erentiation has been outlined. ERK is important for Th2 differentiation, whereas p38 and JNK2 appear to be involved in Th1 development. TCR/CD3 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19798435 stimulation and CD28 stimulation alone are weak activators of T cell signaling. It is generally conceived that CD28 signaling merely acts as a signal potentiator on top of the initiator signal mediated via the TCR/CD3. Ledbetter and June et al. described that CD28 stimulation in the absence of cross-linking on top of PMA stimulation can activate T cells, without increasing calcium flux. This suggests that co-stimulatory pathways synergize with biochemical pathways induced via the TCR. Whether CD28 ligation, in the absence of TCR signaling, leads to activation and differentiation has not been fully explored. These findings show that effective T cell activation and differentiation towards effector subsets is the result of precise integration of multiple signaling routes. To explore the pathways underlying these distinct routes towards T cell activation and differentiation we used comprehensive biochemical characterization and gene expression profiling of Jurkat T cells that were activated with various co-stimulatory signals in the presence of various inhibitors of specific signaling routes. With this approach we identified specific PMA/CD3 and PMA/CD28 signal transduction and genomic fingerprints. PMA/CD3 stimulation induced a Th1 phenotype, dependent on both Lck and PKC, whereas PMA/CD28 stimulation, which is independent of TCR-mediated activation of Lck, resulted in a profound activation of T cells, skewing towards a Th2 phenotype. Results and discussion Activation of Jurkat T cells by various stimuli leads to differential signaling fingerprints Jurkat T cells were activated by anti-CD3, anti-CD28, PMA, or ionomycin or combinations of these single Smeets et al. BMC Immunology 2012, 13:12 http://www.biomedcentral.com/1471-2172/13/12 Page 3 of 17 stimuli, in order to map the contribution of these stimuli towards the activation of proximal, medial and distal signal 
transduction pathways. As shown in reflected in the autophosphorylation of PKC, but was clearly detectable on the phosphorylation of the PKC substrate SCH 58261 web MARCKS. CD3-mediated stimulations and PMA-induced stimulations resulted both in the activation of AP1 family transcription factors c-Jun and ATF2. Analysis of nuclear translocation of NFATc1 and c-Jun /NFB p65, as part of the distal signaling events revealed that indeed CD3-mediated signaling induced both NFAT and c-Jun/NFB, of which the latter pathways were potentiated by CD28-mediated signaling. In line with the calcium release from the ER, PMA or PMA/CD28-mediated signaling did A 55000 PMA/ionomycine CD3/CD28 45000 PMA/CD3 CD3 RFU 35000 CD28 PMA/CD28 PMA Control 25000 15000 0 25 50 75 100 125 150 175 seconds B Proximal signal transduction events Medial signal transduction events Distal signal transduction events C Smeets et al. BMC Immunology 2012, 13:12 http://www.biomedcentral.com/1471-2172/13/12 Page 4 of 17 not induce NFAT nuclear translocation but highly activated the CD28 responsive element transcription factors c-Jun and NFB p65. These results indicate that two distinct co-stimulatory profiles can be identified. A CD3/28 and PMA/CD3 stimulus that signals via Lck, increasing intracellular Ca2+ and activating NFAT, and a PMA/CD28 calcium independent -stimulatory activation signaling via PKC and MARCKS. Next, the molecular mechanisms involved in these signaling pathways were further