In order to gain a deeper insight into the genetic regulation of cytokine-determined and immunoglobulin/ T cell receptor based signaling in lymphocytes, efforts to use RNA interference (RNAi) technology as a screening tool have been undertaken. An shRNA library consisting of all the mouse phosphatases has been prepared in lentivirus vectors with a puromycin resistance element. Unit scientists have chosen to analyze the importance of phosphatases in peripheral CD4 T cell differentiation to Th1 cells. The model utilized takes advantage of an indicator mouse prepared in the Unit in which GFP expression marks the presence of the master Th1 transcriptional regulator T-bet. When T cells differentiate in vitro ino Th1 cells, they express veery large amounts of GFP and are easily detectable and can be purified by cell sorting. The initial screen carried out was aimed at determining what phosphatases were important for the expression or the extinction of T-bet during the Th differentiation process. Nave CD4 T cells were exposed to a library of all shRNAs complementary to phosphatases (over 1000 members), The shRNAs were in lentiviruses so that they could be introduced into nave CD4 T cells. The cels were then differentiated under Th1 conditions for 4 days, rested in IL-2 and exposed to puromycin, to eliminate cells that had not incorporated and expressed a member of the library. At this stage the great majority of the cells were already GFP+. The cells were then shifted to a Th2 culture. At the end of four additional days of culture under Th2 conditions, 1/2 of the cells were T-bet negative. The T-bet negative and positive cells were purified and the incorporated shRENAs were PCR amplified, utilizing a method to avoid the difficulties resulting from the hairpin. The resulting large set of amplified shRNAs were subjected to deep sequencing using an ABI instrument. More than 20 shRNAs were found to be uniquely expressed in the T-bet negative cells and a similar number in the T-bet positives. The technical aspects were were shown to be reproducible using PSR amplification with one extewrnal and one internal primer. We have now identified three phosphastases that catalyze reactions in the PI-3kinase pathway and one related to TCR signaling that either enhance Th1 priming or suppress xuch priming. We will undertake validation of these effects utilizing over or underexpression studies and then will analyze the effects of these phosphatases in vivo.
| Guo, Liying; Martens, Craig; Bruno, Daniel et al. (2013) Lipid phosphatases identified by screening a mouse phosphatase shRNA library regulate T-cell differentiation and protein kinase B AKT signaling. Proc Natl Acad Sci U S A 110:E1849-56 |
| Yamane, Hidehiro; Paul, William E (2012) Cytokines of the ?(c) family control CD4+ T cell differentiation and function. Nat Immunol 13:1037-44 |
