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Blood, Vol. 113, Issue 21, 5125-5133, May 21, 2009 This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef Selective expression of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b) on activated human FOXP3+ regulatory T cells allows for their purification from expansion cultures Blood Tran et al. 113: 5125 Supplemental materials for: Tran et al Files in this Data Supplement: Figure S1. CD25 and CD127 allow for purification of FOXP3+ Tregs (JPG, 213 KB) - (A) Flow cytometric correlation of the expression of different cell surface antigens with FOXP3 expression on freshly explanted CD4+ T cells from three healthy adult donors (D1, D2, D3). (B) Correlation of CD127hi and CD127low with expression of CD25 and FOXP3. Data is from one representative donor of 6. (C and D) Post-sort analysis of the percentage of FOXP3+ T cells purified by FACS-sorting the top 2% CD25+ T cells, by bead-sorting through depletion of CD127+ cells and positive selection of CD25+ cells, and by bead-sorting through positive selection of CD25. Figure S2. Correlation of surface markers on resting and activated Tregs in expansion cultures (JPG, 257 KB) - Day 14 Treg expansion cultures generated in the absence (NONE) or presence of rapamycin were restimulated with anti-CD3/CD28 for 48 h and analyzed for cell surface expression of a panel of Treg “specific” markers. Data are representative of 3 independent experiments. Figure S3. Repurification of Tregs with anti-LAP in expansion culture using one-step CD25 selection method (JPG, 52.1 KB) - (A) Percentage of CD4+FOXP3+ T cells in the starting population and following expansion of CD25+ cells in the absence (NONE) or presence of rapamycin over a 21 d period. (B) Percentage of LAP+ cells after 48 h restimulation of day 21 expansion cultures. (C) Percentage of CD4+FOXP3+ and (D) cell yield following purification of LAP+ cells. Figure S4. Purified LAP+ Tregs can be further expanded to obtain a greater cell yield while maintaining FOXP3 purity (JPG, 73.3 KB) - (A) FOXP3 purity and (B) cell yield of post-sort bead purified LAP+ Tregs on day 14 and on day 21 and 28 with continued expansion using anti-CD3/CD28 Dynabeads and IL-2. (C) In vitro suppression assay of fresh Tregs (CD25hi) and day 28 expanded LAP+ Tregs. Figure S5. Majority of cytokine producing effector T cells are found in the FOXP3− and FOXP3+ non-Tregs (JPG, 196 KB) - (A) Day 14 expansion cultures in the absence (NONE) or presence of rapamycin were restimulated for 5 h with PMA/ionomycin and analyzed by intracellular staining for cytokine production and FOXP3. (B) LAP+ and LAP− cells were purified 48 h after restimulation of day 14 expansion cultures generated in the absence (NONE) or presence (RAPA) of rapamycin. The purified cell populations were then restimulated for 5 h with PMA/ionomycin and production of IL-2 and IFN-γ measured by intracellular staining. (C) LAP+ and LAP− cells were purified 48 h after restimulation of day 14 expansion cultures and analyzed for IL-17 production. Data are representative of 3 independent experiments. This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef

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