-2. Whole cell lysates were prepared, and equal amounts of proteins were resolved by SDS-PAGE and blotted with 
anti-cyclin E mAbs, or with anti-pRb mAbs. A representative experiment out of 3 is shown. Arbitrary densitometric units for the bands were analysed by computing densitometry. Means +/-SEM for pRb and cyclin E expression are represented below the immunoblot. doi:10.1371/journal.pone.0136885.g003 were similar between the lines in the different stimulation conditions. We then measured the expression of mRNA coding for the transcription factors T-bet, GATA-3, RORc and FoxP3 that are master regulators of Th1, Th2, Th17 and Treg cells differentiation, respectively. While PEA-15-deficient and wt T cells exhibited similar levels of T-bet, GATA-3 and FoxP3, RORc seemed to be expressed at higher levels in PEA-15-deficient T cells although this difference did not reach statistical significance. Furthermore, compared to wt cells, PEA-15-deficient T cells expressed and secreted more IL-4-induced gene 1 , a phenylalanine oxidase, whose mRNA expression was recently showed in Th17 to be strictly dependent on RORc expression. We next investigated whether the lower production of IL-4 and IL-10 by TCR-stimulated PEA-15-deficient CD4+ CD62L+ nave T cells in vitro, was associated with an abnormal humoral immune response in vivo. To this aim, we used the previously described red blood cells alloimmunization model in which mice are injected intraperitoneally with poly and then transfused with Hen Egg Lysozyme -conjugated RBC. In order to sensitize the model by preventing potential suppression, prior injection with anti-CD25 mAbs was performed before transfusion, to deplete Treg before alloimmunization . Mice were sacrificed after one week, and serum levels of anti-HEL PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19731547 IgG were measured by cross-matched flow cytometry. While wt mice exhibited readily detectable anti-HEL IgG in their serum, PEA15-deficient mice did not, demonstrating that PEA-15 was necessary for antibody production in this model. Discussion In this paper, we have found that PEA-15 deficiency in CD4+ T cells resulted in constrained T cell cycling and impaired production of IL-2 and IFN by activated mature CD4+ T cells in vitro, as well as impaired production of IL-4 and to a lesser extent IL-10 by TCR-stimulated differentiating CD4+CD62L+ nave T cells. Conversely, higher expression and activity of IL4I1, a Th17-associated phenylalanine oxidase was induced in TCR-stimulated PEA-15-deficient CD4+ CD62L+ nave T cells. These abnormalities were associated Acacetin site 19728767?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=111″ title=View Abstract(s)”>PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19728767 with defective humoral response to RBC alloimmunization in PEA-15-deficient mice in vivo. In parallel, our results showed that absence of PEA-15 resulted in abnormal subcellular compartmentalization of phosphoERK1/2 in resting and activated CD4+ T cells, and this was associated with impaired regulation of classical targets of the ERK1/2 signaling pathway. Although indirectly, our data suggest that PEA-15-dependent regulation of cytokines expression in CD4+ T cells, involves lower ERK1/2-signaling, in accordance with other reports, showing that subcellular compartmentalization of ERK1/2 represents another level of regulation of the enzymes activity, besides phosphorylation of ERK1/2. The resident nuclear ERK1/2 in resting PEA-15-deficient T cells, confirms the data reported by Pastorino et al.; it may be the result of previous in vivo ERK1/2 activation followed by dephosphorylation by nuclear phosphatases and defect of return of ERK1/2 to t
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