Intestinal inflammation or parasitic infection favours the reprogramming of Foxp3⁺ T_REG cells into effector T cells and promotes host immunity.

<p>(<b>A–B</b>) TCRβ^−/− mice received GFPtg CD4⁺CD25⁺ T cells (0.3×10⁶). 14 days later Foxp3 expression within donor GFP⁺CD4⁺ T cells (<b>A</b>) and the frequency of total IFN-γ or IL-17 producing cells (<b>B</b>) in indicated tissues of recipient mice from one of 3 representative experiments (n>3) is shown. (<b>C–G</b>) TCRβ^−/− mice were infected or not (NI) with 5×10⁶ promastigotes of WT or GP63^−/− (KO) <i>L. major</i> into the right footpad 2 weeks prior reconstitution with GFPtgCD4⁺CD25⁺ T_REG cells (0.3×10⁶). (<b>C</b>) 4 weeks later, GFP⁺CD4⁺ T cells from draining (infected) and non-draining popliteal LN, perLN and mesLN were analyzed for Foxp3 expression. Footpad swelling (<b>D</b>), absolute number of infiltrated lymphocytes (<b>E</b>), and frequencies of IFN-γ producing CD4⁺ T cells (<b>F</b>) are shown in infected and non-infected sites. (<b>G</b>) The loss of Foxp3 expression by T_REG cells was compared between mice infected with WT or GP63^−/−<i>L. major</i> strains. Results are representative of 2 independent experiments with n = 4–5.</p

Foxp3^+→− T cells lose their T_REG cell phenotype and reprogram into Th1 and Th17 effector cells in lymphopenic hosts.

<p>(<b>A–B</b>) TCRβ^−/− mice received CD4⁺GFP⁺ T_REG cells (0.5×10⁶) isolated from Foxp3^GFP reporter mice, and 14 days later, donor CD4⁺GFP⁺(Foxp3⁺)/GFP⁻(Foxp3⁻) cells were sorted from recipient mice and their proliferation (<b>A</b>) and suppressive activity (<b>B</b>) were assessed following <i>in vitro</i> activation. Freshly isolated T_REG and T_EFF cells from Foxp3^GFP mice were used as controls. Data from one of three independent experiments is presented as mean ± s.d. of triplicate wells. (<b>C</b>) TCRβ^−/− mice received GFPtgCD4⁺CD25⁺ T_REG cells (0.3×10⁶), and 14 days post transfer, GFP⁺ donor T cells were examined for the production of various cytokines relative to Foxp3 expression. Frequencies and mean fluorescent intensity (MFI) (<b>C</b>) of cytokines produced by Foxp3^+/− cells are shown as mean ± SEM from one out of 4 independent experiments (n = 4).</p

mTOR inhibition stabilizes Foxp3 expression in T_REG cells and strongly inhibits IL-17 but not RORγt expression.

<p>TCRβ^−/− mice were transferred with GFPtg CD4⁺CD25⁺ T_REG cells (0.3×10⁶), and then treated every second day with rapamycin (R) (18 mg/kg) or control vehicle (formulation without rapamycin) (V) as of day 0. 7 and 14 days later, perLN and mesLN of recipient mice were analyzed by FACS. (<b>A</b>) Foxp3 expression within donor GFP⁺CD4⁺ T cells, (<b>B</b>) proportion of IL-17/IFN-γ producing Foxp3^+/− donor T cells and (<b>C</b>) RORγt expression within Foxp3^+/− donor T cells are shown as mean ± SD from one out of 2 independent experiments (n = 4).</p

Predominant IL-17 secretion precedes Foxp3 down-regulation in reprogramming Foxp3⁺ T_REG cells, a process accentuated in mesenteric sites.

<p>(<b>A–E</b>) TCRβ^−/− mice received CFSE-labelled CD4⁺CD25⁺ T cells (0.3×10⁶) isolated from congenic Ly5.1⁺ mice, and, donor T cells were examined for Foxp3 expression at the indicated timepoints post transfer. (<b>A</b>) Representative FACS plots of Foxp3 expression relative to CFSE dilution in donor Ly5.1⁺CD4⁺ T cells are shown at various time points. (<b>B–E</b>) Representative FACS profiles (<b>B,C</b>) and proportion (<b>D,E</b>) of IL-17/IFN-γ-secreting donor Ly5.1⁺CD4⁺Foxp3⁺ (<b>B,D</b>) and Foxp3⁻ (<b>C,E</b>) T cells undergoing expansion at various time points are shown. Results are representative of 2 independent experiments (n = 3). (<b>F</b>) TCRβ^−/− mice received CD4⁺GFP⁺ T cells (0.5×10⁶) isolated from Ly5.1⁺ congenic Foxp3^GFP reporter mice, 14 days post transfer Ly5.1⁺CD4⁺GFP⁻ cells (Foxp3+→−) were sorted from lymphoid tissues of recipient mice, and reintroduced into secondary TCRβ^−/− recipient mice. Freshly-isolated CD4⁺GFP⁻ T cells (Foxp3-) from Ly5.1⁺Foxp3^GFP mice were used as a control. 14 days post secondary transfer, donor Ly5.1⁺CD4⁺ T cells from mesLN of secondary recipients were analyzed for IL-17/IFN-γ secretion relative to Foxp3 expression. Proportion of cytokine producing Foxp3⁻Ly5.1⁺CD4⁺ T cells is shown as mean ± SEM from one out of 2 independent experiments (n = 4).</p

Distinct Translational Control in CD4⁺ T Cell Subsets

<div><p>Regulatory T cells expressing the transcription factor Foxp3 play indispensable roles for the induction and maintenance of immunological self-tolerance and immune homeostasis. Genome-wide mRNA expression studies have defined canonical signatures of T cell subsets. Changes in steady-state mRNA levels, however, often do not reflect those of corresponding proteins due to post-transcriptional mechanisms including mRNA translation. Here, we unveil a unique translational signature, contrasting CD4⁺Foxp3⁺ regulatory T (T_Foxp3+) and CD4⁺Foxp3⁻ non-regulatory T (T_Foxp3−) cells, which imprints subset-specific protein expression. We further show that translation of eukaryotic translation initiation factor 4E (eIF4E) is induced during T cell activation and, in turn, regulates translation of cell cycle related mRNAs and proliferation in both T_Foxp3− and T_Foxp3+ cells. Unexpectedly, eIF4E also affects Foxp3 expression and thereby lineage identity. Thus, mRNA–specific translational control directs both common and distinct cellular processes in CD4⁺ T cell subsets.</p></div

The Foxp3^+→− T_REG cell phenotype is stable and correlates with a methylated Foxp3 promoter.

<p>TCRβ^−/− mice received CD4⁺GFP⁺ T cells (0.5×10⁶) from Ly5.1⁺ congenic Foxp3^GFP reporter mice. Ly5.1⁺CD4⁺GFP⁺ (Foxp3⁺) or GFP⁻ (Foxp3⁻) cells were sorted from lymphoid tissues of recipient mice 14 days post transfer. (<b>A</b>) Sorted populations were activated <i>in vitro</i> with plate-bound anti-CD3 for 5 days or (<b>B–C</b>) re-introduced into secondary TCRβ^−/− recipient mice. Freshly-isolated CD4⁺GFP^+/− T cells from Ly5.1⁺Foxp3^GFP mice were used as controls. 14 days post secondary transfer, donor Ly5.1⁺CD4⁺ T cells from lymphoid tissues of secondary recipient mice were (<b>B</b>) re-analyzed for Foxp3 expression or (<b>C</b>) sorted according to GFP expression and total genomic DNA was subjected to methylation analysis of Foxp3 promoter region. The percentage of Foxp3⁺ or Foxp3⁻ cells within indicated donor T cell populations is shown in (<b>B</b>) as mean ± SEM (n = 3). (<b>C</b>) For each donor T cell population the percentage of methylated CpG motifs within Foxp3 promoter region was examined at eight different sites and averaged.</p

Loss of Foxp3 expression in thymic or peripheral T_REG cells in lymphopenic hosts is modulated by the frequency of T_EFF cells, not T_REG cells.

<p>(<b>A–C</b>) TCRβ^−/− mice received GFPtg CD4⁺CD25⁺ T_REG cells (0.3×10⁶), and 7, 14 and 21 days post transfer, donor GFP⁺ T cells from mesLN were examined for Foxp3 expression. Representative histograms of Foxp3 expression and percentage of Foxp3⁺ cells within GFP⁺CD4⁺ T_REG cells (<b>A</b>) and proportion of cycling cells (Ki-67 expression) within Foxp3⁺ or Foxp3⁻ donor GFP⁺CD4⁺ T cells (<b>C</b>) at various time points are shown. (<b>B</b>) TCRβ^−/− mice received either thymus- or LN- derived GFP⁺CD4⁺CD25⁺ T_REG cells. The percentage of Foxp3^+/− cells within GFP⁺CD4⁺ T cells is shown 14 days post adoptive transfer. (<b>D–E</b>) Recipients received GFPtg CD4⁺CD25⁺ T_REG cells (0.3×10⁶) either alone or in combination with indicated numbers of CD4⁺CD25⁻ T_EFF, CD4⁺CD25⁺ T_REG or total CD4⁺ T cells. 14 days post T cell transfer cells, mesLN were analyzed for Foxp3 by flow cytometry. The percentage of Foxp3⁺ or Foxp3⁻ cells within donor GFP⁺CD4⁺ T cells is shown. Results are representative of 2 to 4 independent experiments (n = 3–4) are shown as mean ± SEM.</p

eIF4E controls proliferation in T cell subsets.

<p>(a) Inhibition of eIF4E activity suppresses T_Foxp3− cell proliferation. eFluor 670-labeled T_Foxp3− cells were IL-2/TCR-activated for 72 h in the presence of increasing concentrations of the eIF4E inhibitor 4ei-1 (K_d = 0.80 µM). Proliferation was determined under each condition by eFluor 670 dilution assessed by flow cytometry (upper panel). The effect on proliferation was also assessed by comparing cell counts after 72 h under each condition (lower panel; the control was set to 100%). (b) Inhibition of eIF4E activity abrogates IL-2-mediated reversal of anergy in T_Foxp3+ cells. IL-2/TCR-activated eFluor 670 labelled T_Foxp3+ cells were cultured in the presence of 4ei-1, and proliferation was determined as described in (a). (c–d) IL-2/TCR-activated eFluor 670 labelled T_Foxp3− cells (c) or T_Foxp3+ cells (d) were cultured in the presence of 4ei-1 or 4ei-4. Proliferation was determined under each condition as described in (a). (a–d) Representative histograms from 4 independent experiments are shown (upper panels; the percentages of proliferating cells are indicated). Means and standard deviations of cell counts from 4 independent experiments are shown (lower panel). (e) Induction of T_Foxp3+ cell proliferation occurs independently of signalling through 4E-BPs. 4E-BPdko T_Foxp3+ and T_Foxp3− cells were plated and counted as described in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003494#pgen-1003494-g005" target="_blank">Figure 5d</a>), and the fold increase in cell number was calculated and associated means and standard deviations (n = 2) are shown. Welch's two sample t-test was used to compare 4E-BPdko T_Foxp3+ cells cultured under different IL-2 concentrations. Also shown is a western blot of total protein extracts probed with antibodies for eIF4E in 4E-BPdko T_Foxp3+ and T_Foxp3− cells. Densitometry was used to quantify protein levels and obtained levels were normalized to β-actin (the normalized values were related to T_Foxp3− 72 h IL-2 100 U/ml which was set to 1 and are indicated above each lane). (f) Ki-67 and eIF4E co-expression in total CD4⁺ T cells isolated directly <i>ex vivo</i> from lymph nodes (left panel). Quantification of eIF4E expression is shown as Δ (eIF4E <i>vs.</i> isotype control) mean fluorescent intensity (MFI). Filled histograms represent staining with an isotype control. Quantification of eIF4E expression (ΔMFI) in Ki-67^+/− T_Foxp3− and T_Foxp3+ cells isolated directly <i>ex vivo</i> (right panel, mean and standard deviation is indicated, n = 3). (g–h) eFluor 670-labeled T_Foxp3− or T_Foxp3+ cells adoptively transferred into separate TCR β−/− mice were isolated from mesenteric (mes) and peripheral (per) lymph nodes (LN) followed by measurement of eFluor 670 and eIF4E expression four days post transfer. (g) Representative dot plots (n = 3) of T_Foxp3− and T_Foxp3+ cell proliferation relative to eIF4E expression in mesLN. Staining with an isotype control are shown as contour plots. (h) Quantification of eIF4E expression (ΔMFI) in cells that have (eFluor 670 low) or have not (eFluor 670 high) undergone cell division (means and standard deviations are indicated after per experiment normalization to T_Foxp3+ cells, n = 4–6). P-value (Welch two sample t-test) is indicated.</p

Inhibition of eIF4E activity results in spontaneous induction of Foxp3 expression in activated T_Foxp3− cells.

<p>T_Foxp3− cells were IL-2/TCR-activated for 72 h in the presence of increasing concentrations of 4ei-1 or the control pro-drug 4ei-4 in undifferentiating conditions, and Foxp3 expression (i.e. GFP) was assessed by flow cytometry. (a) Representative density plots from experiments using T_Foxp3− cells cultured in the presence of 4ei-1 from 4 independent experiments are shown. (b) Percentage Foxp3⁺ cells following treatment with 4ei-1 or 4ei-4 (shown are means and standard deviations, n = 4).</p

Differential levels of eIF4E between T_Foxp3+ and T_Foxp3− cells partly explain their translational signature and correlate with CD4⁺ T cell subset proliferation.

<p>(a) eIF4E is translationally more active in activated T_Foxp3− cells as compared to T_Foxp3+ cells. Shown is the cytosolic mRNA level (x-axis) vs. the polysome-associated mRNA level (y-axis) for each condition; T_Foxp3+ N (blue) and T_Foxp3− N (red) – <i>ex vivo</i> cells; T_Foxp3+ 36 h (green) and T_Foxp3− 36 h (black) – <i>in vitro</i> activated cells. The lines indicate the regressions used by anota to correct the polysome-associated mRNA level for the cytosolic mRNA level. (b) Activated T_Foxp3− cells express higher protein levels of eIF4E, cyclin-E1, cyclin-D3, and Anapc4 as compared to activated T_Foxp3+ cells. Shown are western blots from T_Foxp3+ and T_Foxp3− cells activated for 36 hours. Densitometry was used to quantify protein levels and obtained levels were normalized to β-actin (the normalized values were related to T_Foxp3− 36 h which was set to 1 and are indicated above each lane). (c) Identification of an eIF4E responsive module in the activated T cell translational signature. Fold changes from differentially translated mRNAs from the activated T cell translational signature that also showed a fold change difference for translation in lungs from 4E-BPdko mice are plotted. The number of mRNAs in each quadrant is shown. (d) High IL-2 concentration induces proliferation in T_Foxp3+ cells. Cell numbers were counted when plated and after 72 h of culture with low (100 U/ml) or high (1000 U/ml) IL-2 concentrations. The fold increase in cell number was calculated and associated means and standard deviations (n = 3) are shown. Welch's two sample t-test was used to compare T_Foxp3+ cells cultured under different IL-2 concentrations. (e) High IL-2 concentration induces eIF4E expression in T_Foxp3+ cells. Shown are western blots of total protein extracts probed with antibodies for eIF4E, cyclin-E1, cyclin-D3, and Anapc4 in T_Foxp3+ and T_Foxp3− cells activated as described in (d). Densitometry was used to quantify protein levels and obtained levels were normalized to β-actin (the normalized values were related to T_Foxp3− 72 h IL-2 100 U/ml which was set to 1 and are indicated above each lane; lanes between lanes 3 and 4 in (e) were spliced out but all shown lanes are from the same gel).</p