1. Volume 48, Issue 4, Pages 745-759.e6 (April 2018)
T Cell Receptor-Regulated TGF-β Type I Receptor Expression Determines T Cell Quiescence and Activation 
Eric Tu, Cheryl P.Z. Chia, Weiwei Chen, Dunfang Zhang, Sang A. Park, Wenwen Jin, Dandan Wang, Maria-Luisa Alegre, Ying E. Zhang, Lingyun Sun, WanJun Chen 
Immunity 
Volume 48, Issue 4, Pages e6 (April 2018)
DOI: /j.immuni
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2. Immunity 2018 48, 745-759.e6DOI: (10.1016/j.immuni.2018.03.025)
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3. Figure 1 T Cell Activation Induces TβR Downregulation
(A) Western blot of p-SMAD2 and SMAD2 in naive CD4+ T cells stimulated for indicated time periods with anti-CD3 and anti-CD28.
(B) Western blot of p-SMAD2 and SMAD2 in naive (Naive + TGF-β) or previously activated CD4+ T cells (Activated + TGF-β) stimulated for 24 hr with anti-CD3 and anti-CD28 plus TGF-β.
(C and D) Tgfbr1 and Tgfbr2 mRNA in naive (C) mouse and (D) human CD4+ T cells stimulated for indicated time periods with anti-CD3 and anti-CD28.
(E) Tgfbr1 and Tgfbr2 mRNA in naive CD4+ T cells stimulated for 24 hr with anti-CD3 plus anti-CD28 or anti-CD3 alone.
(F) IL-2, Tgfbr1, and Tgfbr2 mRNA in T cells from DO11.10xRag2−/− mice treated with PBS (Ctrl) or OVA peptide (pOVA).
(G) Western blot of TβRI, TβRII, and GAPDH in naive or CD4+ T cells stimulated for 24 hr with anti-CD3 and anti-CD28.
(H) Tgfbr1 and Tgfbr2 mRNA in naive CD4+ T cells stimulated for 24 hr with 1 μg/mL anti-CD28 plus indicated concentration of anti-CD3.
(I) Tgfbr1 and Tgfbr2 mRNA in T cells from DO11.10xRag2−/− mice treated with indicated doses of pOVA.
(J) Tgfbr1 and Tgfbr2 mRNA in naive 5CC7 T cells cultured for 24 hr with APCs and indicated peptides.
Data are pooled from two (D and I), three (C, F, H, and J), or four (B and E) independent experiments or are representative of three (A and G) independent experiments. In (F) and (I), each circle represents the data from one mouse. Student’s t test was used. Bars, mean; error bars, SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < See also Figure S1.
Immunity  , e6DOI: ( /j.immuni )
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4. Figure 2
Antigen Stimulation Strength Affects T Cell Differentiation and TβRI Expression
(A) Experimental workflow of naive 5CC7 T cells challenged with pMCC. (B) Tgfbr1 and Tgfbr2 mRNA and (C) Treg cell and (D) Th1 cell frequencies in 5CC7 T cells from mice challenged with indicated doses of pMCC. (E) Tgfbr1 and Tgfbr2 mRNA and (F) Th17 cell frequency in 5CC7 T cells from mice challenged with indicated doses of pMCC plus CFA. Data are pooled from three independent experiments. In (C), (D), and (F), each circle represents one mouse. Student’s t test was used. Bars, mean; error bars, SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p <
Immunity  , e6DOI: ( /j.immuni )
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5. Figure 3 NF-κB Downregulates TβRI Expression during T Cell Activation
(A) Total and nascent Tgfbr1 and Tgfbr2 mRNA in naive or CD4+ T cells stimulated for 24 hr with anti-CD3 and anti-CD28 (Activated).
(B) Tgfbr1, Tgfbr2, and IL-2 mRNA in naive CD4+ T cells stimulated for indicated time periods with anti-CD3 and anti-CD28 plus DMSO (Control), NF-κB, or NFAT inhibitor.
(C) Tgfbr1 and Tgfbr2 mRNA in wild-type (WT), Card11−/−, or NfkbiaΔN-Tg naive or CD4+ T cells stimulated for 24 hr with anti-CD3 and anti-CD28 (Activated).
(D) Tgfbr1 mRNA in WT, Nfkb1−/−, Nfkb2−/−, or Rel−/− naive or CD4+ T cells stimulated for 24 hr with anti-CD3 and anti-CD28 (Activated).
(E) Tgfbr1 mRNA in naive CD4+ T cells stimulated for indicated time periods with anti-CD3 and anti-CD28 plus DMSO (Control) or CAPE.
(F) ChIP-coupled real-time PCR analysis of p65 enrichment in the promoter region of Tgfbr1 gene of naive or T cells stimulated for 24 hr with anti-CD3 and anti-CD28 (Activated). p65 enrichment was assessed using p65 antibody and presented relative to input and compared with control IgG.
Data are pooled from two (A, B, and D), three (E), or four (C) independent experiments or are representative of three (F) independent experiments. In (A), (C), (D), and (F), Student’s t test was used. In (B) and (E), two-way ANOVA was used. Bars, mean; error bars, SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < See also Figures S2 and S3.
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6. Figure 4 TGF-β Upregulates TβRI Expression
(A) Tgfbr1 and Tgfbr2 mRNA in naive or CD4+ T cells stimulated for 24 hr with anti-CD3 and anti-CD28 plus different cytokines as indicated.
(B) Tgfbr1 and Tgfbr2 mRNA in naive CD4+ T cells stimulated for indicated time periods with anti-CD3 and anti-CD28 in the presence or absence of TGF-β.
(C) Western blot of TβRI, TβRII, and GAPDH in CD4+ T cells stimulated with anti-CD3 and anti-CD28 for 24 hr in the presence or absence of TGF-β.
(D) Western blot of p-SMAD2 and SMAD2 in T cells stimulated for indicated time periods with anti-CD3 and anti-CD28 plus TGF-β.
(E) Tgfbr1 mRNA in naive Tgfbr2f/fEsr1-cre CD4+ T cells stimulated for indicated time periods with anti-CD3 and anti-CD28 in the presence or absence of TGF-β. Tgfbr2+/+, untreated Tgfbr2f/fEsr1-cre mice; Tgfbr2−/−, tamoxifen-treated Tgfbr2f/fEsr1-cre mice.
(F) Tgfbr1 mRNA in naive Smad3−/− or Smad3+/+ CD4+ T cells stimulated for indicated time periods with anti-CD3 and anti-CD28 in the presence or absence of TGF-β.
(G) Tgfbr1 and Tgfbr2 mRNA in CD4+CD25− non-Treg cells and CD4+CD25+ Treg cells that were unstimulated or stimulated with anti-CD3 and anti-CD28 for 24 hr.
Data are pooled from two (A and E–G) or three (B) independent experiments or are representative of three (C and D) independent experiments. In (B), (E), and (F), two-way ANOVA was used. In (G), Student’s t test was used. Bars, mean; error bars, SD in (A) and (E)–(G) and SEM in (B); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < See also Figure S4.
Immunity  , e6DOI: ( /j.immuni )
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7. Figure 5
TβRI Overexpression in Naive T Cells Suppresses T Cell Proliferation and Autoimmunity
(A) Tgfbr1, Il-2, and Foxp3 mRNA in naive TβRIGFP+ or CtrlGFP+ CD4+ T cells stimulated for 24 hr with anti-CD3 and anti-CD28 in the presence or absence of TGF-β.
(B) Frequency of Ki-67+ in naive TβRIGFP+ or CtrlGFP+ CD4+ T cells stimulated for 3 days with anti-CD3 and anti-CD28 plus TGF-β.
(C–G) Naive CD4+ T cells from TβRIGFP+ or CtrlGFP+ retrogenic mice were transferred into Rag1−/− mice.
(C) Weight gain of recipient mice after T cell transfer.
(D) Quantitation of colon pathology.
(E) Representative histology images of colon sections. Scale bars, 200 μm.
(F) Frequencies of the donor TβRIGFP+ or CtrlGFP+ CD4+ T cells in recipient mice.
(G) Frequencies of Foxp3+ cells in the donor CD4+ T cell population.
Data are pooled from two (A and B) or three (C–G) independent experiments. In (D), (F), and (G), each circle represents the data from one mouse. In (A), (B), (D), (F), and (G), Student’s t test was used. In (C), two-way ANOVA was used. Bars, mean; error bars, SD in (A) and (B) and SEM in (C); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < See also Figures S5 and S6.
Immunity  , e6DOI: ( /j.immuni )
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8. Figure 6
TβRI Restoration in Activated T Cells Reduces T Cell Proliferation
(A) Tgfbr1 and Il-2 mRNA in CtrlGFP+ or TβRIGFP+ activated T cells re-stimulated with anti-CD3 and anti-CD28 for 24 hr.
(B) Experimental workflow of TβRIGFP+- or TβRIICherry+-DO11.10 T cells challenged with pOVA.
(C) Cell proliferation of CtrlGFP+- or TβRIGFP+-DO11.10 T cells challenged with pOVA.
(D) Experimental workflow of TβRIGFP+- or TβRIICherry+-DO11.10 T cells challenged with OVA protein in an airway inflammation model.
(E) Frequencies of CtrlGFP+- or TβRIGFP+-DO11.10 T cells in the lungs and draining lymph nodes (dLN) of recipient mice.
(F) Frequencies of Ki-67+ cells in CtrlGFP+- or TβRIGFP+-DO11.10 T cells in dLN of recipient mice.
(G) Western blot of phosphorylated p65 (p-p65) and p65 in CtrlGFP+ or TβRIGFP+ activated T cells re-stimulated with anti-CD3 and anti-CD28 for 24 hr.
Data are pooled from two (A, E, and F) or three (C) independent experiments or are representative of two (G) independent experiments. In (C), (E), and (F), each circle represents one mouse. Student’s t test was used. Bars, mean; error bars, SEM; ∗p < 0.05 and ∗∗p < See also Figure S7.
Immunity  , e6DOI: ( /j.immuni )
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9. Figure 7 TβRI Expression Is Reduced in Naive T Cells of SLE Patients
Naive CD4+ T cells were isolated from healthy individuals and SLE patients.
(A) TGF-BR1 mRNA in naive or T cells stimulated for 24 hr with anti-CD3 and anti-CD28 (Activated).
(B) Cell proliferation of naive T cells from healthy individuals or SLE patients stimulated for 3 days with indicated concentration of anti-CD3 and APCs.
Each circle represents one individual. Student’s t test was used. Bars, mean; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p <
Immunity  , e6DOI: ( /j.immuni )
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