transfected T cell; Treg cell, CD4+CD25+Foxp3+ T cell.