Figure 1.
Differential CD45RO expression between Foxp3+ T cells differentiated in vivo by chronic viral infection and in vitro by TCR stimulation.
(A) Flow cytometric analysis of Foxp3 expression by CD4+ PBMCs of 4 HBV-infected patients (upper panels) and 4 healthy controls (lower panels). The frequency of cells in each quadrant is displayed as numbers in the quadrants. (B) The average percentages of CD45RO+Foxp3+, CD45RO-Foxp3+ and Foxp3+ cells in the pools of CD4+ cells from 38 HBV-infected patients and 23 healthy controls. (C) Flow cytometric analysis of CD45RO expression by CD4+Foxp3+ PBMCs of 4 HBV-infected patients (upper panels) and 4 healthy controls (lower panels). The frequency of cells in each quadrant cells is displayed as numbers in the quadrants. (D) The average percentages of CD45RO+ and CD45RO- cells among pools of CD4+Foxp3+ cells from 38 HBV-infected patients and 23 healthy controls. (E) Naive CD4+ T cells were sorted from adult peripheral blood by FACS, and the cells were activated with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) in the absence or presence of TGF-β (5 ng/ml) and IL-2 (100 UI/ml) for 5 days. The cells were then collected and analysed by flow cytometry to evaluate the expression of Foxp3 and CD45RO.
Figure 2.
Small portion of human CD4+ memory T cells can be converted to Foxp3+ T cells in vitro.
(A) CD4+ naive (Tn) and memory (Tm) T cells were sorted and activated with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) in the presence or absence of TGF-β (5 ng/ml) and IL-2 (100 UI/ml). Foxp3 expression was analysed by flow cytometry at different time points as indicated. (B) The average percentages of Foxp3 expression in CD4+ naive and memory T cells on day 5 after TGF-β and IL-2 induction. (C) and (E) Flow cytometric analysis of CTLA-4 (extracellular and total), PD-1, and PD-L1 expression in total, freshly sorted, naive (Tn) or memory (Tm) CD4+ T cells. (D) and (F) Sorted CD4+ naive (Tn) or memory (Tm) T cells were activated for 5 days as described above, and the total population was analysed using flow cytometry for the expression of CTLA-4 (extracellular and total), PD-1, and PD-L1.
Figure 3.
More efficient induction of Foxp3+ T cells by CD4+CD62L+ central memory T cells than by other memory T cell subsets.
(A) Sorted CD4+ central memory and effector memory T cells were activated with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) in the presence of TGF-β (5 ng/ml) and IL-2 (100 UI/ml) for 7 days. The induction of Foxp3+ T cells in the CD4+ fraction was analysed by flow cytometry based on the intracellular staining of Foxp3.
(B-D) Tn indicates sorted CD4+ naive T cells, CD62L+ Tcm indicates sorted CD4+CD62L+ central memory T cells, CD62L+ Tem indicates sorted CD4+CD62L+ effector memory T cells, and CD62L- Tem indicates sorted CD4+CD62L- effector memory T cells. The results are one representative of three independent experiments.
(B) Flow cytometric analyses of the CD4+ naive and memory T-cell phenotypes.
(C) Sorted CD4+ naive, CD62L+ central memory, CD62L+ effector memory and CD62L- effector memory T cells were activated for 5 days as above and analysed by flow cytometry for Foxp3 expression in the indicated group of cells.
(D) The average percentages of Foxp3 expression by different subsets of memory T cells after induction by TGF-β and IL-2.
Figure 4.
Suppressive functions and methylation status of Foxp3+ T cells derived from human CD4+CD62L+ central memory cells.
‘Treg’ indicates sorted CD4+CD25high T cells, ‘Foxp3+ Tn’, ‘Foxp3+ Tcm’ and ‘Foxp3+ Tem’ indicate in vitro TGF-β-induced Foxp3+ cells from naive, CD62L+ Tcm and CD62L- Tem CD4+ cells, respectively.
(A) CFSE-labelled allogeneic CD4+CD25- T cells (2.5×104) cultured alone or mixed with different ratios of suppressor cells (1:0, 1:1 and 2:1) were activated with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) in 96-well plates for 5 days. The suppressor cells were derived from naive, CD62+ Tcm and CD62L+ Tem cells that were induced to differentiate by the addition of TGF-β. The results are one representative of three independent experiments.
(B) Electrophoresis of PCR (BSP) products sequenced with bisulphite. Treg indicates sorted CD4+CD25high T cells, Tn indicates sorted CD4+ naive T cells, CD62L+ Tcm indicates sorted CD4+CD62L+ central memory T cells, Foxp3+ Tn indicates in vitro TGF-β-induced Foxp3+ cells from CD4+ naive T cells, and Foxp3+ Tcm indicates in vitro TGF-β-induced Foxp3+ cells from CD4+CD62L+ central memory T cells.
(C) Analysis of FOXP3 TSDR methylation in cultured cells. Each line represents a clone, and every dot represents a CpG site in the FOXP TSDR. A solid black dot indicates a methylated CpG site, while a hollow dot indicates a demethylated CpG site.
Figure 5.
Induction of functional Foxp3+ T cells from mouse CD4+CD62L+ central memory T cells.
(A) FACS-sorted mouse CD4 naive T cells (Tn), CD4 central memory T cells (Tcm) and CD4 effector memory T cells (Tem) were stimulated in vitro (1×105 cells/well) with anti-CD3 (2 μg/ml) and APCs (1×105 cells/well) in the presence of TGF-β (3 ng/ml) and IL-2 (100 U/ml) for 3–5 days. The induction of Foxp3 in the CD4+ fraction was analysed by FACS based on GFP expression.
(B) CD4+ GFP- (Foxp3-) T cells were sorted from Foxp3gfp mice, labelled with CFSE and used as responder cells. The labelled T cells (1×105 cells/well) were mixed with varying numbers of GFP+ (Foxp3+) T cells to achieve the desired Foxp3+ T cells/T effector cell ratios. GFP+ (Foxp3+) T cells were sorted from CD4+ naive or central memory T cells through the induction of Foxp3. ‘iTreg’ indicates in vitro TGF-β-induced GFP+ cells from CD4+ naive T cells, and ‘iTregcm’ indicates in vitro TGF-β-induced GFP+ cells from CD4+CD62L+ central memory T cells. The cell mixture was stimulated with syngeneic APCs (1×105 cells/well) and soluble anti-CD3 (1 μg/ml). Responder T cell proliferation with or without Foxp3+ T cells was assessed by CFSE dilution after 3 days of culture. The results are one representative of three independent experiments.