Smad2 translocation is not enhanced in <i>Drak2-/-</i> T cells compared to wildtype T cells.

<p>Wildtype and <i>Drak2-/-</i> CD4⁺ cells were negatively selected with Miltenyi magnetic beads and stimulated on anti-CD3-coated coverglass slides along with soluble anti-CD28 for 24 hours. Half of the cells were treated with TGF-β for the final 20 minutes of culture. Cells were fixed, permeabilized, and stained with DAPI, Phalloidin, and anti-Smad2. Images were collected via confocal microscopy. n = 2 mice per group. Data are representative of two independent experiments.</p

Enhanced susceptibility to death of <i>Drak2-/-</i> T cells compared to wildtype T cells is independent of TGF-β signaling <i>in vitro</i>.

<p>A) CD4⁺CD25^-CD44^lo or B) CD8⁺CD25^-CD44^lo naïve cells were purified from wildtype, <i>Drak2-/-</i>, <i>DNRII</i>, and <i>DNRII</i>. <i>Drak2-/-</i> mice and stimulated with anti-CD3 and anti-CD28 for 2–3 days. The percent of nonviable CD4⁺ or CD8⁺ T cells is shown. Cells were obtained from one mouse per group and tested in quadruplicate. Data are representative of four separate experiments. <i>**P < 0</i>.<i>01</i>, <i>***P < 0</i>.<i>001</i> (Mann-Whitney <i>U</i>-test).</p

TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and <i>Drak2-/-</i> T cells.

<p>A) CD4⁺CD25^-CD44^lo naïve cells were purified from <i>OT-II</i> and <i>OT-II</i>.<i>Drak2-/-</i> mice and stimulated with irradiated splenocytes loaded with 10μM OVA₃₂₃ peptide in the presence or absence of 10-fold TGF-β titrations for three days. The number of live, divided Foxp3^-CD4⁺ cells are shown for each titration. Cells were obtained from one <i>OT-II</i> or <i>OT-II</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of five separate experiments. B) CD8⁺CD25^-CD44^loCD62L^hi naïve cells were purified from <i>OT-I</i> and <i>OT-I</i>.<i>Drak2-/-</i> mice and stimulated with splenocytes loaded with 100pM OVA₂₅₇ peptide in the presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow cytometry. The number of live, divided CD8⁺ cells are shown for each titration. Cells were obtained from one <i>OT-I</i> or <i>OT-I</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of three separate experiments. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test.</p

Smad2 and Smad2/3 complex phosphorylation is not enhanced in <i>Drak2-/-</i> T cells compared to wildtype T cells.

<p>A) Wildtype and <i>Drak2-/-</i> splenocytes, and FACS sorted naïve CD4⁺ and CD8⁺ T cells were stimulated for 2 hours with anti-CD3 and anti-CD28, with or without 2 ng/ml TGF-β for one additional hour. Cells were lysed and analyzed by western blot with antibodies specific for Smad2, phosphorylated Smad2, and HSP90 as a loading control. Cells were pooled from 9 wildtype and 8 <i>Drak2-/-</i> mice. Data are representative of two independent experiments. B) Wildtype and <i>Drak2-/-</i> splenocytes were stimulated for 2 hours with anti-CD3 and anti-CD28 with or without increasing concentrations of TGF-β for one additional hour. The cells were harvested, stained with antibodies specific for CD4, CD8, and pSmad2/3, and analyzed by flow cytometry. The average mean fluorescence intensity (MFI) of pSmad2/3 expression is shown for 3 mice per group. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test. Data are representative of 3 independent experiments.</p

TGF-β-mediated regulatory T cell induction is not altered in the absence of <i>Drak2</i>.

<p>A) CD4⁺CD25^-CD44^lo naïve cells were purified from wildtype and <i>Drak2-/-</i> mice and stimulated with 1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 days. The A) percent and B) number of Foxp3⁺ cells of electronically gated CD4⁺ cells is shown. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test.</p

TGF-β-mediated responses to opposing cytokines are comparable between wildtype and <i>Drak2-/-</i> T cells.

<p>CD8⁺CD25^-CD44^loCD62L^hi naïve cells were purified from <i>OT-I</i> and <i>OT-I</i>.<i>Drak2-/-</i> mice and stimulated with 100nM OVA₂₅₇–pulsed splenocytes for 2 days. Cells were harvested and replated at equal numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8⁺ cells and B) percent Annexin V⁺ of CD8⁺ cells are shown for each cytokine condition. Cells were obtained from one <i>OT-I</i> or <i>OT-I</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of two independent experiments. <i>*P < 0</i>.<i>05</i> (Mann-Whitney <i>U-</i>test).</p