Figure 3 P140 peptide interacts with major histocompatibility class II (MHCII) molecules and diminishes their in vivo expression. (A) Representative ﬂuorescence microscopy pictures revealing the co-localisation, in permeabilised MRL/lpr dissociated splenocytes, of P140, HSC70 and MHCII staining after a 1 h incubation at 37°C (bar = 5 μm). See other examples in online supplementary ﬁgure S5. (B) Expression of MHCII molecules measured by ﬂuorescence activated cell sorting analysis after daily P140 intraperitoneal administrations. MHCII expression measured in spleen B cells from 12-week-old MRL/ lpr mice at days 0, 2, 6 and 9 after injection, and compared with the expression level in those cells collected from 6-week-old MRL/lpr and CBA/J mice (three mice/group tested individually). Error bars represent SD. Differences versus 12-week-old MRL/lpr mice at day 0 were determined using the Student t test. *p<0.05, ***p<0.001. MFI, mean ﬂuorescence intensity. (C) P140 alters the stability of MHCII dimers in human lymphoma B cells. Raji cells were incubated for 24 h with P140 and then lysed with or without β-mercaptoethanol. Cell lysates were analysed by Western immunoblotting for expression of MHCII DRαβ dimers or MHCII DRα chain, compared with actin-β control. Representative results of two independent experiments. examined in vitro whether P140 could hamper folding proper- ties of the HSC70 molecule. At 40 and 80 μM, P140 inhibited luciferase refolding by 50.2% and 100%, respectively (ﬁgure 4A; ScP140 showed no effect). This effect was reversed by adding an excess of exogenous HSC70 (ﬁgure 4B). Next, we examined P140-treated cells for altered expres- sion of lysosomal or autophagy pathways markers. We studied the conversion in MRL/lpr cells of the soluble form of microtubule-associated protein-1 light chain 3 (LC3-I), a mammalian homologue of yeast Atg8, into the hydrophobic, mostly membrane-associated form LC3-II, which is enriched in the autophagic vacuole fraction but is rapidly degraded by lysosomal proteases when the autophagic ﬂux is intense.19 We observed that splenic B cells puriﬁed from 8-week-old MRL/ lpr mice exhibited almost undetectable levels of LC3-I and rather low LC3-II levels, which were dramatically enhanced in the presence of lysosomal protease inhibitors E64D (cal- pain and cathepsin B inhibitor) and pepstatin A (cathepsin D inhibitor) (ﬁgure 5A). This reﬂects the existence of a consti- tutive autophagic ﬂux in resting MRL/lpr B cells that can be blocked by speciﬁc inhibitors before the autolysosome for- mation, at the fused autophagosome–lysosome step.19 In the presence of P140, the basal accumulation of LC3-II expres- sion was increased in a P140 concentration-dependent man- ner, and strikingly, the autophagic ﬂux was proportionally less intense (ﬁgure 5A). This suggests that in MRL/lpr B cells, P140 downregulates autophagic ﬂux at the autolysosome stage. Interestingly, this stage is also affected by E64D and pepstatin A, for example, as well as by chloroquine,20 21 an antimalarial drug used in lupus treatment. Ann Rheum Dis 2011;70:837–843. doi:10.1136/ard.2010.139832 Extended report Figure 4 P140 peptide alters folding properties of HSC70. (A) Luciferase was denatured at 40°C for 30 min and then refolded during 1 h at 30°C with rabbit reticulocyte lysate in the presence or absence of increasing concentrations of P140 or ScP140. The results (expressed as a percentage) compare activity of renatured luciferase in the presence or absence of peptide. (B) The addition of increasing amounts of HSC70 counterbalances P140 peptide inhibition of luciferase refolding in a dose- dependent manner. Mean (SD) of two independent measurements. This result was conﬁrmed by measuring the levels of p62 (SQSTM1/sequestosome-1), one of the speciﬁc substrates that are degraded through the autophagy-lysosomal pathway. p62 is selectively incorporated into autophagosomes via direct interac- tion with LC3.22 23 In a peptide dose-dependent manner, p62 accumulated in P140-treated MRL/lpr B cells (ﬁgure 5B; the same effect, although at a lower extent, observable in CBA/J B cells), again consistent with an effect of P140 on the steady-state autophagy in these cells. DISCUSSION We present evidence here that P140 seems to interfere with a signalling pathway engaged in lysosomal degradation, lead- ing to instability of MHCII molecules supposed to present endogenous antigens to autoreactive T cells in murine lupus. After intravenous injection into MRL/lpr mice, P140 concen- trates mainly in the lungs and the spleen, and in the latter it co-localises essentially with B cells and resident macrophages of the lymphoid white pulp. Its localisation (if any) is poor in LNs and thymus. This suggests that P140 might possess a cer- tain degree of speciﬁcity for targeting some but not all tissues/ organs containing HSC70+ cells, and within these tissues and organs, again, it seems to target certain, but not all, HSC70+ cell subsets. We found that HSC70, which is present at the surface of splenic and LN MRL/lpr and CBA/J B cells, T cells (activated, 841