Your privacy, your choice

We use essential cookies to make sure the site can function. We also use optional cookies for advertising, personalisation of content, usage analysis, and social media.

By accepting optional cookies, you consent to the processing of your personal data - including transfers to third parties. Some third parties are outside of the European Economic Area, with varying standards of data protection.

See our privacy policy for more information on the use of your personal data.

for further information and to change your choices.

Skip to main content
Figure 6 | BMC Immunology

Figure 6

From: Induction of granzyme B expression in T-cell receptor/CD28-stimulated human regulatory T cells is suppressed by inhibitors of the PI3K-mTOR pathway

Figure 6

Fresh peripheral blood nTregs were purified based on flow cytometric sorting of the CD4+, CD25-bright T cell subset (brightest 1/3 of CD25+ cells; Figure 6A/B) or by a bead based method based on a CD4+, CD25+, CD127- surface antigen expression profile (Figure C/D) then expanded for four days with CD3/CD28 beads and IL-2 with and without 10 ng/mL rapamycin. Both isolation methods gave similar results. CD4 and FOXP3 expression were then evaluated on day 4 and showed relative purity of the resulting Treg populations (90.8% and 90.5% in panel A and 85% and 83% as indicated in panel C). Tregs were then co-cultured at a 3:1 ratio of Tregs (Tr = Tregs expanded without rapamycin; TrRapa = Tregs expanded with rapamycin) to CFSE labeled L428 target cells. Samples were plated in triplicate in media without additional stimuli or inhibitors. After 6 or 48 hours as indicated, apoptosis was assessed in the CFSE labeled L428 target cell population by flow cytometric evaluation of annexin-V binding (early apoptotic cells) or propidium iodide (PI) staining (late apoptotic cells) in the 6 and 48-hour assays, respectively (panels B and D). Control samples consisted of CFSE-labeled L428 cells alone, L428 cells alone exposed to 10 μM staurosporine (6 hour cytotoxicity assays only) and nonactivated Treg-depleted T cells (Tc) plated at a 3:1 ratio with labeled L428 target cells. Representative dot plots for a 6-hour cytotoxicity assay, with annexin-V positive apoptotic target cells highlighted with percentages, are shown in panel B. Replicates from a 48-hour cytotoxicity assays are graphed in panel D. Statistical comparison of the indicated data sets was performed using a t-test (NS-not significant with P > .05, * denotes P < .02, ** denotes P = .003). The data shown in both of the timepoints are representative of three experiments using multiple donors.

Back to article page