IL-7-dependent STAT1 activation limits homeostatic CD4 T cell expansion

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1 Supplemental Material IL-7-dependent STAT1 activation limits homeostatic CD4 T cell expansion Cecile Le Saout 1, Megan A. Luckey 2, Alejandro V. Villarino 3, Mindy Smith 1, Rebecca B. Hasley 1, Timothy G. Myers 4, Hiromi Imamichi 1, Jung-Hyun Park 2, John J O Shea 3, H. Clifford Lane 1 and Marta Catalfamo 1,* 1 CMRS/Laboratory of Immunoregulation, NIAID, NIH, Bethesda, MD, USA. * Department of Microbiology and Immunology. Georgetown Universisty School of Medicine. Washington DC, USA. 2 Experimental Immunology Branch, CCR, NCI, NIH, Bethesda, MD, USA 3 Lymphocyte Cell Biology Section, NIAMS, NIH, Bethesda, MD, USA 4 Genomic Technologies Section, Research Technologies Branch, NIAID, NIH, Bethesda, MD, USA

2 Figure S1. STAT1 Tg mice exhibit similar T cell counts and differentiation profiles than WT littermates. (A) Schematic representation of the transgene construct of mouse STAT1 cdna under the control of the human CD2 enhancer/promoter used to generate transgenic (Tg) mice. (B) Total cellularity in LNs, spleen and thymus from WT (n= 16, black symbols) and STAT1 Tg (n= 16, red synbols) mice. (C) Absolute numbers of CD4 and CD8 T cells were enumerated in the LNs of both groups. (D) Gating strategy and percentages of naïve (Tn: CD44 low CD62L high ), central memory (Tcm: CD44 high CD62L high ), effector/effector memory (Teff/em: CD44 high CD62L low ) on gated CD3 + CD4 + and CD8 + lymphocytes in WT and STAT1 Tg littermates are shown. LN cells from WT and STAT1 Tg mice were analyzed by flow cytometry for expression of (E) IL-7Rα and (F) phosphorylated STAT1 after in vitro stimulation with rmifn-α4 (5 U/ml) or rmifn-γ (1 ng/ml) in CD4 and CD8 T cells. A nonparametric Mann-Whitney test was performed for comparisons between groups. Data from four independent experiments out of four, including an average of 4 mice per group, are presented as box and whisker plots showing the median value bounded by the first and third quartiles in the box, with whiskers extending to the minimum and maximum values. Figure S2. IL-7 induces a similar set of STAT1/IFN associated genes in WT and STAT1 Tg T cells. Sorted WT and STAT1 Tg CD4 (n=5 and n=4 respectively) and CD8 (n=6 and n=5 respectively) naïve T cells were treated and analyzed as described in Figure 3. Venn diagrams illustrate (A) the overlap between WT and STAT1 Tg DEGs in response to IL-7 or IFN-γ in CD4 and CD8 Naïve T cells; (B) the overlap between the DEGs in WT cells stimulated with IFN-γ (green) and the DEGs in STAT1 Tg (red) T cells stimulated with IL-7.

3 Figure S3. Overexpression of t-stat1 leads to an enrichment of STAT1/IFN genes in T cells stimulated with IL-7. Sorted WT (n= 3) and STAT1 Tg (n= 3) CD4 Naïve T cells were stimulated or not overnight with rmil-7 (2 ng/ml) or rmifn-γ (1 ng/ml). RNA was isolated and gene expression was measured by RNA-seq. Libraries were prepared with.25-1 µg of total RNA using the TruSeq RNA Sample Preparation Kit V2 and sequenced for 5 cycles with a HiSeq 25 instrument (Illumina). 5 bp reads were then mapped onto mouse genome build mm9 using TopHat and further processed using Cufflinks (1). Lists of differentially expressed gene transcripts (DEGs) by IL-7 or IFN-γ stimulation in WT and STAT1 Tg were selected based on gene expression changes of at least 2 fold compared to unstimulated cells. (A) Venn diagrams illustrate the overlap between WT (black) and STAT1 Tg (red) DEGs in response to IL-7 in CD4 Naïve T cells. (B) Venn diagrams illustrate the overlap between the DEGs in WT cells stimulated with IFN-γ (green) and the DEGs in WT (black) or STAT1 Tg (red) T cells stimulated with IL-7. (C) Gene expression of Bcl2, Cish, Cxcl1, Ifit1, Socs1, Bik, Ifi27 and Socs2 was measured by quantitative RT-PCR (Quantitect SYBR green PCR system, Qiagen). The primer sequences are presented in Table S9. Gene expression values were normalized to those of Hprt in the same sample and fold changes were calculated relative to unstimulated condition for each mouse. Data represent mean ± SE of the log 2 (Fold change) of triplicate samples in two independent experiments. A nonparametric Mann-Whitney test was performed for comparisons between WT (black symbols) and STAT1 Tg (red symbols) cells. Figure S4. STAT1 overexpression induces a distinct transcriptional program in CD4 T cells stimulated with IL-7. Sorted WT and STAT1 Tg CD4 Naïve T cells were treated and analyzed as described in Figure 3 (9 minutes stimulation) or Figure S3 (overnight stimulation). Lists of

4 DEGs. their log 2 (FC) and adjusted p values were generated for these cells and used as input in IPA software. A positive or negative z-score value indicates that a function is predicted to be increased or decreased in T cells stimulated with IL-7. All categories shown are significant (p <.5). (A) Bar graphs show the overall affected categories of diseases and biological functions predicted by IPA analysis in WT (black) and STAT1 Tg (red) CD4 T cells stimulated 9 minutes with IL-7. Top 1 activated annotations and the number of RNAs (#Mol) differentially expressed in each are listed in the tables. (B) Bar graphs represent the top 1 diseases or functions annotations associated to Cell Death and Survival, Cellular Growth and Proliferation and Hematological System Development and categories predicted by IPA analysis in theses conditions. (C) Bar graphs show the overall affected categories of diseases and biological functions predicted by IPA analysis in WT (black) and STAT1 Tg (red) CD4 T cells stimulated overnight with IL-7. Top 1 activated annotations and the number of RNAs (#Mol) differentially expressed in each are listed in the tables. (D) Bar graphs represent the top 1 diseases or functions annotations associated to Cell Death and Survival categorie predicted by IPA analysis in theses conditions. Figure S5. WT and STAT1 Tg donor T cells exhibit similar responses to IL-7 through STAT5 and to IFN-γ through STAT1 after adoptive transfer into RAG -/- mice. Lymphopenic RAG -/- mice were injected i.v. with 6x1 6 of CTV-labeled T cells isolated from the LNs of congenic WT (n= 9) or STAT1 Tg (n= 9) mice. The levels of IL-7Ra, p-stat5 and p-stat1 of donor T cells were evaluated by flow cytometry in LNs 7 days post-transfer as function of CTV Fluorescence after in vitro stimulation with rmil-7 (1 ng/ml) and IFN-γ (1 ng/ml) respectively. (A) The percentages of donor T cells CTV + IL-7Ra high, CTV + IL-7Ra low, CTV - IL-7Ra high and CTV - IL-7Ra low are indicated. The MFIs of IL-7Ra in CD4 and CD8 donor T cells after adoptive

5 transfer into RAG -/- mice (SP and FP) were compared using a nonparametric Mann-Whitney test. (B) The percentages of donor T cells CTV + p-stat5 high, CTV + p-stat5 low, CTV - p-stat5 high and CTV - p-stat5 low are indicated. The MFIs of p-stat5 in CD4 and CD8 donor T cells before (d) and after adoptive transfer into RAG -/- mice (SP) were compared as described above. (C) The percentages of donor T cells CTV + p-stat1 high, CTV + p-stat1 low, CTV - p-stat1 high and CTV - p-stat1 low are indicated. The MFIs of p-stat1 in CD4 and CD8 donor T cells before (d) and after adoptive transfer into RAG -/- mice (SP) were compared as described above. Data are presented as box and whisker plots showing the median MFI value bounded by the first and third quartiles in the box, with whiskers extending to the minimum and maximum values from three independent experiments out of three, including an average of 3 mice per group and experiment. Supplemental References 1. Villarino A, et al. Signal transducer and activator of transcription 5 (STAT5) paralog dose governs T cell effector and regulatory functions. Elife. 216;5:e8384.

6 t Figure S1 A B Lymph nodes Spleen Thymus C Lymph nodes Cell count (x1 6 ) T WT ns ns ns g STAT1 Tg T WT g STAT1 Tg T WT g STAT1 Tg Cell count (x1 6 ) ns CD4 T cells CD4 T cells ns CD8 T cells CD8 T cells WT STAT1 Tg D CD4 T cells CD8 T cells CD4 T cells CD8 T cells WT STAT1 Tg CD62L Tn Teff /em Tcm CD44 % T cell subsets ns ns ns % Tn % Tcm % Tem-eff % Tn % Tcm % Tem-eff Tn Tcm Teff/ em ns ns ns Tn Tcm Teff/ em WT STAT1 Tg E WT STAT1 Tg CD4 T cells # Cells IL-7Rα CD8 T cells MFI IL-7Rα (x1 3 ) ns CD4 T cells CD4 T cells ns CD8 T cells CD8 T cells WT STAT1 Tg F CD4 T cells CD8 T cells MFI p-stat1 (x1 3 ) g p<.1 T g T ns g p<.1 ns WT STAT1 Tg 3 stimulation: Unstim rmifn-α rmifn-γ Unstim rmifn-α rmifn-γ

7 Figure S2 A IL-7 Stimulation (9 ) WT CD4 Naïve T cells STAT1 Tg WT CD8 Naïve T cells STAT1 Tg IFN-γ Stimulation (9 ) WT STAT1 Tg WT STAT1 Tg B CD4 Naïve T cells CD8 Naïve T cells STAT1 Tg IL-7 stimulation IFN-γ stimulation IL-7 stimulation IFN-γ stimulation

8 Figure S3 A IL-7 stimulation B WT STAT1 Tg WT IL-7 stimulation IL-7 stimulation overnight stimulation overnight stimulation STAT1 Tg IFN-γ stimulation IFN-γ stimulation C Log 2 (Fold change) Log 2 (Fold change) Overnight Stimulation with: Overnight Stimulation with: 7 Socs1 Bik Ifi27 Socs2 7 Bcl2 Cish Cxcl1 Ifit1 g g * * 7 7 g IL-7 IFN-γ IL-7 IFN-γ IL-7 IFN-γ IL-7 IFN-γ * * * * * * * g IL-7 IFN-γ IL-7 IFN-γ IL-7 IFN-γ IL-7 IFN-γ 7 7 g g 7 7 g g WT cells STAT1 Tg cells

9 Figure S4 A Z-score IL-7 Stimulation (9 ) Z-score B Cell Death and Survival IL-7 Stimulation (9 ) Cell Survival Cell Viability Cell Viability of Lymphocytes Cell Viability of T Lymphocytes Cell Viability of Leukocytes Z-score Z-score C WT Diseases or s Annotation #Mol generation of cells 134 proliferation of cells 221 development of blood cells 79 proliferation of epithelial cells 46 cell survival 95 development of leukocytes 77 proliferation of blood cells 89 activation of T lymphocytes 38 transactivation of RNA 48 proliferation of lymphocytes 82 IL-7 Stimulation (Overnight) STAT1 Tg Diseases or s Annotation #Mol generation of cells 133 formation of osteoclasts 15 cell viability of mononuclear leukocytes 28 development of blood cells 76 transactivation of RNA 46 cell survival 93 development of leukocytes 75 cell viability of lymphocytes 27 activation of cells 86 lymphatic node tumor 48 Cellular Growth and Proliferation Hematological System Development and Proliferation of Lymphocytes Proliferation of Mononuclear Leukocytes Proliferation of Immune Cells Cell Proliferation of T Lymphocytes Proliferation of Hematopoietic Cells Proliferation of B Lymphocytes Proliferation of Cells Proliferation of Blood Cells Generation of Cells Proliferation of Epithelial Cells Formation of Osteoclasts T cell Development T cell Homeostasis Quantity of Thymocytes Quantity of Regulatory T Lymphocytes Quantity of Bone Marrow Cells Development of Leukocytes Development of Lymphocytes Development of Blood Cells Development of Mononuclear Leukocytes Development of APCs Development of Bone Marrow Z-score WT STAT1 Tg Z-score -5-1 WT STAT1 Tg Z-score D IL-7 Stimulation (Overnight) Z-score Diseases or s Annotation #Mol organismal death 495 morbidity or mortality 51 cell survival 321 cell viability 296 Diseases or s Annotation #Mol organismal death 55 morbidity or mortality 556 cell survival 357 cell viability 329 Cell Death and Survival Apoptosiss Cell Survival l Cell Viabilityy Cell Viability of Tumor Cell Liness Cell Viability of Blood Cellss Cell Viability of Leukocytes s cellular homeostasis 352 proliferation of cells 89 antibody response 44 cell movement 55 cell viability of tumor cell lines 22 survival of organism 183 Self-renewal of Cellss Cell Viability of Phagocytes s Cell Viability of T Lymphocytes s Cell Viability of Lymphocytes s survival of organism 169 migration of cells 458 cell viability of blood cells 74 cell movement 451 cellular homeostasis 377 proliferation of cells 891 WT STAT1 Tg

10 Figure S5 A Donor: CD4 donor T cells CD8 donor T cells <APC-A>: CD127 IL-7Rα <APC-A>: CD WT STAT1 Tg <APC-A>: CD127 <APC-A>: CD CellTrace Violet MFI IL-7Rα (x1 3 ) CD4 donor T cells CD8 donor T cells p<.1 p<.1 ns p<.1 ns p<.1 ns P P SP FP SP FP P P T cells -> RAG -/- WT STAT1 Tg B Donor: CD4 donor T cells CD8 donor T cells p-stat5 WT STAT1 Tg CellTrace Violet MFI p-stat5 (x1 3 ) 12 3 stimulation with rmil CD4 donor T cells CD8 donor T cells d SP d SP ns ns T cells -> RAG -/- WT STAT1 Tg C CD4 donor T cells CD8 donor T cells Donor: CD4 donor T cells CD8 donor T cells p-stat1 <Alexa Fluor 488-A>: pstat WT 28 <Alexa Fluor 488-A>: pstat STAT1 Tg CellTrace Violet MFI p-stat1 (x1 3 ) 1 3 stimulation with rmifn-γ d SP ns d SP ns T cells -> RAG -/- WT STAT1 Tg

11 Table S1. Overlapping DEGs by IL-7 and IFN-γ stimulation in WT T cells (Extented Table 2) IL-7 stimulation IFN-γ stimulation Gene ID Log 2 (FC) Adjusted p-value Log 2 (FC) Adjusted p-value CD4 Naive T cells 57358B9Rik E E F6Rik E E E14Rik E E P21Rik E E-2 Abcg E E-6 Acvr1c E E-8 Arid5b E E-8 B4336N3Rik E E-8 Bcl E E-13 Fas E E-8 Gadd45g E E-5 Gata E E-11 Gbp E E-16 Gbp11_ E E-16 Gbp E E-19 Gbp E E-21 Gbp6_ E E-12 Gbp6_ E E-15 Gm E E-22 Gm E E-2 Gm E E-2 Gm E E-11 Gpr E E-6 Gvin E E-1 Ifi E E-15 Igtp E E-27 Iigp E E-23 Il12rb E E-2 Il18rap E E-1 Il2rb E E-11 Irf E E-13 Irf E E-23 Irgm E E-25 Jun E E-11

12 Kat2b E E-9 Kit E E-7 Lrrtm E E-2 Ly6a E E-12 Mgat4a E E-11 Ms4a6d E E-3 Nlrc E E-7 Noc4l E E-13 Pim E E-28 Pmepa E E-16 Pole E E-1 Ppa E E-8 Rbpms E E-3 Rgs E E-2 Ripk E E-17 Samhd E E-12 Slamf E E-13 Socs E E-13 Socs E E-18 Tgtp E E-25 Tgtp E E-28 Tnfrsf1b E E-9 Tnfsf E E-6 Treml E E-14 Zfp E E-4 CD8 Naive T cells F6Rik E E E14Rik E E P21Rik E E-2 Abcb E E-5 B4336N3Rik E E-5 Bcl E E-15 Csf E E-11 Cyp2s E E-3 F2rl E E-6 Fas E E-6 Gadd45g E E-5 Gata E E-12 Gbp E E-15 Gbp11_ E E-15 Gbp E E-16 Gbp E E-16 Gbp6_ E E-11

13 Gbp6_ E E-12 Gem E E-5 Gm E E-21 Gm E E-3 Gm E E-3 Gm E E-6 Gm E E-8 Gpr E E-7 Gpr E E-2 Gvin E E-11 Gzmb E E-4 Ifi E E-13 Igtp E E-27 Iigp E E-22 Il12rb E E-5 Il18rap E E-5 Irf E E-8 Irf E E-19 Irgm E E-21 Irgm E E-24 Jun E E-17 Kit E E-1 Lgals3bp E E-12 Lrrtm E E-3 Ly6a E E-16 Map3k E E-2 Mthfd E E-12 Muc E E-5 Nlrc E E-4 Noc4l E E-1 Parp E E-9 Pim E E-28 Pole E E-9 Ppa E E-9 Rgs E E-2 Ripk E E-12 Rnf E E-2 Rtp E E-1 Samhd E E-12 Sema4a E E-5 Serpina3g E E-9 Slamf E E-11 Socs E E-15

14 Stat E E-1 Syt E E-2 Tgtp E E-21 Tgtp E E-25 Tnfsf E E-5 Treml E E-11 Lists of DEGs, their log 2 (FC) and adjusted p values generated for each condition were compared using Venn diagrams to obtain the list of overlapping genes between IL-7 and IFNγ stimulation in WT T cells. Fold changes (FC) were calculated relative to unstimulated condition for each sample.

15 Table S2. Top 15 IPA-predicted activated upstream regulators in WT T cells stimulated for 9 minutes with IFN-γ Upstream Regulator Activation Z-score p-value of overlap CD4 Naive T cells IFNG E-54 IRF E-5 IFNA E-4 TNF E-21 STAT E-43 IL-1B E-21 IFNB E-44 IL E-36 IRF E-3 IRF E-25 IL E-18 IFNL E-22 OSM E-8 IRF E-21 NFATC E-22 CD8 Naive T cells IFNG E-52 IRF E-45 IFNA E-34 STAT E-4 IFNB E-39 IL-1B E-24 IRF E-26 IL E-31 TNF E-17 IRF E-22 IL E-2 OSM E-1 PRL E-19 NFATC E-21 IRF E-17

16 Lists of differentially expressed gene transcripts (DEGs). their log 2 (FC) and adjusted p values were generated for each condition and used as input in IPA software. Activation Z-score are based on a model that assigns random regulation directions for predicted upstream regulators. Z >2 predicts activation of the upstream regulator. The p-value of overlap was used to rank the significance associated for each Upstream Regulator. The p-value indicates the significance of the overlap between the genes targeted by the upstream regulator in the database and the experimental dataset.

17 Table S3. Target genes predicted by IPA with IFNG as activated upstream regulator in WT stimulated for 9 minutes with IL-7 or IFN-γ IL-7 stimulation IFN-γ stimulation Gene ID Log 2 (FC) Adjusted p- value Log 2 (FC) Adjusted p- value CD4 Naive T cells Bcl E E-13 Fas E E-8 Gbp E E-21 Gbp E E-16 Gvin E E-1 Ifi E E-15 Igtp E E-27 Iigp E E-23 Il12rb E E-2 Il18rap E E-1 Irf E E-13 Irf E E-23 Irgm E E-25 Jun E E-11 Ly6a E E-12 Nlrc E E-7 Pim E E-28 Ripk E E-17 Samhd E E-12 Slamf E E-13 Socs E E-13 Socs E E-18 Tgtp E E-25 Tnfrsf1b E E-9 CD8 Naive T cells Bcl E E-15 Csf E E-11 Fas E E-6 Gbp E E-16 Gbp E E-15 Gpr E E-2 Gvin E E-11 Ifi E E-13

18 Igtp E E-27 Iigp E E-22 Il12rb E E-5 Il18rap E E-5 Irf E E-8 Irf E E-19 Irgm E E-24 Irgm E E-21 Jun E E-17 Lgals3bp E E-12 Ly6a E E-16 Map3k E E-2 Nlrc E E-4 Pim E E-28 Ripk E E-12 Rtp E E-1 Samhd E E-12 Sema4a E E-5 Serpina3g E E-9 Slamf E E-11 Socs E E-15 Stat E E-1 Tgtp E E-21 Lists of DEGs, their log 2 (FC) and adjusted p-values generated for each condition were used as input in IPA software to obtain a list of predicted upstream regulators (Figure 4A and Table S2) and their target genes among the DEGs. Fold changes (FC) were calculated relative to unstimulated condition for each sample.

19 Table S4. Target genes predicted by IPA with STAT1 as activated upstream regulator in WT T cells stimulated for 9 minutes with IL-7 or IFN-γ IL-7 stimulation IFN-γ stimulation Gene ID Log 2 (FC) Adjusted p- value Log 2 (FC) Adjusted p- value CD4 Naive T cells Fas E E-8 Gbp E E-21 Gbp E E-16 Ifi E E-15 Igtp E E-27 Il12rb E E-2 Irf E E-23 Jun E E-11 Samhd E E-12 Socs E E-13 Socs E E-18 Tgtp E E-25 CD8 Naive T cells Fas E E-6 Gbp E E-16 Gbp E E-15 Gzmb E E-4 Ifi E E-13 Igtp E E-27 Il12rb E E-5 Irf E E-19 Irgm E E-21 Jun E E-17 Samhd E E-12 Serpina3g E E-9 Socs E E-15 Stat E E-1 Tgtp E E-21 Lists of DEGs, their log 2 (FC) and adjusted p-values generated for each condition were used as input in IPA software to obtain a list of predicted upstream regulators (Figure 4A and

20 Table S2) and their target genes among the DEGs. Fold changes (FC) were calculated relative to unstimulated condition for each sample.

21 Table S5. Top 15 IPA-predicted activated upstream regulators in STAT1 Tg T cells stimulated for 9 minutes with IL-7 Upstream Regulator Activation Z-score p-value of overlap CD4 Naive T cells IL E-32 IRF E-8 IFNG E-27 IL E-34 IL E-2 CSF E-18 STAT E-14 IL E-17 EPO E-8 STAT5B E-13 IL E-7 STAT E-14 IL E-21 IL E-13 IFNA E-1 CD8 Naive T cells IFNG E-29 IL E-34 STAT E-18 IRF E-13 IL-1B E-19 STAT E-16 IL E-19 IL E-13 PRL E-14 IL E-19 IFNA E-14 IL E-5 IL E-9 IL E-25 IL E-18

22 Lists of differentially expressed gene transcripts (DEGs). their log 2 (FC) and adjusted p values were generated for each condition and used as input in IPA software. Activation Z-score are based on a model that assigns random regulation directions for predicted upstream regulators. Z >2 predicts activation of the upstream regulator. The p-value of overlap was used to rank the significance associated for each Upstream Regulator. The p-value indicates the significance of the overlap between the genes targeted by the upstream regulator in the database and the experimental dataset.

23 Table S6. Top 1 DAVID-predicted enriched Biological processes within DEGs unique to CD4 or CD8 Naive T cells stimulated for 9 minutes with IL-7 WT cells stimulated with IL-7 STAT1 Tg cells stimulated with IL-7 CD4 Naive T cells Description Gene counts p-value Description Gene counts p-value Regulation of programmed cell death E-4 Regulation of programmed cell death E-3 Regulation of cell death E-4 Regulation of cell death E-3 Death E-4 Death E-4 Regulation of apoptosis E-4 Regulation of apoptosis E-3 Apoptosis E-4 Apoptosis E-4 Programmed cell death E-4 Programmed cell death E-4 Cell death E-4 Cell death E-4 Positive regulation of macromolecule metabolic process E-2 Phosphate metabolic process E-2 Intracellular signaling cascade E-2 Phosphorus metabolic process E-2 Regulation of cell proliferation E-2 Cell cycle E-2 CD8 Naive T cells Hemopoietic or lymphoid organ development E-2 Hemopoietic or lymphoid organ development E-2 Immune system development E-2 Immune system development E-2

24 Immune response E-4 Homeostatic process E-2 Regulation of small GTPase mediated signal transduction E-2 Regulation of hydrolase activity E-2 Regulation of Ras protein signal transduction 6 3.E-2 Hemopoiesis E-2 Gland development 6 4.8E-2 Small GTPase mediated signal transduction E-2 Tissue homeostasis E-3 Regulation of GTPase activity 5 5.6E-3 Multicellular organismal homeostasis E-3 Homeostasis of number of cells E-3 Anatomical structure homeostasis E-3 Regulation of cytokine production 5 2.E-2 Lymph node development E-4 Regulation of cytokine secretion 4 1.7E-4 Gene Ontology (GO) term enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.7 ( to assess the top biological processes associated with the lists of DEGs by IL-7 in WT and STAT1 Tg T cells. Lists of DEGs. their log 2 (FC) and adjusted p values generated for WT and STAT1 Tg T cells stimulated for 9 minutes with rmil-7 were used as input in DAVID website. Gene counts indicate the number of enriched DEGs in the pathway. The p-value indicates the perturbation of each pathway.

25 Table S7. Overlapping DEGs by IL-7 and IFN-γ overnight stimulation in WT and STAT1 Tg CD4 Naïve T cells WT cells stimulated with IL-7 STAT1 Tg cells stimulated with IL-7 WT cells stimulated with IFN-γ Gene ID CD4 Naive T cells Log 2 (FC) Adjusted p-value Log 2 (FC) Adjusted p-value Log 2 (FC) Adjusted p-value 619L18Rik E E E B21Rik E E E L1Rik E E E F4Rik E E E I7Rik E E E-5 132E11Rik E E E-8 171K19Rik E E E-3 171I14Rik E E E B3Rik E E E A7Rik E E E G4Rik E E E H14Rik E E E-4 173C12Rik E E E-3 173J22Rik E E E-2 173K9Rik E E E N19Rik E E E B19Rik E E E K1Rik E E E A15Rik E E E B1Rik E E E E14Rik E E E H14Rik E E E J17Rik E E E G2Rik E E E O18Rik E E E I18Rik E E E-9 213C2Rik E E E B3Rik E E E-8

26 2312F9Rik E E E A5Rik E E E I22Rik E E E A7Rik E E E P9Rik E E E B7Rik E E E-4 254C2Rik E E E E7Rik E E E L7Rik E E E P9Rik E E E D17Rik E E E N2Rik E E E I1Rik E E E H2Rik E E E O15Rik E E E C18Rik E E E D19Rik E E E K23Rik E E E B14Rik E E E L24Rik E E E N6Rik E E E I4Rik E E E-5 295J15Rik E E E G21Rik E E E I22Rik E E E F17Rik E E E E18Rik E E E O21Rik E E E M2Rik E E E M4Rik E E E L22Rik E E E C13Rik E E E C21Rik E E E H12Rik E E E E17Rik E E E C13Rik E E E L5Rik E E E M22Rik E E E-3

27 493427A7Rik E E E F8Rik E E E N24Rik E E E D5Rik E E E K13Rik E E E A2Rik E E E L24Rik E E E E9Rik E E E E16Rik E E E H18Rik E E E B21Rik E E E L6Rik E E E E8Rik E E E G24Rik E E E F22Rik E E E M22Rik E E E C7Rik E E E P19Rik E E E O12Rik E E E B17Rik E E E J17Rik E E E D14Rik E E E C1Rik E E E K11Rik E E E P16Rik E E E G16Rik E E E H2Rik E E E B9Rik E E E H23Rik E E E F6Rik E E E J6Rik E E E C11Rik E E E K2Rik E E E J24Rik E E E D23Rik E E E L13Rik E E E C15Rik E E E K14Rik E E E-8

28 933151L19Rik E E E L9Rik E E E E14Rik E E E I1Rik E E E E19Rik E E E-4 A2356P14Rik E E E-4 A3317A19Rik E E E-3 A4346D13Rik E E E-9 A4315I19Rik E E E-6 A5372M11Rik E E E-6 A5383I2Rik E E E-8 A6323P12Rik E E E-8 A935H1Rik E E E-8 A937I19Rik E E E-4 A9324E5Rik E E E-5 Aaed E E E-9 Aar E E E-8 Aars E E E-8 Aasdh E E E-8 Abca E E E-6 Abca E E E-4 Abcb E E E-6 Abcb1a E E E-7 Abcb1b E E E-7 Abcb E E E-4 Abcb E E E-7 Abcd E E E-7 Abcg E E E-9 Abcg E E E-3 Abhd14a E E E-5 Abhd14b E E E-7 Abi E E E-7 Abl E E E-9 Acaa1a E E E-8 Acacb E E E-5 Acad E E E-5 Acadsb E E E-5 Acbd E E E-7

29 Acer E E E-9 Acot E E E-7 Acot E E E-8 Acot E E E-5 Acox E E E-9 Acp E E E-9 Acrbp E E E-7 Acsl E E E-9 Acsl E E E-8 Acsm E E E-3 Acss E E E-9 Actb E E E-8 Actrt E E E-3 Acvr2a E E E-8 Acvr2b E E E-2 Acyp E E E-8 Adam E E E-8 Adam E E E-3 Adamts E E E-6 Adamts E E E-5 Adamtsl E E E-7 Adap E E E-2 Adat E E E-9 Adcy E E E-3 Adcy E E E-9 Add E E E-12 Adgrg E E E-4 Adgrg E E E-7 Adgrg E E E-5 Adgrl E E E-7 Adprh E E E-7 Aebp E E E-9 Aff E E E-8 Aff E E E-8 Ager E E E-1 Agfg E E E-8 Aggf E E E-7 Agpat E E E-6

30 Agrp E E E-4 Ahnak E E E-7 AI E E E-11 AI E E E-4 Aida E E E-1 Aig E E E-7 Aim E E E-1 Aim1l E E E-3 Airn E E E-6 Ak E E E-7 Akap E E E-7 Akr1e E E E-5 Akr7a E E E-5 Aldh1l E E E-6 Alg E E E-6 Alkbh E E E-7 Als2cr E E E-4 Amd E E E-8 Amd E E E-5 Amdhd E E E-6 Amigo E E E-6 Amn E E E-8 Ampd E E E-7 Amt E E E-8 Amy E E E-6 Anapc E E E-9 Angptl E E E-6 Ankrd13c E E E-7 Ankrd13d E E E-7 Ankrd E E E-8 Ankrd E E E-4 Ankzf E E E-9 Anln E E E-4 Anxa E E E-4 Anxa E E E-8 Ap1m E E E-5 Ap1s E E E-6 Ap3s E E E-9

31 Ap5b E E E-8 Apbb E E E-5 Apcdd E E E-4 Apex E E E-9 Aph1b E E E-3 Aplf E E E-6 Apmap E E E-9 Apobec E E E-5 Apobec E E E-9 Apol E E E-6 Apol9a E E E-3 Apol9b E E E-5 Appbp E E E-8 Arc E E E-6 Areg E E E-6 Arf E E E-8 Arhgap E E E-9 Arhgap E E E-9 Arhgap E E E-3 Arhgef E E E-4 Arhgef E E E-4 Arid4b E E E-8 Arid5a E E E-9 Arih E E E-9 Arih E E E-9 Arl E E E-8 Arl E E E-7 Arl4d E E E-4 Arl5b E E E-8 Arl8b E E E-8 Armc E E E-5 Armcx E E E-6 Armcx E E E-8 Armcx E E E-5 Armcx E E E-5 Arntl E E E-2 Arpc1a E E E-9 Arrdc E E E-7

32 Arsk E E E-8 Art2b E E E-11 Art E E E-3 Asah E E E-9 Ascl E E E-4 Asf1a E E E-4 Asgr E E E-4 Aspm E E E-6 Asrgl E E E-9 Atad E E E-8 Atad3aos E E E-4 Atad E E E-9 Atcay E E E-4 Atf E E E-8 Atf E E E-6 Atf E E E-8 Atf E E E-4 Atg E E E-7 Atg E E E-8 Atg2a E E E-8 Atg E E E-8 Atl E E E-8 Atp1a E E E-5 Atp6vd E E E-9 Atp6ve E E E-3 Atp6v1e E E E-9 Atp6v1g E E E-4 Atp6v1h E E E-9 Atxn7l1os E E E-8 AU E E E-8 AU E E E-8 Aurka E E E-3 AV E E E-4 Avil E E E-8 Avl E E E-8 AW E E E-4 Axl E E E-4 Azin E E E-8

33 B1334C11Rik E E E-8 B3galnt E E E-7 B3gnt E E E-5 B3gnt E E E-9 B3gnt E E E-2 B3gnt E E E-4 B43212C6Rik E E E-4 B4galt E E E-9 B6319A1Rik E E E-1 Bace E E E-8 Bach E E E-7 Bach2it E E E-3 Bag E E E-5 Bag E E E-5 Baiap E E E-9 Bambi E E E-5 Banp E E E-6 Bap E E E-8 Basp E E E-6 Batf E E E-6 Bbs E E E-8 BC E E E-7 BC E E E-8 BC E E E-5 BC E E E-2 BC E E E-8 BC E E E-7 BC E E E-1 BC E E E-4 BC E E E-8 Bcar E E E-3 Bcat E E E-4 Bcl E E E-8 Bcl2a1a E E E-3 Bcl2a1b E E E-8 Bcl2l E E E-11 Bcl2l E E E-6 Bcl2l E E E-9

34 Bcl2l E E E-4 Bdkrb E E E-3 BE E E E-1 Bend E E E-9 Bend E E E-6 Bend E E E-3 Bhlha E E E-5 Bhlhe E E E-8 Bhlhe E E E-6 Bin E E E-9 Blm E E E-8 Bloc1s E E E-8 Blvrb E E E-5 Bmp2k E E E-8 Bpgm E E E-6 Bpnt E E E-8 Brat E E E-1 Brca E E E-4 Brms1l E E E-8 Bsdc E E E-9 Btaf E E E-9 Btbd E E E-7 Btg E E E-6 Btg E E E-6 Btg E E E-8 Btnl7-ps E E E-7 Bud E E E-8 Bzw E E E-9 C339L3Rik E E E-7 C1336L24Rik E E E-8 C1346K22Rik E E E-5 C1qtnf E E E-3 C2337L18Rik E E E-8 C2338L3Rik E E E-6 C2362I16Rik E E E-5 C2cd E E E-7 C3318D2Rik E E E-8 C3327C9Rik E E E-5

35 C432N11Rik E E E-2 C E E E-4 C929B18Rik E E E-5 C9225E4Rik E E E-7 Cabp E E E-4 Cacna2d E E E-1 Cacnb E E E-7 Calca E E E-5 Calm E E E-8 Camk2n E E E-5 Camsap E E E-9 Car E E E-5 Card E E E-6 Carhsp E E E-8 Carns E E E-1 Casc E E E-8 Casc E E E-5 Casp E E E-8 Casp E E E-9 Cass E E E-4 Casz E E E-8 Cbx E E E-9 Ccbl E E E-7 Ccdc E E E-8 Ccdc E E E-3 Ccdc E E E-6 Ccdc E E E-8 Ccdc E E E-2 Ccdc E E E-7 Ccdc E E E-4 Ccdc E E E-2 Ccdc E E E-5 Ccdc E E E-6 Ccdc E E E-7 Ccdc E E E-5 Ccdc E E E-5 Ccdc E E E-6 Ccdc E E E-2

36 Ccdc E E E-7 Ccin E E E-2 Ccnb E E E-5 Ccne E E E-9 Ccne E E E-6 Ccnk E E E-9 Ccno E E E-4 Ccnt E E E-8 Ccnyl E E E-7 Ccpg E E E-7 Ccr E E E-5 Ccr E E E-7 Ccr E E E-1 Ccr E E E-6 Ccrl E E E-3 Cd164l E E E-7 Cd2r E E E-8 Cd24a E E E-6 Cd E E E-8 Cd E E E-8 Cd E E E-1 Cd E E E-9 Cd E E E-6 Cd E E E-6 Cd E E E-5 Cd E E E-5 Cd E E E-9 Cdc E E E-6 Cdc25b E E E-1 Cdc E E E-8 Cdc42se E E E-11 Cdc E E E-5 Cdca E E E-6 Cdh E E E-4 Cdh E E E-8 Cdh E E E-7 Cdk11b E E E-9 Cdk E E E-8

37 Cdkn1a E E E-6 Cdkn2aip E E E-7 Cdo E E E-4 Cdpf E E E-8 Cdt E E E-8 Ceacam E E E-2 Cebpa E E E-4 Cecr E E E-4 Celf E E E-6 Cenpc E E E-1 Cenpe E E E-5 Cenpi E E E-4 Cenpl E E E-8 Cenpq E E E-6 Cenpw E E E-4 Cep E E E-8 Cep E E E-5 Cep E E E-4 Cep E E E-7 Cep E E E-1 Cep E E E-7 Cep83os E E E-7 Cep85l E E E-7 Cfap E E E-9 Cfap E E E-9 Chaf1a E E E-5 Chd E E E-8 Chd E E E-8 Chd E E E-8 Chdh E E E-7 Chek E E E-6 Chek E E E-8 Chka E E E-8 Chl E E E-5 Chn E E E-5 Chrm E E E-3 Chrna E E E-4 Chrnb E E E-5

38 Chrnb E E E-7 Chrne E E E-5 Chst E E E-7 Chst E E E-6 Ciart E E E-5 Cir E E E-6 Cirh1a E E E-9 Cited E E E-2 Cited E E E-7 Ckap E E E-8 Ckap2l E E E-5 Ckb E E E-8 Cks E E E-8 Clcf E E E-9 Cldn E E E-2 Cldnd E E E-8 Cltc E E E-9 Cmah E E E-11 Cnbp E E E-8 Cnga E E E-1 Cnksr E E E-9 Cnnm E E E-7 Cnot E E E-8 Cnp E E E-1 Cnst E E E-9 Coa E E E-6 Coasy E E E-7 Cobll E E E-5 Col11a E E E-9 Col23a E E E-6 Colq E E E-4 Cops E E E-7 Cops E E E-9 Coq1b E E E-8 Coq E E E-7 Coq E E E-5 Coq E E E-5 Corin E E E-4