Supplementary Figure S1, Schneider et al Fully migrated out of seeded (FM ratio) A medium WT sup total PBMC NS KO sup medium NS NS NS NS WT sup lymphocytes NS KO sup medium monocytes WT sup NS KO sup 0 10 20 30 40 50 FM ratio (%) Fully migrated out of seeded (FM ratio) B medium activated NK cells WT sup NS KO sup 0 10 20 30 FM ratio (%) Fully migrated out of seeded (FM ratio) C medium total PBMC TNF ctrl medium lymphocytes TNF ctrl medium monocytes TNF ctrl medium activated NK cells TNF ctrl 0 5 10 15 20 FM ratio (%) Supernatants from PAEC-WT and PAEC-KO Induce Similar Chemotactic Activity in Human PBMC Materials and Methods: sDMEM-1% or supernatant from TNFa-stimulated PAEC-WT or PAEC-KO was added at 900 mL per well in 24-well plates (BD Falcon, Basel, Switzerland). Empty transparent cell culture inserts (3 mm pores, BD Falcon) were placed into the wells and rapidly followed by addition to the upper compartment of 106 freshly isolated total human PBMC or 5 X105 purified NK cells in 300 mL sDMEM-1%. After incubation in a humidified incubator (37°C, 5% CO2) for 4 h inserts were removed and cells in the lower compartment were collected as the “fully migrated” (FM) cell fraction and counted and analyzed on FACScan or FACSCanto (Becton-Dickinson). Results: The xenogeneic chemotactic activity of supernatants from PAEC stimulated with human TNFa was first tested on freshly isolated human PBMC. At 4 h the FM ratio for the total PBMC population toward supernatants from PAEC-WT and PAEC-KO was 13.2% and 12.0%, respectively, as compared with 4.7% towards medium control (Supplemental Fig. 1A). Nevertheless, significant chemotaxis was only detected in the monocyte fraction with FM ratios of 40.1% and 38.5%, or 4.2 and 4.0 times background, respectively. For the lymphocyte fraction (B, NK, NK/T, and T cells) the FM ratios were only 4.3% and 3.9% towards PAEC-WT and PAEC-KO supernatants, respectively, compared with 3.0% in the medium control. In contrast, supernatants from stimulated PAEC-WT and PAEC-KO induced significant chemotaxis in IL-2 activated NK cells, with FM ratios of 25.2% and 26.4%, or 2.5 and 2.7 times background, respectively (Supplemental Fig. 1B), demonstrating the ability of pEC to induce chemotaxis in the lymphocyte population after preactivation. Addition of human TNF-a (100 U/ml) to the medium control did not increase the background chemotaxis in any of the tested cell populations, excluding a chemotactic effect of residual human TNF-a in the PAEC supernatants (Supplemental Fig. 1C). In conclusion, no significant difference was observed between PAEC-WT and PAEC-KO regarding secretion of factors inducing chemotaxis in human PBMC. SUPPLEMENTAL FIGURE 1. Chemotaxis of human PBMC (A) or activated purified NK cells (B) towards supernatants from human TNF-a-stimulated PAEC-WT (WT sup) or PAEC-KO (KO sup), or medium control was analyzed using empty permeable inserts in 4 h assays. Shown is percent fully migrated (FM) out of seeded for total PBMC, lymphocytes, monocytes (A), and activated NK cells (B), after adding total PBMC (A) or purified activated NK cells (B), respectively. Unspecific chemotactic effect of human TNF-a was investigated by comparing 4 h chemotaxis of human PBMC or activated purified NK cells towards medium containing 100 U/mL human TNF-a or not (C). Bars represent mean percentage (±SEM) as calculated from four (A) to three (B and C) independent experiments.