Onset of ECM after CTLA-4 and PD-1 blockade in <i>Pb</i>A-infected BALB/c mice is associated with parasite accumulation in the brain.

<p>BALB/c mice were infected i.v. with 10⁴<i>PbA</i>luc pRBCs and were either untreated (control) or treated with α-CTLA4 or α-PD-L1 antibodies. (A) Cumulative survival curve: X = Control (n = 10); ▪ = α-CTLA-4 (n = 10); □ = α-PD-L1 (n = 10), P-values (Log-rank (Mantel Cox) test). (B) Cumulative incidence of mice developing ECM, P-values (Fisher's exact) test. Bonferroni correction was used to adjust for multiple comparisons; threshold for significance is P<0.017 for (A) and (B). (<b>C</b>) Parasitaemia, shown as mean ± SD, of <i>PbA</i>-infected mice: X = Control; ▪ = α-CTLA-4; □ = α-PD-L1. Data are representative of two independent experiments performed with 5 mice in each group. (<b>D–G</b>) Kinetics of parasite accumulation in the (<b>D</b>) whole body, (<b>E</b>) head and (<b>F,G</b>) isolated brain as measured by luciferase activity. X = Control; ▪ = α-CTLA-4; □ = α-PD-L1. For D-G, data are shown as mean ± SD. For <b>D</b> and <b>E</b>, ¹ Control vs α-CTLA4 p<0.05, ² Control vs α-PD-L1 p<0.05, and ³ α-CTLA4 vs a-PD-L1 p<0.05 (Kruskal-Wallis Test/Dunn's multiple comparison test). For <b>G</b>, P-values (Mann Whitney U test).</p

CTLA-4 and PD-1 blockade in <i>Pb</i>A-infected BALB/c mice leads to onset of ECM.

<p>BALB/c mice were infected i.v. with 10⁴<i>PbA</i> pRBCs and treated with α-CTLA4 or α-PD-L1 antibodies or with no antibody. (<b>A</b>) Cumulative survival curve: X = Control (n = 31); ▪ = α-CTLA-4 (n = 20); □ = α-PD-L1 (n = 22), P-values (Log-rank (Mantel Cox) test). (<b>B</b>) Cumulative incidence of mice developing ECM, P-values (Fisher's exact) test. Bonferroni correction was used to adjust for multiple comparisons; threshold for significance is P<0.017 for (A) and (B). The incidence of ECM was based on neurological signs, i.e. ataxia and paralysis. Surviving BALB/c mice were euthanized on day 15 due to high parasitaemia and anemia. (<b>C</b>) Parasitaemia levels, shown as mean ± SD, of <i>PbA</i>-infected mice: X = Control; ▪ = α-CTLA-4; □ = α-PD-L1. Data are representative of three independent experiments with four to six mice in each group. (<b>D</b>) Absolute numbers of brain infiltrating CD8 T cell lymphocytes. Data (mean ± SD) are from two experiments, the numbers of animals are shown; P-values (Kruskal-Wallis Test/Dunn's multiple comparison test). (<b>E</b>) Histological examination of H&E stained brain sections from uninfected and day 7 infected mice (control without ECM and treated with ECM). Blue arrows indicate areas of haemorrhages. Magnification = 20X.</p

Course of infection of <i>Plasmodium berghei</i> ANKA (PbA) in C5BL/6 (ECM-susceptible) and BALB/c (ECM-resistant) mice.

<p>Mice were infected i.v. with 10⁴<i>PbA</i> pRBCs. The course of infection in C57BL/6 (n = 12) and BALB/c (n = 16) mice was followed by monitoring: (<b>A</b>) Cumulative survival – C57BL/6 (•) and BALB/c (○), *** P<0.0001 (Log-rank (Mantel Cox) test); and (<b>B</b>) Development of experimental cerebral malaria (ECM; cumulative incidence during the observation period), *** P<0.0001 (Fisher's exact test). The incidence of ECM was based on neurological signs, i.e. ataxia and paralysis. This was confirmed by histopathological examination of the brain. Surviving BALB/c mice were euthanized on day 15 due to the development of high parasitaemia and anemia; these mice were not ataxic or paralysed and they did not have brain lesions. (<b>C</b>) Parasitaemias are shown as mean <u>+</u> SD; representative of three experiments (four to six mice per group in each experiment). (<b>D</b>) H&E histopathology of brains from uninfected (left panel) and day 7 infected (right panel) C57BL/6 mice. Blue arrow indicates an area of haemorrhage. Magnification = 20X.</p

T cell depletion or cytokine blockade abrogates the effects of CTLA-4 and PD-L1 blockade in <i>PbA</i>-infected BALB/c mice.

<p>BALB/c mice were infected i.v. with 10⁴<i>PbA</i> pRBCs and were treated with (<b>A,C</b>) α-CTLA or (<b>B,D</b>) α-PD-L1 antibodies. (<b>A,B</b>) CTLA-4 or PD-L1 blockade without T cell depletion (□,▪); with α-CD8 depletion throughout infection (▽,▾); with α-CD8 depletion early in infection (△,▴); with α-CD4 depletion throughout infection (◊, ⧫); or with α-CD4 depletion early in infection (○,•). Cumulative survival curves (left) and cumulative incidence of ECM (right). (<b>C,D</b>) CTLA-4 or PD-L1 blockade without cytokine blockade (□,▪) or with α-IFN-γ () or α-TNF () treatment on on days −1, 1, 3, 5 and 7. Cumulative survival curves (left) and cumulative incidence of ECM (right). Data are representative of two independent experiments performed with four mice in each group. P values: Log-rank (Mantel Cox) test for the survival curves, and Fisher's exact test for cumulative ECM incidence. Bonferroni correction was used to adjust for multiple comparisons; threshold for significance is P<0.01 for the T cell depletions and P<0.017 for the cytokine neutralisations.</p

Differential expression of CTLA-4 and PD-1 by T cells of <i>Pb</i>A-infected C5BL/6 and BALB/c mice.

<p>Mice were infected i.v. with 10⁴<i>PbA</i> pRBCs. Splenocytes were prepared from uninfected (solid curves) or day 7 infected (clear curves) mice and stained for CD4 (left) or CD8 (right) expression of (<b>A</b>) CTLA-4 or (<b>B</b>) PD-1. For CTLA-4, staining was for both surface and intracellular protein; for PD-1, staining was for surface expression only. Line graphs show the kinetics of expression during the first 7 days of infection: data points (mean ± SD) from C57BL/6 (•) and BALB/c (○); n = 6, ** P<0.01 and *** P<0.001, P-values (Mann Whitney U test). (<b>C</b>) CD4⁺ and CD8⁺ T cells from uninfected and day 7 infected mice were stained for intracellular IFN-γ following stimulation with PMA/Ionomycin in the presence of Brefeldin A. CD4⁺ T cells were directly stained <i>ex vivo</i> for (<b>D</b>) intracellular CTLA-4 and surface PD-1; (<b>E</b>) CTLA-4 and intracellular FoxP3 or (<b>F</b>) surface PD-1 and intracellular FoxP3. Data for (C) and (D) are representative of three independent experiments with three to six mice in each group. Data for (E) and (F) are representative of two independent experiments with three mice in each group.</p

Enhanced cytokine secretion in <i>PbA</i>-infected BALB/c mice treated with α-CTLA and α-PD-L1 antibodies.

<p>(<b>A</b>) Plasma cytokine levels as determined by cytometric bead array: X = Control; ▪ = α-CTLA-4-treated; □ = α-PD-L1-treated. ¹ Control vs α-CTLA4 p<0.05, ² Control vs α-PD-L1 p<0.05, and ³ α-CTLA4 vs a-PD-L1 p<0.05. (<b>B,C</b>) IFN-γ and IL-10 ELISA of culture supernatants of splenocytes (from day 5 and day 7 infected mice) cultured for 48 h with α-CD3/CD28 antibodies. Spleens from 3–5 individual mice in each treatment group were pooled and each supernatant was analysed in triplicate. Mean (SE) values are shown for six wells. Uninfected = bar with horizontal line, Control = bar with vertical line, α-CTLA-4 = black bar, α-PD-L1 = white bar. (<b>D–G</b>) Intracellular IFN-γ staining of splenocytes isolated on day 7 post-infection and stimulated with PMA/Ionomycin for 5 hours in the presence of Brefeldin A. (<b>D,F</b>) Representative plots. (<b>E,G</b>) IFN-γ levels, shown as mean ± SD, are representative of three experiments (three to six mice per group in each experiment).</p

Histological analysis of brain and liver sections.

1<p>Sections from each mouse were examined and the numbers in 50 fields were recorded.</p><p>Control vs α-CTLA-4 p<0.05, Control vs α-PD-L1 p<0.05, α-CTLA-4 vs α-PD-L1 p<0.05.</p>2<p>Vessels plugged with pRBCs and leucocytes/50 fields: +++ = 11–15, ++ = 6–10, + = 1–5.</p>3<p>Control vs α-CTLA-4 p = 0.01, Control vs α-PD-L1 p = 0.02, α-CTLA-4 vs α-PD-L1 p = 0.07.</p

Long-term protection generated by the F3sap vaccine.

<p>Balb/c mice were vaccinated with NH36sap, F1sap, F2sap or F3sap at the indicated time intervals, through the sc route, followed by the intravenous challenge with <i>L. chagasi</i> amastigotes 28 days after the last immunization<b>.</b> Bars represent the mean ± SD of the individual parasite load in liver measured by LDU (one experiment, n = 3–4 mice). <b>*</b>p<0.05 significant differences from the saline controls and ○ from the F2sap vaccine.</p

Development of NH36-specific cellular immune response as disclosed by intracellular staining analysis of splenocytes <i>in vitro</i> cultured with NH36 before and after <i>L. chagasi</i> infection.

<p>Anti-CD4-FITC and anti-CD8-FITC antibodies were used for labeling the cell surfaces and anti-IFN-γ-APC, anti-TNF-α-PE and anti-IL-10-PE for intracellular staining. In order characterize the TH1 response bars represent the ratio of IFN-γ/IL-10 and TNF-α/IL-10 producing cells. Results represent mean + SE of two independent experiments (n = 7–8 mice per treatment). * p<0.05 indicate significant differences from the saline treated controls, from F1sap, ○ from F2sap, and ◆ from the NH36sap vaccine.</p

Nucleoside hydrolase NH36 T cell and antibody epitope mapping.

<p>The peptide sequence of MHC class II-IA^d and IE^d, haplotype H2^d CD4+ T cell epitopes (bold), of MHC class I L^d-CD8+ T cell predicted epitopes (underlined) and of epitopes for antibodies (grey background) in the F1, F2 and F3 fragments of the NH36 Nucleoside hydrolase of <i>Leishmania donovani</i>.</p