Fig 1.
IFNAR in hematopoietic cells is necessary and sufficient for resistance to lethal mousepox.
(A) Survival of the indicated BMCs infected with 3000 pfu of ECTV-GFP in the footpad. Data were pooled from two independent experiments (Log-rank Mantel-Cox compared to B6→ B6 control group). (B) ECTV titers in livers of survivors at endpoint (30 dpi) quantified by plaque assay. The dashed line indicates the detection limit. Positive control: liver sample harvested from infected Ifnar1-/- mice at 5 dpi. (C-D) ECTV titers in the livers and spleens of each BMC group were quantified by plaque assay at 5 (C) and 7 dpi (D). The dashed line indicates the detection limit. (E) Number of live splenocytes in BMCs naïve or at 5 and 7 dpi with ECTV. Data are represented as mean ± SEM from three pooled independent experiments (N = 11 per group, multiple comparison ANOVA with Tukey correction). (F) Isg15 transcripts in the spleen at 5 dpi quantified by qPCR, normalized to Gapdh, and adjusted to fold-change to naïve. Data are represented as mean ± SEM from two independent experiments (N = 6 per group, multiple comparison ANOVA with Tukey correction). (G-H) Cre-driven excision of Ifnar1 gene exon10 in the livers (G) and the spleens (H) of the indicated naïve mice measured by qPCR, normalized by Gapdh transcription, and adjusted to fold-change to B6. (I) Survival of the indicated mice infected as in (A). Data pooled from two independent experiments (Log-rank Mantel-Cox compared to Ifnar1fl/fl mice). (J-K) ECTV titers in the livers and spleens of indicated mice at 5 (J) and 7 dpi (K) quantified as in (C-D). Data are represented as mean ± SEM from two or three pooled independent experiments (N = 8–9 for each Cre+ mice and N = 12–18 for Ifnar1fl/fl mice, ANOVA with Tukey correction compared to Ifnar1fl/fl mice).
Fig 2.
IFNAR in hematopoietic cells is necessary for efficient T cell responses to ECTV.
BMC mice were infected as in Fig 1A, and spleens were harvested at 7 dpi. (A) Representative flow cytometry plots showing the frequency of CD44+ and GzmB+ cells on gated CD8 T cells (NK1.1-TCRβ+CD8+) and on CD4 T cells (NK1.1-TCRβ+CD4+). (B) Percentages and numbers of cytotoxic GzmB+ CD8 and CD4 T cells. (C) Percentages and numbers of antigen experienced CD44+ CD8 and CD4 T cells. Data are represented as mean ± SEM from three pooled independent experiments (N = 11 per group, N = 3 for Ifnar1-/-→ Ifnar1-/-, multiple comparison ANOVA with Tukey correction).
Fig 3.
Intrinsic IFNAR on adaptive lymphocytes is dispensable for efficient T cell responses and resistance to mousepox.
(A) CFSE-labeled WT (F1 [B6.CD45.1 x B6.CD45.2) and Ifnar1-/- (CD45.2) splenocytes mixed in 1:1 ratio were adoptively transferred i.v. into naïve or infected mousepox resistant WT hosts (B6.CD45.1), and spleens were harvested at 7.5 days following ECTV infection and transfer. Created with BioRender.com. (B) Representative flow cytometry histograms of CFSE dilution in gated CD8 T cells (NK1.1-TCRβ+CD8+) in infected and naïve hosts. (C) Percentages of CFSElow CD8 and CD4 T cells (NK1.1-TCRβ+CD4+). (D) Representative flow cytometry plots showing staining with anti-CD44 and -GzmB (top) or anti-CD44 and Kb-TSYKFESV dimers (bottom) of donor WT or Ifnar1-/- CD8 T cells. (E) Percentages of CD44+ GzmB+ CD8 and CD4 T cells. (F) Percentages of CD44+ Kb-TSYKFESV+ CD8 T cells. Data are represented as mean ± SEM from two pooled independent experiments (N = 9 for infected hosts and N = 6 for naïve hosts). (G) Survival of indicated BMC infected with 3000 pfu of ECTV-GFP in the footpad (Log-rank Mantel-Cox compared to Rag1-/- + Ifnar1-/-→ Rag1-/- and to Rag1-/- + B6 → Rag1-/-). (H) ECTV viral titers in the livers of survivors at endpoint (30 dpi) quantified by plaque assay. The dashed line indicates the detection limit. Positive control: liver sample harvested from infected Ifnar1-/- mice at 5 dpi. (I) Concatenated flow cytometry plots showing levels of Kb-TSYKFESV+ CD8 T cells and levels of CD44 expression in CD8 and CD4 T cells in the blood of survivor mice at 22 dpi. The graphs of the left are gated on CD8 T cells. (J) Percentages of CD44+ Kb-TSYKFESV+ CD8 T cells. (K) Percentages of antigen experienced CD44+ CD8 and CD4 T cells. Data are represented as mean ± SEM from three pooled independent experiments (N = 8 for Rag1-/- + B6 → Rag1-/- and N = 11 for Rag1-/- + Ifnar1-/-→ Rag1-/- survivors).
Fig 4.
IFNAR on NK cells is essential for resistance to mousepox (A) Concatenated histograms showing IFNAR1 expression in gated NK (NK1.1+TCRβ-) or T (NK1.1-TCRβ+) cells from spleens of the indicated infected (6 dpi) mice or of Ifnar1fl/fl naïve mice. Concatenated histograms are derived from one independent experiment (N = 3 or four mice for each group). (B) Survival of the indicated mice infected with 3000 pfu of ECTV-GFP in the footpad. Data pooled from two independent experiments (Log-rank Mantel-Cox compared to Ifnar1fl/fl mice). (C-D) ECTV titers the livers and spleens of the indicated mice were quantified by plaque assay at six (C) and eight dpi (D). The dashed line indicates the detection limit. (E-G) Number of live splenocytes (E), frequency of NK cells (F) and numbers of NK cells (G) in the spleens of naïve and indicated infected mice at 6 and 8 dpi. (H) Concatenated flow cytometry plots showing NK and T cells gating and CD44 expression, Kb-TSYKFESV-specific and GzmB expression in CD8 T cells from spleens of Ifnar1fl/fl naïve mice and indicated infected mice at 8 dpi. (I-K) Number of antigen experience CD44+ (I), of CD44+ Kb-TSYKFESV+ (J) and of CD44+ GzmB+ (K) CD8 T cells in the indicated mice at 8 dpi. Data are represented as mean ± SEM from two pooled independent experiments (N = 9 or 10 for each group, ANOVA with Tukey correction compared to Ifnar1fl/fl infected mice).
Fig 5.
NK cells require IFNAR for optimal maturation, activation, and cytolytic killing.
(A) Graphs and concatenated flow cytometry histograms depicting GzmB and Prf expression in gated NK cells (NK1.1+ TCRβ-) from spleens of the indicated naïve or infected mice at six dpi. (B-C) Concatenated flow cytometry plots (B) and frequency (C) of GzmB+ Prf+ NK cells (NK1.1+ TCRβ-) in spleens of the indicated mice at 6 dpi. (D) Mice were infected with 3000 pfu of WT ECTV in the footpad and CFSE-labeled WT + Tap1-/- splenocytes at a 1:1 ratio were transferred i.v. Two hours before organ harvest at 6 dpi. Created with BioRender.com. (E-F) Concatenated flow cytometry plots (E) and proportions (F) of the preferential killing of Tap1-/- cells (CFSEhigh CD45.2) over WT cells (CFSEhigh B6.CD45.1) normalized to donor transfer ratio in spleens of the indicated naïve and infected mice at 6 dpi. Data are represented as mean ± SEM from two pooled independent experiments (N = 6 or 7 for each group, ANOVA with Tukey correction compared to Ifnar1fl/fl infected mice).
Fig 6.
Intrinsic IFNAR in Lysozyme-expressing myeloid cells is necessary for resistance to mousepox.
(A) Concatenated flow cytometry plots showing the gating strategy for cDCs (CD19-TCRβ-NK1.1-CD11chighSiglecH-), pDCs (CD19-TCRβ-NK1.1-CD11c+SiglecH+), eosinophils (CD19-TCRβ-NK1.1-SiglecH-F4-80+SSC-Ahigh), MO (CD19-TCRβ-NK1.1-SiglecH-CD11b+Gr-1+Ly6Clow/high), and neutrophils (CD19-TCRβ-NK1.1-SiglecH-CD11b+Gr-1high) and concatenated histograms showing IFNAR expression in indicated cell populations from spleens of naïve Ifnar1fl/fl and Ifnar1ΔLyz2 mice. (B) IFNAR1 expression in Ly6Clow, Ly6Cint, and Ly6Chigh gated MO. Histogram legend as in (A). Representative data from one out of two independent experiments (N = 6 to 7 for each group). (C) Survival of the indicated mice infected s.c. with 3000 pfu of ECTV-GFP. Data pooled from two independent experiments (N = 10 for Ifnar1ΔLyz2 mice and N = 18 for Ifnar1fl/fl mice, Log-rank Mantel-Cox compared to Ifnar1fl/fl mice). (D-E) ECTV titers in the dLN (D) and the spleen (E) were quantified by plaque assay at the indicated dpi. The dashed line indicates the detection limit. Data are represented as mean ± SEM from two or three pooled independent experiments (N = 5 to 7 for each Cre+ mice and N = 11 to 24 for Ifnar1fl/fl mice; ANOVA with Tukey correction compared to Ifnar1fl/fl mice). (F-G) ECTV titers in the liver and spleen were quantified by plaque assay at 5 (F) and 7 dpi (G). The dashed line indicates the detection limit. Data are represented as mean ± SEM from two pooled independent experiments (N = 8 to 9 for each Cre+ mice and N = 18 for Ifnar1fl/fl mice, ANOVA with Tukey correction compared to Ifnar1fl/fl mice). (H) Representative flow cytometry plots showing levels of CD44 and GzmB expression and levels of Kb-TSYKFESV-specific CD8 T cells in the spleens of infected mice. (I) Total cell numbers, and percentages and total numbers of CD44+ GzmB+ CD8 T cells (NK1.1- TCRβ+CD8+) in spleens of the indicated mice at 7 dpi. (J) Numbers of CD44+ Kb-TSYKFESV-specific CD8 T cells in the spleens of the indicated mice at 7 dpi. Data are represented as mean ± SEM from two pooled independent experiments (N = 8 to 9 for each Cre+ mice and N = 18 for Ifnar1fl/fl mice, ANOVA with Tukey correction compared to Ifnar1fl/fl mice).
Fig 7.
iMOs require intrinsic IFNAR for optimal transcription of IFN-I genes.
(A) Mixed BMC B6.CD45.1+Ifnar1-/-→ F1 [B6.CD45.1 x B6.CD45.2] were infected with 3000 pfu of ECTV-GFP in the footpad and at 3 dpi, uninfected (GFP-) and infected (GFP+) WT and Ifnar1-/- iMOs were sorted from the popliteal dLNs as shown in the representative flow cytometry plots. Created with BioRender.com. (B) IFN-I transcription quantification was performed by RT-qPCR using RNA extracted from each sorted population as a template. Transcript levels of Ifnb1, Ifna4, Ifna5, and all Ifna except Ifna4 (Ifna non-a4) were normalized to Gapdh. (C) Transcript levels of the ECTV EVM36 gene were quantified as in (B). (D) Indicated sorted populations were lysed, and ECTV loads in the indicated iMOs populations were quantified by plaque assay. Data are represented as mean ± SEM from two pooled independent experiments for (B) and (C) and from one independent experiment for (D) (ratio paired Student t-test).