1. Supplementary Figure S1, Schneider et al lymphocytes monocytes medium WT sup KO sup FM ratio (%) 01020304050 medium WT sup KO sup medium WT sup KO sup total PBMC A NS Fully migrated out of seeded (FM ratio) 0102030 activated NK cells medium WT sup KO sup FM ratio (%) B NS Fully migrated out of seeded (FM ratio) 05101520 lymphocytes activated NK cells medium TNF ctrl medium TNF ctrl medium TNF ctrl total PBMC C monocytes medium TNF ctrl FM ratio (%) NS Fully migrated out of seeded (FM ratio) SUPPLEMENTAL FIGURE 1. Chemotaxis of human PBMC (A) or activated purified NK cells (B) towards supernatants from human TNF-  -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-  was investigated by comparing 4 h chemotaxis of human PBMC or activated purified NK cells towards medium containing 100 U/mL human TNF-  or not (C). Bars represent mean percentage (±SEM) as calculated from four (A) to three (B and C) independent experiments. Supernatants from PAEC-WT and PAEC-KO Induce Similar Chemotactic Activity in Human PBMC Materials and Methods: sDMEM-1% or supernatant from TNF  -stimulated PAEC-WT or PAEC-KO was added at 900  L per well in 24-well plates (BD Falcon, Basel, Switzerland). Empty transparent cell culture inserts (3  m pores, BD Falcon) were placed into the wells and rapidly followed by addition to the upper compartment of 10 6 freshly isolated total human PBMC or 5 X10 5 purified NK cells in 300  L sDMEM-1%. After incubation in a humidified incubator (37°C, 5% CO 2 ) 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 TNF  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-  (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-  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.