P140 Peptide in Lupus Figure 2. NMR structure, solubility, and stability of P140 and 131–151 peptides. (A,B) Superposition of the four lowest energy structures of the non-phosphorylated and phosphorylated peptides 131–151 after simulated annealing and restrained MD calculations. For simplicity, peptide residues are numbered from 1 to 21 for the non-phosphorylated peptide (a) and phosphorylated P140 peptide (b). The phosphorylated Ser residue at position 10 (Ser140) is represented by pS. For full details, see Text S1, Tables S1, S2, S3, S4, and Figures S4, S5, S6, S7. (C) Determination of the solubility limit of P140 and 131–151 peptides in distilled water, PBS and RPMI 1640 culture medium. The data show the variation of the mean intensity of light scattered (expressed as kilocounts, Kcps) that occurs when peptide aggregates are formed. In culture medium, P140 peptide aggregates at concentrations equal or superior to 50 mM. The amount and size of aggregates increase with peptide concentrations (413662800 nm at a 100 mM- concentration and 697264200 nm at a 200 mM-concentration). (D) Stability at 37uC of P140 peptide in 150 mM NaCl and PBS, as measured by high- performance liquid chromatography from the area of the peak corresponding to the intact peptide. doi:10.1371/journal.pone.0005273.g002 cells, and notably to cd T cells [9,10]. Earlier studies also showed that cd T cells interact with foreign and self-ligands, including MHC and MHC-like molecules and non-peptidic phosphoanti- gens [8,23]. We thus tested a pathway in which P140 likely ‘‘presented’’ by HSC70 induces apoptosis of potentially harmful lymphocytes by a cd T cell-mediated mechanism. Nine-week-old MRL/lpr mice received two successive intraperitoneal and intravenous administrations of anti-pan TCR cd monoclonal antibodies UC7-13D5 [24] eight and three days before P140 treatment. Efficiency of depletion was analyzed by FACS using anti-pan TCR cd antibody GL3 [25]. In these conditions, the number of viable cd T cells dropped over 54% (3.5 to 1.6%) in the PBL fraction. The viable ab T-cell compartment was marginally affected (62.7 to 69.2%). As shown in Figure 4H, while a single intravenous administration of P140 into MRL/lpr mice induced raised apoptosis of PBLs (27.6 vs. 10.1% annexin V+ cells; Figure 4H1), P140 induced no PBL apoptosis in cd T cell-depleted MRL/lpr mice (16.1 vs. 15.1%; Figure 4H2). Similar results were obtained in two independent experiments including each three mice per group. These data clearly indicate that in vivo P140 induces PBL apoptosis via a mechanism involving cd T cells. Several genes involved in inflammation are rapidly down-regulated in CD4+ T cells from P140-treated mice In previous studies we found that P140 administration into MRL/lpr mice transiently abolished T cell responsiveness to self- CD4+ T cell epitopes present in various spliceosomal proteins [5,6]. Our experiments were thus designed to test whether in vivo exposure to P140 might regulate expression of genes associated with T cell tolerance and anergy. qPCR and dedicated arrays were used to profile the expression of 84 genes related to T cell tolerance (Figure 5A). Total RNA was extracted from LN non- activated (B2202) CD4+ T cells (6-week-old mice) purified by negative selection. Five house-keeping genes were used to normalize the results and the whole experiment was repeated PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5273