Extended report HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus Nicolas Page,1 Frédéric Gros,1 Nicolas Schall,1 Marion Décossas,1 Dominique Bagnard,2 Jean-Paul Briand,1 Sylviane Muller1 ▶ Additional data (supplementary ﬁgures) are published online only. To view these ﬁles please visit the journal online at (http://ard.bmj. com). 1 CNRS UPR9021, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France 2 INSERM U682, Université de Strasbourg, Strasbourg, France Correspondence to Sylviane Muller, CNRS UPR9021, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France; s.muller@ibmc-cnrs.unistra.fr Accepted 11 November 2010 Published Online First 20 December 2010 This paper is freely available online under the BMJ Journals unlocked scheme, see http:// ard.bmj.com/info/unlocked.dtl Ann Rheum Dis 2011;70:837–843. doi:10.1136/ard.2010.139832 ABSTRACT Background The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus- prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells. Objective To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells. Methods P140 biodistribution was monitored in real time using an imaging system and by ﬂuorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic ﬂux markers. Results P140 ﬂuorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/ lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic ﬂux. Conclusion The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto) antigen processing might be greatly affected in MRL/ lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneﬁcial effect of P140 peptide in treated MRL/lpr mice. INTRODUCTION Systemic lupus erythematosus is a multifacto- rial (multigenic) and highly polymorphic systemic autoimmune disorder. It affects multiple organs, including skin, muscle, joints and vital internal organs such as kidneys and heart. Current pharma- cological treatments are largely palliative and result in non-speciﬁc immunosuppression. The goal today is to design more speciﬁc and effective treatments. P140 peptide is a 21-mer fragment of the spli- ceosomal U1-70K small nuclear ribonucleoprotein protein that signiﬁcantly ameliorates clinical and biological manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in lupus-prone mice.1 2 This peptide con- tains a phosphoserine residue at position 140, a modiﬁcation that speciﬁcally occurs at an early stage of apoptosis, before the cleavage of the C-terminal part of the protein by caspase-3 and the dephosphorylation of other serine residues by a PP1 phosphatase-mediated mechanism.3 Originally thought to act solely via the modulation of intrac- ellular signalling induced by αβ autoreactive T-cell receptor (TCR) engagement,2 4 P140 was unexpect- edly found to interact selectively with the consti- tutive heat-shock HSC70/Hsp73 protein.5 By a granzyme-B and caspase-dependent mechanism, it induces in Fas-deﬁcient MRL/lpr lupus mice apopto- sis of B cells and CD4, CD8 or double negative (DN) αβTCR+/CD4/CD8/B220+ T lymphocytes through a regulatory circuit involving γδT cells.5 Intravenous P140 peptide administration into young MRL/lpr mice thus causes T- and B-cell egress from periph- eral blood but does not affect T-cell priming, and does not interfere with the ability of P140-treated mice to resist to an infectious viral challenge.6 P140 also downregulates the expression of programmed death 1 (PD-1/CD279) receptor, a molecule of the CD28 family, which is overexpressed at the sur- face of MRL/lpr CD4 T cells.5 P140 peptide thus displays various effects on the immune system that probably interfere with several, yet unidentiﬁed, molecular pathways leading ﬁnally to a decreased T-cell autoreactivity and to signiﬁcantly lower levels of autoantibodies to native DNA. In the postulated P140 mode of action, bind- ing to the chaperone HSC70 protein seems to be central. The implication of or any defect of the HSC70 pathway in regulating immune functions in lupus is not documented. It is not known either if HSC70-chaperoned molecules, such as other Hsps and major histocompatibility complex (MHC) molecules, are affected by P140–HSC70 binding. To address these major questions, we evaluated the effect of P140 administration on the expres- sion of HSC70 and MHCII molecules, and studied the possible inﬂuence of P140 on autophagosomal pathways implicated in the endogenous MHCII loading. Since HSC70 is involved in certain phases of these pathways, we expected that binding of P140 to HSC70 might alter the delivery of intracel- lular material into the endosomal/lysosomal path- way and therefore thwart the ﬁnal presentation of self-antigens to autoreactive T cells. METHODS Synthetic peptides and biochemical assays Peptides were assembled and puriﬁed as des- cribed.1 5 The P140 peptide was also synthesised 837