Abstract
During infection, antigen-specific T cells undergo tightly regulated developmental transitions controlled by transcriptional and post-transcriptional regulation of gene expression. We found that the microRNA miR-31 was strongly induced by activation of the T cell antigen receptor (TCR) in a pathway involving calcium and activation of the transcription factor NFAT. During chronic infection with lymphocytic choriomeningitis virus (LCMV) clone 13, miR-31-deficent mice recovered from clinical disease, while wild-type mice continued to show signs of disease. This disease phenotype was explained by the presence of larger numbers of cytokine-secreting LCMV-specific CD8+ T cells in miR-31-deficent mice than in wild-type mice. Mechanistically, miR-31 increased the sensitivity of T cells to type I interferons, which interfered with effector T cell function and increased the expression of several proteins related to T cell dysfunction during chronic infection. These studies identify miR-31 as an important regulator of T cell exhaustion in chronic infection.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
02 June 2017
In the version of this article initially published online, the symbols in the key for Figure 2a were reversed. The correct key symbol colors are black for DMSO and white for CyA. The error has been corrected in the print, PDF and HTML versions of this article.
References
Wherry, E.J. et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27, 670–684 (2007).
Muljo, S.A. et al. Aberrant T cell differentiation in the absence of Dicer. J. Exp. Med. 202, 261–269 (2005).
Cobb, B.S. et al. T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer. J. Exp. Med. 201, 1367–1373 (2005).
Zhang, N. & Bevan, M.J. Dicer controls CD8+ T-cell activation, migration, and survival. Proc. Natl. Acad. Sci. USA 107, 21629–21634 (2010).
Wu, T. et al. Temporal expression of microRNA cluster miR-17-92 regulates effector and memory CD8+ T-cell differentiation. Proc. Natl. Acad. Sci. USA 109, 9965–9970 (2012).
Yang, L. et al. miR-146a controls the resolution of T cell responses in mice. J. Exp. Med. 209, 1655–1670 (2012).
Taganov, K.D., Boldin, M.P., Chang, K.J. & Baltimore, D. NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc. Natl. Acad. Sci. USA 103, 12481–12486 (2006).
Gracias, D.T. et al. The microRNA miR-155 controls CD8+ T cell responses by regulating interferon signaling. Nat. Immunol. 14, 593–602 (2013).
Lu, L.F. et al. Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity 30, 80–91 (2009).
Trifari, S. et al. MicroRNA-directed program of cytotoxic CD8+ T-cell differentiation. Proc. Natl. Acad. Sci. USA 110, 18608–18613 (2013).
Bronevetsky, Y. et al. T cell activation induces proteasomal degradation of Argonaute and rapid remodeling of the microRNA repertoire. J. Exp. Med. 210, 417–432 (2013).
Monticelli, S. & Rao, A. NFAT1 and NFAT2 are positive regulators of IL-4 gene transcription. Eur. J. Immunol. 32, 2971–2978 (2002).
Martinez, G.J. et al. The transcription factor NFAT promotes exhaustion of activated CD8+ T cells. Immunity 42, 265–278 (2015).
Scott-Browne, J.P. et al. Dynamic changes in chromatin accessibility occur in CD8+ T cells responding to viral infection. Immunity 45, 1327–1340 (2016).
Alexiou, P., Maragkakis, M., Papadopoulos, G.L., Reczko, M. & Hatzigeorgiou, A.G. Lost in translation: an assessment and perspective for computational microRNA target identification. Bioinformatics 25, 3049–3055 (2009).
Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008).
Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).
Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).
Wu, D., Wang, B., Shang, J., Song, J. & Zhang, H. miR-31 reduces cell growth of papillary thyroid carcinoma by RNA-binding protein HuR. Clin. Lab. 61, 1625–1634 (2015).
Creighton, C.J. et al. Molecular profiling uncovers a p53-associated role for microRNA-31 in inhibiting the proliferation of serous ovarian carcinomas and other cancers. Cancer Res. 70, 1906–1915 (2010).
Ivanov, S.V. et al. Pro-tumorigenic effects of miR-31 loss in mesothelioma. J. Biol. Chem. 285, 22809–22817 (2010).
Liu, X. et al. MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors. J. Clin. Invest. 120, 1298–1309 (2010).
Rutledge, H. et al. Identification of microRNAs associated with allergic airway disease using a genetically diverse mouse population. BMC Genomics 16, 633 (2015).
Chen, J.H. et al. Prostaglandin E2 and programmed cell death 1 signaling coordinately impair CTL function and survival during chronic viral infection. Nat. Med. 21, 327–334 (2015).
Singer, M., Wang, C., Cong, L., Marjanovic, N.D., Kowalczyk, M.S. & Zhang, H. et al. A distinct gene module for dysfunction uncoupled from activation in tumor-infiltrating T cells. Cell 166, 1500–1511 (2016).
Ivashkiv, L.B. & Donlin, L.T. Regulation of type I interferon responses. Nat. Rev. Immunol. 14, 36–49 (2014).
McNally, J.M. et al. Attrition of bystander CD8 T cells during virus-induced T-cell and interferon responses. J. Virol. 75, 5965–5976 (2001).
Marshall, H.D., Urban, S.L. & Welsh, R.M. Virus-induced transient immune suppression and the inhibition of T cell proliferation by type I interferon. J. Virol. 85, 5929–5939 (2011).
Terawaki, S. et al. IFN-α directly promotes programmed cell death-1 transcription and limits the duration of T cell-mediated immunity. J. Immunol. 186, 2772–2779 (2011).
Stefansson, B. & Brautigan, D.L. Protein phosphatase PP6 N terminal domain restricts G1 to S phase progression in human cancer cells. Cell Cycle 6, 1386–1392 (2007).
Kajino, T. et al. Protein phosphatase 6 down-regulates TAK1 kinase activation in the IL-1 signaling pathway. J. Biol. Chem. 281, 39891–39896 (2006).
Lee, M.N. et al. Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach. Nat. Immunol. 14, 179–185 (2013).
Barber, D.L. et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439, 682–687 (2006).
Teijaro, J.R. et al. Persistent LCMV infection is controlled by blockade of type I interferon signaling. Science 340, 207–211 (2013).
Wilson, E.B. et al. Blockade of chronic type I interferon signaling to control persistent LCMV infection. Science 340, 202–207 (2013).
Punkosdy, G.A. et al. Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens. Proc. Natl. Acad. Sci. USA 108, 3677–3682 (2011).
Penaloza-MacMaster, P. et al. Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection. J. Exp. Med. 211, 1905–1918 (2014).
Rouas, R. et al. Human natural Treg microRNA signature: role of microRNA-31 and microRNA-21 in FOXP3 expression. Eur. J. Immunol. 39, 1608–1618 (2009).
Zhang, L. et al. MicroRNA-31 negatively regulates peripherally derived regulatory T-cell generation by repressing retinoic acid-inducible protein 3. Nat. Commun. 6, 7639 (2015).
Xue, F. et al. miR-31 regulates interleukin 2 and kinase suppressor of ras 2 during T cell activation. Genes Immun. 14, 127–131 (2013).
Fan, W. et al. Identification of microRNA-31 as a novel regulator contributing to impaired interleukin-2 production in T cells from patients with systemic lupus erythematosus. Arthritis Rheum. 64, 3715–3725 (2012).
Wang, Y. et al. Timing and magnitude of type I interferon responses by distinct sensors impact CD8 T cell exhaustion and chronic viral infection. Cell Host Microbe 11, 631–642 (2012).
Bahl, K. et al. IFN-induced attrition of CD8 T cells in the presence or absence of cognate antigen during the early stages of viral infections. J. Immunol. 176, 4284–4295 (2006).
Marshall, H.D., Urban, S.L. & Welsh, R.M. Virus-induced transient immune suppression and the inhibition of T cell proliferation by type I interferon. J. Virol. 85, 5929–5939 (2011).
Terawaki, S. et al. IFN-α directly promotes programmed cell death-1 transcription and limits the duration of T cell-mediated immunity. J. Immunol. 186, 2772–2779 (2011).
Xue, F. et al. miR-31 regulates interleukin 2 and kinase suppressor of ras 2 during T cell activation. Genes Immun. 14, 127–131 (2013).
Crow, M.K. Type I interferon in the pathogenesis of lupus. J. Immunol. 192, 5459–5468 (2014).
Fan, W. et al. Identification of microRNA-31 as a novel regulator contributing to impaired interleukin-2 production in T cells from patients with systemic lupus erythematosus. Arthritis Rheum. 64, 3715–3725 (2012).
Welsh, R.M. & Seedhom, M.O. Lymphocytic choriomeningitis virus (LCMV): propagation, quantitation, and storage. Current Protocols in Microbiology Ch 15, Unit 15A 11 (2008).
Stamm, A., Valentine, L., Potts, R. & Premenko-Lanier, M. An intermediate dose of LCMV clone 13 causes prolonged morbidity that is maintained by CD4+ T cells. Virology 425, 122–132 (2012).
Laidlaw, B.J. et al. Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity. PLoS Pathog. 9, e1003207 (2013).
Decman, V. et al. Cell-intrinsic defects in the proliferative response of antiviral memory CD8 T cells in aged mice upon secondary infection. J. Immunol. 184, 5151–5159 (2010).
Mueller, S.N. et al. Qualitatively different memory CD8+ T cells are generated after lymphocytic choriomeningitis virus and influenza virus infections. J. Immunol. 185, 2182–2190 (2010).
Wherry, E.J., Blattman, J.N., Murali-Krishna, K., van der Most, R. & Ahmed, R. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77, 4911–4927 (2003).
Call, M.J. et al. In vivo enhancement of peptide display by MHC class II molecules with small molecule catalysts of peptide exchange. J. Immunol. 182, 6342–6352 (2009).
Martinez, G.J. et al. The transcription factor NFAT promotes exhaustion of activated CD8+ T cells. Immunity 42, 265–278 (2015).
Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).
Rosenbloom, K.R. et al. ENCODE data in the UCSC Genome Browser: year 5 update. Nucleic Acids Res. 41, D56–D63 (2013).
Okamura, H. et al. Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol. Cell 6, 539–550 (2000).
Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).
Reich, M. et al. GenePattern 2.0. Nat. Genet. 38, 500–501 (2006).
Schadt, E.E., Li, C., Ellis, B. & Wong, W.H. Feature extraction and normalization algorithms for high-density oligonucleotide gene expression array data. J. Cell. Biochem. Suppl. 37, 120–125 (2001).
Tusher, V.G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116–5121 (2001).
Miranda, K.C. et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell 126, 1203–1217 (2006).
Friedman, R.C., Farh, K.K., Burge, C.B. & Bartel, D.P. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105 (2009).
Betel, D., Koppal, A., Agius, P., Sander, C. & Leslie, C. Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites. Genome Biol. 11, R90 (2010).
Yang, J.H. et al. starBase: a database for exploring microRNA-mRNA interaction maps from Argonaute CLIP-Seq and Degradome-Seq data. Nucleic Acids Res. 39, D202–D209 (2011).
Krek, A. et al. Combinatorial microRNA target predictions. Nat. Genet. 37, 495–500 (2005).
Acknowledgements
We thank R. Webby (St. Jude Children's Research Hospital) for recombinant influenza virus containing the LCMV GP33–41 epitope inserted into the neuraminidase protein stalk region; J. Maiarana for technical assistance; and the Dana Farber Cancer Institute Molecular Biology Core Facility for RNA sequencing. Supported by the US National Institutes of Health (R01CA173750 to K.W.W.; R01DK102165 and R01CA140986 to C.N.; P01AI056299 to A.S.; and R01AI007386 to H.C.) and the Cancer Research Institute (A.N.R.C.).
Author information
Authors and Affiliations
Contributions
H.F.M. and K.W.W. designed the study and discussed and analyzed data; H.F.M., A.N.R.C. and K.W.W. wrote the paper; H.F.M. and A.N.R.C. performed experiments and analyzed data; H.-J.K. and J.G. helped with infection experiments; J.P. performed immunoblot analysis; T.A., G.J.M. and A.R. provided NFAT chromatin-immunoprecipitation data; J.L. and T.R.G. performed miRNA profiling; H.C., A.H.S. and C.D.N. provided advice on experimental design and data interpretation; and K.W.W. supervised the study.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 miRNA expression profiling.
MiRNA expression profiling of thymocytes (Thy), total T cells, naïve CD8+ T cells and activated CD8+ T cells (24 and 96 hours post stimulation with CD3+ CD28 antibodies). Also included is an analysis of total B cells, bone marrow-derived DC (DC), lipopolysaccharide activated DC (DC-LPS), kidney and liver. Data represent expression relative to the mean value for each row. (a) Pairwise complete linkage hierarchical clustering of all miRNAs expressed at >3 standard deviations over background in at least one of the samples. (b, c) Expression patterns of miRNAs that are upregulated (b) or downregulated (c) at least 2-fold in CD3+CD28 Ab activated CD8+ T cells compared to naive CD8+ T cells (comparison of days 0, 1 and 4). Data are representative of one experiment.
Supplementary Figure 2 Expression of miR-31 after treatment of CD8+ T cells with cytokines or TLR agonists.
(a) Naïve OT-I CD8+ T cells were treated for 24 hours with 1 μg/ml SIINFEKL peptide and 20 ng/ml IL-2, or 20 ng/ml of the indicated cytokines for 48 hours. MiR-31 expression was assayed by qPCR relative to input naïve OT-I. (b) Naïve OT-I T cells were treated for 48 hours with CD3+CD28 antibodies or 20 ng/ml TGF-b for 48 hours. MiR-31 expression was assayed by qPCR relative to input naïve OT-I. (c) CD8+ T cells were treated for 24 hours with PMA/ionomycin, 10 μg/ml LPS, or 3 μg/ml CpG 2006. MiR-31 expression was assayed by qPCR relative to input CD8+ T cells. (d) miR-31 expression relative to the small nucleolar RNA sno234 was measured by qPCR 24 hours following activation of CD4+ or CD8+ T cells (CD3+CD28 antibodies), NK cells (PMA/ionomycin) and B cells (PMA/ionomycin), compared to cells without activation stimulus. (e) miR-31 expression relative to sno-234 in OT-I CD8+ T cells activated by SIINFEKL peptide-pulsed splenocytes for 72 hours, and then cultured in the presence of IL-2 or IL-15 for 8 or 15 days. (f) miR-31 expression relative to sno234 in ex vivo sorted CD4+ and CD8+ T cell subsets: naïve (CD44low CD62L+), effector memory (EM, CD44hi CD62L-), central memory (CM, CD44hi CD62L+), Treg (CD4+ CD25+ GITR+) subsets. Data are representative of two (a-e) experiments. Data show mean ± s.d.
Supplementary Figure 3 Genomic targeting of Mir31.
(a) Diagram of conditional miR-31 targeting construct, showing locations of pre-miR-31, inserted LoxP sites, and flpE flanked neomycin resistance cassette. (b) Mir31flox/flox mice were crossed to Zp3cre mice to generate Mir31-/- mice. CD8+ T cells from WT or Mir31-/- mice were not treated (Unstimulated) or stimulated with aCD3/CD28 for 24 hours. Relative miR-31 expression was determined by qPCR. (c) Mir31flox/flox mice were crossed to Cd4cre mice to generate Mir31flox/flox Cd4cre mice. CD8+ T cells from Mir31flox/flox or Mir31flox/flox Cd4cre mice were not treated (Unstimulated) or stimulated with CD3+CD28 antibodies for 24 hours. Relative miR-31 expression was determined by qPCR. Data are representative of 2 (b-c) experiments. Data show mean ± s.d.
Supplementary Figure 4 Analysis of STAT signaling.
Mir31+/+ and Mir31-/- CD8+ T cells were activated using CD3+CD28 Ab coated beads for 48 hours, then cultured for 5 days in IL-2. (a) STAT1, phospho-tyrosine STAT1, and phospho-serine STAT1 were assessed by Western blotting in CD8+ T cells stimulated with IFN-β for 0, 10, or 30 mins. (b) Quantification of STAT1 total protein relative to total protein (imaged per Biorad protocol). (c) Phospho-tyrosine STAT1 as a ratio to total STAT1 protein. (d) Phospho-Serine STAT1 as a ratio of total STAT1 protein. (e). STAT1, STAT2, IRF9, and SOCS1 gene expression was assessed by qPCR in CD8+ T cells stimulated with IFN-β for 0, 4, or 18 hours. Data are representative of 2 independent experiments consisting 3 cell cultures from 3 individual mice. * p value <0.05, ** p value <0.01 by two-way ANOVA with Sidak corrections for multiple comparisons. F ratios and degrees of freedom are a) 5.18, 17, b) 3.74, 11, and c) 2.23, 17, respectively.
Supplementary Figure 5 Modulation of Ppp6c expression affects sensitivity to type 1 interferons.
(a) Flow cytometry for CD69 expression after IFN-ɣ treatment of T hybridoma cells. 7678 hybridoma cells were infected with lentiviral constructs driving either expression of miR-31 or shRNAs against miR-31 target genes (Lats2, Stk40, Ppp6c, Sh2d1a, luciferase as control). Cells were mixed with uninfected cells, treated with the indicated concentration of IFN-ɣ for 18 hours, and CD69 expression relative to uninfected cells was determined by FACS. (P values from ratio paired two-tailed Students t test: sh-Control vs pre-miR-31= 0.005; sh-control vs sh-Ppp6c = 0.002) (b) Flow cytometry for CD69 expression after IFN-ɣ treatment of Mir31-/- OT-I T cells infected with lentiviral constructs driving expression of miR-31 or shRNAs targeting Ppp6c or luciferase (control). CD69 expression was measured following treatment with the indicated IFN-ɣ for 18 hours. (P values from ratio paired two-tailed Students t test: sh-Control vs pre-miR-31= 0.005; sh-control vs sh-Ppp6c = 0.003) (c) Western blot for Ppp6c protein expression in Mir31+/+ and Mir31-/- OT-I T cells. OT-I T cells from three mice/condition were activated with peptide-pulsed splenocytes for 24 hours and cultured in media containing IL-2 for 15 days; 3x105 cells/lane were lysed and expression of β-actin and Ppp6c was examined by Western blot. Relative expression of Ppp6c normalized to b-actin is indicated. (n=3/group, p value from unpaired two-tailed Students t test=0.0008) (d) Full blot images for Western blot data.. Left panel displays size marker data from SeebluePlus2 pre-stained protein size standards. Middle and right panel display images collected within the linear response range for β-actin (middle) and Ppp6c (right). Data are representative of one (a), three (c), or two (d) experiments. ** p value <0.01, *** p value <0.001)
Supplementary Figure 6 LCMV RNA persistence, Foxp3 gene expression and CD4+ Treg cell populations in wild-type and Mir31–/– mice.
(a) LCMV Gp gene expression relative to Gapdh was quantified by qPCR in total cDNA from liver and kidney of WT and Mir31-/- mice on day 30 following infection with LCMV clone 13 (n= 4, 5 (WT, Mir31-/-), p values: 0.04, 0.02 respectively)). (b) Luciferase reporter assay for reporter genes containing no 3’UTR (control), Ppp6c 3’UTR, or Foxp3 3’UTR in the presence of miR-31. (c) Intracellular staining for Foxp3 expression in CD4+ T cells from blood, spleen, peripheral lymph nodes (PLN) or mesenteric lymph nodes (MLN) from WT and Mir31-/- mice. Data are representative of two experiments.
Supplementary Figure 7 Acute anti-viral responses are not altered in Mir31–/– mice.
Mir31+/- / and Mir31-/- mice were intra-nasally infected with 1.6x105 TCID50 of influenza strain X31-GP33. (a) Body weight was measured daily before (day 0) and following infection. Percentage of original body weight is shown. (b) Quantification of influenza RNA by qPCR in total lung RNA on day 8 following infection. (c) Cells from spleen and lung at 8 dpi and 30 dpi were stained with H-2Db tetramers specific for GP33, NP or PA peptides. Data represent the sum of the three CD8+ CD44+ tetramer+ populations as a fraction of total CD45+ cells (n≥4mice per group). Data are representative of one experiment.
Supplementary Figure 8 IL-2 expression, and ratio of GP33 tetramer to GP33 cytokine response in wild-type and Mir31–/– mice.
(a) Total splenocytes from WT and Mir31-/- mice on day 30 following infection with LCMV clone 13 were treated with Brefeldin A +/- PMA/ionomycin for 5 hours. IL-2 expression by CD4+ and CD8+ T cells was assessed by intracellular staining (n≥4 mice per group). (b) Ratio of cytokine secreting GP33-tetramer+ CD8+ T cells to total GP33-tetramer+ CD8+ T cells per spleen for both IFN⍺ (left panel) or IFNɣ/TNF⍺ (right panel) producers. (n= 4, 5 (WT, Mir31-/-), given p value derived from one-tailed Student’s t test. Graphs represent mean ± s.d. Data are representative of two experiments * p value <0.05.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8 and Supplementary Tables 1 and 2.
Rights and permissions
About this article
Cite this article
Moffett, H., Cartwright, A., Kim, HJ. et al. The microRNA miR-31 inhibits CD8+ T cell function in chronic viral infection. Nat Immunol 18, 791–799 (2017). https://doi.org/10.1038/ni.3755
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.3755
This article is cited by
-
Targeting the epigenome to reinvigorate T cells for cancer immunotherapy
Military Medical Research (2023)
-
Regulation of CD8+ T memory and exhaustion by the mTOR signals
Cellular & Molecular Immunology (2023)
-
Nicotine exhausts CD8+ T cells against tumor cells through increasing miR-629-5p to repress IL2RB-mediated granzyme B expression
Cancer Immunology, Immunotherapy (2021)
-
MicroRNA-181a regulates IFN-γ expression in effector CD8+ T cell differentiation
Journal of Molecular Medicine (2020)
-
miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy
Cancer Immunology, Immunotherapy (2020)