ARTICLES https://doi.org/10.1038/s41590-019-0345-x Interferon-λ enhances adaptive mucosal immunity by boosting release of thymic stromal lymphopoietin Liang Ye1, Daniel Schnepf1,2, Jan Becker1, Karolina Ebert3, Yakup Tanriver3,4,5, Valentina Bernasconi 6, Hans Henrik Gad7, Rune Hartmann 7, Nils Lycke6 and Peter Staeheli 1,4* Interferon-λ (IFN-λ) acts on mucosal epithelial cells and thereby confers direct antiviral protection. In contrast, the role of IFN-λ in adaptive immunity is far less clear. Here, we report that mice deficient in IFN-λ signaling exhibited impaired CD8+ T cell and antibody responses after infection with a live-attenuated influenza virus. Virus-induced release of IFN-λ triggered the synthesis of thymic stromal lymphopoietin (TSLP) by M cells in the upper airways that, in turn, stimulated migratory dendritic cells and boosted antigen-dependent germinal center reactions in draining lymph nodes. The IFN-λ–TSLP axis also boosted pro- duction of the immunoglobulins IgG1 and IgA after intranasal immunization with influenza virus subunit vaccines and improved survival of mice after challenge with virulent influenza viruses. IFN-λ did not influence the efficacy of vaccines applied by sub- cutaneous or intraperitoneal routes, indicating that IFN-λ plays a vital role in potentiating adaptive immune responses that initiate at mucosal surfaces. nterferon-λ (IFN-λ) is an antiviral cytokine produced in response M cells which, in turn, influences germinal center responses by act- to viral infection in a variety of cell types, including airway epithe- ing on migratory dendritic cells (DCs). This previously unknown Ilial cells1,2. IFN-λ acts by binding to a heterodimeric surface recep- IFN-λ–TSLP axis strongly enhances mucosal immunity and confers tor consisting of the ubiquitously expressed IL-10 receptor β-chain enhanced influenza virus resistance. and a second IFN-λ-specific chain designated IFNLR1 (refs. 2–4). IFNLR1 is present on epithelial cells but seemingly absent on most Results immune cells of mice2,3, with the exception of neutrophils5–8. IFN-λ Defective antibody production and weak CD8+ T cell responses in induces an antiviral response in epithelial tissues and thus protects Ifnlr1–/– mice after infection with live-attenuated influenza virus. against infection with viruses that replicate preferentially in the To detect possible impairments in adaptive immunity resulting from respiratory tract9,10 or in the intestine11–13. IFN-λ further protects IFN-λ deficiency, we infected Mx1-WT and Mx1-Ifnlr1–/– mice with against fungal infections of the respiratory tract in mice by activat- a live-attenuated influenza A virus strain, designated hvPR8-ΔNS1. ing neutrophils to produce reactive oxygen species8. These mice carry functional alleles of the IFN-regulated influenza Little is known concerning the role of IFN-λ in adaptive immu- virus resistance gene Mx1 required for revealing the full antiviral nity. The IFN-λ receptor is expressed on B cells from humans7,14 but potential of IFN10. The hvPR8-ΔNS1mutant virus cannot produce not mice6. A study in the human system indicated that IFN-λ is a the NS1 virulence factor and, therefore, fails to suppress the IFN negative regulator of adaptive immunity that can suppress vaccine- response of the host. NS1-deficient influenza viruses are largely induced antibody production by a poorly defined mechanism14. non-pathogenic and trigger strong B and T cell responses in immu- However, another study showed that IFN-λ can stimulate rather nocompetent mice17,18. Mx1-Ifnlr1–/– mice had 8–15-fold reduced than inhibit IgG synthesis by human B cells7. The reasons for these levels of influenza virus hemagglutinin (HA)-specific serum IgG1 discrepant findings remain elusive. In mice, IFN-λ was reported to compared with Mx1-WT mice at 14 and 21 days post-infection with boost CD8+ T cell-mediated immunity by reducing regulatory T cell hvPR8-ΔNS1, whereas serum levels of other IgG subtypes were populations during DNA vaccination15. In another study, IFN-λ was normal (Fig. 1a). HA-specific IgA levels in bronchoalveolar lavage found to enhance neutralizing antibody titers against herpes simplex (BAL) fluids of Mx1-Ifnlr1–/– mice at day 21 post-infection were also virus 2 in mice immunized with a DNA vaccine16, but the mecha- significantly reduced compared with Mx1-WT mice (Fig. 1b). Mx1- nism by which IFN-λ confers adjuvant activity was not resolved. Ifnlr1–/– mice further contained significantly fewer influenza virus We searched for immunomodulatory effects of IFN-λ dur- nucleoprotein-specific CD8+ T cells in the mediastinal lymph nodes ing viral infection of the respiratory tract and found that IFN-λ is at day 7 post-infection with hvPR8-ΔNS1 compared with Mx1-WT required for full-scale production of virus-specific IgG1 and IgA, as mice (Fig. 1c). well as efficient generation of antiviral CD8+ T cells. Mechanistically, In agreement with earlier work6,9,19, we failed to detect functional we show that IFN-λ triggers the synthesis of TSLP in upper airway IFN-λ receptors on naive (Fig. 1d) or antigen-experienced mouse 1Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany. 2Spemann Graduate School of Biology and Medicine, Albert Ludwigs University Freiburg, Freiburg, Germany. 3Institute of Medical Microbiology, Medical Center University of Freiburg, Freiburg, Germany. 4Medical Faculty, University of Freiburg, Freiburg, Germany. 5Department of Internal Medicine IV, Medical Center University of Freiburg, Freiburg, Germany. 6Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden. 7Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. *e-mail: [email protected] NATURE IMMUNOLOgy | VOL 20 | MAY 2019 | 593–601 | www.nature.com/natureimmunology 593 ARTICLES NATURE IMMUNOLOGY a IgG IgG1 IgG2c IgG2b b IgA ) 10 5 5 5 5 WT 1.2 * WT **** –/– –/– 4 4 **** 4 4 Ifnlr1 0.9 Ifnlr1 3 3 3 3 0.6 2 2 2 2 1 1 1 1 0.3 0 0 0 0 0.0 HA-specific Ab (OD) HA-specific Ab (log 7 14 21 7 14 21 7 14 21 7 14 21 21 days post-infection Days post-infection c –/– d Control WT Ifnlr1 Mock IFN-λ IFN-α 105 2 WT 100 500 4 –/– ** 0.02 0.68 0.29 cell (%) Ifnlr1 80 400 10 + 60 300 103 * CD8 1 + 40 200 2 10 20 100 0 p-STAT1 MFI Pentamer-PE 0 0 Normalized to mode 0 3 4 5 3 4 0 10 10 10 Pentamer 0 10 10 CD8-FITC p-STAT1-PE e + + f B220 B cells MFI CD19 B cells MFI –/– WT → WT Ifnlr1 → WT IFN- WT Ifnlr1–/– Ifnlr1–/– Ifnlr1–/– –/– α 155 223 → → IFN- 158 242 ) λ 6 10 Ifnar1 Mock 155 241 5 *** ** IFN-α 303 546 –/– 4 IFN-λ 201 293 Ifnlr1 Mock 214 296 3 IFN-α 324 507 2 IFN-λ 188 265 WT 1 Mock 174 328 HA-specific lgG1 (log 0 01103 104 105 0 103 104 05 21 days post-infection p-STAT1-PE Fig. 1 | Defective immune response in Ifnlr1–/– mice after infection with live-attenuated influenza virus is not rescued by hematopoietic cells from wild type mice. a, HA-specific antibodies in sera of Mx1-WT (n = 8) and Mx1-Ifnlr1–/– (n = 7) mice after infection with hvPR8-ΔNS1. The data are representative of three independent experiments. ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test. b, HA-specific IgA in BAL fluids of Mx1-WT and Mx1-Ifnlr1–/– mice after infection with hvPR8-ΔNS1. Data are representative of two independent experiments with seven mice per group. *P = 0.0117, by unpaired two-tailed Student’s t-test. c, Mx1-WT (n = 5) and Mx1-Ifnlr1–/– (n = 4) mice were infected with hvPR8-ΔNS1 virus. Seven days later, the frequency of influenza virus nucleoprotein-specific CD8+ T cells in mediastinal lymph nodes (mLNs) was measured by flow cytometry using labeled H-2Db/ ASNENMETM pentamer. *P = 0.0157, by unpaired two-tailed Student’s t-test. Data are representative of two independent experiments. d, Isolated spleen cells from naive mice were mock-treated or stimulated in the presence or absence of 1 µg ml−1 IFN-α or IFN-λ2 for 30 min before the levels of phosphorylated STAT1 (p-STAT1) in CD19+ B cells were quantified by flow cytometry. Data are pooled from two independent experiments with six mice per group. **P = 0.0031, by unpaired two-tailed Student’s t-test. e, Mx1-WT (n = 3), Mx1-Ifnar1–/– (n = 3) and Mx1-Ifnlr1–/– (n = 3) mice were vaccinated by intranasal infection with 105 PFU of hvPR8-ΔNS1. At day 21 post-infection, isolated spleen cells were stimulated with 1 µg ml−1 IFN-α or IFN-λ2 for 30 min before levels of p-STAT1 were quantified by flow cytometry. Geometric means of fluorescence intensity (MFI) for p-STAT1 in B220+ or CD19+ B cells are depicted. f, Serum IgG1 levels in BM chimeric mice (n = 6 per group) after infection with hvPR8-ΔNS1. ***P < 0.001 and **P < 0.01, by one-way ANOVA with Tukey’s multiple-comparison test. a–f, Error bars represent s.e.m. centered on the mean. B cells (Fig. 1e). To determine whether IFN-λ receptor expression intranasally to Mx1-WT mice, only low HA-specific serum IgG on other immune cells might be important for adequate immune titers were observed after one priming and two booster immuniza- responses to live-attenuated influenza virus, we generated bone tions (Fig. 2a). However, when this vaccine was enriched by adding marrow (BM) chimeric mice and measured HA-specific IgG1 levels 1 µg of mouse IFN-λ2 (ref. 20), HA-specific total serum IgG levels in serum after infecting these animals with hvPR8-ΔNS1.