c-Myc-induced transcription factor AP4 is required for host protection mediated by CD8+ T cells

Abstract

Although the transcription factor c-Myc is essential for the establishment of a metabolically active and proliferative state in T cells after priming, its expression is transient. It remains unknown how T cell activation is maintained after c-Myc expression is downregulated. Here we identified AP4 as the transcription factor that was induced by c-Myc and sustained activation of antigen-specific CD8+ T cells. Despite normal priming, AP4-deficient CD8+ T cells failed to continue transcription of a broad range of c-Myc-dependent targets. Mice lacking AP4 specifically in CD8+ T cells showed enhanced susceptibility to infection with West Nile virus. Genome-wide analysis suggested that many activation-induced genes encoding molecules involved in metabolism were shared targets of c-Myc and AP4. Thus, AP4 maintains c-Myc-initiated cellular activation programs in CD8+ T cells to control microbial infection.

Figure 1

AP4 is post-transcriptionally regulated in CD8⁺ T cells. (a) Mean microarray signal intensity for TFs differentially expressed (>1.8-fold) in activated CD8⁺ T cells treated with IL-2 (100 U/ml) or anti-IL-2 antibody (2 μg/ml) for 12 h. (n =3). (b) qRT-PCR analysis showing expression levels of indicated genes in CD8⁺ T cells treated as in (a). Error bars, s.d. (n = 2). (c) Immunoblot showing AP4 expression in CD8⁺ T cells treated as in (a). Phosphorylated STAT5 and β-tubulin serve as controls. (n = 4). (d) Immunoblot showing AP4 expression in IL-2-treated or IL-2-deprived Tfap4^−/− CD8⁺ T cells transduced with an empty (EV) or AP4-expressing RV. (n = 2). (e) Immunoblot analysis of AP4 in IL-2-treated or IL-2-deprived CD8⁺ T cells in the presence of cycloheximide (CHX, 10 μM) for indicated time. (n = 2). (f) Immunoblot analysis of AP4 in IL-2-deprived CD8⁺ T cells in the presence of MG-132 (10 μM) for indicated time. (n = 2). (g) Immunoblot analysis of AP4 in CD8⁺ T cells stimulated with different concentrations of anti-CD3 antibody for 12 h (left, n = 2) or with indicated cytokines for 12 h after 2 day stimulation with anti-CD3 antibody (right, n = 2). (h) Immunoblot analysis of AP4 in CD8⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 48 h followed by treatments with indicated chemicals for 6 h (left, n = 2) and in those treated with chemicals after IL-2 stimulation for 24 h following anti-CD3 and anti-CD28 antibody stimulation (right, n = 2). Wort: Wortmannin. (i) Immunoblot analysis of AP4, Blimp-1 and T-bet in adoptively transferred P14 T cells at indicated time points after LCMV-Arm infection. (j) Histogram showing CD25 expression in CD8⁺ CD44⁺ CD62L⁻ T cells in B6 mice on day 4.5 after LCMV-Arm infection (left), immunoblots showing AP4, Blimp-1 and T-bet expression (middle), and qRT-PCR analysis showing Tfap4 expression (right) in CD25^Lo and CD25^Hi cells. (n =2). Error bars, s.d. (k) Immunoblots showing AP4 expression in Il2ra^−/− P14 T cells on day 4 post LCMV-Arm infection. (n = 2). * P < 0.05 by unpaired t-test.

Figure 2. AP4 is required for expansion of Ag-specific CD8⁺ T cells following LCMV-Arm infection. (a,b) Flow cytometry of splenocytes from Tfap4^−/− and WT mice 8 days after LCMV infection showing CD4, CD8, KLRG1 and CD62L expression and binding of an H-2D^b–gp(33–41) tetramer. Percentages of CD8⁺ T cells, CD8⁺ KLRG1⁺ effector cells, and gp(33–41)-specific CD8⁺ T cells of the total splenocytes (top panels) and percentages of CD62L⁺ and KLRG1⁺ cells in gp(33–41)-specific CD8⁺ T cells (bottom panels) are shown with gates indicated as rectangles. Statistical analysis of data from ten WT and eight Tfap4^−/− mice from three experiments is shown in (c) and (d). (e) qRT-PCR analysis of the LCMV GP transcript in spleen, kidney, and liver of Tfap4^F/F CD8-Cre⁺ and control Tfap4^F/F Cre⁻ mice 6 days after LCMV-Arm infection. Transcript levels were normalized against Hprt1 levels. Data from five mice in two experiments were combined. Dotted lines indicate the limit of detection. Error bars, s.d. * P < 0.0001 by unpaired t-test. NS: not significant.

Figure 3

AP4 is required for sustained CD8 clonal expansion but not for initial proliferation. (a) Flow cytometry of Tfap4^−/− (Thy-1.1⁻ CD45.2) and control WT (Thy-1.1⁺ CD45.2) P14 T cells 16 h after co-transfer at a 1:1 ratio into congenic host mice showing expression of Thy-1.1, CD45.2, CD62L and CD44. (n = 3). (b) Frequencies of CD8⁺ T cells in the spleen, mesenteric lymph nodes (MLN), lung, liver, and kidney of Tfap4^−/− and control B6 mice under steady-state conditions. (n = 5). (c) Flow cytometry of CFSE-labeled Tfap4^−/− and WT P14 T cells in host mice three days after LCMV-Arm infection. (n = 2). (d) Flow cytometry showing the frequencies of Tfap4^−/− and WT P14 donor cells at different time points after LCMV-Arm infection in host mice with a starting ratio of 1:1. (e-g) Statistical analysis of numbers of Tfap4^−/− and WT P14 cells in the spleen normalized to 10,000 transferred cells (e), ratios between Tfap4^−/− and WT P14 T cells (f), and percentages of BrdU⁺ cells following two hour pulse labeling (g). (n = 9-13 for day 3, 8-18 for day 4, 8-13 for day 5, 9-19 for day 6, 4-8 for day 7, 4-9 day 8, 3 for day 10, 5 for day 12). (h,i) Flow cytometry of transferred Tfap4^−/− and WT P14 cells and host CD8⁺CD44⁺ cells in the spleen six days after LCMV-Arm infection showing AnnexinV (AnnV) binding. Representative plots (h) and statistical analysis from five mice (i) are shown. (j) Relative frequencies of Tfap4^−/− and WT P14 donor cells in different organs and blood in host mice seven days after LCMV-Arm infection with a starting ratio of 6:1. (n = 8). Error bars, s.d. * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired t-test. NS: not significant by unpaired t-test (b, e, g) and by one-way ANOVA (j).

Figure 4

AP4 is essential for host protection against WNV infection in a CD8⁺ T cell-intrinsic manner. (a) Survival of Tfap4^F/F CD8-Cre⁺ and control Tfap4^F/F Cre⁻ mice following WNV infection. Data from three independent experiments were pooled (n = 22 for CD8-Cre⁺, n = 16 for Cre⁻). * P < 0.001 by the log-rank test. (b,c) Viral titers in the brain (b) and spleen (c) from Tfap4^F/F CD8-Cre⁺ and control Tfap4^F/F Cre⁻ mice on day 9 post-WNV infection. Data were pooled from four (b, n = 18) or two (c, n = 7) independent experiments. Red bars in (b) indicate median values of PFU/gram tissue. ** P < 0.05, NS: not significant by Mann Whitney U-test.

Figure 5

AP4 is induced by c-Myc and sustains glycolysis. (a) Immunoblot and qRT-PCR analysis showing AP4 and c-Myc expression in CD8⁺ T cells following a 4OHT treatment and stimulation for 10 or 24 h in vitro. Myc^+/+: Myc^F/+ Cre⁻, Myc^+/−: Myc^F/+ CreER^T2(+), Myc^−/−: Myc^F/F CreER^T2(+), N: 4OHT-treated WT naive CD8⁺ T cells. (n = 3). (b,c) Flow cytometry of OT-I T cells showing FSC after Lm-Ova infection (b) with statistical analysis (c). (n = 8 for day 4, 4 for day 6). (d) Flow cytometry analysis of c-Myc-GFP and AP4 expression in OT-I T cells following Lm-Ova infection. c-Myc-GFP levels were determined by subtracting the autofluorescence from the GFP fluorescence of Myc^c-Myc-GFP/+ OT-I T cells. FSC of WT OT-I T cells is shown as a reference. (n = 1 for day 0, 3 for days 1 to 3, 2 for days 4 and 5). (e) Immunoblot showing c-Myc and AP4 expression in OT-I T cells after Lm-Ova infection. Lysates from Tfap4^−/− CD8⁺ T cells were used to show antibody specificity. (n = 3). (f) Total RNA content in OT-I T cells four days after Lm-Ova infection. (n = 5 for WT, 4 for Tfap4^−/−). (g) ECAR measurement of CD8⁺ T cells without stimulation (naive), after 24 h stimulation in vitro, or on days 4 and 6 after Lm-Ova infection. Baseline values (UT) and changes following treatment with oligomycin are shown. (n = 6 for naive, 10 for 24 h, 3 for days 4 and 6). (h) qRT-PCR analysis of expression of glycolytic genes in OT-I T cells. Expression levels were normalized against the ERCC-00108 spiked-in control RNA and shown as relative values to WT OT-I T cells (n = 2 for day 4 WT and day 6 Tfap4^−/−, 3 for day 4 Tfap4^−/− and day 6 WT). Error bars, s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, NS: not significant by one-way ANOVA (a), paired (c) or unpaired t-test (f-h).

Figure 6

AP4 is essential for sustained expression of c-Myc target genes. (a, b) Scatter plots showing normalized signal intensity (NSI) of endogenous transcripts in Tfap4^−/− (KO) and WT OT-I T cells on day 4 after Lm-Ova infection (upper panels) and ERCC control RNA (bottom panels). Values of slopes obtained by linear regression (95% CI in parentheses) are shown. The slope of the blue dotted line in each plot is 1. Error bars, 95% CI in (b). (c) Heat maps showing expression of genes in over-represented pathways identified by NIH DAVID ver. 6.7 (n = 2). Genes shown in red are bound by both c-Myc and AP4. (d) Venn diagram showing overlap between AP4-bound genes, c-Myc-bound genes and differentially expressed genes. (e) Expression kinetics of the 479 genes defined in (d) in WT OT-I T cells after Lm-Ova infection. Expression levels were normalized to values in naive CD8⁺ T cells. (n = 2 for naive, days 4 and 6, 3 for day 2). (f) Flow cytometry of Myc^c-Myc-GFP P14 T cells showing c-Myc-GFP expression after LCMV-Arm infection. (n = 4). (g) ChIP-qPCR analysis of AP4 binding to AP4-c-Myc co-targeted genes in P14 T cells on day 5 after LCMV-Arm infection. Data normalized to signals from Tfap4^−/− cells. Error bars, s.d. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significant by unpaired t-test.

Figure 7

Sustained c-Myc expression rescues defects of Tfap4^−/− CD8 T cells. (a) Flow cytometry of Tfap4^−/− OT-I T cells transduced with empty (EV), AP4- or c-Myc(T58A)-expressing RV showing FSC. Co-cultured EV-transduced WT OT-I T cells were used as control. Statistical analysis of FSC ratios between RV-transduced Tfap4^−/− and EV-transduced WT OT-I T cells are shown in (b). (n = 5). (c,d) ECAR (c) and BrdU incorporation rates (d) of RV-transduced Tfap4^−/− and EV-transduced control WT OT-I T cells. (n = 3). (e) qRT-PCR analysis of expression of AP4-c-Myc co-target genes in RV-transduced Tfap4^−/− and EV-transduced WT OT-I T cells. Expression of each gene was normalized against the control ERCC-108 spiked-in RNA and shown as a heat map. (n = 2). (f) Flow cytometry showing frequencies of and expression of Thy-1.1, CD44, KLRG1 and CD62L in adoptively co-transferred RV-transduced Tfap4^−/− (Thy1.1⁻) and EV-transduced WT (Thy-1.1⁺) OT-I T cells on day 6 after Lm-Ova infection. (n = 8). (g-j) Statistical analyses of relative frequencies of RV-transduced Tfap4^−/− to EV-transduced WT OT-I T cells (KO/WT, g) and percentages of KLRG1⁺ cells (h), BrdU incorporation rates (i), and FSC ratios between RV-transduced Tfap4^−/− and co-transferred EV-transduced WT OT-I T cells (j). (n = 8 for g, h, j, 4 for i). Error bars, s.d. * P < 0.05, * P < 0.01, *** P < 0.001 by one-way ANOVA.