Extended report Figure 2 P140 peptide interacts with HSC70 and, in vivo, diminishes HSC70 overexpression in MRL/lpr lymphocytes. (A) Pull-down experiments showing the interaction of P140 peptide with HSC70 at 25°C and 4°C (representative results of ﬁve independent experiments). (B1) Co-localisation in permeabilised MRL/lpr dissociated splenocytes of P140 peptide and HSC70 examined 1 h after retro-orbital injection of 100 μg Alexa Fluor488-labelled P140 in NaCl. (B2) Co-localisation of P140 and HSC70 on spleen sections from MRL/lpr mice that received intravenously 200 μg rhodamine-labelled P140. (C) Co-localisation of P140 and HSC70 staining shown by immunoelectron microscopy (example with a single cell). HSC70, 15 nm gold particles; P140, 6 nm gold particles. (D) Fluorescence activated cell sorting analysis showing HSC70 expression at the surface of (D1) or intracellularly (D2) in different spleen cell subsets from CBA/J and 12-week-old MRL/lpr mice (n=5): B cells (B220+CD3), activated T cells included double negative T cells (B220+CD3), naïve T cells (B220–CD3), granulocytes/macrophages (CD11b+Gr-1+). HSC70 levels were expressed as the mean ﬂuorescence intensity (MFI) for speciﬁc antibody/MFI for isotype-matched control antibody (Ab) ratio. Error bars represent SD. Representative results of at least two independent experiments. (E) Levels of HSC70 mRNA detected by qRT-PCR in lymph node, splenic and thymic cells from 6- and 12-week-old MRL/lpr mice compared with CBA/J mice. Mean of two experiments in triplicate, normalised to actin-β expression. (F) Intracellular staining of HSC70 and Hsp70 in splenic B cells collected from untreated or P140-treated (six times at daily intervals; 100 μg/injection/mouse) 6-week-old female MRL/ lpr mice. *p<0.05, **p<0.01, ***p<0.001 (Student t test). Bars = 100 μm, except in (C) where bars = 500 and 100 nm in the picture and inset, respectively. with observations showing that in B cells HSC70 associates with HLA-DRB1*0401,10 and that HSC70 and MHCII molecules are associated in B-cell exosomes, for example,15 we found that HSC70 and MHCII molecules staining co-localises in per- meabilised MRL/lpr splenocytes (ﬁgure 3A and supplementary ﬁgure S5). After in vitro incubation, co-localisation of P140 and MHCII molecules staining was observed in 25% of cells, and co-localisation of P140, HSC70 and MHCII molecules staining was visualised in 5–10% of cells. Interestingly, we found that after daily intraperitoneal injections of P140 into 12-week-old MRL/lpr mice, overexpression of MHCII molecules in spleen B cells signiﬁcantly decreased (ﬁgure 3B). We thus examined further the effect of P140 on the stability of functional MHC molecules using Raji cells.13 In a dose-dependent manner, P140 hampered the formation of stable HLA-DRαβ dimers in these 840 lymphoblast-like cells (ﬁgure 3C; MHCII monomer expression remained unaffected). Thus, P140 associates with both HSC70 and MHCII molecules and decreases directly or indirectly their surface expression on splenic B cells. P140 alters autophagic processes in MRL/lpr B cells It has been shown earlier in B-cell lines that manipulating APC expression of HSC70 modiﬁes the presentation of epitopes derived from intracellular proteins but not from exogenous anti- gens to CD4 T cells.14 Since HSC70 is involved in autophagy pathways, notably in chaperone-mediated autophagy,14 16–18 leading to endogenous MHCII loading, we explored this mecha- nism, hypothesising that P140 might disrupt normal functions of HSC70 in this catabolic pathway. First, using a refolding test of heat-denatured luciferase by rabbit reticulocyte lysate,7 8 we Ann Rheum Dis 2011;70:837–843. doi:10.1136/ard.2010.139832