Figure 2.

MSC mediate suppression of mitogen-induced T cell proliferation through iNOS. (A) LNC from PVG.7B rats were labeled with CFSE and cultured in the presence of ConA for 3 days. Dilution of CFSE intensity was measured by flow cytometry to quantify lymphocyte proliferation. PVG.7B MSC were added at the start of culture at MSC:LNC ratios of 1:10 (left panel) or 1:100 (right panel), either alone (shaded off-set histograms) or together with inhibitors of IDO (1-MT, 1 mM; dotted line), COX (IMC, 5 μg mL⁻¹; dashed line), or iNOS (L-NMMA, 1 mM; solid line). Proliferation in the absence of MSC is also shown (ConA–LNC, black histograms). MSC addition resulted in a complete or partial inhibition of ConA-induced proliferation at 1:10 and 1:100 MSC:LNC ratios, respectively. The proliferative response was fully restored by addition of L-NMMA while 1-MT or IMC had no effect. (B) In the same experimental set-up as shown in (A), inhibitors were added to ConA-stimulated LNC cultures in the absence or presence (MSC:LNC ratio 1:10) of MSC. The percentage of CFSE^lo cells was used as a measure of cells that had undergone one or several cell divisions. The gate shown in (A) marks the distinction between CFSE^lo and non-dividing CFSE^hi cells. Data are representative of three independent experiments and are shown as mean and SD of triplicates.