PMC

(a) The staining profile of CD62L/CD27 on the cell surface of the WT and Menin KO T_H cells. The cell numbers are indicated in each quadrant. (b) The results of the intracellular FACS analysis of IL-2/IL-6, or IL-2/OPN in the WT and Menin KO T_H cells. The percentages with s.d. of IL-2-, IL-6- and OPN-producing cells in three independent cultures are shown (lower). (c) The ELISA of chemokines in supernatants of the cells in b. (d) The results of the quantitative RT-PCR analysis of Gzm’s and Prf1 in cells in b. The results are presented relative to the expression of Hprt mRNA with the s.d. (e) The results of the intracellular FACS analysis of IL-4 and IFN-γ in cells cultured under IL-2- (top) or neutral-conditions (bottom). The numbers of cells are indicated in each quadrant. (f) The ELISA of cytokines in supernatants of the cells cultured under IL-2 conditions and restimulated with an immobilized anti-TCR-β for 16 h. All experiments were performed with T_H cells cultured under IL-2 conditions for 5 days. At least three independent experiments (a–f) were performed. **P<0.01 (Student’s t-test).

(a) The results of the intracellular FACS analysis of IL-4/IFN-γ and IL-2/IL-6 in the Bach2 KO naive CD4 T cells cultured under IL-2-conditions for 7 days. The percentages with the s.d. of IFN-γ-, IL-2-, IL-4- and IL-6- producing cells in three independent cultures are shown (right). (b) The ELISA of cytokines and chemokines in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h. (c) The results of the quantitative RT-PCR analysis of the Gzm’s and Prf1 of the cells in a. The results are presented relative to the mRNA expression of Hprt with the s.d. (d) SA β-galactosidase (SA β-Gal) staining of T_H cells obtained from Bach2 KO and WT control mice cultured under IL-2-conditions for 10 days (left). The percentages with the s.d. of SA β-Gal-positive cells in three independent cultures are shown (right). (e) Assay of the binding of RelA and RelB to an NF-κB-binding consensus motif in the WT and Bach2 KO T_H cells. The experiment was performed with T_H cells cultured under IL-2 conditions for 5 days. Three independent experiments (a–e) were performed. *P<0.05, **P<0.01 (Student’s t-test).

(a) The results of the quantitative RT-PCR analysis of mRNA encoding Bach2 in the WT and Menin KO naive CD4 T (right) and T_H (left). The results are presented relative to the mRNA expression of Hprt with the s.d. (b) The results of the immunoblot analysis of Bach2 in the nuclear fractions of the WT and Menin KO T_H cells. (c) The results of the intracellular FACS analysis of IL-4/IFN-γ and IL-2/IL-6 in the Menin KO T_H cells transduced with an empty vector (Mock) or with Bach2-expressing retroviral vector (Bach2). The naive CD4 T cells cultured under IL-2-conditions were infected with retroviral vectors and cultured for 3 days in the presence of IL-2. (d) The mock- or Bach2-transduced cells in c were purified using an AutoMACS device and restimulated with immobilized anti-TCR-β for 16 h. The amounts of cytokines in the supernatants were determined using ELISA. (e) The amounts of pro-inflammatory chemokines in the supernatants in d were determined using ELISA. (f) The results of the quantitative RT-PCR analysis of the Gzm’s and Prf1 of cells in d. The results are presented relative to the mRNA expression of Hprt with the s.d. All experiments were performed with T_H cells cultured under IL-2 conditions for 5 days. Three independent experiments (a–f) were performed. **P<0.01 (Student’s t-test).

(a) The global patterns of histones, H3K4me3 (upper), H3K27me3 (middle) and H3K27ac (lower) at the Bach2 locus in the WT and Menin KO T_H cells cultured under IL-2 conditions for 5 days were determined using ChIP-seq. (b) The results of the ChIP assay with a quantitative PCR analysis of the histone modification (H3K4me3, H3K27me3 and H3K27ac) status around the Tss of the Bach2 locus in the WT and Menin KO T_H cells. The relative intensity (input) with the s.d. is shown. (c) ChIP with a quantitative PCR analysis of the binding of HMT2A/B (MLL1, Rbbp5 and Wdr5) and PRC2 (Ezh2 and Suz12) components to the Bach2 Tss region in the Menin KO T_H cells. (d) The results of the quantitative RT-PCR analysis of Bach2 mRNA expression in the Menin KO T_H cells treated with TSA for 24 h. The results are presented relative to the mRNA expression of Hprt mRNA with the s.d. (e) The ChIP data with the results of the quantitative PCR analysis of PCAF binding to the Bach2 Tss region. The relative intensity (/input) with the s.d. is shown. (f) The association between Menin and PCAF in the T_H cells. The nuclear lysates (5 × 10⁶ cells) were precipitated with anti-PCAF mAb or anti-MLL1 pAb, and immunoblotted with anti-Menin pAb. Two (a) and three (b–f) independent experiments were performed. **P<0.01 (Student’s t-test). All experiments were performed with T_H cells cultured under IL-2 conditions for 5 days.

(a) A quantitative RT-PCR analysis of the Bach2 and Menin mRNA expression in senescent effector CD4 T (sT_H) cells. The results are presented relative to the expression of Hprt mRNA with the s.d. (b) An immunoblot analysis of Bach2 and Menin in the nuclear fractions of the primary effector CD4 T (pT_H) and sT_H cells. The α-tubulin proteins in the cytosolic fractions were blotted as a loading control. (c) The global patterns of Menin binding at the Bach2 gene locus in the pT_H and sT_H cells were determined using ChIP sequencing. (d) The global patterns of histones H3K4me3 and H3K27ac at the Bach2 gene locus in the pT_H and sT_H cells were determined using ChIP sequencing. (e) ChIP with quantitative PCR analysis of Menin, RNA polymerase II, KMT2A/2B components and Suz12 in the pT_H and sT_H cells. The relative intensity (input) is shown with the s.d. (f) A quantitative RT-PCR analysis of the Bach2 mRNA expression in the sT_H cells treated with TSA for 24 h. The results are presented relative to the expression of Hprt mRNA with the s.d. (g) A decreased Bach2 protein expression in the elderly T_H cells. Bach2 protein expression in young (6 weeks old) and elderly (30 week old) was determined by immunoblotting. Three (a,b,e,f and g) and two (c,d) independent experiments were performed with similar results. **P<0.01 (Student’s t-test).

(a) The TCR-induced proliferation of the Menin KO naive CD4 T cells in the presence or absence of IL-2. The average of three independent cultures and s.d. are shown. (b) The results of the cell cycle analysis of CD4 T cells from the Menin KO and WT mice cultured under IL-2 conditions for 7 days. The percentages of G0/G1- and S-phase cells in three independent cultures are shown (right). (c) The results of the quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of mRNA encoding Cdk inhibitors (vertical axes) in the WT and Menin KO T_H cells. The results represent the expression levels relative to Hprt with the s.d. (d) The results of the cell death analysis of CD4 T cells from the WT and Menin KO mice on day 7. The percentages of annexin V-positive cells in three independent cultures are shown (right). (e) The results of the quantitative RT-PCR analysis of mRNAs encoding pro-apoptotic proteins (vertical axes) in the WT and Menin KO T_H cells, with the s.d. The results are presented relative to the mRNA expression of Hprt with the s.d. (f) SA β-galactosidase (SA β-Gal) staining of the T_H cells from the Menin KO and WT control mice cultured under IL-2-conditions for 7 days (left). The percentages of SA β-Gal-positive cells in three independent cultures with the s.d. are shown (right). (g) The phosphorylation of RelA (Ser534) in the WT and Menin KO T_H cells cultured for 7 days. (h) Assay of the binding of RelA and RelB to an NF-κB-binding consensus motif in the WT and Menin KO T_H cells cultured under IL-2 conditions for 7 days. Four (panel c and e) and three (panels a,b,d,f,g and h) independent experiments were performed. *P<0.01, **P<0.01 (Student’s t-test).

(a) CD4 T cells from the spleens of the WT or Menin KO mice with a DO11.10 TG background were transferred into syngenic BALB/c mice, and the mice were then immunized with OVA (100 μg) plus LPS (10 μg) (n=5). The transferred OVA-TCR (KJ-1)-positive cells were monitored using FACS on the indicated days. The percentages (upper) and numbers (lower) of KJ1-positive cells are indicated (right). (b) The WT (CD45.1) and Menin KO (CD45.2) T_H cells were mixed 1:1 and transferred into TCR-βδ KO mice (n=5). Representative staining patterns gated on TCR-β-positive cells (upper) and the percentage (lower) of CD45.1 and CD45.2 cells 4 weeks after transfer are indicated. (c) Quantification of eosinophils (Eos), neutrophils (Neu), lymphocytes (Lym), macrophages (Mac) and total cells in the bronchioalveolar (BAL)-fluid from the WT and Menin KO mice (n=5 per group) that were not immunized (ctrl.) or immunized with OVA plus alum (imm.). (d) The microscopic findings of the lungs from mice as in c, fixed and stained with haematoxylin and eosin. (e) The effects of Menin deficiency on L. monocytogenes (LM) infection. The bacterial burden in the spleen was determined 3 days after the primary (n=6) and secondary (n=8) infections, respectively. The data represent the findings of three (a,b) or two (c,d and e) independent experiments. *P<0.05, **P<0.01 (Student’s t-test).