Foxp3⁺ cells upregulate CD69 after TCR activation in an antigen-non-specific manner.

<p>(A) Gating strategy on CD4⁺ enriched T cells after overnight culture without stimulation. Cells are gated in the lymphocyte gate based on forward versus side scatter, then doublets are excluded based on forward scatter height versus amplitude and live cells are gated as negative for the dead cell marker. Finally Foxp3⁺ and Foxp3^- CD4⁺ T cells are gated using CD4 and Foxp3 expression. This gating strategy was applied throughout the analyses unless otherwise stated. (B) CD69 expression on Foxp3^- (top row) and Foxp3⁺ (bottom row) CD4⁺ cells before and after overnight stimulation with or without anti-CD3 stimulation. Staining is representative of at least three independent experiments. (C) CD69 expression on Foxp3^- (left) and Foxp3⁺ (right) enriched CD4⁺ T cells from either wild-type (white bars) or OT-II (black bars) donor mice, after overnight culture with BMDCs. Unstim, control without BMDC; /, unpulsed BMDC; OVA, OVA-pulsed BMDC, anti-CD3, anti-CD3-coated BMDC. Data are representative of four independent experiments with each 3 replicates per group. (D) Normalized MHCII KO BMDC-induced CD69 expression compared to control BMDC-induced CD69. The graphs show the frequency of CD69⁺ cells among CD4⁺ Foxp3^- (left) and Foxp3⁺ (right) T cells after stimulation with MHCII KO BMDC, normalized to CD4⁺ Foxp3^- and Foxp3⁺ T cells stimulated with wild type BMDC for each given condition. 100% represents the same CD69 expression by cells stimulated with control or MHCII KO BMDC. Data show mean + SD, *p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001, ns, non significant. Data are representative of three independent experiments with each n = 3 per group.</p

Foxp3⁺ cells upregulate Nur77 after TCR activation in an antigen-specific manner.

<p>(A) Time course of Nur77 (black bars) and CD69 expression (white bars) on Foxp3⁺ T-cells among sorted CD25⁺ CD4⁺ splenocytes stimulated with plate-bound αCD3 and 1U/ml IL-2. Data are representative of 3 independent experiments with n ≥ 3. (B) Nur77 expression on Foxp3^- (left) and Foxp3⁺ (right) among enriched CD4⁺ T-cells from either wild-type (white bars) or OT-II (black bars) mice, co-cultured overnight with unstimulated, OVA pulsed or αCD3 coated BMDCs. Data are representative of two independent experiments, with each n = 3 per group. unstim, control without BMDC; /, unpulsed BMDC; OVA, OVA-pulsed BMDC, anti-CD3, anti-CD3-coated BMDC. Data show mean + SD, *p ≤ 0.05, *** p ≤ 0.001; ns, non significant. Data are representative of four independent experiments each with n = 3 per group.</p

Nur77 intensity is not modulated by cytokines.

<p>(A) Nur77 expression on Foxp3^- (white bars) and Foxp3⁺ (black bars) CD4⁺ T cells from the culture in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137393#pone.0137393.g002" target="_blank">Fig 2B</a>. Data are representative of at least two independent experiments. (B) Representative plots of Nur77 versus CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells, stimulated for 4 hours (top row) or 16 hours (bottom row) without additional cytokines (/), with IFN-α, TNF-α, or plate-bound αCD3. Data are representative of two independent experiments. (C) Nur77 expression on Foxp3⁺ cells among sorted CD25⁺ CD4⁺ splenocytes stimulated with plate-bound αCD3 with low (1U/ml) or high (1000U/ml) IL-2 for 6 hours. Data are representative of four independent experiments. Data show mean + SD, ns, non significant.</p

Foxp3⁺ cells preferentially upregulate CD69 in response to soluble factors.

<p>(A) CD69 expression on Foxp3^- (left) and Foxp3⁺ (right) enriched CD4⁺ T cells after overnight incubation with or without BMDC-conditioned culture medium. /, control medium; OVA, medium supplemented with OVA; BMDC SN, supernatant from BMDCs cultured overnight with fresh medium; BMDC OVA SN, supernatant from BMDCs cultured overnight with medium supplemented with OVA. Data are representative of at least two independent experiments. (B) CD69 expression on Foxp3^- (white bars) and Foxp3⁺ (black bars) among enriched CD4⁺ T cells cultured overnight without additional cytokines (/) or with IL-1β, IFN-α or TNF-α. Data are representative of at least two independent experiments. (C) CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells cultured overnight without additional cytokines (/) or with IL-33, IL-4, IL-12, IL-27, IL-6, IFN-γ, or GM-CSF. (D) Representative FACS plot and response to stimulation of sorted CD4⁺ Foxp3^RFP+ cells. Right: representative FACS plot showing the purity of Foxp3^RFP+ cells after sort. Cells are gated on forward and side scatter and doublets and dead cells are excluded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137393#pone.0137393.g001" target="_blank">Fig 1A</a>. Plot shows CD4 versus intracellular Foxp3 staining. Left, CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells (white bars) or Foxp3^RFP+ sorted (black bars) CD4⁺ T cells, cultured overnight without additional cytokines (/) or with IFN-α or TNF-α (left). (E) CD69 expression on Foxp3⁺ among enriched CD4⁺ splenocytes from TNFR1 KO, Myd88 KO and ctrl C57BL/6 or from IFNAR1 KO and control 129 mice, cultured overnight with control medium (/) or with supernatant from OVA stimulated BMDCs (BMDC OVA SN). (F) CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells of IFNAR1 <i>KO</i> and control mice cultured overnight with control medium (unstim) or with supernatant from OVA stimulated BMDCs (BMDC OVA SN) with or without addition of different concentrations of blocking anti-TNF-α antibody. Data are representative from two independent experiments. Data show mean + SD, *** p ≤ 0.001 compared to unstimulated control, unless comparison indicated by line below the stars, n ≥ 3 per group.</p