3 research outputs found
Premature Expression of Foxp3 in Double-Negative Thymocytes
<div><p>Peripheral immune regulation depends on the generation of thymic-derived regulatory T (tT<sub>reg</sub>) cells to maintain self-tolerance and to counterbalance overshooting immune responses. The expression of the T<sub>reg</sub> lineage defining transcription factor Foxp3 in developing tT<sub>reg</sub> cells depends on TCR signaling during the thymic selection process of these T cells. In this study, we surprisingly identify Foxp3<sup>+</sup> immature thymocytes at the double-negative (DN) stage in transcription factor 7 (Tcf7)-deficient mice. These Foxp3<sup>+</sup> cells did not express a TCR (β or γδ chains), CD3 or CD5 and therefore these cells were true DN cells. Further investigation of this phenomenon in a transgenic TCR model showed that Foxp3-expressing DN cells could not respond to TCR stimulation <i>in vivo</i>. These data suggest that Foxp3 expression in these DN cells occurred independently of TCR signaling. Interestingly, these Foxp3<sup>+</sup> DN cells were located in a transition state between DN1 and DN2 (CD4<sup>-</sup>CD8<sup>-</sup>CD3<sup>-</sup>TCR<sup>-</sup>CD44<sup>high</sup>CD25<sup>low</sup>). Our results indicate that Tcf7 is involved in preventing the premature expression of Foxp3 in DN thymocytes.</p></div
Analysis of Foxp3<sup>+</sup> DN cells in TEa-Tcf7-deficient mice.
<p>(A) Representative plots showing TCRVβ6 and TCRVα2 expression on CD4SP thymocytes from TEa-Tcf7<sup>+/+</sup> and TEa-Tcf7<sup>-/-</sup> mice in the presence or absence of cognate antigen (Ag). The Tg TCR population is divided into TCR<sup>high</sup> and TCR<sup>low</sup> populations. (B-C) Quantification of the percentage of total (B) or TCR<sup>high</sup> (C) TCRVβ6<sup>+</sup>TCRVα2<sup>+</sup> cells among CD4SP thymocytes (n = 8). (D) Representative plots showing Foxp3 expression in DN TCRVβ6<sup>+</sup>TCRVα2<sup>+</sup> thymocytes from TEa-Tcf7<sup>+/+</sup> and TEa-Tcf7<sup>-/-</sup> mice in the absence of Ag. (E) Quantification of Foxp3<sup>+</sup> DN TCRVβ6<sup>+</sup>TCRVα2<sup>+</sup> thymocytes from TEa-Tcf7<sup>+/+</sup> and TEa-Tcf7<sup>-/-</sup> mice in the presence or absence of Ag (n = 8). (F-G) Representative plots showing TCRVβ6 and TCRVα2 expression on DN Foxp3<sup>+</sup> (F) or CD4SP Foxp3<sup>+</sup> (G) thymocytes from TEa-Tcf7<sup>+/+</sup> and TEa-Tcf7<sup>-/-</sup> mice in the presence or absence of Ag. Cells are pre-gated on TCRVβ6<sup>+</sup>TCRVα2<sup>+</sup>. Each dot represents one individual animal and mean is shown for all quantified data. Numbers show percentages of cells within the indicated box. NS, not significant, *** P < 0.001, **** P < 0.0001 (unpaired t-test).</p
Foxp3 expression at the DN cell stage in Tcf7-deficient mice.
<p>(A) Representative plots and quantification of Foxp3 staining in CD4<sup>-</sup>CD8<sup>-</sup> (DN) thymocytes from Tcf7<sup>+/+</sup> and Tcf7<sup>-/-</sup> mice (n = 8). (B) Left panels: Representative plots showing Foxp3 and intracellular (IC) TCRβ staining in DN thymocytes from Tcf7<sup>+/+</sup> and Tcf7<sup>-/-</sup> mice. Middle panels: TCRγδ and CD3 staining on DN Foxp3<sup>+</sup>TCRβ<sup>-</sup> cells (gate R1). Right panel: Quantification of DN Foxp3<sup>+</sup>TCRβ<sup>-</sup>TCRγδ<sup>-</sup>CD3<sup>-</sup> cells (gate R2) depicted as the percentage of total DN cells (n = 6). (C) Left panel: Representative histograms showing CD5 staining on Foxp3<sup>+</sup> DN, Foxp3<sup>+</sup> DP, and Foxp3<sup>+</sup> CD4SP cells from Tcf7<sup>-/-</sup> mice. Right panel: Quantification of CD5 geometric mean from DN, DP, and CD4SP Foxp3<sup>+</sup> or Foxp3<sup>-</sup> populations (n = 3). Mean + SD are shown for all quantified data. Numbers show percentages of cells within the indicated box. Each symbol represents an individual animal. ** P < 0.01 (unpaired t-test).</p