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The microRNA miR-31 inhibits CD8+ T cell function in chronic viral infection

Nature Immunology volume 18, pages 791–799 (2017)Cite this article

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An Erratum to this article was published on 19 September 2017

This article has been updated

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.

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Figure 1: miR-31 is induced during T cell activation.
Figure 2: mir-31 is induced by signaling via calcium and NFAT.
Figure 3: Microarray profiling reveals targets of miR-31 in primary CD8+ T cells.
Figure 4: miR-31 limits CD8+ T cell effector programs and enhances exhaustion following stimulation with IFN-β.
Figure 5: Mir31−/− mice undergo a faster recovery and show better viral control in a chronic-LMCV-infection model.
Figure 6: Enhanced LCMV-specific CD8+ T cell responses in miR-31 deficient mice.
Figure 7: T cell–intrinsic effect of miR-31 on anti-viral CD8+ T cell responses during chronic infection with LCMV.
Figure 8: Enhanced production of cytokines by LCMV-specific CD8+ T cells in the absence of miR-31.

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  • 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.

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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.).

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  1. Gustavo J Martinez

    Present address: Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA

Authors and Affiliations

  1. Department of Cancer Immunology & Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Howell F Moffett, Adam N R Cartwright, Hye-Jung Kim, Jason Pyrdol, Harvey Cantor, Carl D Novina & Kai W Wucherpfennig

  2. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Jernej Godec, Arlene H Sharpe & Kai W Wucherpfennig

  3. Department of Information and Computer Science, Aalto University School of Science, Aalto, Finland

    Tarmo Äijö

  4. La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Gustavo J Martinez & Anjana Rao

  5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Jun Lu & Todd R Golub

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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.

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Correspondence to Kai W Wucherpfennig.

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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.

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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

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