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Blood, Vol. 109, Issue 5, 2014-2022, March 1, 2007
Altered activation of AKT is required for the suppressive function of human CD4+CD25+ T regulatory cells
Blood Crellin et al.
109: 2014
Supplemental materials for: Crellin et al, Vol 109, Issue 5, 2014-2022
Files in this Data Supplement:
- Figure S1. CD4+CD25+ Tregs have defective AKT phosphorylation in response to stimulation via the TCR in the absence or presence of costimulation via CD28 (PDF, 29.1 KB) -
Ex vivo CD4+ T cells were (A) stimulated with  CD3 or CD3/CD28 mAbs and stained for CD4, CD25, and phospho-AKT (Ser473), or (B) unstimulated cells were stained for CD4, CD25, and total AKT. Panel A is an average of 3 independent experiments (P = .028, CD3 alone); panel B is representative of 3 experiments.
- Figure S2. CD4+CD25+ Tregs and CD4+CD25– T cells have equivalent PI3′K function and levels of the phosphatases PTEN and SHIP (PDF, 49.2 KB) -
(A,C) Ex vivo CD4+ T cells were stimulated with CD3/CD28 mAbs for the indicated times, and MFIs following staining with anti-PIP3 Abs (A) or anti–phospho-SHIP (C) were determined in CD4+CD25high or CD4+CD25– T cells. For panels A and C, a single experiment representative of at least 3 is depicted (P = NS), and the inset (A) is the fold change in MFI from resting to activation (10 minutes) with each point representing a separate experiment. (B,D) Ex vivo, unstimulated CD4+ T cells were stained for CD4 and CD25, and MFIs following staining with anti-SHIP (B) and anti-PTEN (D) were determined in CD4+CD25high or CD4+CD25– T cells. Panel B shows the average of 3 experiments (P = NS); in panel D each point represents a separate experiment (P = NS).
- Figure S3. Defective activation of phospho-AKT and S6 in CD4+CD25+ Treg-cell lines (PDF, 40.6 KB) -
Expanded CD4+CD25+ Tregs and CD4+CD25– T-cell lines were stimulated with CD3/CD28 mAbs for 10 minutes and stained for (A) phospho-AKT (Ser473) or (B) phospho-S6. A single representative experiment of 3 is depicted.
- Figure S4. Transduction efficiency and relative purity of transduced T-cell lines (PDF, 68.4 KB) -
(A) FACS-sorted CD4+CD25+ Tregs and CD4+CD25– T cells were transduced with control (pCLL) or AKTER-encoding lentivirus, and the percent of  NGFR+ cells was determined after 6 days. Transduced cells were purified and stained intracellularly for HA expression to monitor expression of AKT-ER. (B) Control and AKT-ER–transduced cell lines were stimulated with vehicle alone or 4HT (1 µM) for 1 hour and stained for phospho-S6. In both panels, a single representative experiment of 4 is depicted.
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