Fig 1: TNFa induced endothelial expression of costimulatory and coinhibitory molecules. (A) HMEC-1 endothelial cells were stimulated with TNFa (20ng/mL) for 1hr, 3hr, 6hr, 18hr or 24hr. Time course of TNFa induced costimulatory molecule mRNA for TNFSF14, ICOSLG, TNFRSF9, TNFSF4, CD40, and CD276 in HMEC-1. Results are presented as absolute number of normalized mRNA counts for each gene, normalized to housekeeping controls. (B) Expression values of TNFRSF9 from GSE27870 are plotted, for HUVEC stimulated with TNFa for 1hr, 1.5hr, 2hr, 3hr, 4hr, 5hr and 6hr. Values at each time point were compared to baseline (0hr) by One way ANOVA followed by uncorrected Fisher’s LSD t test. *p < 0.05; ****p < 0.0001. Results are shown as mean ± SEM (n=3). (C) Expression values of TNFRSF9 from GSE144810 are plotted, comparing HUVEC (n=39) left untreated or stimulated with TNFa for 24hr. Value distributions were compared by unpaired t test. Results are presented with each measured value, and the line at the median. ****p < 0.0001 compared to control. (D) Primary human (HAEC, HCAEC, HCMVEC, HPAEC, HPMVEC, HLSEC) endothelial cells were treated with TNFa (20ng/mL) for 4hr or 18hr. Cell surface expression of 4-1BB was measured by flow cytometry (n=6). **p < 0.01 by paired t test comparing 4hr to 18hr. (E) Primary human aortic endothelial cells (n=3 donors) were stimulated with TNFa for 24hr and 48hr. Cell surface 4-1BB was measured by flow cytometry. Results are presented as average fold increase in MFI of each molecule relative to untreated cells. (F) Expression values of ICOSLG from GSE27870 are plotted, for HUVEC stimulated with TNFa for 1hr, 1.5hr, 2hr, 3hr, 4hr, 5hr and 6hr. Values at each time point were compared to baseline (0hr) by One way ANOVA followed by uncorrected Fisher’s LSD t test. *p < 0.05; ***p < 0.001; ****p < 0.0001. Results are shown as mean ± SEM (n=3). (G) Expression values of ICOSLG from GSE144810 are plotted, comparing HUVEC (n=39) left untreated or stimulated with TNFa for 24hr. Value distributions were compared by unpaired t test. Results are presented with each measured value, and the line at the median. ****p < 0.0001 compared to control. (H) Primary human (HAEC, HCAEC, HCMVEC, HPAEC, HPMVEC, HLSEC) endothelial cells were treated with TNFa (20ng/mL) for 4hr or 18hr. Cell surface expression of ICOSL was measured by flow cytometry (n=6). **p < 0.01 by paired t test comparing 4hr to 18hr. (I) Primary human aortic endothelial cells (n=3 donors) were stimulated with TNFa for 24hr and 48hr. Cell surface ICOSL was measured by flow cytometry. Results are presented as average fold increase in MFI of each molecule relative to untreated cells. (J) Expression values of TNFSF4 from GSE27870 are plotted, for HUVEC stimulated with TNFa for 1hr, 1.5hr, 2hr, 3hr, 4hr, 5hr and 6hr. Values at each time point were compared to baseline (0hr) by One way ANOVA followed by uncorrected Fisher’s LSD t test. *p < 0.05. Results are shown as mean ± SEM (n=3). (K) Expression values of TNFSF4 from GSE144810 are plotted, comparing HUVEC (n=39) left untreated or stimulated with TNFa for 24hr. Value distributions were compared by unpaired t test. Results are presented with each measured value, and the line at the median. *p < 0.01 compared to control. (L) Primary human (HAEC, HCAEC, HCMVEC, HPAEC, HPMVEC, HLSEC) endothelial cells were treated with TNFa (20ng/mL) for 4hr or 18hr. Left panel shows relative change in OX40L MFI on HAEC (n=3). Right panel shows fold change in cell surface expression of OX40L measured by flow cytometry across EC from different vascular beds (n=6). *p < 0.05 by paired t test comparing 4hr to 18hr. (M) Expression values of TNFRSF14 from GSE27870 are plotted, for HUVEC stimulated with TNFa for 1hr, 1.5hr, 2hr, 3hr, 4hr, 5hr and 6hr. Values at each time point were compared to baseline (0hr) by One way ANOVA followed by uncorrected Fisher’s LSD t test. *p < 0.05. Results are shown as mean ± SEM (n=3). (N) Expression values of TNFRSF14 from GSE144810 are plotted, comparing HUVEC (n=39) left untreated or stimulated with TNFa for 24hr. Value distributions were compared by unpaired t test. Results are presented with each measured value, and the line at the median. ****p < 0.0001 compared to control. (O) Expression values of CD83 from GSE27870 are plotted, for HUVEC stimulated with TNFa for 1hr, 1.5hr, 2hr, 3hr, 4hr, 5hr and 6hr. Values at each time point were compared to baseline (0hr) by One way ANOVA followed by uncorrected Fisher’s LSD t test. *p < 0.05. Results are shown as mean ± SEM (n=3). (P) Expression values of CD83 from GSE144810 are plotted, comparing HUVEC (n=39) left untreated or stimulated with TNFa for 24hr. Value distributions were compared by unpaired t test. Results are presented with each measured value, and the line at the median. *p < 0.01 compared to control. (Q) Expression values of CD276 from GSE144810 are plotted, comparing HUVEC (n=39) left untreated or stimulated with TNFa for 24hr. Value distributions were compared by unpaired t test. Results are presented with each measured value, and the line at the median. (R) Primary human (HAEC, HCAEC, HCMVEC, HPAEC, HPMVEC, HLSEC) endothelial cells were treated with TNFa (20ng/mL) for 4hr or 18hr. Cell surface expression of B7-H3 was measured by flow cytometry (n=6). **p < 0.01 by paired t test comparing 4hr to 18hr. (S) Primary human aortic endothelial cells (n=3-4 donors) were stimulated with TNFa for 24hr and 48hr. Cell surface B7-H3 was measured by flow cytometry. Results are presented as average fold increase in MFI of each molecule relative to untreated cells. ****p < 0.0001 vs untreated.