Fig 2: Analyses of CD8+ T cell memory formation. T cell memory population was investigated in WT or NAC1-/- mice after VACV challenge. The mice were sacrificed on day 35. The spleen and LNs were dissected, smashed, and stained before flow cytometry. (A) Memory CD8+ T cells (CD8+ CD44+). (B) VACV-specific memory CD8+ T cells (CD8+ CD44+ B8R+), gating on memory CD8+ T cells. (C) Tissue-resident memory (CD44hiCD69hi CD197low), central memory (CD44hiCD69low CD197hi) and effector memory (CD44hiCD69low CD197low) T cells, gating on VACV-specific memory CD8+ T cells. The plotting data shown are representative of three identical experiments (n = 5).

Fig 3: Comparison of development of viral Ag-specific CD8+ T cells. The VACV-specific CD8+ T cell frequency was monitored in WT or NAC1-/- mice for 35 days after VACV challenge. (A) Mice were challenged with VACV at 2 × 106 PFU/mouse. The spleen and LNs (superficial cervical, axillary, brachial, and inguinal nodes) were dissected, smashed, and stained with CD8 Ab and B8R tetramer. On day 7, day 14, day 21, and day 35, Ag-specific cell frequencies were analyzed by flow cytometry. (B) VACV-uninfected mice were used as a control. The spleen and lymph nodes were also dissected, smashed, and stained with CD8 Ab and B8R tetramer. The plotting data shown are representative of three identical experiments (n = 5).

Fig 4: Defects in glycolysis and oxidative phosphorylation rate of NAC1-/- CD8+ T cells. Cell metabolism was tested using the Agilent Seahorse Assay on day 3 after activation. (A,C) The cells were activated with coated anti-CD3 and soluble anti-CD28 Abs. Then, 2 × 105 cells were plated in each well of seahorse microplate for glycolytic rate testing. The drugs were injected into each well at the time indicated in the figures. The Extra Cellular Acidification Rate (ECAR) was measured as a proxy for glycolytic rate. The ECAR level after ROT/AA injection was compared between WT and NAC1-/- groups. Sample numbers (n = 4), the p-value is 0.0076 (B,D). The activated 2 × 105 T cells were seeded in a microplate before mitochondrial stress testing. The different drugs were added to the well according to the indicated time points. The Oxygen Consumption Rate (OCR) was tested as a proxy for oxidative phosphorylation. The OCR level was then compared after FCCP injection between the two groups. Sample numbers (n = 4), p-value is 0.0054 (**, p < 0.01).

Fig 5: Enhanced CD8+ T cell memory formation in NAC1-/- mice. Quantification for T cell memory population after 35 days. The mice were sacrificed on day 35. The spleen and lymph nodes were dissected and smashed. The total live cell number for each mouse was calculated with trypan blue staining through a Bio-Rad cell counter. T cell surface markers were stained before flow cytometry. (A) The VACV-specific memory CD8+ T cell (CD8+ CD44+ B8R+) number for each mouse had been calculated and compared between WT and NAC1-/- groups. (B) The VACV-specific memory CD8+ T cell (CD8+ CD44+ B8R+) frequency for each mouse was collected and analyzed. The p-value is 0.040. (C) Tissue-resident memory (CD44hiCD69hi CD197low) T cell frequency had been analyzed between WT and NAC1-/- groups. (*, p < 0.05; ns, p > 0.05).