1. Volume 46, Issue 4, Pages 660-674 (April 2017)
Transforming Growth Factor-β Signaling in Regulatory T Cells Controls T Helper-17 Cells and Tissue-Specific Immune Responses 
Joanne E. Konkel, Dunfang Zhang, Peter Zanvit, Cheryl Chia, Tamsin Zangarle-Murray, Wenwen Jin, Songlin Wang, WanJun Chen 
Immunity 
Volume 46, Issue 4, Pages (April 2017)
DOI: /j.immuni
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2. Figure 1
Deletion of TGF-βR1 in Foxp3+ T Cells Leads to Enhanced T Cell Cytokine Production in the Gastrointestinal Tract with Age
(A) Representative flow cytometry plots show IFN-γ against IL-17 staining on TCRαβ+CD4+ T cells isolated from lung (left) and colonic lamina propria (Lp; right) of young control (Tgfbr1f/+Foxp3-cre+ or Tgfbr1f/fFoxp3-cre−) or Tgfbr1f/fFoxp3-cre+ mice. Lung n = 4–5. Colonic Lp n = 4.
(B) Bar graph shows frequency of Foxp3+ T cells in control and Tgfbr1f/fFoxp3-cre+ mice. n = 4–8.
(C and D) Bar graph shows frequency of CD4+IFN-γ+ (C) and CD4+IL-17+ (D) T cells in control and Tgfbr1f/fFoxp3-cre+ mice. n = 3–8.
(E) Representative flow cytometry plots show IFN-γ against IL-17 staining on TCRαβ+CD4+ T cells isolated from lung and colonic Lp of 1-year-old control and Tgfbr1f/fFoxp3-cre+ mice. n = 4.
(F) Bar graph shows frequency of Foxp3+ T cells in 1-year-old control and Tgfbr1f/fFoxp3-cre+ mice.
(G and H) Bar graph shows frequency of CD4+IFN-γ+ (G) and CD4+IL-17+ (H) T cells in 1-year-old control and Tgfbr1f/fFoxp3-cre+ mice.
∗p < 0.05, as determined by unpaired Student’s t test. Error bars represent mean ± SEM.
See also Figure S1 and S2.
Immunity  , DOI: ( /j.immuni )
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3. Figure 2
Tgfbr1−/− Treg Cells Are Able to Control CNS Inflammation, as well as WT Treg Cells
(A–C) Tgfbr1f/fFoxp3-cre+ (black squares) and littermate controls (Tgfbr1f/+Foxp3-cre+ or Tgfbr1f/fFoxp3-cre−; open circles) were immunized with pMOG+CFA to induce EAE. (A) Graph shows EAE disease scores. (B and C) Bar graphs show frequency of Foxp3+ Treg cells and cytokine+ CD4+ T cells in the (B) brain and (C) spinal cords of Tgfbr1f/fFoxp3-cre+ and control mice.
(D and E) Bar graphs show the frequency of Foxp3+cytokine+ Treg cells in the (D) brain and (E) spinal cords of Tgfbr1f/fFoxp3-cre+ and control mice.
∗p < 0.05, as determined by unpaired Student’s t test. Data shown are representative of two independent experiments with three or four mice per group. Error bars represent mean ± SEM.
Immunity  , DOI: ( /j.immuni )
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4. Figure 3
Tgfbr1−/− Treg Cells Are Better at Suppressing T Cell Proliferation In Vitro
(A and B) Cells were cultured with anti-CD3 and anti-CD28. Graph shows percent of suppression of effector T cell proliferation when co-cultured with Treg cells flow cytometry sorted from controls (Tgfbr1f/+Foxp3-cre+; open circles) or Tgfbr1f/fFoxp3-cre+ (closed squares) mice. Flow cytometry plots show effector T cell CFSE dilution when co-cultured with Treg cells at a ratio of 1:1 (top) or 1:2 (bottom). Percent indicates frequency of undivided effector T cells. Data representative of five experiments.
(C and D) Representative flow cytometry plots show percent IL-17+ and IFN-γ+ cells gated on effector T cells in cultures with Treg cells at a ratio of 1:1. T cells were stimulated with anti-CD3 and anti-CD28 (C) alone or (D) with the addition of IL-12 and IFNγ. Data representative of two experiments.
(E) Bar graph shows percent change in suppressive capability of Tgfbr1−/− Treg cells compared to controls. In different polarizing conditions, the ability of Treg cells to suppress IFN-γ, IL-17, and IL-4 production by effector CD4+ T cells was examined. Graph shows data for two or three separate experiments per culture condition.
(F and G) Suppression assays were established, stimulating cells with anti-CD3 and anti-CD28 in the presence of IL-6. (F) Representative flow cytometry plots show percent IL-17+ and IFN-γ+ cells gated on effector T cells in cultures with Treg cells at a ratio of 1:1. (G) Bar graph shows frequency of IL-17+ effector CD4+ T cells cultured with control Treg cells (white bar), Tgfbr1−/− Treg cells (black bar), or no Treg cells. Data representative of two experiments.
∗p < 0.05, as determined by unpaired Student’s t test. Error bars represent mean ± SEM. See also Figure S3.
Immunity  , DOI: ( /j.immuni )
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5. Figure 4
Tgfbr1−/− Treg Cells Produce Enhanced IL-10 and Exhibit Elevated Expression of T-bet and Gata-3
(A and B) Representative flow cytometry plots and bar graphs show percent of (A) IL-10+Foxp3+ and (B) T-bet+Foxp3+ Treg cells from spleens of control (Tgfbr1f/+Foxp3-cre+ or Tgfbr1f/fFoxp3-cre−) or Tgfbr1f/fFoxp3-cre+ mice. n = three or four mice per group.
(C) Sorted Tgfbr1−/− Treg cells were stimulated in vitro for 24 hr with anti-CD3, anti-CD28, and IL-2. Expression of GATA-3 was determined by qPCR. Data, representative of two experiments, are presented relative to Gata-3 expression in control Treg cells. ∗p < 0.05, as determined by paired Student’s t test.
(D) Representative flow cytometry plots and bar graph show percent GATA-3+Foxp3+ Treg cells in the colonic Lp of control and Tgfbr1f/fFoxp3-cre+ mice (n = 3).
(E) Treg cells were flow cytometry sorted from Tgfbr1f/fFoxp3-cre+ mice and transfected with either an RFP+ vector or RFP+ vector containing a dominant-negative T-bet. Transfected Treg cells were then sorted again and cultured with congenic CD4+ T cells in Th1 polarizing conditions at a ratio of 1:1. Graph shows percent suppression of IFN-γ production by effector CD4+ T cells. Flow cytometry plots show CD45.1 and IFN-γ staining gated on effector CD4+ T cells cultured with either control or dominant-negative T-bet transfected Treg cells at a ratio of 1:1. Data representative of two experiments.
(F) Treg cells were flow cytometry sorted from Tgfbr1f/fFoxp3-cre+ and Tgfbr1f/+Foxp3-cre+ mice. IL-10Ra mRNA transcripts were examined ex vivo (−) or following 24 hr culture with anti-CD3, anti-CD28, and rIL-2 (+). Data presented relative to control. Bar graphs show data from three experiments combined.
(G) Representative flow cytometry plots and bar graphs show percent of RORγt+Foxp3+ Treg cells from spleens of control or Tgfbr1f/fFoxp3-cre+ mice. n = five mice per group.
(H) Treg cells were flow cytometry sorted from Tgfbr1f/fFoxp3-cre+ and Tgfbr1f/+Foxp3-cre+ mice. RORc mRNA transcripts were examined directly ex vivo (−) or following 24 hr culture with anti-CD3, anti-CD28, and rIL-2 (+). Data presented relative to control. Bar graphs show data from three experiments combined.
∗p < 0.05, ∗∗p < as determined by unpaired Student’s t test, unless otherwise indicated. Error bars represent mean ± SEM.
See also Figure S4.
Immunity  , DOI: ( /j.immuni )
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6. Figure 5
Tgfbr1−/− Treg Cells Are Unable to Control Colitis in a Transfer Model of the Disease
(A) Rag1−/− mice received CD4+CD45RBhiCD45.1+ cells in combination with GFP+ Treg cells from CD45.2+ Tgfbr1f/fFoxp3-cre+ (black squares) or Tgfbr1f/+Foxp3-cre+ (open circles) mice. Mice were weighed weekly post cell transfer. p < 0.05 comparing Area Under the Curve for weight of each mouse. n = three or four mice per group.
(B) Bar graph shows total number of monocytes and neutrophils per colon from two experiments combined.
(C and D) Foxp3+ cells were examined in the colons of recipient mice. Bar graph shows frequency of Tgfbr1f/fFoxp3-cre+ and Tgfbr1f/+Foxp3-cre+ CD4+Foxp3+ Treg cells in the colons of recipient mice. Representative flow cytometry panels show Foxp3 versus CD45.2 staining on colonic TCRβ+CD4+ cells.
(E and F) CD103 expression was examined on Foxp3+ cells in the colons of recipient mice. Bar graph shows frequency of Tgfbr1f/fFoxp3-cre+ or Tgfbr1f/+Foxp3-cre+ Foxp3+ Treg cells that express CD103. Representative flow cytometry panels show Foxp3 versus CD103 staining on CD45.2+CD4+Foxp3+ cells in the colon. Data are representative of two experiments with three or four mice per group.
(G and H) GFP+ Treg cells were flow cytometry sorted from Tgfbr1f/fFoxp3-cre+ and Tgfbr1f/+Foxp3-cre+ mice. mRNA transcripts of (G) GPR174 and (H) GPR15 were examined, data presented relative to control. Bar graphs show data from three separate experiments combined.
∗∗∗p < , ∗∗p < were determined by unpaired two-tailed Student’s t test, unless otherwise indicated. Error bars represent mean ± SEM.
See also Figure S5.
Immunity  , DOI: ( /j.immuni )
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7. Figure 6 Tgfbr1−/− Treg Cells Fail to Persist within the Colon
(A) Example experimental outline; Rag1−/− mice received CD45.1+CD4+CD45RBhi cells. At the same time, they also received congenically distinct GFP+ Treg cells from Tgfbr1f/fFoxp3-cre+ and Tgfbr1f/+Foxp3-cre+ mice, mixed at a 1:1 ratio, allowing three different T cell populations to be identified. Treg cells were examined 4 weeks post transfer.
(B) Bar graph shows frequency of Tgfbr1f/fFoxp3-cre+ Treg cells relative to controls; ratio of 1 indicates control and Tgfbr1−/− Treg cells were present at a ratio of 1:1. ∗∗p < 0.002, ∗∗∗p < (paired two-tailed Student’s t test). Representative flow cytometry plots show CD45.1 versus CD45.2 staining gated on transferred CD45.2+ Treg cells.
(C–E) Bar graphs show frequency of control or Tgfbr1f/fFoxp3-cre+ Treg cells which are (C) Ki67+, (D) Bcl2+, and (E) ICOS+.
(F) Bar graph shows CD103+Foxp3+ cells in the colon of recipient mice, and representative flow cytometry plots show CD103 versus CD45.1 staining gated on Foxp3+CD45.2+ T cells. ∗∗∗p < (unpaired two-tailed Student’s t test).
Data representative of two to three independent experiments (n = 4–5). Error bars represent mean ± SEM.
See also Figure S6.
Immunity  , DOI: ( /j.immuni )
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8. Figure 7
TGF-β Signals Are Constantly Required to Maintain Treg Cell CD103 Expression
(A) Foxp3GFP Treg cells were flow cytometry sorted and cultured with anti-CD3, anti-CD28, and IL-2 either with or without recombinant TGF-β. After 24 and 48 hr of culture, GPR174 expression was examined by qPCR. Data are presented relative to expression in cells not treated with TGF-β.
(B and C) CD103+ Treg cells were flow cytometry sorted and cultured with irradiated antigen-presenting cells, IL-2, anti-CD3, and either recombinant TGF-β (+TGF-β; black bars), or anti-TGF-β1, 2, and 3 plus TGF-β inhibitors (+inhibitors; gray bars), or media alone (control; white bars). CD103 expression was then examined. (B) Histogram shows CD103 expression following 48 hr in culture. (C) Bar graphs show frequency of CD103+ Treg cells. Data representative of four experiments.
(D) CD103+ Treg cells were flow cytometry sorted and cultured with anti-CD3, anti-CD28, IL-2, and either recombinant TGF-β (+TGF-β; black bars), or anti-TGF-β1, 2, and 3 plus TGF-β inhibitors (+inhibitors; gray bars), or media alone (control; white bars). After 18 hr, CD103 mRNA transcripts were examined. Data presented relative to control sample and representative of two experiments.
(E) CD103+ Treg cells were flow cytometry sorted from WT or Smad3−/− mice and cultured with irradiated antigen presenting cells, IL-2, anti-CD3, and either recombinant TGF-β (+TGF-β), or anti-TGF-β1, 2, and 3 plus TGF-β inhibitors (+inhibitors), or media alone (control). CD103 expression was then examined. Data representative of two experiments.
(F) CD103− Treg cells were flow cytometry sorted and cultured with irradiated antigen presenting cells, IL-2, anti-CD3, and recombinant TGF-β for 48 hr, after which cells were washed and replated with either TGF-β (+TGF-β; black bars) or anti-TGF-β1, 2, and 3 plus TGF-β inhibitors (+inhibitors; white bars). Representative flow cytometry plots show CD25 versus CD103 staining. Bar graph shows data from two experiments combined. ∗p < 0.05, ∗∗p < (unpaired two-tailed Student’s t test).
Error bars represent mean ± SEM.
See also Figure S7.
Immunity  , DOI: ( /j.immuni )
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