Abstract
Foxp3+ regulatory T cells (Treg cells) maintain immunological tolerance, and their deficiency results in fatal multiorgan autoimmunity. Although heightened signaling via the T cell antigen receptor (TCR) is critical for the differentiation of Treg cells, the role of TCR signaling in Treg cell function remains largely unknown. Here we demonstrated that inducible ablation of the TCR resulted in Treg cell dysfunction that could not be attributed to impaired expression of the transcription factor Foxp3, decreased expression of Treg cell signature genes or altered ability to sense and consume interleukin 2 (IL-2). Instead, TCR signaling was required for maintaining the expression of a limited subset of genes comprising 25% of the activated Treg cell transcriptional signature. Our results reveal a critical role for the TCR in the suppressor capacity of Treg cells.
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Acknowledgements
We thank M. Schmidt-Supprian (Technical University Munich) and K. Rajewsky (Max Delbrück Center) for TracFL mice. Supported by the US National Institutes of Health (R37AI034206 to A.Y.R.), the Ludwig Cancer Center at Memorial Sloan-Kettering Cancer Center (A.Y.R.) and the Howard Hughes Medical Institute (A.Y.R.).
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A.G.L. and A.Y.R. designed the experiments; A.G.L. conducted experiments and wrote the manuscript; A.Y.R. supervised the research and edited the manuscript; and W.J. prepared samples for microarray analysis, and A.A. and A.G.L. conducted these analyses.
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Integrated supplementary information
Supplementary Figure 1 Treg cell surface phenotype and proliferative capacity in the absence of a functional TCR.
(a) CD4+eGFP+ TCRβ+ and TCRβ- cells were sorted from TracFL/WTFoxp3Cre-ERT2 mice 9 days after tamoxifen treatment on days 0 and 1 (a), labeled with CellTrace Violet and plated in 96-well plates coated with anti-CD3ɛ (clone 2C11) (b), anti-TCRβ (clone H57-597) (c) or PMA and Ionomycin (d) in the presence of 25U/mL IL-2 for 84 hours before analysis. All conditions were performed in triplicate wells with similar results. (e) Expression of Treg cell markers on TCRβ+ and TCRβ- CD4+Foxp3+ cells in lymph nodes of TracWT/WTFoxp3Cre-ERT2 (WT/WT), TracFL/WTFoxp3Cre-ERT2 (FL/WT) and TracFL/FLFoxp3Cre-ERT2 (FL/FL) mice. Gray histograms are gated on CD4+Foxp3- cells in TracWT/WTFoxp3Cre-ERT2 mice. (f) Percentages and absolute numbers of total Foxp3+ among CD4+ cells in spleens and lymph nodes of the indicated mice. (g) Gating strategy for analysis of TCRβ+ and TCRβ- cells among CD4+Foxp3+ lymph node cells. For (e-g), mice were analyzed on day 9 following tamoxifen treatment on days 0 and 1. Mice in (f-h) are indicated as in (e). (h) BrdU incorporation vs. TCRβ expression on CD4+eGFP+ cells sorted to >99% purity from pooled spleens and lymph nodes of the indicated mice on day 9 following tamoxifen treatment on days 0 and 1 and i.p. injection of BrdU on day 8. Data is representative of two experiments with four or more mice per group each (e-g) or three experiments with two or more mice per group each (h). P-value in (f) was calculated using a two-tailed unpaired t-test.
Supplementary Figure 2 Constitutive ablation of the TCR in TracFL/FLFoxp3YFP-Cre mice impairs Treg cell population expansion and precipitates early-onset autoimmunity.
(a-c) TCRβ expression on CD4+Foxp3+ cells in the thymi (a) and lymph nodes (a,b) of 2.5 wk old mice, and Foxp3 median fluorescence intensity (MFI) in CD4+Foxp3+ lymph node cells (c) in 2.5 wk old TracWT/WTFoxp3YFP-Cre (WT/WT), TracFL/WTFoxp3YFP-Cre (FL/WT) and TracFL/FLFoxp3YFP-Cre (FL/FL) mice. (d) Representative expression of Treg markers on the indicated TCRβ+ and TCRβ- populations. Gray histogram is gated on CD4+Foxp3- cells in TracWT/WTFoxp3YFP-Cre mice. (e,f) Activation status (e), spleen and lymph node cell numbers, and percent cytokine producers in the spleen (f) of CD4+Foxp3- and CD8+ cells. (g) Absolute numbers of TCRβ+ and TCRβ- CD4+Foxp3+ cells in 2.5 week old TracWT/WTFoxp3YFP-Cre (WT), TracFL/WTFoxp3YFP-Cre (Het), and TracFL/FLFoxp3YFP-Cre (KO) mice. (h) Absolute numbers of CD4+TCRβ+Foxp3- (left) and TCRβ+ and TCRβ- CD4+Foxp3+ cells (right) in liver (top) and lungs (bottom) of indicated mice. Data in (b-e) are representative of three experiments with a total of six or more mice per group. Data in (f,g) are aggregated from three experiments with a total of six or more mice per group. Data in (a) and (h) are representative of two experiments with three or more mice per group each. ***, P < 0.001; **, P < 0.01; *, P < 0.05. P-values were calculated using a two-tailed unpaired t-test.
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Supplementary Figure 3 Partial depletion of Treg cells in Foxp3DTR mice recapitulates activation of the immune system in TracFL/FLFoxp3Cre-ERT2 mice.
(a) Experimental protocol. TracWT/WTFoxp3Cre-ERT2 (TracWT/WT), TracFL/FLFoxp3Cre-ERT2 (TracFL/FL) and Foxp3DTR mice were gavaged with tamoxifen on days 0, 3, 7 and 10. Foxp3DTR mice were injected i.p. with PBS, 0.065ug DT, or 0.1ug DT and TracWT/WTFoxp3Cre-ERT2 and TracFL/FLFoxp3Cre-ERT2 mice were injected with 0.065ug DT on days 4, 7 and 10. (b) Percent TCRβ+Foxp3+ cells among CD4+ cells in spleens of TracWT/WTFoxp3Cre-ERT2 and TracFL/FLFoxp3Cre-ERT2 (black squares) and Foxp3DTR (gray squares) mice. Total Foxp3+ cells among CD4+ cells in TracFL/FLFoxp3Cre-ERT2 mice are shown as white squares. (c,d) Activation of (c) and cytokine production by (d) splenic CD4+Foxp3- cells. Fold-changes are shown. All data are representative of two experiments with four or more mice per group each.
Supplementary Figure 4 Maintenance of TCR-deficient Treg cell responsiveness to IL-2 and proliferation in the presence of activated DCs in vitro.
(a) p-STAT5 was assessed in eGFP+ (Treg) cells among bulk CD4+ cells purified on day 9 from TracFL/WTFoxp3Cre-ERT2 mice treated with tamoxifen on days 0 and 1, stained for CD4 and TCRβ, and cultured with increasing concentrations of IL-2 for 20 minutes before fixation. Data is representative of two experiments with a total of two mice. (b) In vitro IL-2 capture assay. TracFL/WTFoxp3Cre-ERT2 mice were administered tamoxifen on days 0 and 1, and on day 9 CD4+eGFP+TCRβ+, CD4+eGFP+TCRβ-, and CD4+TCRβ+eGFP- cells were sorted and cultured in media with increasing concentrations of IL-2. PE median fluorescence intensity (MFI) directly correlates with IL-2 remaining in the media at the time of analysis. (c) CD80 expression on DC populations in lymph nodes of TracWT/WTFoxp3Cre-ERT2 (black lines, left, and white circles, right) and TracFL/FLFoxp3Cre-ERT2 (red lines, left, and black circles, right) mice on day 13 following tamoxifen administration on days 0, 3, 7 and 10. (d) In vitro proliferation of Treg cells was assessed as follows: CD4+eGFP+ cells were sorted on day 9 from TracFL/WTFoxp3Cre-ERT2 mice treated with tamoxifen on days 0 and 1, labeled with CellTrace Violet and cultured for 84 hrs with IL-2 in the presence or absence of DCs with or without LPS. Cells were stained for CD4, TCRβ and Foxp3 for analysis. All conditions were performed in triplicate with similar results. **, P < 0.001; *, P = 0.002. P-values were calculated using a two-tailed unpaired t-test.
Supplementary Figure 5 IL-2–anti-IL-2 complexes do not 'rescue' autoimmunity in TracFL/FLFoxp3Cre-ERT2 mice.
(a) Schematic of the experimental setup. (b) Percent TCRβ+Foxp3+ (black squares) and total Foxp3+ (white squares) cells among CD4+ cells in lymph nodes of TracWT/WTFoxp3Cre-ERT2 (WT/WT) and TracFL/FLFoxp3Cre-ERT2 (FL/FL) mice treated with IL-2-anti-IL-2 complexes (IL2-c) or PBS. (c,d) CD4+ T cell activation (c) and cytokine production (d) in the spleens of the indicated mice, as in (b). **, P < 0.01; *, P < 0.05. P-values were calculated using a two-tailed unpaired t-test.
Supplementary Figure 6 Altered in vitro conjugate formation between Treg cells and DCs in the absence of the TCR.
(a) Conjugate formation was assessed by culturing 6 x104 CellTrace Violet-labeled DCs overnight with 104 CFSE-labeled TCR+ or TCR- Treg cells in the presence of 500 U/mL IL-2. Percent CellTrace Violet+ cells among CFSE+ cells is plotted. TCR+ cells and DCs were cultured in the presence of 2.5 ug/mL Concanavalin A (ConA) as a positive control. (b) Assessment of LFA-1 expression on CD44hiCD62Llo vs. CD44loCD62Lhi CD4+Foxp3+ and Foxp3- cells by flow cytometric analysis of CD11a (ITGAL) expression. *, P < 0.01. P-value in (a) was calculated using a two-tailed unpaired t-test.
Supplementary Figure 7 IRF4 expression in Treg cells is restricted to CD44hi cells, is TCR dependent and can be inducibly ablated in Irf4FL/FLFoxp3Cre-ERT2 mice.
(a) Histograms showing IRF4 protein levels in TCR+ Foxp3+ cells in TcraWT/WTFoxp3Cre-ERT2 (WT/WT; CD44loCD62Lhi in green and CD44hiCD62Llo in black) and in TcraFL/FLFoxp3Cre-ERT2 (FL/FL; CD44loCD62Lhi in orange and CD44hiCD62Llo in blue) mice (above), and in TCR- Foxp3+ cells in TcraFL/FLFoxp3Cre-ERT2 (FL/FL; CD44loCD62Lhi in green and CD44hiCD62Llo in red) mice (below). (b) Histograms demonstrating an increase in eGFP median fluorescence intensity (MFI) in eGFP+ cells following tamoxifen administration to Irf4FL/FLFoxp3Cre-ERT2 (red line) vs. Irf4WT/WTFoxp3Cre-ERT2 (black line) mice (left); quantitation of eGFP median fluorescence intensity of eGFP+ cells in Irf4WT/WTFoxp3Cre-ERT2 (white squares) and Irf4FL/FLFoxp3Cre-ERT2 (black squares) mice (right). Mice were administered tamoxifen on days 0, 3, 7 and 10 and were analyzed on day 13. Data is representative of two experiments with three or more (a) or four or more (b) mice per group each. P-value in (b) was calculated using a two-tailed unpaired t-test.
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Levine, A., Arvey, A., Jin, W. et al. Continuous requirement for the TCR in regulatory T cell function. Nat Immunol 15, 1070–1078 (2014). https://doi.org/10.1038/ni.3004
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DOI: https://doi.org/10.1038/ni.3004
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