Volume 27, Issue 4, Pages 1205-1220.e4 (April 2019) Noc4L-Mediated Ribosome Biogenesis Controls Activation of Regulatory and Conventional T Cells  Xueping Zhu, Wei Zhang, Jie Guo, Xuejie Zhang, Liping Li, Ting Wang, Jinghua Yan, Fuping Zhang, Baidong Hou, Ning Gao, George F. Gao, Xuyu Zhou  Cell Reports  Volume 27, Issue 4, Pages 1205-1220.e4 (April 2019) DOI: 10.1016/j.celrep.2019.03.083 Copyright © 2019 The Author(s) Terms and Conditions

Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions

Figure 1 Noc4L Is Highly Expressed in Activated Tregs (A) Noc4L mRNA expression in unstimulated or stimulated (anti-CD3 and anti-CD28, 72 h) of mouse CD4+GFP-CD62Lhi Tconvs or CD4+GFP+CD62Lhi Tregs. (B) Noc4L mRNA expression in sorted mouse CD44loCD62Lhi and CD44hiCD62Llo Tregs. (C) Schematic of Noc4lmCherry reporter mice (top); representative flow cytometry analysis of mCherry expression in Tregs (CD4+CD25+) of the non-transgenic mice (non-Tg) or Noc4lmCherry reporter mice (bottom). (D) Quantifications of the mCherry MFI in CD44loCD62Lhi and CD44hiCD62Llo Tconvs and Tregs. (E) In vitro suppression assays mediated by sorted mCherryhi and mCherrylo Tregs based on mCherry and CD44 expression from lymph nodes and spleen of Noc4lmCherry reporter mice. Data represent the mean ± SD. Results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, paired t test. Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions

Figure 2 Selective Ablation of Noc4L in Tregs Leads to a Severe Autoimmune Disease (A) Schematic of Noc4L floxed mice. Arrows represent primers used for genomic PCR analysis (top). To examine Cre sensitivity, DNA was extracted from sorted CD4+GFP− and CD4+GFP+ cells of Foxp3CreNoc4l+/fl mice, and genomic PCR analysis were performed. The wild-type band and loxP site were identified using the primers (384 bp/502 bp, left) indicated by the arrows (bottom). (B) Body weight of Foxp3CreNoc4l+/fl (Het) and Foxp3CreNoc4lfl/fl (cKO) mice (4–10 weeks old). (C) Survival curve of mice as in (B). (D) H&E staining of liver and lung from mice as in (B). (E) Flow cytometry analysis of CD44 and CD62L expression in CD4+ Foxp3− T cells (left) and frequency of CD44hiCD62Llo cells (right) in mice as in (B). (F) Flow cytometry analysis of IFN-γ, IL-4, IL-17A, and IL-10 cytokines expression in CD4+ T cells (left) and frequency of IFN-γ+, IL-4+, IL-17A+, and IL-10+ in lymph nodes and splenic CD4+ T cells (right) of mice as in (B). Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 3 h before intracellular staining. Data represent the mean ± SD. Results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, paired t test. Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions

Figure 3 Noc4L-Mediated Ribosome Biogenesis Is Dispensable for Global Translation in Tregs (A) Flow cytometry analysis of GFP and Foxp3 expression in CD4+ T cells (left) and frequency of Foxp3+GFP+ and Foxp3+GFP− cells in lymph nodes and splenic CD4+ T cells (right) of Foxp3CreNoc4l+/fl (Het) or Foxp3CreNoc4lfl/fl (cKO) mice. (B) Quantification of Foxp3 MFI in lymph nodes and splenic Foxp3+GFP+ cells of mice as in (A). (C) Flow cytometry analysis of YFP and Foxp3 expression in CD4+ T cells (left) and frequency of Foxp3+YFP−, Foxp3+YFP+, and Foxp3−YFP+ cells in lymph nodes and splenic CD4+ T cells (right) of Foxp3CreNoc4l+/flRosa26YFP (Het) and Foxp3CreNoc4lfl/flRosa26YFP (cKO) mice. (D) Quantification of Foxp3 MFI in lymph nodes and splenic Foxp3+YFP+ cells of mice as in (C). (E) YFP expression and MFI in lymph nodes CD4+YFP+ cells from mice as in (C). (F) Schematic of co-receptor re-expression assay (left). Surface expression of CD4 in Tregs (YFP+) was detected by flow cytometry analysis (right). Data represent the mean ± SD. Results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, paired t test (ns, not significant). Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions

Figure 4 Noc4L-Mediated Ribosome Biogenesis Specifically Regulates Activation of Tregs (A) Flow cytometry analysis of ICOS, CD69, CD44, KLRG1, CD25, CD62L, CTLA4, and Bcl2 in GFP+ and GFP− cells of CD4+Foxp3+ Tregs (right) and percentages or MFI of the indicated markers (left) in GFP+ and GFP− cells from Foxp3CreNoc4lfl/fl (cKO) mice. (B) Flow cytometry analysis of GFP and Ki-67 expression in CD4+Foxp3+ cells (left) and frequency of Ki-67+ cells in GFP+ and GFP− cells (right) in mice as in (A). (C) CellTrace-violet-labeled CD4+CD62LhiGFP+ Tregs sorted from lymph nodes and spleen of Foxp3CreNoc4l+/fl (Het) or Foxp3CreNoc4lfl/fl (cKO) mice were stimulated with Dynabeads in presence of rhIL-2 for 6 days. Flow cytometry analysis of cell size (forward scatter area [FSC-A]), ICOS expression, and CellTrace dilution in Tregs. (D) Flow cytometry analysis of CCR7, CCR4, CCR5, and CXCR3 expression in GFP+ and GFP− cells among CD4+Foxp3+ cells (right) and percentages or MFI of the indicated markers (left) in mice as in (A). (E) In vivo labeling assay, flow cytometry analysis of CD4 RM4-5 and CD4 RM4-4 expression in splenic Tregs (left), and frequency of CD4 RM4-4+ cells in Foxp3+YFP+ and Foxp3+YFP− Tregs (right). (F) Quantification of the ratio of GFP+ cells to CD4+Foxp3+ cells in the lymph node, spleen, lung, and liver of Foxp3CreNoc4l+/fl (het) and Foxp3CreNoc4lfl/fl (cKO) mice. (G) Cell number of GFP+ cells in the lymph node, spleen, lung, and liver of mice as in (F). Data represent the mean ± SD. Results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, paired t test (ns, not significant). See also Figure S1. Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions

Figure 5 Noc4L-Mediated Ribosome Biogenesis Dictates Treg Activation by Specifically Regulating Translation (A) Flow cytometry analysis of p-S6, CD71, and CD98 expression in unstimulated or anti-CD3 and anti-CD28 stimulated CD4+YFP+ Tregs from Foxp3CreNoc4l+/flRosa26YFP (Het, red line) and Foxp3CreNoc4lfl/flRosa26YFP (cKO, blue line) mice (left). Graphs represent the MFI of indicated markers (right). (B) Phosphorylation of STAT5 at Try694 (pSTAT5 [Y694]) in lymph node CD4+YFP+ cells stimulated with rhIL-2 for 5, 15, and 30 min at 37°C from mice as in (A) (left). Graphs represent MFI of pSTAT5 (Y694) (right). (C) Flow cytometry analysis of c-Myc expression in unstimulated or anti-CD3 and anti-CD28 stimulated CD4+YFP+ Tregs from mice as in (A) (left). Graphs represent MFI of c-Myc (right). (D) Flow cytometry analysis of YFP and IRF4 or BATF expression in CD4+Foxp3+ cells from Foxp3CreNoc4lfl/flRosa26YFP mice, and quantification of IRF4 or BATF MFI in YFP+ and YFP− Tregs (left). Foxp3+ cells were also gated on the IRF4 or BATF low (lo), intermediate (int), and high (hi) gates. YFP and ICOS expression in the indicated population is shown (middle). The frequency of ICOS+ cells in YFP+ and YFP− cells from each subset is shown (right). (E) Protein and mRNA level of ICOS, CTLA4, CXCR3, and CD69 in CD4+YFP+ Tregs from mice as in (A). Data represent the mean ± SD. Results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, paired t test (ns, not significant). See also Figure S2. Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions

Figure 6 Deletion of p53 Does Not Rescue Treg Activation and Autoimmune Phenotype (A) Flow cytometry analysis of p53 and c-Myc expression in unstimulated or anti-CD3 and anti-CD28 stimulated CD4+YFP+ Tregs from Foxp3CreNoc4l+/flRosa26YFP (Het) and Foxp3CreNoc4lfl/flRosa26YFP (cKO) mice (left). Graph represents MFI of p53 (right). (B) Body weight of 10-week-old Trp53+/+Foxp3CreNoc4l+/fl (Trp53+/+ Het), Trp53−/−Foxp3CreNoc4l+/fl (Trp53−/− Het), Trp53+/+Foxp3CreNoc4lfl/fl (Trp53+/+ cKO), and Trp53−/−Foxp3CreNoc4lfl/fl (Trp53−/− cKO) mice. (C) H&E staining of liver and lung of mice as in (B). (D) Frequency of IFN-γ+, IL-4+, and IL-17A+ in splenic CD4+Foxp3− T cells from mice as in (B). Cells were stimulated with PMA and ionomycin for 3 h before intracelluar staining. (E) Flow cytometry analysis of GFP expression in lymph node and splenic CD4+Foxp3+ Tregs (left) and ratios of GFP+ cells relative to CD4+Foxp3+ Tregs from Trp53+/+Foxp3CreNoc4lfl/fl (Trp53+/+ cKO) and Trp53−/−Foxp3CreNoc4lfl/fl (Trp53−/− cKO) mice (right). (F) Flow cytometry analysis of CTLA4, Ki-67, Bcl2, ICOS, CD25, and CCR7 expression in splenic CD4+Foxp3+ Tregs from mice as in (E) (left), and percentages or MFI of the indicated markers in GFP− and GFP+ cells (right). Data represent the mean ± SD. Results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05 and ∗∗p < 0.01, paired t test (ns, not significant). See also Figure S3. Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions

Figure 7 Complete Knockout of Noc4L in Tregs Results in a Scurfy Phenotype (A) Images of 21-day-old male Foxp3YFP-CreNoc4l+/fl and Foxp3YFP-CreNoc4lfl/fl mice. Shown are peripheral lymph nodes, mesenteric lymph nodes, spleen, and thymus (left) and total cellularity of the indicated organ (right). (B) Survival curve (left) and body weight of mice in (A). (C) H&E staining of liver, lung, skin, and colon of mice in (A). (D and E) Flow cytometry analysis of CD44 and CD62L expression in CD4+Foxp3− (D) and CD8+ (E) T cells (left) and frequency of CD44hiCD62Llo cells (right) of mice as in (A). (F) Flow cytometry analysis of IFN-γ, IL-4, IL-17A, and IL-10 expression in lymph nodes CD4+Foxp3− T cells (left) and frequency of IFN-γ+, IL-4+, IL-17A+, and IL-10+ in lymph nodes and splenic CD4+Foxp3− cells (right) from mice as in (A). Cells were stimulated with PMA and ionomycin for 3 h before intracelluar staining. Data represent the mean ± SD. Results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, paired t test. See also Figure S4. Cell Reports 2019 27, 1205-1220.e4DOI: (10.1016/j.celrep.2019.03.083) Copyright © 2019 The Author(s) Terms and Conditions