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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.

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Figure 1: AP4 is regulated post-transcriptionally in CD8+ T cells.
Figure 2: AP4 is required for the population expansion of antigen-specific CD8+ T cells following infection with LCMV-Arm.
Figure 3: AP4 is required for the sustained clonal expansion of CD8+ T cells but not for their initial proliferation.
Figure 4: AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.
Figure 5: AP4 is induced by c-Myc and sustains glycolysis.
Figure 6: AP4 is essential for the sustained expression of genes that are targets of c-Myc.
Figure 7: Sustained c-Myc expression 'rescues' defects of Tfap4−/− CD8+ T cells.

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Acknowledgements

We thank M.J. Bevan (University of Washington, Seattle) for LM-OVA; I. Taniuchi (RIKEN, Yokohama) and D.R. Littman (New York University) for CD8-Cre mice; B.P. Sleckman (Washington University School of Medicine) for c-Myc–GFP knock-in mice; F.W. Alt (Harvard Medical School) and D.R. Green (St. Jude Children's Research Hospital) for MycF mice; T. Hansen (Washington University School of Medicine) for the H-2Kb–OVA single-chain tetramer; S. Hsiung, J. Govero and M.J. Sunshine for technical assistance; and E. Oltz, C.-S. Hsieh and D.R. Littman for discussions and critical reading of the manuscript. Supported by the Lucille P. Markey Pathway Program (C.C.), the Burroughs Wellcome Fund (B.T.E.), the McDonnell International Scholars Academy at Washington University (C.-C.L.), the US National Institutes of Health (P30 AR048335 to the Rheumatic Diseases Core Center; R01 AI097244 to T.E.; and U54 AI081680 to M.S.D.), the Edward Mallinckrodt Jr. Foundation (T.E.) and the Washington University Institute of Clinical and Translational Sciences of the National Center for Advancing Translational Sciences of the US National Institutes of Health (UL1 TR000448).

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Authors

Contributions

T.E. designed the study with help from M.Ce., P.M.A., M.Co., E.L.P. and M.S.D.; C.C., A.K.P., J.D.C., S.P.P., C.-C.L., B.T.E. and T.E. did experiments; C.C., E.L.P., M.S.D. and T.E. analyzed data; and T.E. wrote the manuscript with editorial comments from the other authors.

Corresponding author

Correspondence to Takeshi Egawa.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 AP4 is required in a cell-autonomous way for the population expansion of CD8+ T cells.

(a) A gene-targeting strategy shown with representative restriction enzyme sites used for screening by Southern blot with a 5’ external probe (gray square). LoxP sites were inserted to flank exons 2 to 4 of Tfap4 in ES cells. The loxP-neo cassette was deleted in vitro by transient Cre transfection in ES cells prior to blastocyst injection. (b) Quantitative RT-PCR analysis of RNA of CD4+ and CD8+ T cells purified from Tfap4F/F Cre or Tfap4F/F CD8-Cre+ mice showing the abundance of the transcript encoding Tfap4. Error bars, s.d. (n = 3) (c,d) Flow cytometry analysis showing expression of CD4, CD8α, KLRG1, CD62L and H-2Db–gp(33–41)-specific TCR in peripheral blood mononuclear cells (PBMC) from Tfap4–/– (n = 7), Tfap4F/F CD8-Cre+ (n = 7) and control C57BL/6 mice (n = 15) on day 8 after LCMV-Arm infection. Statistical analysis is shown in (d). Each symbol in d represents an individual mouse; small horizontal lines indicate the mean (± s.d.). * P < 0.05, ** P < 0.01, *** P < 0.001, NS: not significant by unpaired t-test (b) or one-way ANOVA (d). Data pooled from three experiments.

Supplementary Figure 2 AP4 is required for the population expansion of antigen-specific CD8+ T cells in response to infection with LM-OVA.

(a) Flow cytometry analysis showing expression of CD8α, KLRG1, CD62L and H2-Kb–OVA-specific TCR in splenocytes of Tfap4–/– and age-matched C57BL/6 mice on day 7 after LM-OVA infection. OVA-specific CD8+ T cells, and KLRG1+ CD62L effectors and KLRG1 CD62L+ memory precursors are shown with rectangular gates with percentages. (b) Statistical analysis of total, KLRG1+ and CD62L+ H2-Kb–OVA-specific CD8+ T cells. (n = 6 for C57BL/6, 8 for Tfap4–/– mice in two independent experiments). (c) Statistical analysis of ratios between Tfap4–/– and Tfap4+/+ OT-I T cells. (n = 15 for day 3, 18 for day 4, 6 for day 5, 8 for day 6, 8 for day 7). (d) Statistical analysis of percentages of BrdU+ OT-I T cells following 2 h pulse labeling. (n = 9 for day 3, 5 for day 4, 7 for day 5, 6 for day 6, 8 for day 7). (e) Flow cytometry analysis of OT-I T cells showing levels of phosphorylated serine 235/236 of S6 ribosomal protein (p-S6) on day 4 after LM-OVA infection shown as histogram overlays. (n = 5). (f) Statistical analysis of ratios of Tfap4–/– and Tfap4+/+ OT-I T cells co-cultured with OVA peptide-pulsed irradiated splenocytes. Percentages of Tfap4–/– OT-I cells in total OT-I T cells are shown. (g) Flow cytometry analysis of Tfap4–/– and Tfap4+/+ OT-I T cells showing FSC after co-culture in the presence of irradiated splenocytes pulsed with 0.1 nM of OVA peptide. (n = 3). (h) Statistical analysis of percentages of IFN-γ-producing Tfap4–/– and Tfap4+/+ OT-I cells that had been initially activated by anti-CD3 and anti-CD28 and re-stimulated with OVA-peptide pulsed irradiated splenocytes. Values were normalized to the maximum percentage of cells producing IFN-γ. (n = 2). (i) Statistical analysis of LM-OVA bacterial titers in spleens and livers of Tfap4F/F CD8-Cre+ and Tfap4F/F Cre mice on days 4 and 6 after infection. (n = 4 for Tfap4F/F Cre, 6 for Tfap4F/F CD8-Cre+ in two experiments). Error bars, s.d. Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.d.). * P < 0.05, ** P < 0.01, *** P < 0.001, NS: not significant by unpaired t-test. Data are representative of three experiments (a-d), two experiments (e-i).

Supplementary Figure 3 AP4 is essential for the secondary population expansion of memory CD8+ T cells.

(a) Flow cytometry analysis of splenocytes showing expression of CD8α, CD127, CD62L and H-2Db–gp(33–41)-specific TCR on day 60 after LCMV-Arm infection. (n = 4). (b,c) Statistical analysis of absolute numbers of gp(33–41)-specific total memory CD8+ T cells and CD62L+ central memory CD8+ T cells in the spleen on day 60 after LCMV-Arm infection (b) and frequencies of gp(33–41)-specific memory CD8+ T cells in peripheral blood mononuclear cells (PBMC) in Tfap4–/– (n = 6), Tfap4F/F CD8-Cre+ (n = 7) or control C57BL/6 mice (n = 14) on day 60 after LCMV-Arm infection (c). (d) IFN-γ ELISPOT assay showing frequencies of OVA-specific memory CD8+ T cells in splenocytes of Tfap4–/– (n = 4) and control C57BL/6 mice (n = 4) on day 45 after LM-OVA infection. All mice had less than 11 spots per million splenocytes when no antigen was included. (e,f) Flow cytometry analysis showing expression of CD8α, CD45.2, CD62L, KLRG1 and H-2Db–gp(33–41)-specific TCR in splenocytes of CD45.1 wild-type host mice on day 5 after LCMV-Arm infection. Before infection, the host mice received CD8+ T cells from Tfap4–/– or C57BL/6 mice, which were infected with LCMV-Arm 60 days before. (n = 8 in two independent experiments). Statistical analysis is shown in (f). (g) Flow cytometry analysis of PBMC of Tfap4–/– (n = 15), Tfap4F/F CD8-Cre+ (n = 9), or control C57BL/6 mice (n = 14) 5 days after secondary challenge with LM-OVA. (h) Statistical analysis of numbers of total CD8+ T cell and OVA-specific CD8+ T cells numbers in blood and spleen 5 days after secondary challenge with LM-OVA is shown. Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.d.). * P < 0.01, ** P < 0.001, *** P < 0.0001, NS: not significant by unpaired t-test (b,d,f) or one-way ANOVA (c,h). Data are representative of three experiments.

Supplementary Figure 4 AP4 is required for the population expansion of antigen-specific CD8+ T cells after infection with WNV.

Statistical analysis showing absolute numbers of total CD8+ T cells (a), H-2Db–NS4B-specific T cells as determined by H-2Db–NS4B tetramer binding (b) or intracellular staining for IFN-γ following NS4B peptide stimulation (c) in the spleen on day 7 after WNV infection. Data is from one experiment using five Tfap4–/–, four Tfap4F/F CD8-Cre+, and four control age- and sex-matched C57BL/6 mice. * P < 0.05, NS: not significant by Mann Whitney U-test. Each symbol represents an individual mouse; small horizontal lines indicate median.

Supplementary Figure 5 AP4 is required for maintenance of T cell activation during acute CD8+ T cell responses.

(a) Quantitative RT-PCR and immunoblot analysis showing c-Myc and AP4 expression at mRNA and protein levels in CD8+ T cells following stimulation with anti-CD3 and anti-CD28. (n = 2). (b) Flow cytometry of CD8+ T cells from OT-I Mycc-Myc–GFP/+ mice showing c-Myc–GFP expression after two-hour stimulation with anti-CD3 and anti-CD28 and quantitative RT-PCR analysis of transcripts encoding Myc or Tfap4 in purified GFPLo and GFPHi cells. (n = 2). (c) Quantitative PCR analysis of anti-c-Myc immunoprecipitated chromatin of activated CD8+ T cells showing enrichment of the Tfap4 intronic fragments containing c-Myc consensus binding sequences. PCR primers for Cd4 promoter (Cd4Pro), Hoxa13 promoter (Hoxa13) and Cd4 silencer (Cd4Sil) were used as negative control (n = 2). (d) Flow cytometry of CD8+ T cells showing FSC in the naive state and following 24 h stimulation with anti-CD3 and anti-CD28 antibodies in vitro. (n = 3). (e) ECAR measurement showing glycolytic rates of Tfap4–/– and C57BL/6 CD8+ T cells after stimulation with anti-CD3 and anti-CD28 for 24 h. Data represent three independent experiments with three to four biological replicates in each experiment. (f) qRT-PCR analysis of RNA from CD8+ T cells showing 18S rRNA-normalized expression of Myc, Hif1a and genes encoding glycolytic enzymes at different time points following stimulation with anti-CD3 and anti-CD28 antibodies in vitro. (n = 3). (g) Flow cytometry of co-transferred Tfap4–/– and Tfap4+/+ P14 T cells showing FSC after infection of recipient mice with LCMV-Arm. Representative histogram overlays at each time point are shown in the left panel. (n = 3). (h) Microarray gene expression analysis of C57BL/6 CD8+ T cells in the naive state, or after activation with anti-CD3 and anti-CD28 in vitro for 3 days (n = 2), and OT-I T cells 2 days after LM-OVA infection in vivo (n = 3). Expression levels of the 479 AP4/Myc co-regulated genes identified in Fig. 6f are shown by a heat map. Expression levels are normalized to those of naive CD8+ T cells. Error bars, s.d. Each symbol in g represents an individual mouse; small horizontal lines indicate the mean (± s.d.).* P < 0.05, ** P < 0.01, NS: not significant by one-way ANOVA (b), unpaired t-test (e) or paired t-test (g). Data are representative of two experiments (a-c,f), three experiments (d-g), and one experiment (h).

Supplementary Figure 6 ChIP-sequencing analysis identifies AP4 and c-Myc target genes in CD8+ T cells.

(a-d) Annotation and motif analysis of genomic sequences bound by AP4 (a,b) or c-Myc (c,d) in activated C57BL/6 CD8+ T cells. (e) Examples of AP4 and c-Myc binding to genes that are differently expressed between Tfap4–/– and Tfap4+/+ OT-I T cells in vivo. BedGraph histograms are shown with statistically significant peaks as red rectangles. P < 1 × 10-10 for anti-AP4 ChIP, P < 5 × 10-9 for anti-c-Myc ChIP by Poisson distribution. ChIP-seq data for RNA polymerase II C-terminal domain (polII-CTD) serine phosphorylations (S2P and S5P) are shown as reference to transcribed regions. Data are from one experiment.

Supplementary Figure 7 AP4 and c-Myc have distinct biological functions.

(a) An experimental scheme to retrovirally express AP4 in Myc-deficient CD8+ T cells. MycF/+Cre (Myc+/+) or Myc+/+Rosa26-Cre-ERT2 and MycF/FRosa26-Cre-ERT2 (Myc–/– after 4OHT treatment) CD8+ T cells were transduced with empty-, FLAG-tagged AP4-, or Myc-expressing RV after activation with anti-CD3 and anti-CD28 for 24 h, and subsequently cultured in media containing IL-7 (10 ng/ml) and 4-hydroxytamoxifen (4OHT, 500 nM) for 2 days. Transduced cells (GFP+) were sorted, labeled with the division-tracking dye CellTraceTM Violet (CTV), and restimulated with anti-CD3 and anti-CD28 for two days. (b) Immunoblot showing c-Myc and AP4 expression 48 h after re-stimulation under the same conditions. n = 2. (c) Quantitative RT-PCR analysis showing the abundance of transcripts encoding Myc, Tfap4, or Il2ra after re-stimulation for 2 h. (n = 3). (d) Flow cytometry showing CTV fluorescence and FSC of RV-transduced Myc+/+, Myc+/+ Cre-ERT2(+) and Myc–/– CD8 T cells shown as histogram overlays. (n = 3). (e) Microarray analysis of Myc+/+ and Myc–/– CD8+ T cells transduced with AP4- or c-Myc-expressing RV shown as a heat map. Genes that were differently expressed by >2-fold between EV-transduced Myc+/+ and Myc–/– cells are shown with the abundance normalized to Myc+/+ + EV samples. Datasets were analyzed by hierarchical clustering using Euclidean distance matrix with average linkage. (n = 2). Error bars, s.d. * P < 0.0001, NS: not significant by one-way ANOVA. Data are representative of two experiments.

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Chou, C., Pinto, A., Curtis, J. et al. c-Myc-induced transcription factor AP4 is required for host protection mediated by CD8+ T cells. Nat Immunol 15, 884–893 (2014). https://doi.org/10.1038/ni.2943

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