The Spliceosomal Phosphopeptide P140 Controls the Lupus Disease by Interacting with the HSC70 Protein and via a Mechanism Mediated by cd T Cells Nicolas Page1, Nicolas Schall1, Jean-Marc Strub 2 , Marc Quinternet3, Olivier Chaloin1, Marion De´ cossas1, Manh Thong Cung 3 , Alain Van Dorsselaer2, Jean-Paul Briand1, Sylviane Muller1* 1 CNRS UPR9021, Institut de biologie mole´culaire et cellulaire, Strasbourg, France, 2 CNRS UMR7178, laboratoire de spectrome´trie de masse BioOrganique-IPHC-DSA- Universite´ de Strasbourg, Strasbourg, France, 3 CNRS-INPL UMR7568, Laboratoire de Chimie-Physique Macromole´culaire, Nancy Universite´, ENSIC, Nancy, France Abstract The phosphopeptide P140 issued from the spliceosomal U1-70K snRNP protein is recognized by lupus CD4+ T cells, transiently abolishes T cell reactivity to other spliceosomal peptides in P140-treated MRL/lpr mice, and ameliorates their clinical features. P140 modulates lupus patients’ T cell response ex vivo and is currently included in phase IIb clinical trials. Its underlying mechanism of action remains elusive. Here we show that P140 peptide binds a unique cell-surface receptor, the constitutively-expressed chaperone HSC70 protein, known as a presenting-protein. P140 induces apoptosis of activated MRL/lpr CD4+ T cells. In P140-treated mice, it increases peripheral blood lymphocyte apoptosis and decreases B cell, activated T cell, and CD42CD82B220+ T cell counts via a specific mechanism strictly depending on cd T cells. Expression of inflammation-linked genes is rapidly regulated in CD4+ T cells. This work led us to identify a powerful pathway taken by a newly-designed therapeutic peptide to immunomodulate lupus autoimmunity. Citation: Page N, Schall N, Strub JM, Quinternet M, Chaloin O, et al. (2009) The Spliceosomal Phosphopeptide P140 Controls the Lupus Disease by Interacting with the HSC70 Protein and via a Mechanism Mediated by cd T Cells. PLoS ONE 4(4): e5273. doi:10.1371/journal.pone.0005273 Editor: Jacques Zimmer, Centre de Recherche Public de la Sante´ (CRP-Sante´), Luxembourg Received March 3, 2009; Accepted March 23, 2009; Published April 23, 2009 Copyright: 2009 Page et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by the Centre National de la Recherche Scientifique (CNRS), Region Alsace, and ImmuPharma France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: s.muller@ibmc.u-strasbg.fr Introduction The U1-70K small nuclear ribonucleoparticle protein is a major spliceosomal autoantigen recognized in systemic lupus erythema- tosus (SLE). We previously identified an epitope between residues 131–151 present within its RNA recognition motif and targeted early during the progression of the disease by IgG antibodies and CD4+ lymph node cells (LNCs) from H-2k MRL/lpr and H-2d/z (NZBxNZW)F1 lupus-prone mice [1,2]. A peptide analogue phosphorylated on Ser140 (named P140) was also recognized by LN and peripheral MRL/lpr CD4+ T cells [3]. Intravenous administration into Fas(CD95)-deficient MRL/lpr mice of P140 significantly improved their clinical and biological manifestations and prolonged their survival, while the non-phosphorylated analogue did not [3]. Moreover, when incubated with lupus patient’s peripheral blood lymphocytes (PBLs), P140 generated secretion of high levels of regulatory cytokine IL-10 in cell cultures without proliferation of CD4+ T cells, suggesting that P140 (and not the non-phosphorylated analogue, which induces CD4+ T cell proliferation) possesses specific immunomodulatory functions on lupus T cells [4]. This assumption was supported by showing that repeated administration of P140 into MRL/lpr mice transiently abolishes T cell reactivity to other regions of the U1-70K protein and to epitopes from other spliceosomal proteins [5,6] without altering the capacity of P140-treated mice to mount a normal protective antiviral immune response [6]. P140 was successfully included in a phase IIa clinical trial [7], and is currently evaluated in a phase IIb, double-blind, placebo-controlled dose-ranging study. The present study was performed to decipher the P140 mode of action. We sought putative receptor(s), different from the MHC molecules, which might explain the remarkable in vivo efficacy of P140, either alone or synergistically with class II MHC-peptide-T cell receptor (TCR) interaction. This led us to identify at the surface of spleen cells and LNCs a very specific P140-receptor, the heat-shock cognate HSC70 protein, and to further investigate whether the P140 phosphopeptide acts via cd T cells. These regulatory T cells, which control ab T cells, activated B cells and NK cells, preferentially respond to phospholigands [8] and interact with HSC70 [9,10]. They are abnormally regulated in human and murine lupus [11,12]. We also examined the genes that are differentially expressed rapidly after P140 administration into MRL/lpr mice. The results indicate that P140 controls the lupus disease by a unique mechanism involving pathways of both innate and acquired immune responses. Results P140 recognizes cell surface-expressed HSC70 protein To identify putative cell-surface receptor(s) of P140, we undertook a series of experiments based on a previously described method [13,14] using spleen cells and LNCs from MRL/lpr mice and biotin-labeled P140. The purified proteins were subjected to SDS-PAGE in denaturing conditions and the resolved gel was PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5273