Type III interferon (IFN-λ) is important for innate immune protection at mucosal surfaces and has therapeutic benefit against influenza A virus (IAV) infection. However, the mechanisms by which IFN-λ programs adaptive immune protection against IAV are undefined. Here we found that IFN-λ signaling in dendritic cell (DC) populations was critical for the development of protective IAV-specific CD8+ T cell responses. Mice lacking the IFN-λ receptor (Ifnlr1−/−) had blunted CD8+ T cell responses relative to wild type and exhibited reduced survival after heterosubtypic IAV re-challenge. Analysis of DCs revealed IFN-λ signaling directed the migration and function of CD103+ DCs for development of optimal antiviral CD8+ T cell responses, and bioinformatic analyses identified IFN-λ regulation of a DC IL-10 immunoregulatory network. Thus, IFN-λ serves a critical role in bridging innate and adaptive immunity from lung mucosa to lymph nodes to program DCs to direct effective T cell immunity against IAV.
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We would like to thank the Cell Analysis Facility Flow Cytometry and Imaging Core in the Department of Immunology at the University of Washington and E. Smith for technical assistance. We would like to thank A.M. Kell and M.E. Long for their critical reading of this manuscript. E.A.H. is supported by American Heart Association award 17POST33660907. This work was also supported by National Institutes of Health grants T32AI007509 (E.A.H.), AI132962 (R.A.L.), AI108765 (R.S.), AI104002 (M.G.), AI118916 (M.G.), AI083019 (M.G.) and AI127463 (M.G.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 IFN-λ signaling contributes to controlling viral load, but not disease severity, during primary IAV infection. WT and Ifnlr1−/− mice were mock- or IAV-infected with 40 PFU of the mouse-adapted H1N1 strain A/PR/8/34 (PR8)
. a. On days 1, 3, 5, 7, and 9 p.i., lungs were harvested, homogenized, and pulmonary virus titer was determined by plaque assay. Each point represents an individual animal, and data from two, pooled independent experiments are shown. Bars shown mean ± SEM. Day 0 n = 4/group, Day 1 n = 6/group, Day 3 n = 7/group, Day 5 n = 6/group, Day 7 n = 7 (WT) or 6 (Ifnlr1−/−)/group, Day 9 n = 12/group. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. *indicates p<0.05 b & c. Weight loss (b) and illness score (c) was monitored over 12 days. Dashed line at illness score of 4 indicates presence of respiratory symptoms. n=2 (mock), 7 (WT IAV), or 6 (Ifnlr1−/− IAV) Symbols show mean and error bars represent SD.
Supplementary Figure 2 IFN-λ signaling is not required for development of antibody responses in bronchoalveolar lavage fluid following IAV.
WT and Ifnlr1−/− mice were mock- or IAV-infected with PBS or 40 PFU PR8. Bronchoalveolar lavage fluid was harvested on D35 post infection, and IAV-specific IgM, IgG, IgG1, IgG2a, and IgA antibody levels were determined by ELISA. n=2 (mock) or 4 (IAV) mice/group. Error bars show mean ± SD. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test between WT IAV and Ifnlr1−/− IAV. n.s. indicates p=0.0504, * indicates p<0.05, ** indicates p<0.0001.
Supplementary Figure 3 IFN-λ signaling is dispensable for effector IAV-specific CD4+ T cells responses in the lung.
WT and Ifnlr1−/− mice were mock- or IAV-infected with PBS or 40 PFU PR8 expressing a 2W epitope, respectively. Lungs were harvested on day 9 p.i. and frequency (% of CD4+ T cells) and numbers of NP311- (a) and 2W1S- specific (b) CD4+ T cells were determined by flow cytometry. One representative of two experiments with similar results shown. Bars shown mean ± SD. n=2 (mock) or 3 (IAV) mice/group. c. On day 9 post infection, a portion of the whole lung homogenates were stimulated with media, IAV (NP311) peptide, or PMA + ionomycin for 6 hrs in the presence of brefeldin A. Following stimulation, IFN-γ, TNF, IL-17A, and IL-10 levels were determined by flow cytometry. Two pooled independent experiments are shown with n=6 (WT IAV), or 7 (Ifnlr1−/− IAV) mice/group. Bars show mean ± SEM. Statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparisons test. * indicates p<0.01.
a. WT, Ifnar1−/−, or Ifnlr1−/− BMDCs were left unstimulated or stimulated with 500 ng recombinant murine IFN-λ3 or 10 ng recombinant murine IFNα2 for 1 hr. b. WT BMDCs were stimulated with increasing doses of recombinant murine IFN-λ3 for 30 min. For a and b, following stimulation cells were washed, collected in RIPA buffer, and western blots were performed for phosphorylated STAT1 (Y701), STAT1, and actin. Data shown from one of three representative experiments. c. WT BMDCs were left unstimulated or stimulated with 500 ng recombinant murine IFN-λ3 for 12 hrs. Following stimulation, relative gene expression of Ifi44, Ifit1, Isg15, and Oas3 was determined. Data from three, pooled independent experiments shown where bars represent the mean ± SEM. Statistical significance was determined using unpaired two-sided t-test. * indicates p<0.05, ** indicates p<0.01, *** indicates P<0.001.
Supplementary Figure 5 IFN-λ signaling in LysM+ cells is dispensable for effector IAV-specific CD8+ and CD4+ T cells responses in the lung.
Ifnlr1fl/fl and Ifnlr1fl/flLyz2-cre mice were administered 40 PFU PR8 containing a 2W epitope i.n. a. Weight loss was monitored through day 9 p.i. Symbol shows mean ± SD. b–d. Lungs were harvested on day 9 p.i. and frequency (% of CD8+ or CD4+ T cells) and numbers of PA224-specific CD8+ T cells (b), 2W1S- (c) and NP311-specific (d) CD4+ T cells were assessed by flow cytometry. Data from two pooled experiments shown. For b and c n = 6 mice/group. For d n=5 (Ifnlr1fl/fl) or 6 (Ifnlr1fl/flLyz2-cre) mice/group. For b–d bars show mean ± SEM.
Supplementary Figure 6 IFN-λ signaling in DCs is required for optimal DC function and generation of CD8+ T cell responses capable of mediating protection against heterosubtypic IAV re-challenge.
Following IAV infection, DCs become activated and migrate to initiate IAV-specific CD8+ T cell responses capable of mediating protection against heterosubtypic IAV re-challenge. In Ifnlr1−/−, DC migration and activation are aberrant, and DCs produce IL-10, which correlates with a reduced IAV-specific CD8+ T cell responses that is unable to protect against heterosubtypic IAV re-challenge.