Interferon-λ enhances adaptive mucosal immunity by boosting release of thymic stromal lymphopoietin

Abstract

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 production 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 subcutaneous or intraperitoneal routes, indicating that IFN-λ plays a vital role in potentiating adaptive immune responses that initiate at mucosal surfaces.

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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.
Fig. 2: IFN-λ enhances IgG1 and IgA production after intranasal application of influenza subunit vaccines.
Fig. 3: The immune-enhancing effect of IFN-λ depends on TSLP.
Fig. 4: Upper airway M cells produce TSLP after IFN-λ stimulation.
Fig. 5: IFN-λ promotes TSLP-dependent germinal center reactions.
Fig. 6: IFN-λ-triggered TSLP acts on CD103+ migratory DCs.
Fig. 7: Mucosal immunization in the presence of IFN-λ results in enhanced protection from influenza virus-induced disease.
Fig. 8: IFN-λ-adjuvanted M2e vaccine reduces influenza virus transmission.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank A. Ohnemus for technical support, and H. Pircher, G. Gasteiger and O. Haller for helpful discussions and comments on the manuscript. Funding for this work was provided by the European Union’s Seventh Framework Program grant agreement 607690 (to P.S. and N.L.), the Deutsche Forschungsgemeinschaft grant agreement STA 338/15-1 (to P.S.) and TA 436/4-1 (to Y.T.), the Else Kröner-Fresenius Stiftung grant agreement 2017_EKES.34 (to Y.T.) and the Danish Council for Independent Research, Medical Research grant agreement 11‐107588 (to R.H.).

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L.Y. designed and performed most of the experiments, analyzed and interpreted the data. D.S. designed and performed experiments, analyzed and interpreted the data. J.B. performed experiments and analyzed samples. K.E. and D.S. generated bone marrow chimeric mice. V.B. and H.H.G. provided essential materials. Y.T., R.H. and N.L. analyzed and interpreted data, and gave advice. P.S. conceived the project, acquired funding for the study, designed experiments and interpreted the data. P.S., L.Y. and D.S. wrote the manuscript with input from all authors.

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Correspondence to Peter Staeheli.

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Integrated supplementary information

Supplementary Figure 1 IFN-λ exhibits no immunostimulatory effect when antigen is applied by the intraperitoneal or subcutaneous route.

a-b, Titers of HA-specific IgG subclasses on day 10 after the first and second booster immunization in sera of Mx1-WT mice immunized by the intraperitoneal (a) or subcutaneous (b) route with Influsplit Tetra® in the presence or absence of IFN-λ2. Data are representative of two independent experiments with n=6 (a) and n=7 (b) animals per group. Error bars represent SEM centered on the mean. Each symbol represents an individual animal. c-d, Titers of M2e-specific IgG subclasses on day 10 post booster immunization in sera of Mx1-WT mice immunized by either the intraperitoneal (c) or subcutaneous (d) route with M2e vaccine in the presence or absence of IFN-λ2. Data are representative of two independent experiments with n=6 animals per group. Error bars represent SEM centered on the mean. Each symbol represents an individual animal.

Supplementary Figure 2 IFN-λ-triggered enhanced antibody synthesis depends on TSLP produced by upper airway M cells.

a, upper panels, Representative flow cytometry histograms of TSLP staining in cell subsets from snouts of Mx1-WT and Mx1-Ifnlr1–/– mice infected with hvPR8-ΔNS1 for 24 h. a, lower panels, MFI of TSLP signals in Mx1-WT (n=6 per group) and Mx1-Ifnlr1–/– mice (n=6 in mock group, n=5 in infected group) infected with hvPR8-ΔNS1 for 24 h. Data are pooled from two independent experiments. Error bars represent SEM centered on the mean. **p=0.01, *p=0.0166 by unpaired two-tailed Student’s . b, upper panels, Representative flow cytometry histograms of TSLP staining in cell subsets from snouts of Mx1-WT and Mx1-Ifnlr1–/– mice treated with IFN-λ2 for 24 h. b, lower panels, MFI of TSLP signals in Mx1-WT (n=6 in mock group and n=9 in infected group) and Mx1-Ifnlr1–/– mice (n=7 per group) treated with IFN-λ2 for 24 h. Data are pooled from two independent experiments. Error bars represent SEM centered on the mean. ****p<0.0001 by unpaired two-tailed Student’s t test. c, Levels of Tslp and Mx1 mRNA in purified snout cell subsets from IFN-λ2-treated Mx1-WT mice. Cells originating from 12 animals per group were pooled prior to cell sorting. Data are pooled from three independent experiments. Error bars represent SEM centered on the mean. *p=0.0252 by unpaired two-tailed Student’s t test.

Supplementary Figure 3 Gating strategy used to isolate M cells from the upper airways of mice.

a, Snout cells were gated based on forward and side scatters, then single cells, live cells, CD45+ cells (CD45+ EpCAM-), epithelial cells (CD45- EpCAM+), M+ cells (CD45- EpCAM+ NKM16-2-4+) and M- cells (CD45- EpCAM+ NKM16-2-4-) were gated. b, Strategy for sorting CD45+ cells (CD45+ EpCAM-, 99% purity), M+ cells (CD45- EpCAM+ NKM16-2-4+, 93.9% purity) and M- cells (CD45- EpCAM+ NKM16-2-4-, 98.9% purity) from Mx1-WT mice treated with IFN-λ2 for 4 h.

Supplementary Figure 4 M cells in mouse airway epithelial cells at the air–liquid interface of cultures do not substantially upregulate TSLP synthesis upon stimulation with IFN-λ.

a-b, In vitro differentiated airway epithelial cells from Mx1-WT mice were treated with or without IFN-λ2 for 24 h before expression of TSLP in M+ (EpCAM+ NKM16-2-4+) (a) and M- (EpCAM+ NKM16-2-4-) cells (b) was analyzed by flow cytometry. Each symbol represents an individual replicate. a-b, n=7 and n=9 independent cell culture wells for mock and IFN-λ2-treated groups, respectively. Data are pooled from two individual experiments and shown as mean ± SEM. *p=0.0149 (a) and p=0.0146 (b) by unpaired two-tailed Student’s t test.

Supplementary Figure 5 IFN-λ increases the frequency of Tfh cells and GC-B cells in the spleen of immunized mice in a TSLP-dependent manner.

a-c, WT (n=3 for M2e group and n=4 for M2e+IFN-λ group) and Tslpr–/– (n=4 per group) mice intranasally immunized with M2e in the presence or absence of IFN-λ2 were sacrificed on day 5 post boosting, and the frequencies of CXCR5+ PD1+ Tfh cells among CD19- CD4+ CD44+ cells (a), GL7+ Fas+ GC-B cells among CD4- B220+ cells (b) and IgG1+ Fas+ GC-B cells among CD4- B220+ cells (c) in the spleen were determined. a-c, left panels, Representative flow cytometry histograms. a-c, right panels, data are from one of two representative experiments. Each symbol represents an individual animal. Shown are mean ± SEM. ****P<0.0001 (a, left), ****P<0.0001 (a, right), **p=0.0012 (b, left), **p=0.0011 (b, right), ***p=0.0001 (c, left), ***p=0.0006 (c, right) by unpaired two-tailed Student’s t-test.

Supplementary Figure 6 IFN-λ does not induce GC reactions in the absence of antigen.

a-c, WT and Tslpr–/– mice were mock-immunized in the presence or absence of IFN-λ2. Animals were sacrificed on day 5 post boosting, and the frequencies of CXCR5+ PD1+ Tfh cells among CD19- CD4+ CD44+ cells (a), GL7+ Fas+ GC-B cells among CD4- B220+ cells (b) and IgG1+ Fas+ GC-B cells (c) among CD4- B220+ cells in the spleen and draining LN were determined. a, n=5 mice per group. b-c, left panel, n=5 mice per group. b-c, right panel, n=4 in mock group, n=5 in IFN-λ group. Each symbol represents an individual animal. Error bars represent SEM centered on the mean.

Supplementary Figure 7 Mucosal immunization in the presence of IFN-λ results in enhanced suppression of the replication of influenza virus in lungs.

Mx1-WT mice were intranasally immunized with M2e vaccine in the presence (n=7) or absence (n=6) of IFN-λ2. Ten days after booster immunization, the animals were challenged with hvPR8. Viral titers were measured by plaque assay at day 2 post-infection. Mock-immunized mice (PBS) served as additional controls (n=6). Each symbol represents an individual animal. Data are shown as mean ± SEM. ****p<0.0001 by one-way ANOVA with Tukey’s multiple-comparison test.

Supplementary Figure 8 TSLP-adjuvanted M2e vaccine reduces the transmission of influenza virus from infected mice to naive cage mates.

BALB/c mice were intranasally immunized with M2e vaccine in the presence (n=6) or absence (n=6) of TSLP. Mock-immunized animals (PBS) served as additional controls (n=3). Eight weeks after booster immunization, the animals were infected with influenza virus strain A/Udorn. Twenty-four hours later, each infected BALB/c mouse was co-housed with one naive DBA/2J mouse for 4 days. Virus transmission to contact mice was assessed by measuring infectious virus in the snouts of immunized BALB/c (Index) and exposed DBA/2J mice (Contact) on day 4 post cohousing. Each symbol represents an individual animal. Small horizontal lines indicated the mean. The dotted line indicates the detection limit of the assay.

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Ye, L., Schnepf, D., Becker, J. et al. Interferon-λ enhances adaptive mucosal immunity by boosting release of thymic stromal lymphopoietin. Nat Immunol 20, 593–601 (2019). https://doi.org/10.1038/s41590-019-0345-x

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