Journal: Nature immunology
Article Title: The activation trajectory of plasmacytoid dendritic cells in vivo during a viral infection
doi: 10.1038/s41590-020-0731-4
Figure Lengend Snippet: a , YFP vs CCR7 expression in splenic pDCs during MCMV infection. Data from one representative Ifnb1 Eyfp mouse is shown for each time points. b , Frequency of YFP + CCR7 + cells within splenic pDCs during MCMV infection. The data represent individual mice with n = 5 at 0h, 7 at 33h, 10 at 36h, 5 at 40h, and 3 at 44h and 48h, from one experiment for 44h and 48h, and pooled from 2 (resp. 3) independent experiments for 33h and 40h (resp. 0h and 36h). c , Mean fluorescence intensity of IFN-α/β on YFP + CCR7 + vs YFP + CCR7 − pDCs isolated from 36h MCMV-infected mice. d , Relative median fluorescence intensity (MFI) of indicated markers on pDC subpopulations isolated from 36h MCMV-infected Ifnb1 Eyfp mice. The data in c and d are from n = 10 (resp. 5) mice from two independent experiments (c) or one experiment representative of two (d). e , Flow cytometry sorting strategy for splenic pDC subpopulations from uninfected (UN) or 38h MCMV-infected Ifnb1 Eyfp mice, starting from live single CD11b neg Lin neg cells. f , Expansion of naïve CD4 OT-II cells upon co-culture with the indicated OVA peptide-pulsed pDC subpopulations. The graph shows individual data points pooled from 3 independent experiments, each with 2-4 replicate co-cultures for each pDC subpopulation. g , Immunohistological analysis of spleen sections from uninfected (UN) or MCMV-infected Ifnb1 Eyfp mice harvested at the indicated time points. Top right image, 5x zoom of the region delimited in the previous image. h , Definition of spleen zones for cell quantification (see ). i , Distribution of YFP + cells across the different spleen zones during the course of MCMV infection. j , Detail of the individual data collected and used for generating the graph of panel I. k , A similar analysis was performed as in panel (j) for the percentages of WP YFP + pDCs residing in the TCZ. For g-k, data were analyzed from 3 different mice, with 5 different whole splenic sections per mouse (i.e. 15 images per time point). In b-d,f,i-k, data are shown as mean±s.e.m. and P values are from One-way ANOVA with Tukey’s post hoc test, with *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001.
Article Snippet: Cells were spun down, resuspended in complete RPMI with 5 μg/mL OVA peptide (323-339; H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-OH) (Sigma-Aldrich) and plated at 2,000 cells in 100 μL per well in a V-bottom plate for 20 min at 37°C.
Techniques: Expressing, Infection, Fluorescence, Isolation, Flow Cytometry, Co-Culture Assay
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