P140 Peptide in Lupus Figure 5. P140-induced changes in expression of inflammation/tolerance/anergy-related genes in MRL/lpr mice. T cell anergy and tolerance RT-PCR-Array were used to evaluate expression of a selected set of genes expressed by non-activated T CD4+ cells from 6 week-old CBA/J mice, MRL/lpr mice and P140-treated MRL/lpr mice. (A) Heat map of differentially-expressed genes between 6-week-old normal CBA/J and MRL/lpr mice and between non-treated MRL/lpr and MRL/lpr mice that received each a single intravenous injection of 100 mg P140 peptide (evaluated 6 h and 24 h post-administration). A scale of colour intensity is presented for each heat diagram. Red, up-regulation; green, down-regulation; black, no change. Genes included in the microarray are listed in the table. Results are the average in gene expression for four independent experiments, with three mice per group in each experiment. (B) Changes in gene expression between non-activated T CD4+ cells from CBA/J and non-treated MRL/lpr. Out of the 84 genes tested, four were significantly up-regulated and one significantly down-regulated. The other changes were not statistically- significant. (C) Changes in gene expression between non-activated T CD4+ cells purified from non-treated MRL/lpr mice and MRL/lpr mice collected 6 and 24h post-treatment. (D) PBL surface expression of PD-1 studied in the three groups of mice by flow cytometry. Mean fluorescence intensity (MFI) values are indicated. doi:10.1371/journal.pone.0005273.g005 primary receptor. Interestingly, the immunosuppressive com- pound 15-deoxyspergualin has been shown to specifically bind to HSC70, which among other effects inhibits NF-kB nuclear translocation. It suppressed the progression of polyclonal B cell activation and lupus nephropathy in MRL/lpr mice [26]. Ex vivo, it altered the effective presentation of antigen to T cells by antigen- presenting cells and changed the levels of cytokines produced after engagement [27]. In a short trial, two of three treated SLE patients showed non-severe infectious episodes after 15-deoxyspergualin treatment [28]. Both P140 and 131–151 peptides (but not ScP140) bind to HSC70 showing that the phosphoryl moiety does not alter recognition and that sequence specificity exists. It has been shown that HSC70 undergoes conformational changes upon binding peptides [29]. An interesting issue would be to determine if HSC70 displays different conformations upon binding P140 and 131–151 peptides with a possible effect on recognition properties of the resulting complex. HSC70 plays an important role in binding and protecting peptide for MHC class II molecules [30]. It interacts with MHC molecules and particularly with HLA-DR4 [31]. Whether this association is of central importance remains to be determined. If the phosphoryl moiety has no obvious effect on the recognition of peptide 131–151 by HSC70, it apparently represents an important check-point in the P140 pathway for cd T cell recognition. Thus, only P140 phosphoantigen presented in the context of HSC70 could activate cd T cells. It is not known yet if all cd T cell subsets can be activated by this new ligand. cd T cells present an altered homing in lupus patients with an increased PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5273