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eji2305-sup-0001-figures1.pdf297KSuppl. figure 1 Experimental design and cell purities A scheme of the experimental approach is depicted in Figure (A). Viable CD4+ T cells amongst (B): PBMC, (D): ‘Tmem’, (F): ‘Tnaive’ are shown according to their expression of memory markers CD45RA/RO, and according to their expression of Treg markers FOXP3 and CD127 (C: PBMC, E: ‘Tmem’, G: ‘Tnaive’, I: ‘APC’ and K: ‘Treg’). Compared to undepleted PBMC ‘Tmem’ were depleted of > 98% of CD4+ CD45RA+ and > 74% of CD4+ FOXP3+ CD127lo/- T cells. ‘Tnaive’ cells were depleted > 97% of CD4+ CD45RO+ and > 80% of CD4+ FOXP3+ CD127lo/- T cells compared to PBMC. (H): the percentage of viable cells expressing HLA-DR amongst ‘APC’ is shown. The ‘APC’ fraction comprised of a more than 77% pure HLADR+ population, of which > 94% of CD4+ FOXP3+ CD127lo/- T cells were depleted compared to PBMC. (J): Isolated ‘Treg’ were uniformly labeled with CFSE and contained at least 54% CD4+ FOXP3+ CD127lo/- cells (K). Note, for better comparability, percentages depicted in figures B-K refer to the proportion amongst viable cells, not CD4+ cells. The proportion of viable CD4+ T cells expressing a Treg phenotype in the cell mix containing (L): ‘Tnaive’ + ‘APC’ + ‘Treg’, or (N): ‘Tmem’ + ‘APC’ + ‘Treg’ is shown; The CFSE+ cells are depicted in blue, CFSE- cells are shown in red. The histograms in (M) and (O) show the proportion of CFSE+/- cells amongst viable CD4+FOXP3+CD127lo/- cells shown in (L) and (N). ‘Tnaive’ mix (L) and ‘Tmem’ mix (N) contained a mean of 14.73% and 4.35% CD4 cells expressing a Treg phenotype, respectively, out of which a mean of 17.6% (in the mix with ‘Tnaive’) and 23.6% (in the mix with ‘Tmem’) were CFSE neg, indicating the presence of contaminating Treg from either of the non-labelled T cell compartments (‘Tmem’ or ‘Tnaive’) or added ‘APC’.

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