Volume 23, Issue 3, Pages 796-807 (April 2018) KAP1 Regulates Regulatory T Cell Function and Proliferation in Both Foxp3-Dependent and -Independent Manners  Shigeru Tanaka, Christian Pfleger, Jen-Feng Lai, Florence Roan, Shao-Cong Sun, Steven F. Ziegler  Cell Reports  Volume 23, Issue 3, Pages 796-807 (April 2018) DOI: 10.1016/j.celrep.2018.03.099 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 23, 796-807DOI: (10.1016/j.celrep.2018.03.099) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Treg-Specific KAP1-Deficient Mice Develop Autoimmune Disease (A) Enlarged lymph nodes and spleen in 8-week-old TregΔKAP1 mice. (B) Total cell number in the spleen and the lymph nodes. (C) Representative lung histology stained with H&E. (D) Total CD45+ cell number in the lung. (E–G) Flow-cytometric analysis of CD4+ T cells from TregΔKAP1 and control mice in the spleen and the lung for (E) CD44 and CD62L, (F) IFNγ, IL-13, and IL-17, and (G) Foxp3. (H) Flow-cytometric analysis of thymic CD4+ Foxp3+ cells. Data are expressed as mean ± SD of n = 6–8 mice pooled from 2–3 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. NS, not significant. See also Figures S1 and S2. Cell Reports 2018 23, 796-807DOI: (10.1016/j.celrep.2018.03.099) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 KAP1-Deficient Tregs Are Less Proliferative but Not Apoptotic (A) Flow-cytometric analysis of CD4+ T cells from Foxp3cre/wtKAP1+/+ females and Foxp3cre/wtKAP1f/f females for Foxp3 and YFP. Data are expressed as mean ± SD of n = 5 mice from 2 independent experiments. (B) Apoptotic cell analysis. FVD, fixable viability dye. (C) Flow-cytometric analysis of CD4+ Foxp3+ T cells from Foxp3cre/wtKAP1f/f females for Ki-67. (D) Dilution of CTV of splenic Tregs from TregΔKAP1 mice and control mice. (E) BrdU uptake assay was determined by flow cytometry. Data shown in (B)–(E) are expressed as mean ± SD of n = 3–4 from 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01. See also Figure S3. Cell Reports 2018 23, 796-807DOI: (10.1016/j.celrep.2018.03.099) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 KAP1-Deficient Tregs Are Less Suppressive in Both In Vitro and In Vivo (A–C) In vitro suppression assay. Tregs from Foxp3creKAP+/+ mice or Foxp3creKAPf/f mice were co-cultured with CFSE-labeled CD4+ CD25− T cells in the presence of anti-CD3/CD28 for 4 days. (A) The dilution of CFSE was determined by flow cytometry. (B) Representative histogram of CFSE dilution (Treg:responder T cell ratio = 1:2) and cumulative data (mean ± SD) from 3 independent experiments at the indicated ratio of Tregs to responder T cells. (C) The cell number of Tregs 4 days after incubation. (D and E) Adoptive transfer colitis model. (D) Changes in body weight after cell transfer. Data were expressed as mean ± SEM from 3 independent experiments (n = 7–9). (E) Total CD45.1+ responder cells in the lamina propria. Data are expressed as mean ± SD from 3 independent experiments (n = 7–9). ∗p < 0.05, ∗∗p < 0.01. See also Figure S4. Cell Reports 2018 23, 796-807DOI: (10.1016/j.celrep.2018.03.099) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Dysregulated Gene Expression in KAP1-Deficient Tregs (A) The distribution of H3K9me3 marks along the gene body. Tregs from TregΔKAP1 mice and littermate control mice were subjected to ChIP experiment. (B) Scatterplot of the fold change of transcripts determined by RNA-seq against the fold change of H3K9me3 marks around TSS between KAP1-sufficient and -deficient Tregs. The correlation coefficient was determined by spearman’s test. (C) Changes in gene expression and adjusted p values in KAP1-sufficient Tregs versus KAP1-deficient Tregs as a volcano plot. (D) Heatmap of 770 differentially expressed genes whose fold change is less than 2 in KAP1-sufficient Tregs versus KAP1-deficient Tregs. See also Figure S5. Cell Reports 2018 23, 796-807DOI: (10.1016/j.celrep.2018.03.099) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 KAP1 Directly Regulates Treg-Associated Gene Expression together with Foxp3 (A) Venn diagram showing the overlap of the genes bound by KAP1 and the genes bound by Foxp3 (left panel). Enriched GO terms of 2,004 genes that were co-occupied with KAP1 and Foxp3 (right panel). (B) ChIP-seq of KAP1 and Foxp3 tracks at Prdm1 locus (left panel). Highlighted is the peak called by MACS2. KAP1 binding in the intronic region by ChIP-qPCR is shown in right panel. Data are expressed as mean ± SD of n = 3 from 3 independent experiments. (C) Representative histogram of Blimp-1 expression in KAP1-sufficient and -deficient Tregs in the spleen and the lung and cumulative MFI of Blimp-1 (lower panel) (n = 5 from 2 independent experiments). (D) Representative flow plots (gated on Foxp3+ cells) of IL-10 in the spleen and the lung (upper panel) and cumulative data (lower panel) (n = 6–7). (E) Proportion of KAP1 binding sites that contain the indicated motifs. Data are expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. NS, not significant. See also Figure S6. Cell Reports 2018 23, 796-807DOI: (10.1016/j.celrep.2018.03.099) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 KAP1 Positively Regulates Slc1a5 Expression Followed by mTORC1 Activation in a Foxp3-Independent Manner (A) ChIP-seq of KAP1 and Foxp3 tracks at Slc1a5 locus (left). Highlighted is the peak called by MACS2. KAP1 binding in the conserved non-coding sequence of Slc1a5 by ChIP-qPCR is indicated (right). Data are expressed as mean ± SD of n = 3 from 3 independent experiments. ∗p < 0.05. (B) Slc1a5 expression in KAP1-sufficient and -deficient Tregs. Data are representative of 3 independent experiments. (C and D) Phosphorylation of S6 ribosomal protein. Splenocytes from TregΔKAP1 mice and control mice were stimulated with (C) anti-CD3/CD28 antibody or (D) 5 mM L-glutamine. Data are representative of 3 independent experiments. (E) Cell proliferation of Slc1a5-expressing KAP1-deficient Tregs determined by CellTrace Far Red (CTFR) dilution. The histograms (gated on YFP+CFP+) are representative of 4 independent experiments, and the cumulative data are also shown (n = 6 each). Data are expressed as mean ± SD. ∗p < 0.05 (paired t test). NS, not significant. Cell Reports 2018 23, 796-807DOI: (10.1016/j.celrep.2018.03.099) Copyright © 2018 The Author(s) Terms and Conditions