previous studies where the possible functional role of TMS chemical information SPINT2 in human lymphocytes is unraveled, however, SPINT2 was recently found to be a STAT6 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19798435 target in human macrophages as well as in human Th2 cells. We, hence, chose to experimentally validate the specificity of SPINT2 in primary human Th2-polarizing cells. We tested the specificity of SPINT2 expression at protein level on the cell surface of the Th cells with flow cytometry. At 24 hours after activation and induction of polarization the Th2 cells were found to express significantly more SPINT2 than the Th1 polarizing cells or the activated Th0 cells. As some of the human SPINT2 transcripts do not harbor the coding signal for the 
transmembrane domain, we therefore also investigated if SPINT2 would be secreted A SPINT2 B C DUSP6 Th0 Th1 Th2 D PPP1R14A Th0 Th1 Th2 GAPDH GAPDH ij et al. BMC Genomics 2012, 13:572 http://www.biomedcentral.com/1471-2164/13/572 Page 9 of 20 from the Th subsets. The SPINT2 concentrations were measured from the culture supernatants by enzymelinked immunosorbent assay at 48 hours after activation and polarization, and the Th2 cells were observed to secrete significantly more SPINT2 than Th0 or Th1 cells. The Th2 specific hits included also PPP1R14A, a phosphorylation-dependent inhibitor of smooth muscle myosin phosphatase, involved in regulation of smooth muscle contraction as well as DUSP6, responsible for dephosphorylation of ERK1/2. Recently, IL-4 induced RNA expression of signaling molecules PPP1R14A and DUSP6 have been reported. As the regulation of phosphorylation of the signaling intermediates is known to be highly important in defining the cell differentiation, we wanted to experimentally validate the subset specific expression of these two signaling molecules at protein level. We detected a clear Th2 specific PPP1R14A and DUSP6 protein expression at 72 hours time point post activation and initiation of the polarization, and very little or no expression in Th0 and Th1 lineages. Reciprocal regulators of lineage commitment In context of determination of T cell subset identity, a key group of genes is the one where the expression kinetics differ between all the lineages. The list of these significantly different genes is shown in identified to function in mouse splenic T cells as a regulator of IL-2 induced proliferation, however, no specific link to Th1 cells has been observed. Also, the significance of ATP9A, LPAR3 functioning in G-protein coupled receptor signaling, XRN1, BSPRY, MCTP2 or PTPRO in Th1 cells is yet to be studied. Recent data indicate that in B cells, PTPRO dephosphorylates Syk, a kinase that is critical in signal transduction of B-cell receptor. The Th2 up-regulated genes, PDE7B, SETBP1, C9orf135, TPRG1, IGSF3, or PPP1R14A have not been linked to CD4+ Th cell function, although their IL-4 mediated upregulation has been published, and furthermore, SETBP1, TPRG1 and PPP1R14A have been identified as direct targets of STAT6. Interestingly, we observed that most of the genes whose expression differs between all the three lineages behave in a similar manner, i.e., they are upregulated in Th1 and down-regulated in Th2. Among the reciprocally regulated genes we found 34 genes up-regulated in Th1 condition and only six genes behaved in the opposite manner. The hierarchical clustering of the kinetic profiles is depicted in Transcription factor binding sites in Th2 lineage To extend our transcriptional analysis into transcriptional regulation, we
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