P140 Peptide in Lupus Figure 3. Ex vivo effect of P140 peptide on MRL/lpr PBL apoptosis. (A) Induction of MRL/lpr and CBA/J PBLs apoptosis as measured by flow cytometry after annexin V- allophycocyanin labeling. The effect of increasing concentrations of the three peptides P140, 131–151 and ScP140 was assayed 20h after peptide incubation with cells. The results are expressed as the mean percentage (6SD) of specific apoptosis measured in three independent experiments. The decreased effect visualized when using 80 mM P140 probably results from peptide aggregation. (B) The same experiment was performed in measuring apoptosis of MRL/lpr PBLs by the decrease of mitochondrial Dym, as measured by a reduction of DIOC6 dye uptake, in the presence of increasing concentrations of P140 peptide. (C) Induction of CD4+ and CD8+ T cell apoptosis by P140 peptide and effect of granzyme-B, perforin and caspase inhibitors. Twenty hours after P140 incubation, cell death was determined by flow cytometry using an annexin V- allophycocyanin labeling (see also insert c1). Granzyme-B inhibitor I (20 mMZ-AAD-CMK; see also insert c2), perforin inhibitor (5 nM concanamycin A) and caspase inhibitors (20 mM Z-VAD-FMK) were added 30 min post-P140 incubation to analyze the involvement of these pathways. The results are expressed as the mean percentage (6SD) of specific apoptosis measured in three independent experiments. P values were (1) ,0.0001; (2) 0.0002; (3) non significant (ns); (4) ns; (5) 0.0003; (6) 0.0090. (D) Effect of P140 and 131–151 peptides on the viability of CD138+B2202 plasmocytes as measured by flow cytometry 20h after MRL/lpr PBLs treatment. Plots are representative of three independent experiments. (E) Viability of murine lymphoma T cells T29 studied by measuring mitochondrial reduction of 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium dye 24h post-treatment with increasing concentrations of P140 peptide and two unrelated peptides X and Y used as positive controls. (F) Effect of P140 and 131–151 peptides on the B220 marker expression by CD4+ and CD8+ T cells. Expression of B220 was analyzed on these cells 20h after adding P140 or 131–151 peptides. The results are expressed as the mean percentage of cell subpopulations measured in three independent experiments. P values comparing CD4+B2202 and CD8+B2202 cells without or after P140 treatment were 0.059 and 0.0432, respectively. P values comparing CD4+B220+ and CD8+B220+ cells without or after P140 treatments were 0.0364 and 0.1707, respectively. The other comparisons were statistically non-significant. doi:10.1371/journal.pone.0005273.g003 four times. Compared to CBA/J CD4+ T cells, pdcd1, il2rb, stat3 and ctla4 gene expression was significantly up-regulated and tnfrsf4 gene expression significantly down-regulated in MRL/lpr CD4 T+ cells of the same age (Figure 5A and 5B). Expression of the 79 other genes was not significantly affected (P.0.05). After a single intravenous administration of P140 into MRL/lpr mice, expression of several genes was down-regulated (Figure 5A and 5C). Thus, pdcd1 gene expression, which was increased in MRL/ lpr mice, was significantly down-regulated 6h only after P140 administration (P = 0.03). This feature was confirmed by FACS in showing an increased number of CD4+ T cells expressing PDCD1/PD-1 in MRL/lpr mice, as compared to CBA/J mice (Figure 5D), and a decreased expression level of PDCD1/PD-1 subsequent to a single P140 injection into MRL/lpr mice (Figure 5D). Post-P140 injection, other genes were either significantly down-regulated (il5 and il15, csf2, tnfsf10, cdk4, icam1, foxp2, and itch) or up-regulated (NFkb1 and stat6) in MRL/lpr mice (Figure 5C). PLoS ONE | www.plosone.org 5 April 2009 | Volume 4 | Issue 4 | e5273