The microRNA miR-155 controls CD8+ T cell responses by regulating interferon signaling

Journal name:
Nature Immunology
Volume:
14,
Pages:
593–602
Year published:
DOI:
doi:10.1038/ni.2576
Received
Accepted
Published online

Abstract

We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8+ T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8+ T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8+ T cells, as miR-155-deficient CD8+ T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8+ T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8+ T cells had enhanced type I interferon signaling and were more susceptible to interferon's antiproliferative effect. Inhibition of the type I interferon–associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8+ T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8+ T cell responses to pathogens in vivo.

At a glance

Figures

  1. Expression of miR-155 in CD8+ T cells.
    Figure 1: Expression of miR-155 in CD8+ T cells.

    (a) RT-PCR analysis of miR-155 expression in splenic CD8+ T cells sorted from wild-type C57BL/6 mice and stimulated in vitro for 1, 3 and 5 d with anti-CD3 and anti-CD28; results were normalized to 18S rRNA expression and are presented relative to those of unstimulated CD8+ T cells. (b) RT-PCR analysis of miR-155 expression in naive CD44CD62L+ CD8+ T cells sorted from spleens of naive OT-I mice (Naive) and in donor effector CD44+CD62L CD8+ T cells and donor central memory CD44+CD62L+ or effector memory CD44+CD62L CD8+ T cells sorted from the lungs (day 10; effector) or spleen (day 60; central memory and effector memory) of congenic mice given adoptive transfer of OT-I cells and infected with influenza virus WSN-OVA; results were normalized as in a and are presented relative to those of naive CD8+ T cells. Data represent three independent experiments with five mice per group in each (a; mean and s.e.m.) or two independent experiments with three to five mice per group in each (b; mean and s.e.m.).

  2. CD8+ T cell responses require miR-155.
    Figure 2: CD8+ T cell responses require miR-155.

    (a) Flow cytometry of cells from lungs of wild-type (WT) mice and miR-155-deficient (KO) mice at day 10 after infection with influenza virus. Numbers above outlined areas indicate percent NP(366–374)-specific CD8+ T cells among lymphocytes. (b) Quantification of NP(366–374)-specific CD8+ T cells as in a. *P < 0.002 (Student's t-test). (c) Viral loads in lungs of mice as in a. TCID50, half-maximal tissue culture infectious dose. *P < 0.05 (Student's t-test). (d) Flow cytometry of cells from the lungs of congenic wild-type (CD45.1+) mice given adoptive transfer of OT-I or miR-155-deficient OT-I (CD45.2+) CD8+ T cells (above plots) and then infected with influenza virus WSN-OVA. Numbers adjacent to outlined areas indicate percent CD45.2+CD8+ T cells among lymphocytes. (e) Quantification of donor OT-I cells in lungs, mediastinal lymph nodes (MLN) and spleens of recipient mice as in d at day 10 after infection. *P < 0.001 (Student's t-test (lungs and spleen) or Mann-Whitney U-test (mediastinal lymph nodes)). (f) Kinetics of the change in abundance of donor OT-I cells in the lungs of mice as in d. (g) Flow cytometry of cells from the spleens of Thy-1.2+ congenic mice given adoptive transfer of Thy-1.1+ OT-I or miR-155-deficient OT-I cells and then infected with OVA-expressing L. monocytogenes. Numbers above outlined areas indicate percent IFN-γ+Thy-1.1+ T cells (responding donor cells) among lymphocytes. (h) Quantification of IFN-γ+ donor OT-I cells in the spleen and mesenteric lymph nodes (Mes LN) of recipient mice at day 7 after infection as in g, assessed by flow cytometry. *P < 0.001 (Student's t-test). (i) Flow cytometry of splenocytes from CD45.1+ mice given adoptive transfer of CD45.2+ OT-I or miR-155-deficient OT-I cells and then infected with influenza virus WSN-OVA, assessed at day 60 after infection. Numbers above outlined areas indicate percent CD45.2+CD8+ T cells among lymphocytes. (j) Quantification of donor memory OT-I cells in spleens from recipient mice as in i at day 60 after infection. *P < 0.05 (Mann-Whitney U test). Each symbol (b,c,e,h) represents an individual mouse; small horizontal lines indicate the mean. Data are from three experiments (ac), four experiments (d,e), two experiments with four mice per group (f), two experiments (g,h) or two experiments with six mice per group (i,j; mean and s.e.m. in f,j).

  3. Overexpression of miR-155 augments CD8+ T cell responses.
    Figure 3: Overexpression of miR-155 augments CD8+ T cell responses.

    (a) Population expansion of donor OT-I cells from the lungs of mice given adoptive transfer of OT-I cells retrovirally transduced to express GFP alone (MigR1 OT-I) or GFP plus miR-155 (MigR1–miR-155 OT-I), followed by infection of recipient mice with influenza virus WSN-OVA, assessed at day 10 after infection. *P < 0.05 (Mann-Whitney U-test). (b) Expression of IFN-γ and TNF in donor OT-I cells obtained from mice treated as in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (Student's t-test). (c) Clearance of influenza virus from mice treated as in a. Data are from three independent experiments with eight to ten mice per group (a; mean and s.e.m.), two experiments with five mice per group (b) or three experiments with eight mice per group (c; mean and s.e.m.).

  4. Deficiency in miR-155 impairs the proliferation of CD8+ T cells and enhances the antiproliferative effect of IFN-[beta].
    Figure 4: Deficiency in miR-155 impairs the proliferation of CD8+ T cells and enhances the antiproliferative effect of IFN-β.

    (a) Intracellular Ca2+ flux in wild-type and miR-155-deficient CD8+ T cells stimulated with anti-CD3 (downward arrow). (b) CFSE dilution in purified and CFSE-labeled OT-I and miR-155-deficient OT-I CD8+ T cells left unstimulated (US) or stimulated for 4 d with OVA(257–264)-pulsed irradiated splenocytes (Stim). (c) Absolute number of live CD8+ T cells in each division peak among the cells in b. *P < 0.015 (Student's t-test). (d) Incorporation of [3H]thymidine by purified wild-type and miR-155-deficient CD8+ T cells stimulated for 5 d with anti-CD3 plus various concentrations (horizontal axis) of IL-2. (e) BrdU incorporation by OT-I and miR-155-deficient OT-I cells stimulated with OVA(257–264)-pulsed irradiated splenocytes and left untreated (UT) or treated with IFN-β (1,000 U/ml) throughout culture, assessed by flow cytometry at day 5. Numbers above outlined areas indicate percent CD8+ T cells with BrdU incorporation. (f) Frequency of BrdU+ CD8+ T cells among OT-I and miR-155-deficient OT-I cells treated with increasing doses of IFN-β (horizontal axis), assessed on day 3 (D3) and day 5 (D5) of culture. *P < 0.05, OT-I versus miR-155-deficient OT-I, and **P < 0.05, IFN-β-treated versus untreated (0) miR-155-deficient OT-I (Student's t-test). Data are from one experiment representative of three experiments (a), four experiments (d; mean and s.e.m. of triplicates) or five independent experiments (e), are from five independent experiments with five mice per group (b,c; mean and s.e.m. in c) or are pooled from five independent experiments (f; mean and s.e.m.).

  5. Molecular signature of activated miR-155-deficient CD8+ T cells shows enrichment for genes associated with type I interferon signaling.
    Figure 5: Molecular signature of activated miR-155-deficient CD8+ T cells shows enrichment for genes associated with type I interferon signaling.

    (a) Significance analysis of microarray of the difference in gene expression in wild-type versus miR-155-deficient CD8+ T cells left unstimulated (US; left) or activated in vitro (Act; right), presented as the 'd score' for gene 'i' (d(i)). Numbers in plots indicate number of genes. (b) Network of type I interferon–related genes upregulated in activated miR-155-deficient CD8+ T cells. Color intensity indicates amount of upregulation (red) or downregulation (blue); lines indicate direct (solid lines) and indirect (dotted lines) relationships between genes. (c) Gene-set enrichment analysis showing enrichment of previously defined transcriptional signatures associated with interferon and IL-12 signaling pathways in activated wild-type and miR-155-deficient CD8+ T cells. NES, normalized enrichment score. Colors below indicate the transcriptional profile of wild-type cells (red) and miR-155 deficient cells (blue). Data are from three experiments with three to four mice per group.

  6. Deficiency in miR-155 in CD8+ T cells leads to dysregulated expression of potential miR-155 target genes.
    Figure 6: Deficiency in miR-155 in CD8+ T cells leads to dysregulated expression of potential miR-155 target genes.

    (a) Heat map of the expression of putative miR-155 target genes in unstimulated (US) and in vitro–activated (Act) wild-type and miR-155-deficient CD8+ T cells. Color intensity indicates degree of upregulation (red) or downregulation (blue), row normalized. (b) All predicted miR-155 target genes in miR-155-deficient and wild type CD8+ T cells (left; dark brown); inset (light brown wedge), subset of those at left downregulated in activated versus unstimulated wild type CD8+ T cells. Right, predicted miR-155 targets downregulated in activated wild type CD8+ T cells; inset (light blue wedge), subset of those upregulated in activated miR-155-deficient CD8+ T cells relative to their expression in wild-type CD8+ T cells. Numbers indicate percent genes. (c) RT-PCR analysis of Ikbke and Bach1 (miR-155 target genes) in miR-155-deficient OT-I CD8+ T cells stimulated for 4 d with OVA(257–264)-pulsed irradiated splenocytes; results normalized to expression of GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase) are presented relative to expression in their wild-type counterparts. Data are from three experiments with three to four mice per group (a,b) or two independent experiments with five mice per group (c; mean and s.e.m.).

  7. STAT1 expression is regulated by miR-155 and type I interferon signaling contributes to the proliferative defect of miR-155 deficiency.
    Figure 7: STAT1 expression is regulated by miR-155 and type I interferon signaling contributes to the proliferative defect of miR-155 deficiency.

    (a) STAT1 expression in OT-I and miR-155-deficient OT-I cells activated for 4 d with OVA(257–264)-pulsed irradiated splenocytes. Isotype, isotype-matched control antibody. (b) Mean fluorescence intensity (MFI) of total STAT1 in miR-155-deficient OT-I cells in vitro, relative to that in OT-I cells, set as 1. *P < 0.03 (Student's t-test). (c) Mean fluorescence intensity of total STAT1 in vivo in donor OT-I or miR-155-deficient OT-I cells from mice given adoptive transfer of those cells and infected with influenza virus WSN-OVA, assessed at day 10 after infection ex vivo and presented relative to that of their OT-I counterparts. *P < 0.006 (Student's t-test). (d) Phosphorylation of STAT1 at Tyr701 (p-STAT1(Y701)) in OT-I and miR-155-deficient OT-I cells activated as in a and left untreated or treated with IFN-β (+ IFN-β). (e) STAT5 phosphorylation in activated wild-type and miR-155-deficient CD8+ T cells left unstimulated (filled curves) or stimulated (lines) with IL-2 (left) or IL-15 (right). (f,g) In vivo population expansion of donor OT-I cells in mice given adoptive transfer of OT-I or miR-155-deficient OT-I cells transduced with retrovirus expressing control vector (Ctrl) or DN-STAT1 (f) or DN-IRF7 (g), followed by infection of recipient mice with influenza virus WSN-OVA, assessed day 10 of infection.*P < 0.02 and **P < 0.03 (Student's t-test). Data are from five experiments (a,b), two experiments with five mice per group (c), one experiment representative of four experiments (d) or three experiments (e), two experiments with eight to ten mice per group (f), or three experiments with seven to nine mice per group (g; mean and s.e.m. in b,c,f,g).

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Author information

  1. These authors contributed equally to this work.

    • Donald T Gracias &
    • Erietta Stelekati

Affiliations

  1. Department of Microbiology and Immunology, Center for Immunology and Vaccine Science, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.

    • Donald T Gracias,
    • Erietta Stelekati,
    • Jennifer L Hope,
    • Alina C Boesteanu,
    • Jillian Norton,
    • Yvonne M Mueller,
    • Joseph A Fraietta &
    • Peter D Katsikis
  2. Department of Microbiology and Institute for Immunology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Travis A Doering &
    • E John Wherry
  3. Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK.

    • Martin Turner

Contributions

D.T.G., infection with influenza virus, adoptive transfer, in vitro proliferation, immunoblot analysis, siRNA transfection, retroviral transduction and RT-PCR; E.S., infection with L. monocytogenes, adoptive transfer, in vitro proliferation, siRNA transfection and RT-PCR; J.L.H., A.C.B., J.A.F. and J.N., infection with influenza virus, flow cytometry, BrdU assays, RT-PCR and mouse breeding; T.A.D., E.S. and E.J.W., microarray data analysis; Y.M.M., adoptive transfer and data analysis; E.S., D.T.G., A.C.B., E.J.W., M.T. and P.D.K., study design, data analysis and manuscript authorship; and all authors, discussion of results and comments on the manuscript.

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The authors declare no competing financial interests.

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