Isolation of Treg cells and transwell migration assay of CCL28

Human CD4+CD25+CD127DIM cells were prepared using the CD4+CD25+CD127DIM T-Cell Kit (Miltenyi Biotec, Inc., Germany) according to the manufacturer's protocol. The transwell assay was performed as the following: (1) Normoxic group (upper chamber – Treg cells, lower chamber – normoxic medium); (2) Hypoxic group (upper chamber – Treg cells, lower chamber – hypoxic medium); (3) CCL28 antibody group (upper chamber – Treg cells, lower chamber – hypoxic medium + CCL28 antibody [50 μg/mL]); and (4) CCR10 antibody group (upper chamber – Treg cells + CCR10 antibody [2.5 μg/mL], lower chamber – hypoxic medium). The assay was allowed to proceed for 4 h before cell counting by flow cytometry.