P140 Peptide in Lupus Figure 4. In vivo effect of P140 peptide on MRL/lpr PBL apoptosis. (A–H) Female MRL/lpr and CBA/J mice received peptide P140 intravenously (100 mg/mouse in saline) at weeks 5, 7, 9 and 13, according to our standard procedure [3]. Blue = CBA/J mice, orange = MRL/lpr mice, red = P140-treated MRL/lpr mice. (A) The number of leukocytes/ml was evaluated by counting cells at weeks 7, 14 and 21. Each symbol represents the absolute number of cells in one individual mouse (5 mice in each of the three groups). Horizontal bars represent the respective average values6SD. (B–F) Influence of P140 therapy on the cell distribution. Detailed data observed at weeks 9, 12, 14 and 21 weeks are shown in the Figure S11. (B) Different lymphocyte subpopulations present in the PBL fractions from CBA/J, MRL/lpr and P140-treated MRL/lpr mice evaluated by flow cytometry. The gates used in the studies are shown in the inserts. (C) Number of B2202TCRb+ cells/ml in the peripheral PBL fraction collected from CBA/J, MRL/ lpr mice and P140-treated MRL/lpr mice, respectively at 14 and 21 weeks of age. P values were (1) 0.0011; (2) ns. (D) Number of B220+TCRb+ T cells/ml in the peripheral PBL fraction collected from CBA/J, MRL/lpr mice and P140-treated MRL/lpr mice, respectively at 14 and 21 weeks of age. P values were (1) 0.0084; (2) ns. (E) Absolute numbers of B220+TCRb2 cells in the peripheral PBL fraction collected from CBA/J, MRL/lpr mice and P140-treated MRL/lpr mice, respectively at 14 and 21 weeks of age. P values were (1) 0.0059; (2) ns. (F) Number of CD4+ and CD8+ T cells/ml in the peripheral PBL fraction collected from CBA/J, MRL/lpr mice and P140-treated MRL/lpr mice, respectively at 14 and 21 weeks of age. The number of CD4+ and CD8+ T cells bearing the B220 marker is shown in gray. (G) Percentage of annexin V-allophycocyanin+ PBLs collected from 9, 12, 14 and 21 week-old CBA/J mice, MRL/lpr mice and P140-treated MRL/lpr mice. P values were (1) 0.0344; (2) 0.0051; (3) 0.0047; (4) ns, 0.8668. (H) PBL apoptosis measured after a single intravenous administration of P140 peptide into MRL/lpr mice that received control hamster IgG (H1) or depleting cd T cell hamster IgG eight and three days before P140 treatment (H2). cd T cell-depletion was achieved by two consecutive injections of 500 mg (1st administration) and 1 mg (2nd administration) of monoclonal antibody UC7-13D5 (half intraperitoneally and half intravenously). Sham depletion was performed with the same amount of hamster IgG isotype. Efficacy of cd T cell depletion was measured by flow cytometry using anti-pan TCR cd hamster monoclonal antibody GL3. Data reflect 3 mice per condition, and are representative of two separate experiments. doi:10.1371/journal.pone.0005273.g004 Discussion This report defines a previously undescribed pathway taken by a peptide to immunomodulate autoimmune response (diagrammed in Figure 6). Key features of this pathway, as identified in MRL/ lpr mice, are (i) P140 binding to a unique receptor, the constitutively-expressed HSC70 chaperone protein; (ii) involve- ment of cd T cells in a granzyme B- and caspase-dependent mechanism, leading directly or indirectly via cytotoxic effectors to apoptosis of distinct (activated) T and B lymphocytes/blasts. In parallel, an important effect is also observed on non-activated CD4+ T cells, as shown at the level of several genes involved in inflammation, which are rapidly down- or up-regulated upon P140 treatment. Several check-points are decisive in this scheme and might explain the remarkable protecting efficacy of P140 in lupus. First, HSC70 but not inducible Hsp70 seems to operate as a PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5273