Interactions between CD8⁺ T cells and MBECs.

<p>(A) IFNγ-stimulated MBECs were incubated with (or without) 3 × 10⁶ thawed PbA mature iRBCs for 24 h. After washing, we added 10⁶ CD8⁺ T cells from either a naïve mouse or one infected with PbA 6 days previously. The wells were washed gently and photographed (DIC) after 20 h. Images are representative of triplicate wells. (B) An olfactory bulb smear from a mouse with ECM was fixed and stained with antibodies against von Willebrand factor (green) and CD8 (red). CD8⁺ T cells were present within blood vessels (arrows), on the abluminal face of endothelial cells (arrow head) and in the parenchyma (asterisks), 40× objective. Inset: a perivascular CD8⁺ T cell in close contact with the endothelium (100× objective).</p

Uptake and cross-presentation efficiency of different rodent malaria parasites by MBECs <i>in vitro</i>.

<p>Mature parasite stages were isolated from the blood of mice infected with PbA, PbNK65 or Py17X by Percoll gradient centrifugation. (A) Various numbers of frozen-thawed parasites were added to IFNγ-stimulated MBECs in triplicate wells. After 24 h, the wells were washed and Pb1 cross-presentation was assayed using LR-BSL8.4a reporter cells. *<i>P</i><0.05, ***<i>P</i><0.001, ****<i>P</i><0.0001, 2-way ANOVA with Bonferroni’s post test, comparing each of the non-ECM strains against PbA at the same dose. The dotted line indicates the background spot count from MBECs without parasites. (B) Equal numbers of frozen-thawed parasites from the three strains were labeled with PKH26 and added to unstimulated or IFNγ-stimulated MBECs in quadruplicate wells. After overnight incubation, the wells were washed and imaged by confocal microscopy (6 fields per well with 40× objective) in the presence of trypan blue to quench extracellular PKH26. The number of red pixels per field that exceed a defined brightness threshold was quantified with ImageJ. Bars represent medians and interquartile ranges. ns not significant, **<i>P</i><0.01, ****<i>P</i><0.0001, Kruskal Wallis test with Dunn’s post test comparing against the IFNγ + PbA group.</p

Activated Brain Endothelial Cells Cross-Present Malaria Antigen

<div><p>In the murine model of cerebral malaria caused by <i>P</i>. <i>berghei</i> ANKA (PbA), parasite-specific CD8⁺ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation <i>in vivo</i>, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells <i>in vitro</i> confers susceptibility to killing by CD8⁺ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation <i>in vivo</i> and <i>in vitro</i>, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA <i>in vitro</i>. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed <i>P</i>. <i>falciparum</i> merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.</p></div

Endothelial cells and leukocytes are the cross-presenting cells in the brain during PbA infection.

<p>(A–B) Brains from naïve (<i>n</i> = 5) and PbA-infected mice exhibiting signs of ECM (<i>n</i> = 11) were subjected to automated dissociation using papain. After myelin removal, the single cell suspensions were stained and sorted into four populations: CD45⁺ microglia and leukocytes, CD45^-CD31⁺ endothelial cells, CD45^-CD140b⁺ cells including pericytes, and cells negative for all three markers. Each sorted population was tested for cross-presentation by incubating with LR-BSL8.4a cells and staining with X-gal. Data were pooled from 3 independent experiments. (A) Raw spot counts. **<i>P</i>< 0.01, separate Mann-Whitney U tests comparing naïve vs infected for each population. (B) Only data from infected mice are shown, after background subtraction (the mean of all naïve wells) and normalization by the number of sorted cells added. Negative data are plotted as zeroes. (C) Brains from naïve (<i>n</i> = 5) and PbA-infected mice (<i>n</i> = 10) were manually dissociated and digested with collagenase. After myelin removal, the single cell suspensions were stained and sorted for microglia (CD45^intCD11b⁺) and peripheral leukocytes together with PVM (CD45^hi), which were assayed for cross-presentation as above. Data were pooled from 2 independent experiments. **<i>P</i>< 0.01, Mann-Whitney U test. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004963#ppat.1004963.s001" target="_blank">S1 Fig</a> for representative FACS plots.</p

IFNγ-stimulated MBECs cross-present PbA antigen <i>in vitro</i>.

<p>(A) After culturing mouse brain microvessels for 10 days including puromycin selection, the resulting cells are >97% CD45^-CD31⁺ endothelial cells. Dot plots are representative of 3 independent culture experiments. (B) MBECs were fixed and stained for the endothelial marker von Willebrand factor (green) and counterstained with DAPI (blue). (C) MBECs in quadruplicate wells of a 48-well plate were stimulated (or not) with 10 ng/ml IFNγ for 24 h, then 5 × 10⁶ thawed PbA mature iRBCs were added (or not) for 24 h. The wells were washed and co-cultured with LR-BSL8.4a reporter cells overnight prior to X-gal staining. ****<i>P</i><0.0001 compared to every other group, ANOVA with Bonferroni’s post test on log-transformed blue spot counts. (D) Cross-presentation by IFNγ-stimulated MBECs was similarly assayed after pulsing with 10⁶ freshly isolated or freeze-thawed PbA mature stages (<i>n</i> = 4). ****<i>P</i>< 0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed blue spot counts.</p

Optimisation of effector (THP-1) to target (erythrocytes) ratio.

<p>The effectors used in these experiments were (<b>A</b>) THP-1 monocytes, (<b>B</b>) PMA-differentiated THP-1 macrophages and the targets were fresh uninfected erythrocytes (uRBC) or ring-staged <i>Plasmodium falciparum</i> culture (ring culture) at 10% parasitemia. ____Percentage of effectors that have engulfed at least one erythrocyte (uninfected or infected) at effector : target (E:T) ratios from 1∶10 to 1∶260 after 4 h incubation at 37°C with pure effectors as the control. The experiments were done in duplicates at least 3 times with data expressed as mean ± SEM. Statistical analyses compared the number of effectors which had ingested erythrocytes from the uRBC culture with that from ring culture at the same E:T ratio. (*represents p<0.05, **represent p<0.005, ***represent p<0.001).</p

Human brain endothelial cells phagocytose Pf merozoites <i>in vitro</i>.

<p>PKH26-labeled Pf merozoites were incubated with IFNγ-stimulated hCMEC/D3 cells for 24 h. The cells were then washed thoroughly and stained with Lysotracker Green DND-26 and Hoechst 33342 prior to confocal imaging. Extracellular PKH26 fluorescence was quenched with trypan blue.</p

Cytokine requirements and timing of brain microvessel cross-presentation during PbA infection.

<p>(A–C) WT mice and mice deficient for IFNγ (A), TNFα (B), and LTα (C) were infected with PbA. After 7 days, brain microvessels were isolated from the infected mice as well as naïve WT mice, and incubated overnight with LR-BSL8.4a reporter cells. The total number of blue cells (counted as blue spots by the ELISOT reader) resulting from each brain after X-gal staining was quantified. *<i>P</i><0.05, ****<i>P</i><0.0001, ns not significant, ANOVA with Bonferroni’s post-test on log-transformed numbers. (D) Brain microvessels from uninfected mice and WT mice infected with PbA 5, 6, or 7 days previously were tested for cross-presentation as above. **<i>P</i><0.01, ****<i>P</i><0.0001, ns not significant as compared to uninfected mice by ANOVA with Bonferroni’s post-test on log-transformed numbers.</p

Cross-presentation of PbA antigen by MBECs occurs by the cytosolic pathway.

<p>(A) MBEC cultures were established from WT mice as well as TAP1-deficient mice and stimulated with IFNγ. PbA mature iRBCs were added to some wells in triplicate. After 24 h, the wells were washed and cross-presentation of Pb1 was detected using LR-BSL8.4a cells and X-gal staining. ****<i>P</i><0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed data. Results are representative of 2 independent experiments. (B) PbA mature iRBCs were added (or not) to IFNγ-stimulated MBECs. Lactacystin (10 μM) was added to some wells 6 h later. After another 6 h, all wells were washed and assayed for Pb1 cross-presentation using LR-BSL8.4a cells. <i>n</i> = 3, *<i>P</i><0.05, **<i>P</i><0.01, ANOVA with Bonferroni’s post test on log-transformed data. Results are representative of 2 independent experiments. (C, D) PbA mature iRBCs were added to IFNγ-stimulated MBECs 1 h after either 10 μg/ml chloroquine diphosphate (C) or 10 μM cytochalasin D (D) were added to some wells. The MBECs were washed and co-incubated with LR-BSL8.4a cells 24 h later to measure Pb1 cross-presentation. <i>n</i> = 3, ***<i>P</i><0.001, ****<i>P</i><0.0001, unpaired t-test on log-transformed spot counts.</p

Phagocytosis of fresh uninfected erythrocytes (uRBC), ring-staged (ring culture) and schizont-staged cultures (schizont culture) at 10% parasitemia under different conditions by THP-1 differentiated macrophages.

<p>In standard conditions, incubation of erythrocytes with macrophages was carried out for 4 h at 37°C with an E:T ratio of 1∶100. To prevent phagocytosis, macrophages were pretreated with 5 µM cytochalasin D for 1 h before the phagocytic incubation of 4 h at 37°C. To increase phagocytosis, erythrocytes were opsonised with serum from a <i>Plasmodium falciparum</i> (+) patient at room temperature for 30 min before incubation with macrophages. A) Macrophages that have engulfed at least one uRBC and B) macrophages that have engulfed at least one iRBC. Data expressed as mean ± SEM. (bvc, p<0.05; BvE, p<0.005; AvF, CvD, FvI, GvH, evh, fvg, p<0.001; avd, p = 0.098; n = 3 separate experiments, each in duplicates) C) Representative dotplots of the respective conditions when exposed to THP-1 macrophages.</p