explored in genomics studies. Differential regulation of genes after PMA/CD3 and CD3/ 28 Vs PMA/CD28 stimulation Differentially BQ123 web regulated genes CCL1 and IL-2 are profilespecific secreted proteins In order to further characterize the different signal transduction events induced by different -stimulatory signals, we performed a first gene expression experiment with Jurkat T cells that were stimulated for 1 or 8 hours with PMA/CD3, CD3/28 and PMA/CD28. It appeared that after 1 hour a limited response on transcription level was seen, whereas after 
8 hours of stimulation, several hundreds of genes were regulated. Furthermore, PMA/ CD3 and PMA/CD28 regulate more genes compared to CD3/28, reflecting the strength of the stimuli used. Multivariate analysis by principal component analysis and hierarchical clustering showed that the 3 stimuli lead to clearly distinct gene expression profiles. At both time points the profile induced by PMA/CD28 is clearly distinct from the profiles induced by PMA/CD3 and CD3/28. Gene profiles of the differential stimuli were ranked on the level of induction and evaluated on whether or not the translated protein is secreted. This resulted in the identification of the PMA/CD28-specific transcript CCL1, the CD3-specific transcripts IL-2 and XCL1/2. Small but significant inductions of these genes were observed after 1 hour of stimulation for both CCL1 and IL-2. However, both genes were highly induced after 8 hours of stimulation. The secretion of the protein 24 hours after stimulation reveals an identical profile compared to the mRNA. Pathway profiling with multiple stimuli and inhibitors To investigate the contribution of proximal, medial and distal signaling events on the CD3/28, PMA/CD3 and PMA/CD28 stimuli, we performed a second gene expression profiling experiment with different selective inhibitors, including proximal kinase inhibitors Lck, PKC, medial MAPK inhibitors PD98059, SP600125, Org 48762-0 and the distal Calcineurin inhibitor Cyclosporin A. Jurkat T cells were stimulated with PMA, CD28 and CD3 alone and combinations thereof in the presence of the above mentioned inhibitors. Based on the results of the first gene expression profiling experiment, we chose evaluated gene expression after 8 hours of stimulation. Overlap with Number of probe sets CD3_PMA PMA_CD28 CD3_CD28 CD3_PMA PMA_CD28 T= 1 hrs CD3_CD28 338 194 319 1567 1853 786 1.00 0.92 0.88 1.00 0.60 0.84 0.53 1.00 0.54 T= 8 hrs 0.83 0.89 1.00 0.42 0.30 1.00 CD3_PMA PMA_CD28 CD3_CD28 0.72 1.00 0.70 PMA + CD28 PMA + CD3 CD3 + CD28 Smeets et al. BMC Immunology 2012, 13:12 http://www.biomedcentral.com/1471-2172/13/12 Page 5 of 17 A B A principal component analysis on the ratio data set is shown in Additional file 1: whereas A420983, CsA and AEB071 regulate many genes. A420983 and CsA only show a significant effect on the PMA/CD3 and CD3/CD28 pathways, in which the effect of CsA is smaller than the effect of A420983. AEB071 is the only compound that shows also a significant effect on the PMA/CD28 induced pathway. These analyses were rerun with different settings for the thresholds used for gene selection. In all cases similar results were obtained indicating that the results were not critical dependent on the thresholds settings that were used. Inspection of the profiles of CCL1 and IL-2 revealed that CCL1 mRNA is highly induced via the PMA/CD28 pathway. This induction is depending PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19797474 on PKC signaling and negatively regulated via Lck signaling. Apparently thi