Extended report with a cysteine residue added at its N-terminus. The luciferase renaturation assay was as described7 8 using either intact or heat-denatured luciferase and rabbit reticulocyte lysate in the presence of increasing concentrations of P140 peptide. Luciferase activity was determined using Bright-glo reagent (Promega, Charbomières, France ). In vivo experiments and cell studies Protection experiments and clinical monitoring have been described previously.1 All experimental protocols were carried out with the approval of the local Institutional Animal Care and Use Committee (CREMEAS). The renal pathology was evaluated by measuring proteinuria levels and observing vasculitis corre- sponding to the area of cell inﬁltrates. For in vivo imaging, Alexa Fluor633-labelled P140 was injected through the retro-orbital venous sinus of anaesthetised mice. Mice were immediately transferred into the cabinet of the NightOwl Bioimager (Berthold Technologies, Thoiry, France ) and images were taken periodi- cally for 3 h. Mice were then killed to collect organs for ﬁnal ﬂuorescence acquisition and validation of biodistribution. Signal intensity was evaluated using the Indigo software. Histology was carried out on frozen section of organs collected from MRL/lpr mice that received rhodamine-labelled P140 intra- venously. For co-localisation experiments, spleen sections were permeabilised and incubated with either ﬂuorescein isothiocya- nate (FITC)-labelled anti-B220 antibody or biotin-labelled anti- CD3e antibodies, followed by Alexa Fluor647-labelled streptavidin, or biotin-labelled anti-MOMA-1/CD169 antibodies speciﬁc for metallophilic macrophages, followed by Alexa Fluor488-labelled streptavidin. Other antibodies tested were: biotin-labelled anti- CD11b and biotin-labelled anti-F4/80 antibodies. Immunoﬂuorescence and immunoelectron microscopy experiments were performed on peripheral blood lymphocytes (PBLs) and splenocytes collected from MRL/lpr mice that had received Alexa Fluor488-labelled P140 intravenously 1 h before examination. Some experiments used splenocytes incubated for 1 h at 37°C with 5 μM Alexa Fluor488-labelled P140. HSC70 and MHCII molecules were revealed with speciﬁc antibodies (clone 1B5; Stressgen (Enzo Life Sciences, Villeurbanne, France) and 2G9; BD Biosciences (Le Pont de Claix, France), respectively). Fluorescence activated cell sorting analysis was as described previously.5 The antibodies were anti-HSC70 antibody labelled with R-phycoerythrin (clone 1B5) and anti-IA/IE antibody labelled with FITC (clone 2G9). For speciﬁc analysis of cellular subtypes, antibodies used were: anti-CD3e-FITC, anti-CD3e- allophycocyanin, anti-B220-PerCP, anti-B220-allophycocyanin, anti-CD4-allophycocyanin, anti-CD8-allophycocyanin, anti- CD11b-PerCP and anti-Gr-1-FITC. HSC70 and MHCII lev- els were expressed as the ratio of relative mean ﬂuorescence intensity for speciﬁc antibody/mean ﬂuorescence intensity for isotype-matched control antibody. For measuring lymphocyte proliferation, MRL/lpr and CBA/J mice (four to ﬁve/group) were given P140 peptide or ScP140 (100 μg/mouse) or saline only, daily for 10 consecutive days via the intraperitoneal route. Ciclosporin A (20 mg/kg/day) and prednisolone (feeding at 4 mg/kg/day) were tested in parallel. A lymphocyte proliferation assay was performed as described previously6 using 3×105 cells/well. Statistics Statistical analyses were performed using parametric (Student t test) and non-parametric (Mann–Whitney U) test. For all tests, p<0.05 was considered statistically signiﬁcant. 838 RESULTS In vivo P140 peptide effects and biodistribution We reported previously that intravenous administration of P140 to MRL/lpr mice signiﬁcantly improves their clinical and biological manifestations and prolongs their survival.1 We show here that P140 signiﬁcantly decreases vasculitis with fewer perivascular inﬂammatory inﬁltrates in kidneys (ﬁgure 1A; p=0.0079), slows down glomerulonephritis (ﬁg- ure 1B; p<0.0001) and reduces the appearance of dermatitis (ﬁgure 1C). P140 biodistribution after intravenous administration to MRL/lpr mice was monitored in real time by bioimaging in liv- ing mice. Alexa Fluor633-labelled P140 accumulated particularly in the lungs and spleen, a distribution that was not observed with an unrelated peptide (ﬁgure 1D; online supplementary ﬁgure S1; similar results were obtained in 6- and 12-week-old MRL/lpr mice). Fluorescence in the spleen was detectable 10–15 min after P140 injection and remained for at least 90–120 min. Similar results were observed in MRL/lpr mice that received rhodamine-labelled P140 intravenously (supplementary ﬁgure S2A). In the spleen, ﬂuorescence was mainly localised in the lymphoid white pulp, mostly in the mantle and marginal zones (supplementary ﬁgure S2B). It co-localised essentially with splenic B cells and resident macrophages (ﬁgure 1E) as well as with CD11b- and F4/80-positive cells. The macrophage F4/80 antigen is absent from macrophages located in T-cell areas of the spleen and lymph nodes (LNs). Thus, P140 preferentially homes in organs enriched in activated B cells and, more gener- ally, in antigen-presenting cells (APCs). P140 administration signiﬁcantly decreased peripheral hypercellularity in MRL/lpr mice (supplementary ﬁgure S3; no effect was seen with the ScP140 scrambled analogue). B cells, T cells (including γδT cells and DN T cells) and granulocytes/ monocytes were affected. In CBA/J control mice, however, even an overdosage of P140 did not produce any observable effect while, in contrast, the glucocorticoid prednisolone, which is active in MRL/lpr mice,9 and immunosuppressant ciclosporin A induced a decrease of PBL counts in both CBA/J and MRL/lpr mice (ﬁgure 1F). Peripheral B and T cells from non-treated MRL/lpr mice and remaining peripheral B and T cells from P140-treated mice responded equally well ex vivo to B-cell mitogen and slightly less well (p=0.024) to T-cell mito- gen (ﬁgure 1G). No effect of P140 was observed in CBA/J mice. This was not the case for ciclosporin A, which signiﬁcantly affected the abilities of peripheral B and T cells from MRL/ lpr and CBA/J mice to respond to mitogens, and prednisolone, which affected the abilities of CBA/J B and T cells to respond to mitogens. In the spleen, the proportion of T cells (including activated T cells) was signiﬁcantly decreased after P140 treat- ment (p=0.0022; ﬁgure 1H). The percentage of B cells remained unchanged. HSC70 and MHCII overexpression in MRL/lpr mice is reduced after P140 treatment P140 binds to HSC70 protein both at 25°C and 4°C (ﬁgure 2A), suggesting that P140 recognises both intracellular and cell surface-expressed HSC70 (result obtained with splenocytes, LN cells and PBLs). Fluorescent staining of P140 and HSC70 co-localises in the cytoplasm of freshly isolated, permeabi- lised MRL/lpr PBLs 1 h after intravenous administration of Alexa Fluor488-labelled P140 (ﬁgure 2B1). This co-localisation was conﬁrmed by immunoﬂuorescence and immunoelectron microscopy (ﬁgure 2B2, C), and was mainly cytoplasmic. Ann Rheum Dis 2011;70:837–843. doi:10.1136/ard.2010.139832