MCMV activates Ly49H⁺ NK cells and expands Ag-specific CD8⁺ T cells from Siglec-H^−/− and wt mice with similar viral clearance in the primary and secondary organs.

<p>Siglec-H^−/− and wt chimeric mice were infected with 5×10⁴ PFU of wt MCMV or mock treated with PBS. (<b>A, B</b>) IFNγ serum concentrations were determined at 36 h p.i. and the MFI of CD69 expression on NK1.1⁺ blood NK cells was quantified by flow cytometry. (<b>C, D</b>) Representative histogram overlays and quantification of KLRG-1 expression on splenic NK1.1⁺ Ly49H⁺ cells at day 8 p.i. (<b>E</b>) Representative FACS plots for H-2D^b M45 tetramer staining and CD62L expression among CD8⁺ splenocytes. (<b>F, G</b>) Quantification of frequencies and absolute numbers of tetramer⁺ CD8⁺ T cells from the tetramer staining shown in (E). (<b>H</b>) Shows frequencies of IFNγ⁺ cells among CD8⁺ T cells after restimulation with the indicated concentrations of H-2D^b M45 peptide. (<b>I</b>) Viral load was measured in the spleen, liver and salivary glands of MCMV infected Siglec-H^−/− and wt mice at day 3, 6 and 8 p.i. Dashed line indicates the limit of detection. Data shown are pooled from 2–3 individual experiments. **<i>p</i><0.01 Students t-test, ns = not significant, nd = not detectable.</p

Characterization of the pDCre x RFP reporter line shows terminal targeting of pDCs.

<p>(<b>A, B</b>) BM and spleen cells from pDCre x RFP mice were stained for Siglec-H, CD11c, MHCII, CD3ε, CD19 and NK1.1 (<b>A</b>) Characterization of the reporter expression (RFP) by different cell types: pDCs were gated as Siglec-H⁺, B cells as Siglec-H⁻ CD19⁺ CD3ε⁻ NK1.1⁻, T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁻, NK-T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁺, and NK cells as Siglec-H⁻ CD19⁻ CD3ε⁻ CD11c^int NK1.1⁺. FACS plots are representative for two individual experiments. (<b>B</b>) Characterization of the RFP reporter expression by CD11c^hi MHCII^hi cDCs in spleen and CD11c^int CD19⁻ CD3ε⁻ NK1.1⁻ Siglec-H⁻ cells in BM. (<b>C</b>) Quantification of (A, B) showing pooled data from 2 independent experiments using 4–5 mice/group. (<b>D</b>) Phenotypic comparison of pDC markers expressed by RFP⁻ and RFP⁺ pDCs from BM (top panel) and spleen (bottom panel). pDCs were gated as Siglec-H⁺ CD11c^int. Histogram overlays display the isotype controls as dashed line, and the marker expression by RFP⁻ pDCs as grey filled histogram and by RFP⁺ pDCs as bold line. Data shown are from one representative experiment out of two using 4 mice/group. (<b>E</b>) Splenic <i>ex vivo</i> pDCs were purified by FACS sorting from B16-Flt3L treated pDCre x RFP mice and incubated with the indicated MOIs of MCMV <i>in vitro</i>. IFNα/TNFα concentrations were quantified in the supernatants after 24 h incubation by ELISA or cytometric bead assay. Data shown are from one representative experiment out of two using a pool of 3 mice. The differences between RFP⁻ and RFP⁺ pDCs were not significant as calculated by Students t-test. Data displayed in (D, E) are from one out of two individual experiments with similar results. Data are displayed as mean ± SD.</p

IFNα serum levels are elevated in the absence of Siglec-H upon MCMV infection.

<p>Lethally irradiated CD45.1⁺ wt mice were reconstituted with CD45.2⁺ wt or Siglec-H^−/− BM followed by infection with 5×10⁴ PFU of wt MCMV. (<b>A</b>) FACS plots show efficient donor reconstitution in the blood eight weeks after BM transfer (upper panel). Siglec-H and B220 stainings of splenocytes confirm the lack of Siglec-H expression in wt mice reconstituted with Siglec-H^−/− BM (lower panel). (<b>B</b>) The kinetics of serum IFNα levels 1.5, 3 and 6 days p.i. compared between Siglec-H^−/− and wt mice, n = 4–5 mice/group. Data are from one of two individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p

Siglec-H is downregulated upon MCMV infection <i>in vivo</i>.

<p>pDCre x RFP mice were infected with 5×10⁴ PFU MCMV <i>in vivo</i>. (<b>A</b>) Representative FACS plots show CD11c versus Siglec-H of live splenocytes at 36 h p.i. (<b>B</b>) Gating strategy showing exclusion of B-, T-, NK-T, and NK cells by CD19, CD3ε, NK1.1 in a default channel. RFP⁺ pDCs were gated as CD11c^int B220⁺ MHCII^int to exclude MHCII⁻ DC precursors. Histogram overlays show RFP⁺ pDCs from mock (bold line) and MCMV infected (dashed line) pDCre x RFP mice at 36 h p.i. Isotype staining is displayed as grey histogram. Data are representative of 2 independent experiments using 4–5 mice with comparable results.</p

Siglec-H receptor does not play a role in pDC infection.

<p>Flt3-L derived CD11c⁺ B220⁺ pDCs and CD11c⁺ B220⁻ cDCs were sorted from BMDC cultures of wt and Siglec-H^−/− mice. DCs were mock treated with PBS or MCMV-GFP infected at MOI 2. (<b>A</b>) Representative FACS plots show Siglec-H expression and MCMV-GFP expression at 24 h p.i. (<b>B</b>) Quantification of the viral titers per 10⁶ DCs or MEFs at 24 h p.i. (<b>C</b>) Representative histogram plots of CD86 expression on wt and Siglec-H^−/− sorted pDCs (live cell gate) at 24 h p.i. (gray bar = isotype control, gray dashed line = mock treatment, gray solid line = CpG-A treatment, dark bold line = MCMV infection). Data are from one of three individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p