Fig. 3
From: JunB regulates homeostasis and suppressive functions of effector regulatory T cells
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 Foxp3CreJunbfl/fl and Foxp3CreJunb+/+ mice (8–12-week-old). Graphs show percentages and numbers of CD4+Foxp3+ Treg cells. Error bars indicate s.d. (n = 5 for Foxp3CreJunb+/+ mice, n = 6 for Foxp3CreJunbfl/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 Foxp3CreJunbfl/fl and Foxp3CreJunb+/+ mice (8–12-week-old). Graph shows percentages of CD62hiCD44lo cTreg cells and CD62lo eTreg cells b, and MFIs of CD44 c. Error bars indicate s.d. (n = 7 for Foxp3CreJunb+/+ mice, n = 6 for Foxp3CreJunbfl/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 CD62hiCD44lo cTreg cells and CD62lo eTreg cells among CD4+Foxp3+ Treg cells isolated from spleens of male Foxp3CreJunbfl/fl and Foxp3CreJunb+/+ mice (8–12-week-old). Representative flow cytometry profiles show CD62hiCD44lo cTreg cells and CD62lo eTreg cells d, and CD62lo 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 Cd4CreJunbfl/fl and Junbfl/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 Cd4CreJunbfl/fl and Junbfl/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
