Figure 2

Effect of Pam3Cys titration on proliferation of fresh versus cultured Tregs and Teff cells. Teff and Treg were freshly isolated from spleen or cultured for weeks and stimulated with different concentrations of Pam3Cys in the presence of 1 μg/ml anti-CD3. After 4 days proliferation was determined using thymidine incorporation (relative expression compared to anti-CD3 alone). The condition without Pam3Cys is set to 1 and the conditions with Pam3Cys are related to this condition. The absolute cpm levels of the cells with (5 ug/ml) and without Pam3Cys are for cultured Teffs 13383 cpm vs 7456 cpm, for fresh Teffs 31076 cpm vs 6693 cpm, for fresh Treg 15590 cpm vs 3776 cpm, and cultured Treg 16642 cpm vs 4936 cpm. Freshly isolated Tregs and Teff cells showed increased proliferation upon TLR2 stimulation, while only cultured Tregs (not cultured Teff) remained sensitive to TLR2 triggering. Representative results of 3 independent experiments are shown. The error bars represent standard deviations of triplicate measurements.