Fig 2: Preparation of Vaccinia virus Western Reserve strain (VACV-WR) stock and defects in the survival of NAC1-/- CD8+ T cells. (A) Confluent plates of HeLa cells were infected at an optimal multiplicity of infection (MOI) of 2 PFU/cell. Cellular morphology was monitored during the first two days post-infection. (B) When the HeLa cells reached 100% confluence, the virus stock was serially diluted and added to each 6-well. After two days of incubation, the plaque numbers were counted after crystal violet staining. The viral plaque number was reliable at the dilution factor 107. (C) CD8+ T cells were isolated from WT and NAC1-/- mice, then further stained with CFSE and cultured for 2 days before flow-cytometric analysis. (D) Isolated T cells were seeded into 48-well plates and split at confluence. The live cell number was counted with trypan blue staining. Repeat n = 4. The p-values in the figure are 0.0043 (day 3), 1.5 × 10-5 (day 4), and 1.4 × 10-6 (day 5). (**, p < 0.01; ***, p < 0.001).

Fig 3: Sustained VACV-specific CD8+ T cell survival in NAC1-/- mice. The VACV-specific CD8+ T cell number and cell frequency changed within 35 days post-infection. (A) Mice were challenged with VACV at 2 × 106 PFU/mouse. The spleen and lymph nodes (superficial cervical, axillary, brachial, and inguinal nodes) were dissected and smashed. The total live cell number for each mouse was calculated with trypan blue staining using a Bio-Rad cell counter. T cells were stained with CD8 Ab and B8R tetramer and analyzed by flow cytometry. Total Ag-specific CD8+ T cells were calculated for each mouse (n = 5). The p-values are 0.00038 (day7), 8.2 × 10-5 (day 14), and 0.0066 (day 21). (B) The Ag-specific CD8+ T cell frequency was monitored for 35 days. P-values are 0.011 (day 7), and 0.016 (day 21). (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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: Regulation of IRF4 in CD8+ T cells by NAC1. CD8+ T cells were isolated from WT or NAC1-/- mice and analyzed for expression of IRF4. (A) Protein expression by Western blot. For the day 0 sample, T cells were not activated. For the day 3 sample, T cells were activated and cultured for 3 days. (B) mRNA expression by Q-PCR. CD8+ T cells had been cultured for 3 days, and then RNA was extracted, and qPCR was performed (ns, p > 0.05). (C) CHIP-seq analysis. WT CD8+ T cells were isolated from mice and CHIP was performed with anti-NAC1 and anti-IgG Abs (Input control). The sequenced data was visualized by IGV.