Research ArticleVaccines

Influenza vaccines differentially regulate the interferon response in human dendritic cell subsets

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Science Translational Medicine  22 Mar 2017:
Vol. 9, Issue 382, eaaf9194
DOI: 10.1126/scitranslmed.aaf9194
  • Fig. 1. TIV-09, but not MIV-09, activates moDCs.

    (A) moDCs were stimulated with TIV-09 (6 μl/ml), MIV-09 (6 μl/ml), or media for 18 hours. Surface expression of moDC activation markers was assessed by flow cytometry (n = 7). DC-SIGN, DC-specific intercellular adhesion molecule grabbibng nonintegrin. (B) moDCs were cultured in the presence of TIV-09 or MIV-09 (6μl/ml) for 0, 6, 12, 24, and 48 hours. Supernatants were harvested for quantification of cytokines by Luminex (n = 10). Statistics shown represent the comparison between TIV-09 and MIV-09 at each time point. (C) moDCs were stained with annexin V and propidium iodide (PI) to examine cell death at 4, 8, 12, and 24 hours after vaccine treatment (n = 6). (D) Transcriptional analysis of IFNs and other cytokines in moDCs cultured with TIV-09 or MIV-09 for 6 hours. Data were normalized to medium control (n = 6) (*P < 0.05; **P < 0.01; ***P < 0.001, Mann-Whitney U test).

  • Fig. 2. TIV-09 is a strong inducer of type I IFNs.

    (A) moDCs were treated with increasing doses of TIV-09 with or without anti-IFNAR antibody (10 μg/ml) for 8 hours. Supernatants were assessed by Luminex for the presence of type I IFNs and inflammatory cytokines. Statistics shown represent the comparison between TIV-09 and TIV-09 with IFN-α/βR2 antibody (Ab) at each concentration (*P < 0.05; **P < 0.01; ***P < 0.001, Mann-Whitney U test). IgG, immunoglobulin G; LPS, lipopolysaccharide. (B) moDCs were stimulated with TIV-09 (6 μl/ml) with or without anti-IFNAR antibody for 18 hours. Surface expression of DC markers was assessed by flow cytometry. The figure is representative of experiments in four independent donors.

  • Fig. 3. MIV-09 inhibits type I IFNs.

    (A) moDCs were treated with increasing doses of influenza vaccines used during the 2006–2007, 2008–2009, 2009–2010, and 2010–2011 seasons. The graph summarizes data from 10 donors. (B) moDCs were treated with TIV-09 (25 μl/ml) combined with increasing doses of MIV-09 for 8 hours. Supernatants were harvested for enzyme-linked immunosorbent assay (ELISA). The graph summarizes data from seven donors. (C) moDCs were activated with TIV-09 (6 μl/ml), LPS (50 ng/ml), CD40L (1 μg/ml), R848 (3 μg/ml), or CL097 (5 μg/ml) alone or with MIV-09 (10 μl/ml) with or without recombinant IFN-α (2000 U/ml). Supernatant cytokine concentrations were assessed by Luminex. The graph summarizes data from three donors. (D) moDCs were stimulated with TIV-09, LPS, CD40L, R848, or CL097 with or without MIV-09. The surface expression of moDC activation markers was examined by flow cytometry. The figure is representative of experiments in four independent donors. (E) IPA of the transcripts unique to each vaccine and most highly expressed after vaccine exposure. The top three canonical pathways enriched in the transcripts induced by each vaccine are shown (n = 3) (***P < 0.001, Mann-Whitney U test). eIF2, eukaryotic initiation factor 2; mTOR, mechanistic target of rapamycin; PRRs, pattern recognition receptors; JAK1, Janus kinase 1; TYK2, tyrosine kinase 2.

  • Fig. 4. TIV-09 activates cDCs.

    (A) Sorted blood cDCs were stimulated in vitro with media alone, TIV-09, or MIV-09 for 18 hours. Surface expression of activation markers was examined by flow cytometry. The figure is representative of experiments in four independent donors. (B) Transcriptional analysis of IFNs in cDCs stimulated with TIV-09 or MIV-09 for 6 hours. Data were normalized to media control (n = 3). (C) cDCs were activated in vitro with media, TIV-09, or MIV-09 for 0, 6, 24, and 48 hours. IP-10 levels were measured by ELISA. The graph summarizes data from six donors. (D) cDCs were treated with TIV-09 (6 μl/ml) combined with different doses of MIV-09 for 8 hours. Supernatants were harvested, and IP-10 was measured by ELISA. The graph summarizes data from six donors. (E) cDCs were stimulated with TIV-09 in the presence or absence of MIV-09. Surface expression of CD80, CD86, CD83, and HLA-DR was measured by flow cytometry. The figure is representative of experiments in four independent donors (*P < 0.05; Mann-Whitney U test).

  • Fig. 5. TIV-09 activates pDCs.

    (A) Sorted plasmacytoid DCs (pDCs) were activated in vitro with TIV-09 or MIV-09 or control media for 18 hours, and surface expression of activation markers was measured by flow cytometry. The figure is representative of experiments in four independent donors. (B) Transcriptional analysis of IFNs in pDCs stimulated with TIV-09 or MIV-09 for 6 hours. Data were normalized to medium control (n = 3). (C and D) pDCs were activated in vitro with media, TIV-09, or MIV-09 for 0, 6, 24, and 48 hours. IP-10 and IFN-β levels were measured by ELISA. (E) pDCs were treated with TIV-09 (6 μl/ml), combined with different doses of MIV-09 for 8 hours. Supernatants were harvested, and IP-10 levels were measured by ELISA. Figure 5 (C to E) summarizes data from six donors. (F) pDCs were stimulated with TIV-09 in the presence or absence of MIV-09. Surface expression of CD80, CD86, CD83, and HLA-DR was measured by flow cytometry. The figure is representative of experiments in four independent donors (*P < 0.05; Mann-Whitney U test).

  • Fig. 6. TIV-09, but not MIV-09, induces an IFN signature in the blood of vaccinated individuals at day 1.

    Healthy individuals were vaccinated at t = 0 hour with saline, TIV-09, or MIV-09 (n = 3 per group), and blood was drawn at 0, 1.5, 3, 6, 9, 12, 15, 24, and 36 hours and 2, 3, and 7 days. Data were normalized to the median of the t = 0 hour samples across donors. Transcripts from three IFN blood modules (M1.2, M3.4, and M5.12) (36) were selected for analysis. Both transcript- and module-level analyses are displayed. Module expression is calculated as the percentage of transcripts from a specific module that are over- or underexpressed under a specific condition as compared to the t = 0 hour reference control.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/382/eaaf9194/DC1

    Fig. S1. Dose-dependent induction of IFN-β and IP-10 by TIV-09 and MIV-09 in moDCs.

    Fig. S2. IP-10 production by moDCs challenged with various inactivated viral vaccines.

    Fig. S3. Gating strategy for moDC and pDC sorting.

    Table S1. Vaccines used for comparative studies in moDCs.

    Table S2. Primary data.

  • Supplementary Material for:

    Influenza vaccines differentially regulate the interferon response in human dendritic cell subsets

    Shruti Athale, Romain Banchereau, LuAnn Thompson-Snipes, Yuanyuan Wang, Karolina Palucka, Virginia Pascual, Jacques Banchereau*

    *Corresponding author. Email: jacques.banchereau{at}jax.org

    Published 22 March 2017, Sci. Transl. Med. 9, eaaf9194 (2017)
    DOI: 10.1126/scitranslmed.aaf9194

    This PDF file includes:

    • Fig. S1. Dose-dependent induction of IFN-β and IP-10 by TIV-09 and MIV-09 in moDCs.
    • Fig. S2. IP-10 production by moDCs challenged with various inactivated viral vaccines.
    • Fig. S3. Gating strategy for moDC and pDC sorting.
    • Table S1. Vaccines used for comparative studies in moDCs.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Primary data.

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