Extended report Figure 1 In vivo effect and biodistribution of P140 peptide in MRL/lpr mice. (A, B) MRL/lpr mice received saline alone or 100 μg P140 peptide/ injection/mouse on four occasions. Kidneys were collected from 25-week-old MRL/lpr mice and ﬁxed in formalin. Haematoxylin and eosin-stained kidney parafﬁn sections were scored for (A) vasculitis (n=5/group; p=0.0046 between the two groups) and (B) glomerulonephritis grade (ﬁve mice/ group, corresponding to 132–177 glomeruli). (C) Observation of skin lesions in both groups of 16-week-old mice. (D) In vivo biodistribution of Alexa Fluor633-labelled P140 peptide following retro-orbital injection of 100 μg peptide in NaCl (complete data in online supplementary ﬁgure S1). The yellow circle designates the position of the spleen. At the end of the experiment (180 min), organs were collected and ﬂuorescence was measured in the isolated organs (1, thymus; 2, spleen; 3, lung; 4, salivary gland; 5, axillary lymph node). (E) Spleen sections were co-stained with antibodies to B220 and MOMA-1/CD169 to identify B cells and marginal zone metallophilic macrophages, respectively. (F) Peripheral blood lymphocyte (PBL) counts measured in 10-week-old MRL/lpr and CBA/J mice (n=3–4 mice tested individually/group) that received at daily intervals 10 intraperitoneal injections of P140 (100 μg/mouse), ScP140 (100 μg/mouse), ciclosporin A (20 mg/kg) or saline or that received prednisolone orally at a 4 mg/kg dose. (G) Effect of P140 overdosage on the ability of PBLs to respond to T- and B-cell mitogens. Cultures for 48 h in the presence of 3 μg/ml concanavalin A (ConA) or 5 μg/ml; lipopolysaccharide (LPS). NT, not tested. (H) P140 peptide administration reduces the percentage of total CD3 T cells among splenocytes. Splenocytes from 6-week-old MRL/lpr mice were collected 3 days after a single intraperitoneal injection of 200 μg P140 or saline (n=6/group). Representative results of two independent experiments are shown. *p<0.05, **p<0.01, ***p<0.001. NS, not signiﬁcant (Mann–Whitney U test in panels A, B, F and H; Student t test in panel G). Bars, 100 μm. HSC70 is present at the surface of non-permeabilised MRL/ lpr and CBA/J peripheral B and T cells (notably B220+/TCRβ+ activated T cells and DN T cells)5 as well as on splenic MRL/lpr B220–/CD3/CD11b+/MAC-1/Gr-1– monocytes/macrophages and B220–/CD3/CD11b+/Gr-1+ granulocytes (ﬁgure 2D1). In the spleen, surface and intracellular HSC70 expression lev- els were raised in MRL/lpr mice (ﬁgure 2D and supplementary ﬁgure S4; observed also in LN cells, notably at the surface). The HSC70 expression increase observed in the spleen of MRL/lpr mice appeared to be correlated with an increased HSC70 mRNA expression in MRL/lpr splenocytes (ﬁgure 2E; not visualised in LN cells and thymocytes). Interestingly, in MRL/lpr mice that received intravenous injections of 100 μg P140 daily over 6 days, HSC70 expression in splenic B cells was decreased by 32% (ﬁgure 2F; expression of inducible Hsp70/Hsp72 protein was not affected). Interaction of HSC70 with antigen is an important component of the MHCII antigen processing pathway.10–14 In accordance Ann Rheum Dis 2011;70:837–843. doi:10.1136/ard.2010.139832 839