conjugated to 0.8 nm ultra-small gold particles (Aurion) in TBS containing 2% acetylated BSA (Aurion) and 0.2% WFSG. After washing in TBS, cells were fixed in 1% glutaraldehyde, washed again in TBS, and gold particles were enhanced using a silver kit (HQ silver; Nanoprobes). After further washing in 0.1M PB, cells were postfixed for 10min in 0.5% OsO4, dehydrated in graded series of ethanol, treated with propylene oxide and embedded in epon (Inland Europe). Ultrathin sections were stained with uranyl acetate and lead citrate and examined by TEM using a Hitachi H600 microscope. Images were acquired using a CCD camera (Hamamatsu). Cellular assays and FACS analysis Lymphocytes from normal and lupus mice were purified by density separation (Lympholyte-M, d = 1.0875; Cedarlane). Periph- eral blood lymphocytes (PBLs) were collected, washed 3 times in L- alanyl-L-glutamine-enriched RPMI 1640 medium (Biomedia) containing 10% fetal calf serum (FCS; Biomedia), 10 mg/mL gentamycin, 10 mM HEPES, and 561025 M b-mercaptoethanol, and resuspended at 16106 cells/mL in the above-described medium. Cell death occurring in the presence of peptides was measured in triplicate in a time scale of 30 min to 20 h using 16105 cells and 0–80 mM peptide/well. Apoptosis was measured by double staining with allophycocyanin (APC)-labeled annexin V (BD Bioscience) and propidium iodide using a FACSCalibur flow cytometer (BD Biosciences) [56]. Apoptosis was also studied by a decrease in mitochondrial transmembrane potential as detected by the reduction of DiOC6(3) dye uptake [56]. For specific labeling the following antibodies were purchased from BD Bioscience: FITC- conjugated-B220- and TCRb-antibodies; PE-conjugated-CD4- and CD138-antibodies; PerCP-conjugated-CD8- and B220-antibodies. FITC-conjugated TCRcd and PD-1 antibodies were purchased from eBioscience. PE-conjugated-HSC70-antibodies were pur- chased from Stressgen. For testing the influence of apoptosis inhibitors, the following inhibitors were incubated with cells for 30 min at 37uC: Granzyme B inhibitor I (20 mMZ-AAD-CMK; Calbiochem), perforin inhibitor (5 nM concanamycin A; Calbio- chem) and caspases inhibitors (20 mM Z-VAD-FMK; Promega). Data acquisition and analysis were performed using BD Bioscience CellQuest 3.3 software. Depending on the cell subsets studied, between 20,000 and 100,000 events were acquired. Experiments were repeated at least 3 times. Data are presented as mean6SD. Statistical significance was determined by Student’s t-test. Differ- ences were considered significant at P ,0.05. FACS analysis of cell surface HSC70 expression Splenocytes were washed in PBS and incubated for 20 min at 4uC with biotinylated peptide diluted in PBS containing 2% (v/v) FCS at the indicated concentrations. After washing, cells were incubated with APC-labeled streptavidin at 1 mg/mL in PBS containing 2% (v/v) FCS at 4uC for 15 min before another wash and subsequent analysis by flow cytometry. At least 20,000 events were acquired. Transcriptomic assays For monitoring the effect of P140 peptide on the expression of focused gene panels linked to inflammation, P140 peptide (or PBS as control) was administered intravenously into MRL/lpr and CBA/J mice (100 mg in PBS/mouse/injection), and 6 and 24 h post-treatment, T CD4+ cells mRNA levels for 84 genes involved in T cell anergy and 5 constitutive genes were measured using the SuperArray quantitative PCR assay (SuperArray Cell Anergy and Tolerance RT2 ProfilerTM PCR Array, Bioscience Corp). Briefly, inguinal, popliteal, and periaortic LN from 6-week-old MRL/lpr or CBA/J mice were removed 6 or 24 h after intravenous injection with 100 mg P140 in PBS (3 mice) or PBS alone and washed in P140 Peptide in Lupus L-alanyl-L-glutamine enriched RPMI 1640-Glutamax I (Life Technologies) containing 10% FCS, 10 mg/mL gentamicin, 10 mM HEPES, and 561025 M 2-mercaptoethanol. LN cells were then enriched for CD4+ T cells by negative selection. LN cells were depleted of macrophages, granulocytes, B cells, and CD8+ T cells by incubation with anti-CD11b (Mac-1), anti-GR1 (8C5), anti-CD19 (1D3), anti-B220 (6B2) and anti-CD8 (Lyt-2) monoclonal antibody purified in-house and with magnetic beads coupled to anti-rat immunoglobulins (Dynal). After negative selection, greater than 95% of the resulting cells were CD4+ positive by fluorescence-activated cell sorting analysis. Total RNA was extracted from approximately 56106 T CD4+ cells using Qiagen RNeasyH mini kits with inclusion of a DNase treatment step. First strand cDNA synthesis and quantitative real- time PCR with SYBR green master mix was performed in Stratagene MX4000 Real Time PCR SystemH. In brief, cDNA was prepared from 500 ng total RNA by using a RT2 PCR array first strand kit. A total volume of 25 mL of PCR mixture, which included 12.5 mL of RT2 Real-Time SYBR Green/ROX PCR master mix from SuperArray Bioscience containing HotStart DNA polymerase, SYBR Green dye, and the ROX reference dye (Tebu-bio), 11.5 mL of double-distilled H2O, and 1 mL of template cDNA, was loaded in each well of T cells Anergy and Tolerance RT2 Profiler PCR array (SuperArray BioscienceH), according to the manufacturer’s instruc- tions. PCR amplification was conducted with an initial 10 min step at 95uC followed by 45 cycles of 95uC for 15 s and 60uC for 1 min. The fluorescent signal from SYBR Green was detected immediately after the extension step of each cycle, and the cycle at which the product was first detectable was recorded as the cycle threshold. Data were imported into an Excel database and analyzed using the comparative cycle threshold method with normalization of the raw data to housekeeping genes including b-glucuronidase, hypoxanthine gua- nine phosphoribosyl-transferase1, b-actin, Hsp90 and glyceralde- hyde-3-phosphate dehydrogenase. Data were expressed as the mean6SD of four independent experiments. In vivo depletion of cd T cells Hamster anti-pan TRC cd monoclonal antibodies (UC7-13D5 clone) were purified from hybridoma culture supernatants using HiTrap protein G column (Pharmingen). cd T cell-depletion was achieved by two consecutive injections of hamster monoclonal antibodies (500 mg for the first administration and 1mg for the subsequent one; half intraperitoneally and half intravenously) 8 and 3 days before P140 administration. Sham depletion was performed with the same amount of hamster IgG isotype (The Jackson Laboratory). MRL/lpr mice received P140 peptide (100 mg/mouse) in saline via the intravenous route [3]. Flow cytometry was used to monitor cd T cell depletion and ab T cell number, and to measure PBL apoptosis. Animal welfare All experimental protocols were carried out with the approval of the local Institutional Animal Care and Use Committee (CREMEAS). Supporting Information Figure S1 Found at: doi:10.1371/journal.pone.0005273.s001 (0.18 MB TIF) Figure S2 Found at: doi:10.1371/journal.pone.0005273.s002 (0.14 MB TIF) Figure S3 Found at: doi:10.1371/journal.pone.0005273.s003 (0.31 MB TIF) PLoS ONE | www.plosone.org 11 April 2009 | Volume 4 | Issue 4 | e5273