<i>Plasmodium</i>-primed CD8⁺ T cells induce monocyte accumulation in MacGreen×RAG^−/− mice.

<p>PbA-infected MacGreen×RAG^−/− mice that had received saline, CD8⁻ splenocytes, naïve or primed CD8⁺ T cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004236#ppat-1004236-g004" target="_blank">Fig. 4A</a> underwent intravital imaging on day 7 p.i. (n = 3 mice/group). Representative snapshots show (<b>A</b>) nil, (<b>B</b>) moderate and (<b>C</b>) severe levels of monocyte accumulation in the blood vessels. Scale bars represent 60 µm. Migratory paths of monocytes are shown as purple tracks. Blood vessels are marked by infusion of TRITC-conjugated dextran. White arrows indicate direction of blood flow. (<b>D</b>) % blood vessels with nil, moderate and severe levels of monocyte accumulation in MacGreen×RAG^−/− and MacGreen mice. Average number of (<b>E</b>) adherent and (<b>F</b>) rolling leukocytes per mm² of endothelium. A total 46, 123, 82 and 104 blood vessels were assessed for recipients of saline, CD8⁻ splenocytes, naïve and primed CD8⁺ T cells respectively. Number of vessels assessed per mouse: saline (n = 10, 12, 24), CD8⁻ splenocytes (n = 45, 28, 25), naive CD8⁺ T cells (n = 60, 11, 11), primed CD8⁺ T cells (n = 49, 33, 22). Blood vessel numbers for MacGreen mice are as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004236#ppat-1004236-g002" target="_blank">Fig. 2</a>. Data are a mean of 2–3 independent experiments. Bars represent mean ± SEM, ns, not significant, *p<0.05, **p<0.001, ***P = 0.0001 (ANOVA, Tukey's multiple comparisons test).</p

An adoptive transfer model to study the regulation of monocytes during ECM.

<p>(<b>A</b>) CD8⁺ T cells or CD8⁻ splenocyte fraction (Sp) isolated by MACS from uninfected or PbA-infected C57BL/6 donor mice on day 7 p.i. were adoptively transferred into PbA-infected MacGreen×RAG^−/− recipient mice as depicted. Only primed CD8⁺ T cells (red circle) isolated from PbA-infected C57BL/6 donor mice induce NS (orange recipient). Representative data of 3 independent experiments is shown. (<b>B</b>) Dot plot shows purity of CD8⁺ T cells routinely obtained by MACS sorting. (<b>C</b>) MacGreen×RAG^−/− recipient mice receiving saline (n = 4 mice), naïve (n = 4 mice) or primed CD8⁺ T cells (n = 5 mice) or CD8⁻ splenocytes (n = 4 mice) were infected with PbA and clinical scores monitored daily, *p = 0.01 (unpaired <i>t</i> test). (<b>D</b>) Clinical score of each mouse on day 5–7 p.i. Each symbol represents one mouse. Recipients of naïve and primed CD8⁺ T cells are shown in green and red respectively. (<b>E</b>) Percent survival of MacGreen×RAG^−/− recipients after adoptive transfer, *p<0.05 (Mantel-Cox test).</p

Endothelium-interacting GFP⁺ leukocytes in the brain microvasculature are monocytes.

<p>PbA-infected MacGreen mice with NS were injected i.v. with 5 µg of either (<b>A</b>) an isotype control, (<b>B</b>) anti-Ly6C (n = 2) or (<b>C</b>) anti-Ly6G antibody (n = 2) 1 hour prior to mice undergoing intravital imaging of the brain microvasculature. Representative snapshots from each group are shown. Scale bars represent 30 µm in (A), 100 µm in (B) and 58 µm in (C). Blue represents SHG signals from extracellular matrix fibres. White arrows indicate direction of blood flow. To aid orientation, vascular walls are outlined. Cells stained with Ly6C antibody show an orange punctate stain that co-localises with GFP (red arrowheads = adherent cells; yellow arrowheads = rolling cells). A total of 37, 29 and 45 blood vessels were assessed for the isotype control, Ly6C and Ly6G groups respectively. Number of blood vessels per mouse: Isotype (n = 37), Ly6C (n = 14, 15) and Ly6G (n = 9, 36). Representative snapshots of intravital imaging of the dermis of Lysozyme-GFP mice after injecting 5 µg of (<b>D</b>) an isotype control, (<b>E</b>) anti-Ly6C or (<b>F</b>) anti-Ly6G antibody, as reported <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004236#ppat.1004236-Ng1" target="_blank">[34]</a>. Number of blood vessels per mouse: Isotype (n = 12), Ly6C (n = 11) and Ly6G (n = 11). Cells stained with Ly6C show a punctate stain as in the brain. Cells stained with Ly6G have a red surface stain. Scale bars represent 60 µm in (D), 30 µm in (E) and 20 µm in (F). (<b>G</b>) Changes in blood monocyte populations during ECM. Blood monocytes were quantified by flow cytometry after collection from either uninfected (UI) or PbA-infected mice that had developed ES or NS (n = 4–5 mice/group). Symbols represent individual animals and bars represent means. *P<0.05, **p<0.01 (ANOVA with Dunnett's post-test vs. UI control). (<b>H</b>) Representative flow cytometric plots showing blood monocyte populations in UI, ES and NS mice. The percentage of Ly6C^lo and Ly6C^hi monocytes are indicated. Data are from a single experiment (n = 4–5 mice/group).</p

Visualizing the behaviour of GFP⁺ leukocytes in the brain microvasculature.

<p>PbA-infected MacGreen (n = 16) and MacGreen×RAG^−/− mice (n = 4) were monitored for (<b>A</b>) % survival to pre-defined clinical endpoint, (<b>B</b>) clinical score of each mouse on day 5–7 p.i., *p<0.05, ns, not significant (Mann-Whitney test), or (<b>C</b>) % parasitemia (n = 4 mice/group), ns, not significant, *p<0.05, **p<0.005 (One way ANOVA, Tukey's multiple comparisons test). Each symbol in (B) represents one mouse. Data are a mean of 3–6 independent experiments. MacGreen mice underwent intravital imaging of the brain microvasculature at various stages including when mice were uninfected (UI) (n = 4 mice), during ES on day 5–6 p.i. (n = 5 mice) and after they developed NS on day 7 p.i. (n = 3 mice). (<b>D</b>), (<b>E</b>), (<b>F</b>) Representative snapshots of 3–5 independent intravital imaging experiments shows nil, moderate and severe levels of leukocyte accumulation. Migratory paths of GFP⁺ leukocytes are shown as purple tracks. Blood vessels are marked by infusion of TRITC-conjugated dextran. Blue represents second harmonic generation (SHG) signals from extracellular matrix fibres. White arrows indicate direction of blood flow. Perivascular myeloid cells are indicated by red arrows. Scale bars represent 100 µm in (D), 60 µm in (E) and 28 µm in (F). (<b>G</b>) % blood vessels that have nil, moderate and severe levels of leukocyte accumulation in UI, ES and NS. (<b>H</b>) Average number of rolling and adherent leukocytes per mm² of endothelium. A total 89, 90 and 43 blood vessels were assessed for UI, ES and NS groups, respectively. Number of blood vessels assessed per mouse: UI (14, 30, 28, 17), ES (14, 15, 23, 22, 16) and NS (12, 25, 6). Bars represent mean ± SEM. ns, not significant, *p<0.05, **p<0.005, ****P<0.0001 (Kruskal-Wallis-Test, Dunn's multiple comparisons test).</p