P140 peptide, and 606 M21s21 and 4.0761023s21 for the peptide 131–151, respectively, using Langmuir’s one site model, which gave the best fit at all concentrations used (x2 = 3.2/Rmax = 75 RU and 28.0/Rmax = 236 RU, for the P140 and 131–151 peptides, respectively). The equilibrium dissociation constant (Kd) calculat- ed from the ratio of the kinetic rate constants (kon/koff), was 7.32 mM and 6.72 mM, respectively, for the P140 and 131–151 peptides. Similar data were obtained when biotinylated P140 was on the chip and HSC70 in solution. Thus, in this in vitro system, both peptides bind with medium affinity (mM range) to HSC70. ScP140 did not bind HSC70. Of significant importance, no binding was observed between P140 and Hsp70. In good agreement with previous data showing that HSC70 is located at the plasma membrane, in the cytoplasm and nucleus [16,17], a membrane and punctuated intracellular fluorescent labeling of anti-HSC70 antibodies was observed on permeabilized MRL/lpr (Figure S2) and CBA/J splenocytes and PBLs. By electron microscopy, HSC70 was found localized in association with the plasma and nuclear membranes, the endoplasmic reticulum, small vesicles and the nucleus (Figure 1C). After incubating non-permeabilized splenocytes of both strains with P140, HSC70 and P140-staining co-localized at the plasma membrane (Figure 1D; Figure S3). Binding of P140 at the cell surface was confirmed by FACS analysis using freshly purified MRL/lpr splenocytes, increasing concentrations of biotin-labeled P140 and allophycocyanin-labeled streptavidin to reveal interaction. Surface P140 staining increased in a concentration-dependent manner at 4uC (Figure 1E) and 20uC (Figure S4), while very little staining was seen at 37uC, which is consistent with the internalisation of P140 associated with its HSC70 receptor. No fluorescence staining was visualized in the presence of biotin-labeled ScP140. HSC70 staining was present at the surface of MRL/lpr and CBA/J TCRb+B220+/2 and TCRb 2 B220+/2 cells corresponding to B cells, T cells and CD42CD82B220+ double negative (DN) T cells that progressively invade MRL/lpr mice (Figure 1F). Levels of HSC70 expression at the surface of normal and MRL/lpr PBLs were similar (6-week-old mice). Structural and physical properties of P140 peptide The above-described results show no difference regarding HSC70 recognition by the phosphorylated and non-phosphorylated peptides. Knowing that only P140 controls lupus disease in MRL/ lpr mice, we evaluated conformational characteristics of both peptides by 1H-NMR spectroscopy and molecular dynamics calculation. The detailed determination of the peptide structures is described in the supporting information (Tables S1, S2, S3, S4, and Figures S5, S6, S7, S8). As shown in Figure 2A and 2B, the conformation of the non-phosphorylated and phosphorylated peptides appears only slightly different (see Text S1). It should be emphasized in particular that in both peptide analogues, the side- chain of the Ser140 and pSer140 residues is directed towards the exterior medium and is part of the hairpin loop, which is probably important for recognition properties. The solubility of P140 and 131–151 peptides was determined at 20 and 37uC using dynamic light scattering measurements [18]. While a 200 mM-solution of peptide 131–151 remained highly soluble in water, phosphate-buffered saline (PBS) and culture medium (Figure 2C), aggregates form in the P140 solution at concentrations equal or superior to 50 mM in culture medium and PBS. In water, P140 was highly soluble (Figure 2C). The integrity of P140, as measured in saline and PBS by high-performance liquid chromatography from the area of the peak corresponding to P140 Peptide in Lupus the intact peptide, remained intact during at least 60 days at 37uC (Figure 2D). Ex vivo, P140 induces apoptosis of specific T cell subsets via a granzyme B-dependent mechanism We wondered whether the beneficial P140 effect was mediated by apoptosis of specific T cell subsets. We found ex vivo that after P140 co-incubation, in a peptide dose- and time-dependent manner, MRL/lpr PBLs displayed an apoptotic phenotype (annexin V positivity, reduction of DIOC6 dye uptake; Figure 3A and 3B; Figures S9, S10). Both MRL/lpr CD4+ and CD8+ T cells underwent specific apoptosis. Specific lysis of MRL/lpr CD4+ and CD8+ T cells was granzyme-B and caspase-dependent, while perforin inhibitor had very little effect (Figure 3C; inserts c1,c2). The number of viable B2202CD138/Syndecan-1+ plasma cells of the B cell lineage also decreased in the MRL/lpr PBL fraction (Figure 3D). Virtually no effect was observed when MRL/lpr PBLs were incubated with the non-phosphorylated or the ScP140 peptides, and no P140 effect was detectable with CBA/J PBLs (Figure 3A). No effect either was observed on murine CD4+CD8+ lymphoma T cells T29 (Figure 3E) and NIH 3T3 murine fibroblasts (not shown). It is noticeable that after a 20h-incubation of PBLs with P140 peptide, the proportion of preapoptotic CD4+ activated blasts (bearing the B220/CD45R marker [19]) significantly increased in the culture (Figure 3F). No effect was measurable with peptide 131–151. The results shown with CD8+B220+ activated blasts were not statistically significant (Figure 3F). In vivo, P140 induces PBLs apoptosis via a mechanism mediated by cd T cells Next, we investigated in vivo the P140 effect on lymphocyte fate. It is well documented that compared to normal situation, MRL/ lpr T and B cells are less sensitive to apoptosis, a feature resulting from defective activation-induced cell death, which is a FasL- dependent pathway [20–22]. It was therefore important to examine in vivo the effect of P140 administration on the apoptosis level of different lymphocyte subsets. After P140 administration into MRL/lpr mice according to our standard protection protocol [3], we found that at week 14 the absolute number of total peripheral white cells, the number of which is raised in MRL/lpr mice compared to CBA/J mice, was similar to the one measured in non-autoimmune mice (Figure 4A). At week 7 (i.e. at the onset of the disease) no difference was visible; at week 21 (eight weeks after the last administration) the effect was no longer statistically significant and this was true for all cell subsets axamined. The proportions of living (annexin V2) and apoptotic (annexin V+) cells were measured by FACS in haplotype-matched normal CBA/J mice, MRL/lpr prone mice and P140-treated MRL/lpr mice (Figure 4B). Compared to untreated MRL/lpr mice, the absolute number of TCRb+B2202 and TCRb+CD4 2 CD8 2 B220+ viable T cells, as well as that of B220+TCRb2 viable B cell population, was diminished, at least until week 14 (Figure 4C–4E; Figure S11). Regarding peripheral viable CD4+B220+ T cell blasts and CD8+ T cell population, their absolute number, which are increased in 14- week-old MRL/lpr mice, clearly decreased in treated mice (Figure 4F). In the PBL fraction the proportion of apoptotic cells (diminished in MRL/lpr mice, as expected) increased upon P140 peptide treatment (Figure 4G). This effect was no longer visible at week 21. Previous studies have shown that HSC70 expressed on the cell surface of tumor cells might act as a loading molecule complexed with cellular peptides for presentation to certain immune effector PLoS ONE | www.plosone.org 3 April 2009 | Volume 4 | Issue 4 | e5273