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Results: 5

1.
Fig. 5.

Fig. 5. From: Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

GARP expression is not increased proportionate to FOXP3+ T cells in HIV+ individuals. (A) Representative phenotypic staining of HIV− and HIV+ sorted CD4+ T cells for GARP, CD25, and FOXP3 expression. CD4+ T cells were sorted from both HIV+ and HIV− subjects and were stained both immediately as resting T cells for CD25 and FOXP3 and after 1 day of TCR activation for GARP and FOXP3 expression. (B) Analysis of FOXP3+CD25+ T cells from multiple HIV− and HIV+ donors. (C) Analysis of percentage of T cells that express GARP and FOXP3 postactivation from same donors shown in Fig. 5B.

Rui Wang, et al. Proc Natl Acad Sci U S A. 2009 August 11;106(32):13439-13444.
2.
Fig. 2.

Fig. 2. From: Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

Silencing FOXP3 and GARP expression in Tregs. (A) Silencing FOXP3 in Tregs through shRNAs. Purified TN or TNreg cells were stimulated and transduced with control or FOXP3 shRNA encoding lentiviruses. Cells were expanded in vitro for 2 weeks, sorted based on GFP expression (expressed by vectors) and FOXP3 levels, and induction of GARP expression was determined 2 days post–TCR stimulation. (B) Knockdown of GARP by shRNAs in expanded Tregs. Cells were transduced with viruses encoding control or GARP shRNA, expanded for 2 weeks in vitro, and sorted for GFP expression encoded by the same lentiviral vector. Cells were then stained for FOXP3. GARP was stained 24 hours post–TCR reactivation. Data shown are representative of three healthy donors. (C) Suppressive activity of control or shRNA-expressing Teff and Tregs were assessed as described in Fig. 1. Representative data using a 1-to-4 ratio of Treg/Teff to target T cells are shown.

Rui Wang, et al. Proc Natl Acad Sci U S A. 2009 August 11;106(32):13439-13444.
3.
Fig. 4.

Fig. 4. From: Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

Suppressive activity and IL-17 secretion by CD25+ T cells based on GARP expression. (A) Experimental setup was as follows: CD4+ T cells were first sorted into CD25+ and CD25− subsets. The cells were then activated with anti-CD3/28 beads for 2 days, stained with GARP antibody, and sorted into GARP− and GARP+ cells. These cells were further expanded and rested in culture for an additional 10 days. (B) Suppressive activity of CD25+GARP+ and GARP− cells. Suppressive activity was performed and calculated as described in Fig. 1. Representative CFSE data are shown in Fig. S4. (C) Cells activated and sorted as shown in Fig. 4A were cultured in IL-2 for 8–10 days and then reactivated with PMA and ionomycin for 5 hours, followed by intracellular staining with IL-17–FITC, FOXP3.APC, and IFNγ-PE.Cy7 antibodies. (D) Expression of IFNγ versus IL-17 from the same staining shown in (C). Results are representative of three separate experiments from different donors.

Rui Wang, et al. Proc Natl Acad Sci U S A. 2009 August 11;106(32):13439-13444.
4.
Fig. 3.

Fig. 3. From: Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

Expression of GARP in TGFβ-treated or FOXP3-overexpressing T cells. (A) Induction of FOXP3 by TGFβ in TCR-stimulated T cells. TN cells were activated through TCR in the presence or absence of 20 ng/ml TGFβ. FOXP3 expression was determined by intracellular staining at different time points postactivation. Data shown are representative of day 6 postactivation. (B) Expression of GARP on TGFβ-treated cells. TGFβ-treated or untreated cells were restimulated through TCR at different time points between 6 and 12 days of culture post–original activation, and GARP expression was determined after 2 days. A representative GARP staining performed post–day 6 of first activation is shown. (C) Suppressive activity of TGFβ-treated T cells. Activated T cells treated with or without TGFβ were expanded in vitro, and their suppressive activity was assayed at different days postactivation. Representative CFSE data are shown using a 1-to-4 suppressor-to-target ratio. (D) Expression of FOXP3 on overexpressing T cells. Purified CD4+ T cells were activated and transduced with FOXP3 lentiviruses that also express RFP as a marker. After 12–14 days postculture, cells were sorted based on RFP expression, restimulated through TCR for 2 days, and expression of FOXP3 and GARP determined on FOXP3-overexpressing cells (FOXP3+), vector control-transduced cells (Control), or Tregs. GARP expression was determined for FOXP3+ and Tregs after gating on FOXP3 expression. (E) Expression of GARP on FOXP3-overexpressing T cells analyzed from multiple donors.

Rui Wang, et al. Proc Natl Acad Sci U S A. 2009 August 11;106(32):13439-13444.
5.
Fig. 1.

Fig. 1. From: Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

GARP is specifically expressed on activated Tregs and defines suppressor T cells. (A) Surface expression of GARP on TN (naive), TM (memory), TNreg, and Tregs. Different T-cell subsets were isolated based on CD25 and CD45RO expression as previously described (10). Cells were stimulated with anti-CD3 and anti-CD28 beads (TCR stimulation) overnight and stained for surface GARP. (B) GARP expression restricted to FOXP3+ cells. TN and TNregs were activated and expanded in vitro for 2 weeks to obtain Teff and expanded Tregs. Cells were activated through TCR and stained for GARP/isotype and FOXP3. (C) Kinetics of GARP expression on T-cell subset postactivation. Data represent percentage of GARP+ T cells at different time points postactivation. (D) Induction and sorting of GARP+ T cells. CD4+ T cells were stimulated through TCR for 2 days and stained for GARP and CD25. (E) Suppressive function of GARP+ T cells. GARP+CD25+ and GARP-CD25+ populations were sorted 2 days post–TCR activation, rested in culture for 8–10 days, and assayed for suppressive activity using CFSE-labeled resting target T cells and different ratios of GARP+ suppressor or GARP− effector T cells. The cells were then stimulated with anti-CD3 antibody (50 ng/ml) in the presence of dendritic cells for 4 days, and proliferating cells were determined based on decrease in CFSE expression as shown in Fig. S1D. Percent suppression was calculated based on CFSE dilution of target T cells in suppression culture as described before (10). Briefly, the percentage of target cells that undergo division in response to the stimuli alone, in the presence of expanded Tregs, or control Teff cells, was determined. The percent suppression was then calculated by percent reduction in proliferation of the target cells with suppressors as compared with target cells alone. All data shown are representative of at least three experiments performed with T cells from different healthy donors.

Rui Wang, et al. Proc Natl Acad Sci U S A. 2009 August 11;106(32):13439-13444.

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