Fig. 3: Following sequestration of infected DCs, T. gondii is retrieved inside neurons.

A. Confocal micrographs show the localization of GFP-expressing RH tachyzoites (GFP⁺, green), DCs (CMTMR⁺, blue) and neurons (NeuN⁺, gray) in relation to the vascular marker Evans blue (red). Infected CMTMR pre-labeled DCs (20 × 10⁶ DCs / 10 × 10⁶ cfu Tg) were inoculated via the ICA and brains extracted at indicated time points. Upper panels show 3D projections from confocal stacks and lower panels show confocal planes. Arrows indicate infected NeuN⁺ cells. White and red dotted lines delineate neuronal cytoplasm and cortical capillary, respectively. Asterisks indicate GFP⁺ tachyzoites localized inside NeuN⁺ cells. Scale bars: 10 µm. B Confocal planes show displacement of NeuN signal (asterisks) by T. gondii tachyzoites in corresponding confocal planes in (A). C Confocal micrographs and corresponding 3D surfaces show the extravascular localization of RH wild type (GFP⁺, green) and non-replicative RH-CPS tachyzoites (mCherry⁺, red) in relation to the vascular marker Evans blue (red in left panels and white in right panels). 20 × 10⁶ cfu of freshly egressed tachyzoites were inoculated via the ICA and brains were extracted 1 hpi. Arrows indicate extravascular tachyzoites. Magnifications are shown in insets A and B for RH and RH-CPS, respectively, with distances the nearest blood vessel. Scale bars: 10 µm. D 3D surface analyses illustrate extravascular and intravascular localization of tachyzoites (type I RH, GFP⁺, green and type II ME49, RFP⁺, cyan) in relation to the vascular marker Evans blue (red). 20 × 10⁶ cfu of freshly egressed tachyzoites were inoculated via the ICA and brains were collected 16 hpi. Distance to the nearest vessel, vessel lumen and distance to the nearest branching point (yellow dot) are shown. E Graph shows the absolute numbers (mean ± SEM) of type I and II T. gondii foci per cm² brain tissue at 16 hpi. Data are from 59 (TgI) and 56 (TgII) cortical sections per condition from three independent experiments (n = 4 mice per condition). F Luminal diameter of cortical microvessels containing T. gondii type I and type II tachyzoites, respectively. In box plots, center line indicates median. Box limits: 25th and 75th percentiles. Whiskers: maximum and minimum values. Data are from 89 (TgI) and 33 (TgII) T. gondii foci in cortical tissue per condition from three independent experiments (TgI: n = 4 mice; TgII n = 3 mice). G Distribution of distances to nearest vascular branching point (medians with 25th and 75th percentiles) of type I and II T. gondii retrieved in the cortical microvasculature. Data are from 59 (TgI) and 39 (TgII) T. gondii foci per condition from three independent experiments (n = 3 mice per condition). H Relative expression (qPCR) of T. gondii TgB1 gene at 16 hpi in cortices of mice challenged 20 × 10⁶ freshly egressed type I or type II tachyzoites inoculated via the ICA. Data are expressed as mean ( ± SEM) from three independent experiments (n = 3 mice per condition). I Graphs show the absolute numbers (mean ± SEM) of T. gondii foci or infected DCs per cm² brain tissue at 16 hpi. Mice were inoculated via the ICA with 10 × 10⁶ cfu of freshly egressed tachyzoites of type I RH (Free TgI), type II ME49 (Free TgII) or 10 × 10⁶ cfu of DCs pre-challenged with type I RH (DC/TgI) or type II ME49 (DC/TgII). Data are from 60 (free TgI), 30 (DC/TgI), 55 (free TgII) and 28 (DC/TgII) cortical sections per condition from three independent experiments (TgI: n = 4 mice; TgII n = 3 mice). J Ratios (mean ± SEM) between the relative numbers of infected DCs (from mice inoculated with DC/Tg) and T. gondii foci (from mice inoculated with Free Tg) retrieved in cortices for infections with RH (TgI) and ME49 (TgII), respectively. Data are from three independent experiments (n = 4 mice per condition). E–J 2-tailed Mann–Whitney U-test, (H) 2-tailed unpaired Student’s t-test, I Kruskal–Wallis followed Dunn’s multiple comparison test, numeric p-values are indicated, ns: non-significant, p > 0,05. Source data are provided as a Source Data file.