Abstract

Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3′ untranslated region (3′ UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3′ UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.

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Acknowledgements

We thank the animal caretakers of the NKI and the FACS facility of Sanquin Research and the Babraham Institute for excellent assistance. We thank D. Zehn (TU Munich) for the LM-OVA strain, J. Rohr (University of Freiburg) for Listeria cultures; H. Meijer for sharing the sucrose cushion protocol; G. Stoecklin (University of Heidelberg) for the 4xS1m aptamer construct; R. Arens (Leiden University), T. Schumacher (Netherlands Cancer Institute) and J. den Haan (Free University, Amsterdam) for providing mice; S. Libregts, B. van Steensel, K. Moore and B. Nicolet for technical help and advice; and D. Amsen, M. Nolte and R. van Lier for critical reading of the manuscript. M.T. and S.E.B. are supported by the Biotechnology and Biological Sciences Research Council. D.L.H. and H.A.Y. are funded through the intramural research program of the US NCI, NIH. The use of materials and reagents does not imply any endorsement of these products by the US government. This research was supported by the Dutch Science Foundation (VENI grant 916.76.127/VIDI grant 917.14.314, to M.C.W.).

Author information

Affiliations

  1. Sanquin Research, Department of Hematopoiesis, and Landsteiner Laboratory, Academic Medical Centre (AMC), University of Amsterdam, Amsterdam, the Netherlands

    • Fiamma Salerno
    • , Sander Engels
    • , Aurelie Guislain
    • , Wanqi Zhao
    • , Marieke von Lindern
    •  & Monika C. Wolkers
  2. Sanquin Research, Department of Plasma Proteins, Amsterdam, the Netherlands

    • Maartje van den Biggelaar
    •  & Floris P. J. van Alphen
  3. Laboratory of Experimental Immunology, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    • Deborah L. Hodge
    •  & Howard A. Young
  4. Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham, UK

    • Sarah E. Bell
    •  & Martin Turner
  5. Laboratory of Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, the Netherlands

    • Jan Paul Medema

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Contributions

F.S. and M.C.W. designed experiments, F.S., S.E., M.v.d.B., F.P.J.v.A., A.G., W.Z., S.E.B. and M.C.W. performed experiments and analyzed the data, D.L.H. and H.A.Y. contributed the ARE-Del mice; S.E.B. and M.T. contributed the CD4cre-Zfp36l2flox/flox mice; M.v.L., M.T., H.A.Y. and J.P.M. provided intellectual input; M.C.W. directed the study; F.S. and M.C.W. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Monika C. Wolkers.

Integrated supplementary information

  1. Supplementary Figure 1 Ifng 3′-UTR-dependent regulation of GFP expression in T cells.

    (a-b) Blood from LM-OVA infected mice (Fig. 1) was analyzed for GFP-MFI of GFPcontrol or GFP-Ifng3’UTR-expressing OTI cells. Data are represented as percentage of day 6 (n = 10 mice/group). (a) [One-way ANOVA with Dunnett’s multiple comparison to day 6 time point; *p < 0.05; **p < 0.005; ****p < 0.0001]. (b) [Unpaired Student t-test; **p < 0.005; ****p < 0.0001]. (c-h) T cells from (c-f) C57BL/6 J mice or (g-i) OTI mice were transduced with GFP-Ifng3’UTR or GFPcontrol. (c-f) Representative dot plots of GFP expression of unstimulated resting CD8+ T cells and CD4+ T cells (c,e), or reactivated for 4 h with PMA + ionomycin (d,f). Numbers in (c,e) indicate GFP-MFI. Graphs depict data compiled from 3 independently performed experiments (n = 4 mice/group). (c,e) [Unpaired Student t-test; **p < 0.005; ****p < 0.0001]. (d,f) [Paired Student t-test; *p < 0.05; **p < 0.005]. (g) Intracellular IFN-γ staining of resting (upper panel), or reactivated (OVA257-264 peptide; lower panel) GFP-Ifng3’UTR- or GFPcontrol-expressing OTI cells. Numbers indicate percentage of GFP+ and/or IFN-γ+ T cells. (h) Representative dot plot of GFPcontrol and GFP-Ifng 3’UTR expression levels under the minimal murine Ifng promoter in resting (left) and reactivated OTI cells (right). Numbers indicate GFP-MFI.

  2. Supplementary Figure 2 All conserved AREs contribute to the Ifng 3′-UTR-mediated post-transcriptional regulation.

    (a-b) GFP expression was measured in resting (gray histograms), and reactivated (black lines) OTI cells expressing wild-type GFP-Ifng 3’UTR or GFP-Ifng 3’UTR with indicated ARE mutants. Data are representative of three independently performed experiments. (c) Multiple sequence alignment of the Ifng 3’UTR of 9 representative species performed as described (Di Tommaso, P. et al. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 39, W13–W17; 2011), combined with Boxshade analysis. For the alignment with the mRNA from gorilla, chimpanzee, green monkey, and Yangtze river dolphin, predicted sequences (Pubmed) have been used. ARE sequences are highlighted in yellow, underlined and numbered as in Fig. 2b. (d) Activated human T cells were transduced with wild-type human IFNG 3’UTR (WT), or with indicated ARE mutants. Graphs indicate GFP-MFI during resting phase (top) or GFP fold increase after reactivation with PMA + ionomycin (bottom). [One-way ANOVA with Dunnett’s multiple comparison to the control vector; n = 4 donors per group; *p < 0.005].

  3. Supplementary Figure 3 Chronic IFN-γ production occurs in all TM subsets of IFN-γ-ARE-Del cells.

    (a) Relative distribution of TEFF, TEM and TCM, and of SLEC/MPEC OTI cells 35 days post LM-OVA infection as defined in Fig. 3b (n = 8 mice/group). (b) Percentage (left panel) and MFI (right panel) of spontaneous IFN-γ production at day 48 post LM-OVA infection of spleen- and liver-residing wild-type and IFN-γ-ARE-Del TM cells. (c-d) IFN-γ MFI of spleen- (c) and IFN-γ MFI and percentage of IFN-γ producing T cells of liver- (d) residing wild-type (gray) and IFN-γ-ARE-Del (white) T cell subsets at day 35 post LM-OVA infection. (e) Ifng mRNA levels of sorted spleen-derived naive CD44lowCD62LhiCD8+ T cells and memory-like CD44 hiCD8+ T cells of 6–8 weeks old wild-type OTI mice. Results (mean ± SD) are pooled from 7 mice and five independently performed experiments. (f) Intracellular IFN-γ and TNF cytokine staining after 4h incubation with 1μg/ml brefeldin A. (g,h) IFN-γ production (g) and Ifng mRNA levels (h) of paired sorted and unstimulated CD44hiCD62Llow TEFF/EM and CD44hiCD62Lhi TCM CD8+ T cells. (a-h) [Unpaired Student t-test; ns = not significant; *p < 0.05; **p < 0.005; ***p < 0.0005].

  4. Supplementary Figure 4 Identification of IFNG 3′ UTR ARE-binding proteins ZFP36L1 and ZFP36L2.

    (a-c) Volcano plots represent RBPs quantified by mass spectrometry with a pull-down from human T cell lysates using 4xS1m mRNA aptamers expressing 189nt of the WT human IFNG 3’UTR (4xS1m-WT), the ARE mutant (4xS1m-MUT1-5) or the empty 4xS1m mRNA control. RBPs were eluted by on bead digestion using 50 mM Ammonium Bicarbonate solution (pH 8) containing 1 M urea and 5 mM DTT. Samples were further processed as described in ‘4xS1m RNA aptamer-protein pull-down’. Dotted lines indicate the cutoff of ± 1.75 fold change. (d) Heat map depicts Z-scored log2 LFQ values of proteins that were significantly enriched in the pull down shown in Fig. 4, and present in all three replicates of the second pull-down with fold change > 1.75. (e) ZFP36L1, ZFP36L2 and RhoGDI expression of naive CD44lowCD62LhiCD8+ T cells and memory-like CD44hiCD8+ T cells sorted from spleens of C57BL/6 J mice. CD44hi T cells were left untreated or stimulated for 2 h with PMA/ionomycin.

  5. Supplementary Figure 5 Distribution of TM subsets in wild-type and Zfp36l2cKO mice, and mRNA expression levels in wild-type and IFN-γ-ARE-Del T cells.

    (a) Relative distribution of CD8+ (left) and CD4+ (right) TEFF, TEM and TCM cells from 8-week-old Zfp36lcKO mice and wild-type littermates. (b-e) Tnf (b-c) and 18S (d-e) mRNA decay in in vitro generated resting T cells (b,d), and in memory-like CD44hiCD8+ OTI cells (c,e) upon treatment with 1μg/ml ActD for indicated time points. Results ± SD are pooled from 4 independently performed experiments. (f-g) ActD treatment and analysis of Ifng mRNA decay of CD44hiCD8+ T cells or CD44hiCD4+ T cells from IFN-γ-ARE-Del (g) or Zfp36l2cKO (h) mice and their respective wild-type controls, as in Fig. 5g-h. (h) miR-29a, miR-29b, tbx21 and eomes mRNA expression levels of sorted CD44 hiCD8+ OTI cells analyzed by RT-PCR. Results ± SD are pooled from two independently performed experiments [Unpaired Student t-test; n = 4 mice].

  6. Supplementary Figure 6 Protein expression of T cells treated with ActD and CHX, and analysis of ribosome-bound mRNA.

    (a) Percentage of live cells (defined as Near-IR-) and Geo-MFI of CD44 and CD8 expression of splenic CD44hi sorted from wild-type and IFN-γ-ARE-Del OTI cells, that were incubated for 4h with BFA alone (-), or with BFA together with 1μg/ml ActD or 10μg/ml CHX. (b) Total RNA distribution as determined by the RNAnano Chip assay of the cytosolic fraction of CD44hi T cells that were pre-treated or not with 20 mM EDTA, and that precipitated through a 20% sucrose cushion. (c,d) Tnf (c) and 18S (d) mRNA levels analyzed before (input) and after (ribosomes) sucrose cushion of cytoplasmic RNA of CD44hiCD8+ wild-type and IFN-γ-ARE-Del OTI cells. Results were pooled from 3 independently performed experiments (mean ± SD). [Unpaired Student t-test; n = 3–6 mice; ns = not significant].

  7. Supplementary Figure 7 ZFP36L2 expression and function of CD44hi memory-like T cells.

    (a) Heat map represents Z-scored log2 LFQ values of proteins that were significantly upregulated or downregulated in the proteomics analysis depicted in Fig. 7a. Gray indicates no detection of peptides in the mass spectrometry analysis. Red stars depict proteins encoded by ARE-containing transcripts (Hierarchical clustering, k-means = 4). (b) Bar graphs display log2-normalized counts of transcripts encoding the proteins in panel a. Red indicates ARE-containing transcripts. Of note, Gm3839 and Mtnd4 gene expression was missing in the RNAseq data set (n.a.). (c) Zfp36l2 mRNA expression of CD44hiCD8+ or CD44hiCD4+ T cells from C57BL/6 J mice directly ex vivo, or upon 2 h PMA/ionomycin stimulation [Unpaired Student t-test; *p < 0.005; **p < 0.0005]. (d) Western blot analysis of ZFP36L2 and GAPDH expression in wild-type and in Zfp36l2cKO CD44hiCD8+ T cells that were stimulated for indicated time with PMA + ionomycin, or left untreated. Data are pooled (c) or representative of (d) 4 mice and 3 independently performed experiments.

  8. Supplementary Figure 8 Flow cytometry gating strategies.

    Representative gating strategies of spleen-derived OTI cells. T cell analysis from blood, liver, and bone marrow was performed in a similar manner. (a) Analysis of GFP+CD45.1+CD8+ OTI cells at day 35 post LM-OVA infection (as in Fig. 1). (b) Naive wild-type/CD45.1 and IFN-γ-ARE-Del/CD45.2 OTI cells were co-transferred into recipient mice. IFN-γ production at day 35 post LM-OVA infection (as in Fig. 3). (c) Expression levels of effector and memory markers of transferred OTI cells 48 days upon LM-OVA infection. (d) Example of sorting strategy of naive CD44lowCD62Lhi and memory-like CD44hi CD4+ or CD8+ T cells.

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