Fig 2: Short priming with TNFa followed by withdrawal/rest in the absence of cytokine. (A) HMEC-1 endothelial cells were treated with TNFa (20ng/mL) for 3hr, 6hr, 18hr, or 24hr. In some conditions, medium was replaced after 3hr with medium without TNFa, and cells were allowed to rest for an additional 3hr, 15hr or 21hr. mRNA counts were measured by Nanostring and normalized to housekeeping genes and controls. Heat map shows relative gene expression over time, with normalization across genes/rows. Hierarchical clustering of rows by one minus Pearson correlation. (B-I) Primary human aortic endothelial cells were treated with TNFa (20ng/mL) for 3hr, 24hr or 48hr; or primed with TNFa for 3hr followed by an additional 21hr or 45hr without cytokine. Cell surface expression of (B) HLA-ABC, (C) 4-1BB, (D) ICOSL, (E) CD40, (F) PD-L2, (H) HLA-DR, and (I) PD-L1 was measured by flow cytometry (n=2). Secreted IL-15 was measured in the supernatants by ELISA (G). Quantitated results are presented as average fold increase in MFI or cytokine of each molecule relative to untreated cells. Filled bars show values for the continuous presence of TNFa; open bars show results at the end of the rest period after 3hr priming.

Fig 3: 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.