Pre-treatment of hMSCs with CD200 antibody was performed to study the role of CD200-CD200r axis in hMSC mediated immunosuppression.

<p><b>A</b>) The ability of CD200Hi/Me BMMSCs to inhibit TNF-α secretion of IFN-γ activated THP-1 macrophage-like cells can be prevented by pre-treatment with anti-human CD200 antibody whereas the pre-treatment with anti-human CD200 antibody had no effect on <b>B</b>) UCBMSC in presence of IFN-γ and <b>C</b>) CD200Hi/Me BMMSCs when co-cultured with LPS activated THP-1 macrophage-like cells. THP-1 macrophage-like cells were activated in co-cultures with 100 ng/ml LPS or 100 ng/ml IFN-γ for 24 hours and after that supernatants were collected. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031671#s3" target="_blank">Results</a> are represented as a mean±SD of co-cultures with three different CD200Hi/Me hMSC lines and mean±SD of three independent replicates with one UCBMSC line. The blocking of hMSCs with anti-human CD200 antibody was performed by pre-incubating part of the hMSCs with CD200 antibody for 20 min at room temperature and then washed twice with PBS whereas other part of the cells remained untreated. TNF-α concentration in supernatants from LPS or IFN-γ activated THP-1 macrophage-like cells alone were indicated as 100% and TNF-α concentration in supernatants from co-cultures with different hMSC lines, anti-human CD200 pre-treated and control ones, were compared to that. Before co-cultures, THP-1 macrophage-like cells were differentiated 48 hours in PMA (100 ng/ml) supplemented medium as described in materials and methods. ** indicates two-sample t-test p-value p<0.01.