Another interesting Th2 specific top hit was SPINT2 encoding a transmembrane serine peptidase inhibitor Kunitz type 2. SPINT2 was originally named after its homology to hepatocyte growth factor activator inhibitor 1 and its first isolation from human placenta. The Kunitz inhibitory domains display potent inhibitory ac tivity towards several trypsin like serine selleck chemicals proteases and mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis. In ad dition, SPINT2 may function as a tumor suppressor gene, as its mRNA levels are down regulated in several human cancers and a deficiency in SPINT2 expression is linked with poor prognosis of breast cancer. There are no previous studies where the possible functional role of SPINT2 in human lymphocytes is unraveled, however, SPINT2 was recently found to be a STAT6 target in human macro Inhibitors,Modulators,Libraries phages 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 spe cificity 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 Inhibitors,Modulators,Libraries 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 from the Th subsets.
The SPINT2 concentrations were measured from the culture supernatants by enzyme linked immunosorbent assay at 48 hours after activation and polarization, and the Th2 cells were ob served to secrete significantly more SPINT2 than Th0 or Th1 cells. The Th2 specific hits included al so PPP1R14A, a phosphorylation dependent inhibitor of smooth muscle myosin phosphatase, Inhibitors,Modulators,Libraries involved in regula tion of smooth muscle contraction as well as DUSP6, responsible for depho sphorylation of ERK1 2. Recently, IL 4 induced RNA expression of signaling molecules PPP1R14A and DUSP6 have been reported. As the regulation of phos phorylation of the signaling intermediates is known to be highly important in defining the cell differentiation, we wanted to experimentally validate the subset specific ex pression 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 ex pression in Th0 and Inhibitors,Modulators,Libraries Th1 lineages. Reciprocal Inhibitors,Modulators,Libraries regulators of lineage commitment In context of determination of T cell subset identity, a key group of genes is the one where the expression kin etics differ between afatinib synthesis all the lineages. The list of these significantly different genes is shown in Table 2.