1. Volume 47, Issue 3, Pages 538-551.e5 (September 2017)
The Kinase mTORC1 Promotes the Generation and Suppressive Function of Follicular Regulatory T Cells 
Lifan Xu, Qizhao Huang, Haoqiang Wang, Yaxing Hao, Qiang Bai, Jianjun Hu, Yiding Li, Pengcheng Wang, Xiangyu Chen, Ran He, Bingshou Li, Xia Yang, Tingting Zhao, Yanyan Zhang, Yifei Wang, Juanjuan Ou, Houjie Liang, Yuzhang Wu, Xinyuan Zhou, Lilin Ye 
Immunity 
Volume 47, Issue 3, Pages e5 (September 2017)
DOI: /j.immuni
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2. Immunity 2017 47, 538-551.e5DOI: (10.1016/j.immuni.2017.08.011)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
mTOR Signaling Is Elevated in Tfr Cells following OVA/CFA Immunization
(A) Gating strategy of Tfr, Treg, Tfh, and Teff cells in the detection of phosphorylated signaling proteins.
(B–D) Comparison of S6 and 4E-BP1 phosphorylation (p) (B), CD71 and CD98 (C), and p-AktS473 and p-FoxO1/3a (D) between Treg cells (CD4+Foxp3+CXCR5–), Tfh cells (CD4+Foxp3–CXCR5+), Teff cells (CD4+Foxp3–CD44+CXCR5–), and Tfr cells (CD4+Foxp3+CXCR5+) 8 days after immunization with OVA/CFA. Blue, green, orange, and red lines in the flow cytometry data represent the gating of Treg, Tfh, Teff, and Tfr cells, respectively, and the solid gray histograms denote the isotype control. The levels of p-S6 and p-4E-BP1 (B), CD71 and CD98 (C), and p-AKTS473 and p-FoxO1/3a (D) summarized beside were calculated by subtracting the mean fluorescence intensities (MFIs) of the isotype controls. Data are representative of two (D) or three (B and C) independent experiments with three mice per group. Center values (B–D) indicate mean. Unpaired t test. ∗p < 0.05, ∗∗p < 0.01; ns, not significant. See also Figure S1.
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4. Figure 2
Transient Inhibition of mTORC1 Signaling Impairs the Formation of Tfr Cells without Retardation on Treg Cells
(A) Flow cytometry analysis of donor CD45.2+ Treg cells (gated in CD4+Foxp3+, top) and Tfr cells (bottom) in dLNs obtained from recipient mice transferred with rapamycin (Rapa)- or vehicle control (Ctrl)-treated CD45.2+CD4+GITR+CD25+CXCR5– Treg cells, assessed at day 8 after OVA/CFA immunization. The numbers adjacent to the outlined areas indicate the proportion of each population.
(B) Summary of the proportion and total cell number of Tfr cells and total Fxop3+ Treg cells and cell number of Fxop3+CXCR5– non-Tfr cells as described in (A).
(C and D) Expression (C) and quantification (D) of CXCR5, GITR, and CTLA4 in Tfr cells as described in (A). Blue and red lines represent the gating of control Tfr cells and rapamycin-treated Tfr cells, respectively, and the solid gray histograms denote the isotype control.
The data are representative of two independent experiments with four (A–D) mice per group. Center values (B and D) indicate mean. Unpaired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. See also Figure S2.
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5. Figure 3
The Abolishment of mTORC1 Signaling Affects the Differentiation of Tfr Cells
(A) Flow cytometry analysis of CD4+Foxp3+ Treg cells (top) and Tfr cells (bottom) in dLNs obtained from Foxp3Cre control and Rptorfl/+Foxp3Cre mice, assessed at day 8 after NP-OVA/CFA immunization. The numbers adjacent to the outlined areas indicate the proportion of each cell type.
(B) Summary of the proportion of Tfr cells and total cell number of Tfr cells, Foxp3+ Treg cells, and Foxp3+CXCR5– cells as described in (A).
(C) Quantification of the MFIs of CXCR5, PD-1, CTLA4, ICOS, and CD73 in Tfr cells as described in (A).
(D and E) Flow cytometry analysis of CD4+Foxp3–CXCR5+ Tfh cells (D) and PNAhiFAShi GC-B cells cells (E) in dLNs obtained from Foxp3Cre control and Rptorfl/+Foxp3Cre mice, assessed at day 8 after NP-OVA/CFA immunization. Summary of the frequency and total number of Tfh (D) and GC-B (E) are shown on the right.
(F) Titers of NP-specific IgG in sera obtained from Foxp3Cre control and Rptorfl/+Foxp3Cre mice, assessed at day 8 after NP-OVA/CFA immunization.
The data are representative of three independent experiments with three (A–F) mice per group. Center values (B–F) indicate mean. Unpaired t test. ∗p < 0.05, ∗∗p < 0.01; ns, not significant. See also Figures S3–S5.
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6. Figure 4
Rapamycin Treatment Inhibits the Suppressive Function of Tfr Cells
(A and B) Flow cytometry analysis of PNAhiFAShi GC-B cells (A) gated on the B220+CD19+ population and CD138hiB220lo plasma cells (B) in spleens from recipient mice on day 7 after cell transfer.
(C and D) Summary of the proportion and total number of GC-B cells (C) and plasma cells (D) as described above.
The presented data are representative of two independent experiments with two to four mice per group. Center values (C and D) indicate mean. Unpaired t test. ∗p < 0.05, ∗∗p < 0.01.
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7. Figure 5 mTORC1-Dependent Transcriptional Profiles of Tfr Cells
(A) GSEA analysis of gene signatures in WT and Raptor–/– Tfr cells sorted on day 8 after OVA/CFA immunization.
(B) Heatmap of the expression of genes described in (A). Red, upregulated gene expression; blue, downregulated gene expression.
(C) RT-qPCR of selected genes listed in (A) normalized to their expression level in Rptor–/– Tfr cells.
(D) Quantification of TCF-1 and Bcl-6 in CD4+Foxp3+CXCR5+ Tfr cells of different origins (WT or Rptorfl/fl Foxp3Cre) in dLNs obtained from BM chimeras on day 8 after OVA/CFA immunization.
The data presented are representative of two independent experiments (C and D) with three mice (D) or two technical replicates pooled from at least four mice per group (C). Error bars (C) indicate the mean ± SEM. (C) by unpaired t test. (D) by paired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S6A.
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8. Figure 6
The mTORC1-p-STAT3-TCF-1-Bcl-6 Axis Regulates Tfr Differentiation
(A) Binding of TCF-1 to conserved motifs in the Bcl6 promoter region (Bcl6 −500) and to a region in Bcl6 without TCF-1-binding motifs (Bcl6 +2.9 kb) (negative control) in CD4+GITR+CD25+ T cells sorted from WT mice at day 8 after OVA/CFA immunization, analyzed by ChIP with antibody to TCF-1 (anti-TCF-1) or isotype-matched control antibody (IgG), followed by quantitative PCR (normalized to their expression level in IgG control).
(B) Flow cytometry analysis of CXCR5+ Tfr cells (gated in CD4+Foxp3+ cells) in the dLNs of WT and Tcf7–/– mice on day 8 after OVA/CFA immunization. The proportion and total number of Tfr cells are summarized beside. Quantification of Bcl-6 MFI in Tfr cells is also shown on the right.
(C) Expression of p-STAT3 in Treg cells and CD4+Foxp3+CXCR5+ Tfr cells of different origins in dLNs obtained from BM chimeras (WT and Rptorfl/fl Foxp3Cre) on day 8 after OVA/CFA immunization. Blue, green, and red lines on flow cytometry data represent the gating of WT Tfr cells, WT Treg cells, and Rptorfl/fl Foxp3Cre Tfr cells, respectively, and the solid gray histograms denote isotype control (left).
(D) Quantification of the MFIs of p-STAT3 in Treg cells and Tfr cells from different origins as described in (C). Lines connect data points for the same mouse.
(E) Binding of p-STAT3 to conserved motifs in the Tcf7 5′-regulatory region (Tcf7 −31.8 kb, −17.5 kb) and to a region in Tcf7 without p-STAT3-binding motifs (Tcf7 +0.2 kb) (negative control) in CD4+GITR+CD25+ T cells sorted from WT mice at day 8 after OVA/CFA immunization, analyzed by ChIP with antibody to p-STAT3 (anti-p-STAT3) or isotype-matched control antibody (IgG), followed by quantitative PCR (normalized to their expression in IgG control).
(F and G) Flow cytometry analysis of Tfr population of different origins in spleens obtained from splenic chimeras on day 8 after LCMV-Arm+ infection (gated on CD4+CD25+ cells). The numbers adjacent to the outlined areas indicate the proportion of Tfr cells. The proportion of Tfr cells described in (F) is summarized in (G), and lines connect data points for same mouse.
(H) Quantification of the MFIs of TCF-1 and Bcl-6 in Tfr cells of different origins as described in (F).
The data are representative of two (A and E–H) or three (B–D) independent experiments with at least three (B–D and F–H) mice or three technical replicates (A and E) per group. Error bars (A and E) indicate the mean ± SEM. Center values (B) indicate mean. (A), (B), and (E) by unpaired t test; (D)–(H) by paired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < ; ns, not significant. See also Figures S6B and S6C.
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9. Figure 7
TCF-1 and Bcl-6 Overexpression Restores Defective Tfr Differentiation in Rptor–/– Treg Cells
Flow cytometry analysis of Tfr population of different origins in spleens obtained from TCF-1 overexpression (A) or Bcl-6 overexpression (D) splenic chimeras on day 8 after LCMV-Arm+ infection (gated on CD4+CD25+ cells). The numbers adjacent to the outlined areas indicate the proportion of Tfr cells, which are summarized in (B) and (E). Lines connect data points for the same mouse. Quantification of the MFIs of CXCR5, CD73, ICOS, GITR, and CTLA4 in Tfr cells of different origins are summarized in (C) and (F). The data are representative of three independent experiments with three mice per group. Central values (B, C, E, and F) indicate mean. Paired t test. ∗p < 0.05, ∗∗p < 0.01; ns, not significant.
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