Fig. 2: Replication and egress of T. gondii from sequestrated DCs precedes parenchymal localization.

A Experimental set up. CMTMR pre-labeled DCs were challenged in vitro with T. gondii (RH-GFP, MOI 1) to obtain a DC infection frequency of ∼50%. Infected DCs (20 × 10⁶ DCs / 10 × 10⁶ cfu Tg) were inoculated in the brain circulation via the ICA and brains collected 4, 16, 28 or 48 hpi. B Confocal micrographs, with corresponding 3D surface analyses, show the localization of RH (GFP⁺, green)-infected DCs (CMTMR⁺, blue) in relation to the vascular marker Evans blue (red) at 4, 16, 28 and 48 hpi. White boxes (insets) show infected DCs (CMTMR⁺ GFP⁺) and non-DC associated T. gondii foci with replicating tachyzoites (CMTMR^- GFP⁺). Asterisks indicate extravascular non-DC associated T. gondii. Scale bars: 10 µm. C, D Graphs show the absolute numbers (mean ± SEM) of T. gondii-associated (infected) DCs (CMTMR⁺ GFP⁺) (C) and non-DCs associated T. gondii foci (CMTMR^- GFP⁺) (D), respectively, per cm² of cortical tissue. Data are from 24 (4 h), 36 (16 h), 37 (28 h) and 37 (48 h) cortical sections per timepoint from three independent experiments (n = 3 mice per condition). E Graph shows the Log₁₀ ratio between infected DCs (CMTMR⁺ GFP⁺) and non-DCs associated T. gondii (CMTMR^- GFP⁺) from (C, D). F Graphs show the absolute numbers (mean ± SEM) of non-infected DCs (CMTMR⁺; GFP^-) per cm² of cortical tissue. Data are from 28 (4 h), 34 (16 h), 36 (28 h) and 29 (48 h) cortical sections per timepoint from three independent experiments (n = 3 mice per condition). G, H Graphs show the mean ( ± SEM) ratio (G) and Log₁₀ ratio (H) between non-infected DCs (CMTMR⁺; GFP^-) and infected DCs (CMTMR⁺; GFP⁺), respectively. Data are from 19 (4 h), 34 (16 h), 32 (28 h), 26 (48 h) cortical sections per timepoint from three independent experiments (n = 3 mice per condition). I Confocal 3D surface analyses show intravascularly located (Evans blue, cyan) RH-CPS infected DCs (CFSE⁺; mCherry⁺) at 16 and 48 hpi (left panels) and extravascular RH-WT tachyzoites (GFP⁺) at 48 hpi (right panel). Arrows indicate infected DCs. Asterisk indicates DC CFSE⁺ remnant (ghost, red). Scale bars: 10 µm. J Bar graph shows, for RH-WT and RH-CPS, the absolute numbers (mean ± SEM) of T. gondii-infected DCs retrieved in cortical sections at 16 hpi. Data are from 30 cortical sections from 3 independent experiments (n = 3 mice). C–H For each graph, cell and/or parasite cartoons indicate measured condition(s). C–G Kruskal–Wallis followed by Dunn’s multiple comparisons test, (J) 2-tailed Mann–Whitney U-test, numeric p-values are indicated, ns: non-significant, p > 0,05. Source data are provided as a Source Data file.