Fig. 2

Transcriptome analysis showing changes in gene expression pattern during varlilumab costimulation of T cells. T cells were costimulated continuously for 72 h (Set 1) or first preactivated with anti-CD3 (OKT3) for 46 h and costimulated briefly (4 h) with anti-CD3 + varlilumab or anti-CD3 + control Ab (hIgG; Iso; Set 2). Treated cells were subjected to RNA extraction and hybridization on whole genome arrays as described. a Heatmap showing log2 fold-changes as described under Methods shows the top 25 genes in red (upregulated) or blue (downregulated) during varlilumab costimulation compared to isotype IgG stimulated controls in Set 1 protocol (n = 3). Log2 expression values were rescaled by calculating a mean log2 expression for each gene and then subtracting that value from each individual gene expression value. This approach effectively centers the gene expression values around zero so that actual fold changes are easily discerned from the heatmap (p < 0.05). b Select inflammatory panel gene list queried to map transcripts coding for costimulatory (B7 family, TNFRSFs) and coinhibitory (immune checkpoint) molecules post varlilumab stimulation (Set 1). Expression rescaling approach described above was also employed here. Gene symbols with asterisks are not significant (p > 0.05). All other genes are significant according to non-adjusted p < 0.05. c Congruence plot showing varlilumab stimulation resulted in similar gene expression signatures between Set 1 (n = 3) and Set 2 (n = 4) with 47 overlapped genes. Shown are log2 fold changes with an FDR-adjusted p < 0.05. d Top 10 genes up/downregulated in response to Set 1 and Set 2 stimulation protocols, Shown are log2 fold changes with an FDR-adjusted p < 0.05. e Fold-change in transcripts corresponding to cytokines, chemokines and growth factors. Heatmap shows log2 fold change in gene expression comparing Set 1 and Set 2 type varlilumab stimulations. Heatmap representation was generated from an ordered gene list so not all input genes are significant (shown with black asterisks) at pFDR < 0.05. Labels with red asterisks (IL6 and CCL5) were significant in Set 1 with a direction of change being identical in both studies. All genes highlighted in (b, c and e) are significantly different in T cells stimulated with varlilumab compared to isotype control Ab stimulations