Expression of JunB is upregulated in eTreg cells. a–d Flow cytometry analysis of JunB in Foxp3⁺ (Treg) or Foxp3⁻ (Tconv) cells isolated from spleen a and lung b, Treg cells bearing CD62L^hiCD44^lo phenotypes (cTreg) or CD62L^lo phenotypes (eTreg) c, and ICOS⁺ or ICOS⁻ eTreg cells d isolated from spleen of wild-type C57BL/6 mice (7–10-week-old). Cd4^creJunb^fl/fl mice were used as negative controls. e CD62L^hiCD44^lo cTreg and CD62L^lo eTreg cells were sorted by FACS, and Junb mRNA expression was analyzed by qRT-PCR. a–e Error bars indicate s.d. (n = 4). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). MFI, mean fluorescence intensity. f JunB expression was analyzed by flow cytometry in CD4⁺CD25⁺ Treg cells activated with indicated stimuli for 72 h. Error bars indicate s.d. (n = 3). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). Data represent two independent experiments

Mice deficient for JunB in Treg cells develop autoimmune disease. a Body weight changes of male Foxp3^CreJunb^fl/fl mice and control mice. Error bars indicate s.e.m. (n = 11 for controls, n = 10 for Foxp3^CreJunb^fl/fl mice). b Survival of male Foxp3^CreJunb^fl/fl mice and control mice. Error bars indicate s.d. (n = 11 for controls, n = 22 for Foxp3^CreJunb^fl/fl mice). c Total cell numbers in spleens, superficial cervical lymph nodes (scLN), inguinal LN (iLN), and mesenteric LN (mLN) of male Foxp3^CreJunb^fl/fl mice and control mice (8–12-week-old). Error bars indicate s.d. (n = 5). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). d Hematoxylin and eosin staining of lung, colon, liver, and skin from 12-week-old male Foxp3^CreJunb^fl/fl mice and control mice. Scale bars, 50 μm (lung, colon, and liver) or 100 μm (skin). Bar graph shows histopathological inflammation scores. Error bars indicate s.d. (n = 5). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). e Flow cytometry analysis of CD62L and CD44 in CD4⁺Foxp3⁻ Tconv cells isolated from various tissues of male Foxp3^CreJunb^fl/fl mice and Foxp3^CreJunb^+/+ mice (8–12-week-old). Representative flow cytometry profiles show CD4⁺Foxp3⁻ Tconv cells isolated from the spleen. The graph shows percentages of CD62L^loCD44^hi activated cells among CD4⁺Foxp3⁻ cells. Error bars indicate s.d. (n = 5 for spleens, n = 3 for lymph nodes). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). f Mass cytometry analysis of leukocytes isolated from spleens of Foxp3^CreJunb^fl/fl mice and Foxp3^CreJunb^+/+ mice (8–12-week-old). Mo: monocytes, Mϕ: macrophages, DC: dendritic cells. Size of each node represents cell numbers. Expression levels of JunB is color-coded. g ELISA analysis of immunoglobulin isotypes in sera of 12-week-old male Foxp3^CreJunb^fl/fl mice and Foxp3^CreJunb^+/+ mice. Error bars indicate s.d. (n = 6 for Foxp3^CreJunb^fl/+ mice, n = 9 for Foxp3^CreJunb^fl/fl mice). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). h Flow cytometry analysis of intracellular IL-17A, IFN-γ, IL-4, and IL-13 in CD4⁺Foxp3⁻ cells isolated from spleens of 8–12-week-old male Foxp3^CreJunb^fl/fl mice and Foxp3^CreJunb^+/+ mice. Error bars indicate s.d. (n = 5). *P < 0.05 (unpaired two-tailed Student’s t test). Data represent two independent experiments

JunB is required for accumulation and effector functions of Treg cells. a Flow cytometry analysis of Foxp3 in CD4⁺ T cells in spleens, lungs, and colons of male Foxp3^CreJunb^fl/fl and Foxp3^CreJunb^+/+ mice (8–12-week-old). Graphs show percentages and numbers of CD4⁺Foxp3⁺ Treg cells. Error bars indicate s.d. (n = 5 for Foxp3^CreJunb^+/+ mice, n = 6 for Foxp3^CreJunb^fl/fl mice). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). b, c Flow cytometry analysis of CD44 and CD62L in CD4⁺Foxp3⁺ Treg cells isolated from spleens of male Foxp3^CreJunb^fl/fl and Foxp3^CreJunb^+/+ mice (8–12-week-old). Graph shows percentages of CD62^hiCD44^lo cTreg cells and CD62^lo eTreg cells b, and MFIs of CD44 c. Error bars indicate s.d. (n = 7 for Foxp3^CreJunb^+/+ mice, n = 6 for Foxp3^CreJunb^fl/fl mice). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). d, e Flow cytometry analysis of CTLA4, CD25, and GITR d, and ICOS, TIGIT, KLRG1, and ST2 e in CD62^hiCD44^lo cTreg cells and CD62^lo eTreg cells among CD4⁺Foxp3⁺ Treg cells isolated from spleens of male Foxp3^CreJunb^fl/fl and Foxp3^CreJunb^+/+ mice (8–12-week-old). Representative flow cytometry profiles show CD62^hiCD44^lo cTreg cells and CD62^lo eTreg cells d, and CD62^lo eTreg cells e. Graphs show MFIs of CTLA4, CD25, and GITR d, and percentages of cells expressing indicated molecules e. Error bars indicate s.d. (n = 4). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). f CD4⁺CD25⁺ Treg cells were isolated from Cd4^CreJunb^fl/fl and Junb^fl/fl mice. Treg cells freshly isolated or activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 for 3 days were used for in vitro suppression assay. CD4⁺CD25⁻ Tconv cells (CD45.1) labeled with cell trace violet (CTV) were cultured with different numbers of CD4⁺CD25⁺ Treg cells in the presence of anti-CD3-/anti-CD28-coated beads for 2 days, and CTV dilution was analyzed by flow cytometry. g In vivo suppression assay using CD4⁺CD25⁺ Treg cells isolated from Cd4^CreJunb^fl/fl and Junb^fl/fl mice. In all, 6–8-week-old sex-matched Rag1^−/− mice were injected with wild-type naive CD4⁺ T cells and CD4⁺CD25⁺ Treg cells. The graph shows body weight changes. Colonic histopathology was analyzed on day 40 after injection. Error bars indicate s.e.m. (n = 5). Data represent two independent experiments

JunB regulates expression of eTreg-related molecules. a Flow cytometry analysis of CD4⁺Foxp3⁺ Treg cells in various tissues isolated from Cd4^CreJunb^fl/fl and Junb^fl/fl mice. Graphs show percentages and numbers of CD4⁺Foxp3⁺ Treg cells. Error bars indicate s.d. (n = 4). *P < 0.05 (unpaired two-tailed Student’s t test). b, c Flow cytometry analysis of CD44 and CD62L in CD4⁺Foxp3⁺ Treg cells isolated from spleens of Cd4^CreJunb^fl/fl and Junb^fl/fl mice (8–12-week-old). Graphs show percentages of CD62^hiCD44^lo cTreg and CD62^lo eTreg cells b, and MFIs of CD44 c. Error bars indicate s.d. (n = 4). *P < 0.05 (unpaired two-tailed Student’s t test). d, e Flow cytometry analysis of CTLA4, CD25, and GITR d, and ICOS, TIGIT, KLRG1, and ST2 e in CD62^hiCD44^lo cTreg cells and CD62^lo eTreg cells among CD4⁺Foxp3⁺ Treg cells isolated from spleens of Cd4^CreJunb^fl/fl and Junb^fl/fl mice (8–12-week-old). Representative flow cytometry profiles show CD62^hiCD44^lo cTreg cells and CD62^lo eTreg cells d, and CD62^lo eTreg cells e. Graphs show MFIs of CTLA4, CD25, and GITR d, and percentages of cells expressing indicated molecules e. Error bars indicate s.d. (n = 4). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). f) Flow cytometry analysis of ICOS in Nrp1⁺ and Nrp1⁻ Treg cells isolated from spleens of Cd4^CreJunb^fl/fl and Junb^fl/fl mice (8–12-week-old). The graph shows percentages of ICOS-expressing cells among Nrp1⁺ and Nrp1⁻ Treg cells. Error bars indicate s.d. (n = 4). *P < 0.05 (unpaired two-tailed Student’s t test). g Flow cytometry analysis of ICOS, TIGIT, and KLRG1 in CD4⁺Foxp3⁺ Treg cells isolated from spleens of 1-week-old Cd4^CreJunb^fl/fl and Junb^fl/fl mice. Graphs show percentages of cells expressing indicated molecules among CD4⁺Foxp3⁺ Treg cells. Error bars indicate s.d. (n = 3 for Junb^fl/fl, n = 7 for Cd4^CreJunb^fl/fl). *P < 0.05 (unpaired two-tailed Student’s t test). Data represent two independent experiments

JunB is indispensable for eTreg homeostasis under competitive conditions. a Schematic of generation of bone marrow (BM) chimera mice. b–g Lethally irradiated 6–8-week-old sex-matched Rag1^−/− mice were transferred with BM cells from Cd4^CreJunb^fl/fl (CD45.2) and Junb^fl/fl (CD45.2) mice, in combination with equal numbers of WT BM cells from B6SJL (CD45.1), generating WT: Cd4^CreJunb^fl/fl and WT: Junb^fl/fl BM chimera mice, respectively. Two to three months later, cells were isolated from thymuses, spleens, and lymph nodes, and ratios of CD45.2⁺/CD45.2⁻ in total CD4⁺Foxp3⁺ Treg cells b, CD62L^hiCD44^lo cTreg cells and CD62L^lo eTreg cells among CD4⁺Foxp3⁺ Treg cells c were determined by flow cytometry analysis. Error bars indicate s.d. (n = 6). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). d–g Flow cytometry analysis of ICOS d, TIGIT e, Ki67 f, and Annexin-V g in CD62L^hiCD44^lo cTreg cells and CD62L^lo eTreg cells among CD4⁺Foxp3⁺ Treg cells isolated from spleens of WT: Cd4^CreJunb^fl/fl and WT: Junb^fl/fl BM chimera mice. Graphs show percentages of cells expressing indicated molecules or stained with Annexin-V. Error bars indicate s.d. (n = 4). *P < 0.05; N.S., not significant (unpaired two-tailed Student’s t test). Data represent two independent experiments

JunB diversifies targets for BATF and IRF4 in eTreg cells. a–c RNA-seq analysis of CD4⁺CD25⁺ Treg cells isolated from spleens of Cd4^CreJunb^fl/fl and Junb^fl/fl mice. a Volcano plot comparing gene expression of Treg cells isolated from Cd4^CreJunb^fl/fl mice versus those from Junb^fl/fl mice. b Venn diagram of genes upregulated by JunB (JunB-dependent genes) and BATF (BATF-dependent genes), and genes expressed in ICOS-expressing CD62L^lo eTreg cells. c ChIP-seq analysis of CD4⁺CD25⁺ Treg cells isolated from spleens of Cd4^CreJunb^fl/fl and Junb^fl/fl mice. Cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 for 60 h and used for ChIP-Seq analysis with antibodies against JunB, BATF, and IRF4. The genomic region containing Ctla4 and Icos genes was shown. The red box indicates a region located 28 kb downstream of transcription start site of Ctla4 (Ctla4 TSS + 28 kb) in which the level of IRF4-binding was significantly decreased by JunB deficiency. Arrows indicate the direction of gene transcription. d ChIP-PCR analysis of CD4⁺CD25⁺ Treg cells isolated from spleens of Cd4^CreJunb^fl/fl and Junb^fl/fl mice. Cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 for 72 h and used for ChIP with anti-IRF4 antibody. Immunoprecipitated DNA was analyzed by qPCR using primers to detect the IRF4-binding site located in the Ctla4 TSS + 28 kb region. Error bars indicate s.d. (n = 4). *P < 0.05 (unpaired two-tailed Student’s t test). Data represent two independent experiments