Fig 2: The glutamine-a-KG axis is involved in the dysfunction in menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were activated and cultured for 3 days under normal (Ctrl), glutamine-deprived (dGln), or glutamine-deprived supplemented with DM-a-KG (dGln/a-KG) conditions for 3 days, and then the cells were further expanded with IL-2 under normal conditions for an additional 4 days. An analysis was performed on day 7 after the initial anti-TCR-ß/CD28 stimulation. b Representative staining profiles of CD226 and PD-1 on the cell surface of the cells in a. c The ELISA results for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-ß for 16 h are shown with standard deviation (n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a. The results are presented relative to the expression of Cd3e mRNA with the standard deviations (n = 3: technical replicates). e The percentages of SA ß-galactosidase (SA ß-Gal)-positive cells on day 12 after the initial anti-TCR-ß/CD28 mAb stimulation are shown with the standard deviation (n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1+)/menin KO OT-1 Tg effector CD8 T cells under normal conditions (Thy1.2+) or WT (Thy1.1+)/menin KO under glutamine-deprived conditions (Thy1.2+) was adoptively transferred into WT congenic (Thy1.1+ Thy1.2+) mice. Twenty days after the transfer, the mice were infected with Lm-OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm-OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). *p < 0.05, **p < 0.01 (Student’s t-test)

Fig 3: Demethylation of histone H3K27 is involved in CD8 T-cell dysfunction. a The result of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT CD8 T cells cultured under the indicated conditions for 48 h. The protein amount of histone H3 was used as a loading control. b The results of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT or menin KO CD8 T cells cultured under normal conditions for 3 days. c A representative staining profile of CD62L/CD27 on the cell surface of the WT, utx KO menin KO or menin/utx-double KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. d Representative staining profile of PD-1 on the cell surface of the cells in c. e The ELISA results for IL-6, IL-10, OPN, and IFN-? in the supernatants of the cells in c restimulated with immobilized anti-TCR-ß for 16 h are shown with the standard deviation (n = 3: biological replicates). f The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes of the cells in c. The results are presented relative to the expression of Cd3e mRNA with the standard deviations (n = 3: technical replicates). g The percentages of SA ß-galactosidase (SA ß-Gal)-positive cells on day 12 after the initial anti-TCR-ß/CD28 stimulation are shown with the standard deviation (n = 3: biological replicates). *p < 0.05, **p < 0.01 (Student’s t-test)

Fig 4: Rapamycin inhibits dysfunction of menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were stimulated with anti-TCR-ß mAb plus anti-CD28 mAb with IL-2 in the presence or absence of rapamycin for 2 days, and then the cells were further expanded with IL-2 in the absence of rapamycin for an additional 5 days. b Representative staining profiles of CD226 and PD-1 on the surface of the cells in a. c The ELISA for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-ß for 16 h are shown with the standard deviation (n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a. The results are presented relative to the expression of Cd3emRNA with the standard deviations (n = 3: technical replicates). e The percentages of SA ß-galactosidase (SA ß-Gal)-positive cells on day 12 are shown with the standard deviation (n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1+)/menin KO OT-1 Tg effector CD8 T cells (Thy1.2+) or WT (Thy1.1+)/rapamycin-treated menin KO (Thy1.2+) was adoptively transferred into WT congenic (Thy1.1+ Thy1.2+) mice. Twenty days after the transfer, the mice were infected with Lm-OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm-OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen was indicated (mean ± SD, n = 4 per group: biological replicates). g WT (Thy1.1+ or Thy1.2+), menin KO or rapamycin-treated menin KO OT-1 Tg memory CD8 T cells (Thy1.2+) were mixed and transferred into WT congenic mice (Thy1.1+ Thy1.2+) as in f. The mice were infected with Lm-OVA the next day and analyzed as in f. The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). *p < 0.05, **p < 0.01 (Student’s t-test)

Fig 5: CD153 is essential for functional SAT cell induction and TLT formation.Analysis of aged CD153-deficient (CD153–/–) mouse kidneys subjected to ischemic reperfusion injury (IRI). (A) Periodic acid–Schiff staining and (B) Cd4, Cd19, Ifng, Cxcl13, Ccl19, and Il21 mRNA levels (n = 4/group) in WT and CD153–/– mice 45 days after IRI. (C) Cumulative TLT sizes per cortex, and the number of (D) total and (E) advanced-stage TLTs following IRI induction (n = 4/group). (F) OPN production by CD4+ T cells from aged kidneys of WT and CD153–/– mice 45 days after IRI (n = 4/group). Cells were stimulated in bulk culture with PMA and ionomycin. (G) Representative FACS plots and frequencies of ABCs and germinal center B (GCB) cells in WT and CD153–/– mice (n = 3–4/group). (H) Scatter plot comparing normalized RNA-Seq read counts of ABCs from WT and CD153–/– mouse kidneys 45 days after IRI (n = 3/group). Colored dots indicate transcripts with FDR < 0.01. Red and blue dots indicate genes that are significantly upregulated and downregulated, respectively. (I) Experimental protocol of bone marrow transplantation experiments. Bone marrow cells from aged WT and CD153–/– mice were transplanted into lethally irradiated, aged Rag2-deficient (Rag2–/–) mice. After a 6-week recovery period, mice were subjected to IRI and sacrificed on day 45. (J) Immunofluorescence of CD3e and B220; and CD21, Ki67, and DAPI. (K) Cumulative TLT sizes per cortex and the number of total and advanced-stage TLTs per section (n = 3/group). The arrowheads in A and arrows in J indicate the localization of TLTs. Scale bars: 300 µm (A) and 50 µm (J). Data are presented as mean ± SD, and statistical significance was determined by an unpaired, 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.