Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype

Abstract

Thymic-derived natural T-regulatory cells (nTregs) are important for the induction of self-tolerance and the control of autoimmunity. Murine CD4+CD25(-)Foxp3(-) cells can be induced to express Foxp3 after T-cell receptor (TCR) activation in the presence of transforming growth factor beta (TGFbeta) and are phenotypically similar to nTregs. Some studies have suggested that TCR stimulation of human CD4+CD25(-) cells results in the induction of transient expression of FOXP3, but that the induced cells lack a regulatory phenotype. We demonstrate here that TCR stimulation alone was insufficient to induce FOXP3 expression in the absence of TGFbeta, whereas high levels of FOXP3 expression could be induced in the presence of TGFbeta. Although FOXP3 expression was stable, the TGFbeta-induced FOXP3+ T cells were neither anergic nor suppressive and produced high levels of effector cytokines. These results suggest that even high levels of FOXP3 expression are insufficient to define a human CD4+ T cell as a T-regulatory cell.

Figure 1

Induction of FOXP3 in naive human CD4⁺ T cells was TGFβ-dependent. (A) Flow cytometric analyses of FOXP3 with 259D on postsorted and day 5-activated CD45RA⁺ cells treated without exogenous TGFβ1 with or without anti-TGFβ mAb or with TGFβ1. (B) FOXP3 expression on postsorted and day 5-activated CD45RA⁺ cells and CD25^hi nTregs treated with and without TGFβ1. The level of FOXP3 expression for each population is represented by the mean fluorescence intensity (MFI) gating on FOXP3⁺ cells. (C) FOXP3 expression on postsorted and day 5-activated CD45RA⁺ cells cultured in the presence of serum without exogenous TGFβ or in the absence of serum with exogenous TGFβ1 or without exogenous TGFβ1 with or without anti-TGFβ. Data above are representative of 3 independent experiments. The numbers in each quadrant represent the percentage of cells expressing the antigen.

Figure 2

Memory T cells were resistant to TGFβ induction of FOXP3 expression. (A) Flow cytometric analyses of FOXP3 with 259D on postsorted and day 5-activated CD45RA⁺ or CD45RA⁻ cells treated with and without exogenous TGFβ1. Numbers in each quadrant as in Figure 1. (B) The average percentage and range of FOXP3 induction on day 5-activated CD45RA⁺ cells from 30 different healthy adult donors and CD45RA⁻ cells from 15 of these donors treated with and without exogenous TGFβ1.

Figure 3

Anti-FOXP3 mAb, PCH101, was nonspecific for FOXP3 on activated CD4⁺ T cells. (A) Day 5-activated CD45RA⁺ cells treated with anti-TGFβ or with exogenous TGFβ1 were costained with PCH101 and 259D anti-FOXP3 mAbs or isotype controls. (B) Flow cytometric with 259D and real-time PCR analyses of FOXP3 protein and mRNA on unstimulated CD45RA⁺ cells (CD45RA + d0), unstimulated CD25^hi nTregs (CD25hi d0), day 5 non–TGFβ-treated CD45RA⁺ cells (CD45RA + d5 αTGFβ), day 5 TGFβ-treated CD45RA⁺ cells (CD45RA + d5 + TGFβ), and day 5-stimulated CD25^hi nTregs (CD25hi d5). Numbers in each quadrant as in Figure 1. (C) Flow cytometric analyses of FOXP3 with PCH101 and isotype control (shaded histogram) on CD45RA⁺ cells transfected with nonsilencing (NS, ——) or silencing (S, - - -) FOXP3 siRNA and activated for 5 days without TGFβ1 (left panel) or with TGFβ (center panel). Right panel is an overlay of TGFβ-treated CD45RA⁺ cells (- - -) and non-TGFβ-treated CD45RA⁺ cells (——) transfected with silencing FOXP3 siRNA. Data above are representative of 3 independent experiments.

Figure 4

TGFβ-induced FOXP3 expression was stable and requires IL-2 or other common γ cytokines for maintenance. (A) Flow cytometric analyses of FOXP3 with 259D on TGFβ-treated CD45RA⁺ cells over a 30-day period in the presence of IL-2. (B) FOXP3 expression on TGFβ-treated CD45RA⁺ cells at day 7 and 48 hours later after cultured with IL-2 with or without anti-TGFβ or with neutralizing anti-IL2 mAb with or without IL-1β, TNFα, IFNγ, IL-4, IL-6, IL-7, IL-10, or IL-15. The MFI values are derived from the gating of the FOXP3⁺ population. Data above are representative of 3 independent experiments. Numbers in each quadrant as in Figure 1.

Figure 5

TGFβ-induced FOXP3⁺ T cells were neither anergic nor suppressive. (A) Suppression assay of allogeneic CD4⁺CD25⁻ responder cells cultured alone (CD4, □) or with non-TGFβ-treated CD45RA⁺ cells (CD45RA + d7, ♦), TGFβ-treated CD45RA⁺ cells (CD45RA + d7 + TGFβ, ■), activated CD25^hi nTregs (CD25hi d7, ▴), or fresh CD25^hi nTregs (CD25hi d0, ●). The top panel represents the percentage and MFI of the FOXP3⁺ cells. (B) Suppression assay of CFSE-labeled allogeneic CD4⁺CD25⁻ cells cultured alone (right column, clear histogram) or at 1:1 ratio (right column, shaded histogram) with fresh CD25^hi nTregs (CD25hi d0, right top panel), non–TGFβ-treated CD45RA⁺ cells (CD45RA + d7, right middle panel) or TGFβ-treated CD45RA⁺ cells (CD45RA + d7 + TGFβ, right bottom panel). Center column represents proliferation of CFSE-labeled fresh CD25^hi (top panel), non–TGFβ-treated CD45RA⁺ (center panel), or TGFβ-treated CD45RA⁺ cells (bottom panel). Left column represents the FOXP3 expression of each population. Data above are representative of 3 independent experiments. Numbers in each quadrant as in Figure 1.

Figure 6

TGFβ-induced FOXP3⁺ T cells produced IL-2 and IFNγ. Flow cytometric analyses of FOXP3, IFNγ, IL-2, and IL-10 on day 7 activated adult CD4 + CD25^hi nTregs, adult TGFβ-treated CD45RA⁺ cells, and cord-blood TGFβ-treated CD45RA⁺ cells restimulated with PMA and ionomycin. Data are representative of 3 independent experiments. Numbers in each quadrant as in Figure 1.

Figure 7

Induction of FOXP3 in CD45RA⁺ cells activated with dendritic cells required TGFβ but was insufficient to suppress IL-2 production. (A) Flow cytometric analyses of FOXP3 with 259D on day 5-activated CD45RA⁺ cells stimulated with allogeneic monocyte-derived immature dendritic cells (iDC), mature dendritic cells (mDC), or CD3-depleted PBMC (APC) in the presence or absence of TGFβ1. The iDC and mDC were characterized based on their expression of CD86 and CD83. (B) Flow cytometric analyses of FOXP3, IFNγ, and IL-2 on day 7 TGFβ-treated CD45RA⁺ cells activated with iDC or mDC restimulated with PMA and ionomycin. Data are representative of 3 independent experiments. Numbers in each quadrant as in Figure 1.