Extended report Figure 5 P140 peptide alters the autophagic pathway by slowing down the lysosomal activity in MRL/lpr B cells. (A) Western immunoblotting analysis with anti-LC3-II antibodies reveals increased appearance of lipidated LC3-II in B cells after 24 h of in vitro P140 treatment. Representative results of two independent experiments. (B) Accumulation of autophagy-selective substrate p62 visualised by Western immunoblotting in MRL/lpr or CBA/J B cells treated in vitro with P140. Representative results of two independent experiments. (C) A model by which P140 peptide interacts with HSC70, alters its properties and inﬂuences activation of autoreactive lymphocytes in lupus. (1) HSC70 is overexpressed both at the surface and intracellularly on lupus antigen-presenting cells (APC; B cells). (2) P140 hampers/ inhibits chaperone properties of HSC70 protein. P140 (3a) disturbs a complex of co-chaperones involved in the regulation of macroautophagy (putative) and slows down the autophagic ﬂux, and/or (3b) affects chaperone-mediated autophagy (CMA) leading to the alteration of (auto) antigens processing by lysosomal hydrolysis. (4) Subsequent delivery of antigens into MIIC compartments for class II presentation is altered and class II MHC molecules are destabilised, therefore compromising autoreactive CD4 T-cell response in a beneﬁcial pathway for the P40- treated individual. non-activated and DN T cells) and granulocytes/monocytes is progressively overexpressed in MRL/lpr spleen and LN cells. Intravenous administration of P140 into lupus-prone MRL/lpr mice leads to a decreased expression of HSC70. MHCII mol- ecule levels are also diminished. These data reveal that after downstream P140 binding to HSC70, a series of molecular events take place affecting the APC functions of MRL/lpr B cells, leading ﬁnally to a decreased expression or stability of MHCII molecules and a potential reduction of autoreactive T-cell signalling and priming. Importantly, normal lymphocytes seem to be saved from this process, possibly because the density of HSC70 molecules is insufﬁcient at the surface of normal APCs. The number of peripheral cells is not affected in P140-treated CBA/J mice, 842 and B and T cells proliferate normally in response to speciﬁc mitogens. We also found that the remaining B and T cells from P140-treated mice responded normally to mitogens. These data add to earlier studies showing that P140-treated and untreated MRL/lpr mice mount equivalent immune responses to a ﬂu virus challenge.6 HSC70 protein displays numerous housekeeping chaperoning functions. It is notably involved in nascent protein folding, prevention of protein aggregation under stress conditions, modulation of the assembly/disassembly of protein complexes and stabilisation of RNA messengers. HSC70 protein acts with a series of co-chaperones including Nag-4, CHIP, hip, Bcl-2-associated athanogene BAG-1, BAG-3 and Hsp40. Studies have suggested that depending on the cytokine environment, HSC70 preferentially associates with certain of these co-chaperones leading to different effects. We found that P140 inhibits the ability of HSC70 to refold denatured protein. It might therefore disrupt certain interac- tions of HSC70 with cofactors and chaperones. Future studies should provide insight into this topic and determine if this is relevant to some effects described in this study and lupus phenotype. HSC70 is central in protein translocation across membranes and lysosomal transport of proteins.24 25 It plays a pivotal role in chaperone-mediated autophagy in acting as a chaperone for unfolded substrate protein in association with the membrane receptor Lamp2a.14 16–18 26 In certain conditions, such as an elevation of co-chaperone BAG-3 level, HSC70 can also associ- ate with p62.27 Proper turnover of p62 by autophagy is critical, notably to prevent spontaneous aggregate formation.23 28–31 This protein is regarded as a key signalling player in the cell survival/death balance. Thus, certainly the most remarkable information raised in this work, is the striking effect of P140 on lysosomal degradation and class II antigen presentation (ﬁgure 5C). P140 directly impairs the molecular chaperoning properties of HSC70 by altering its ability to promote proper folding of polypeptide. In MRL/lpr B cells, P140 increases the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulation of lysosomal degradation at the autolysosome stage or with a lysosomal dysfunction. Moreover, the expression of stable MHCII mol- ecules is dramatically affected. This might explain our earlier observations showing a decreased T-cell response to various T-cell autoepitopes in P140-treated MRL/lpr mice6 32 and the absence of lupus patient’s PBL proliferation ex vivo in the presence of P140.4 Autophagy processes have not been explored in lupus. Although much has still to be determined to fully understand the impact of this complex pathway in MHCII presentation, inﬂammation and central or peripheral tolerance processes, our studies show that a peptide that can modulate lysosomal deg- radation can be beneﬁcial in MRL/lpr mice by reducing autore- active T-cell and B-cell activation. Of importance, P140 peptide exerts its effects without affecting normal B and T cells, possibly thanks to the expression levels of HSC70, which seems to act as a sensor receptor on MRL/lpr B cells. Acknowledgements The authors are indebted to Monique Duval for valuable assistance. The authors acknowledge the Imaging facility (Plateforme Inter-Unités de Microscopie et d’Imagerie Cellulaire Strasbourg-Esplanade). Funding This research was funded by the Centre National de la Recherche Scientiﬁque (CNRS), Région Alsace and ImmuPharma France. Competing interests None. Provenance and peer review Not commissioned; externally peer reviewed. Ann Rheum Dis 2011;70:837–843. doi:10.1136/ard.2010.139832