PMC

(A) Plasma levels of soluble TNFRII were elevated in malaria with significant differences between AC, UM, and SM patients. (B) Plasma TNF levels in AC, UM, and SM patients. (C) The ratio of plasma levels of sTNFRII to TNF in AC, UM, and SM patients. (D) In UM, soluble TNFRII levels decreased in convalescence (p = 0.0005; Wilcoxon-rank test). AC, malaria-exposed asymptomatic controls; UM, uncomplicated P. falciparum malaria; SM, Severe P. falciparum malaria.

PBMC of 3 AC, 6 UM, and 5 SM patients were stained for CD4, CD25, Foxp3, and CD127, or CD45RO, CD69, CCR7, or TNFRII. (A) Mean fluorescent intensities (MFI) are shown for CD4⁺CD25⁻Foxp3⁻ (solid line) and CD4⁺CD25⁺Foxp3⁺ (dark grey) cells or CD4⁺Foxp3⁺CD127^lo (light grey) cells. Dotted lines represent the isotype controls. One representative donor is shown, and activated T cells with high expression of CD4 were gated out. (B) Pooled TNFRII MFI (3 AC, 6 UM, 5 SM). Horizontal lines show the median (p = 0.005 by Kruskal-Wallis test, and in post-hoc pairwise comparison of UM and SM: p = 0.008, and between SM and AC: p = 0.04). (C) Foxp3 expression is shown for TNFRII positive Treg cells (top panel) and TNFRII negative Treg cells (bottom panel) for one representative UM and one representative SM sample. The dotted line shows isotype control staining. AC, malaria-exposed asymptomatic controls; UM, uncomplicated P. falciparum malaria; SM, severe P. falciparum malaria.

PBMC from healthy malaria-unexposed blood donors (n = 4) were cultured overnight in the presence of P. falciparum–infected red blood cells (pRBC) at pRBC∶PBMC ratios of 2∶1, 1∶2.5, and 1∶10, or uninfected red blood cells (uRBC) at a uRBC∶PBMC ratio of 2∶1 or without RBC. (A) CD4⁺CD25⁺Foxp3⁺CD127^lo Treg cells are represented as percentage of CD4 T cells. (B) TNFRII MFI on CD4⁺ Foxp3⁺CD127^lo Treg cells. (C) sTNFRII (left panel), TNF (middle panel), and IL-10 (right panel) concentration in culture supernatants. (D) Following overnight culture with P. falciparum–infected red blood cells (pRBC) or uninfected red blood cells (uRBC) at a RBC∶PBMC ratio of 1∶2.5, PBMC were sorted into CD4⁺CD25⁻ responder cells and CD4⁺CD25⁺TNFRII⁺ or CD4⁺CD25⁺TNFRII⁻ Tregs and stained intracellularly for Foxp3. Foxp3 expression is shown for CD4⁺CD25⁺TNFRII⁺ or CD4⁺CD25⁺TNFRII⁻ Treg cells (grey line) and CD4⁺CD25⁻ responder cells (dotted line). (E) Sorted cells were tested for suppressive activity. 10⁴ CD4⁺CD25⁻ responder T cells sorted after uRBC exposure were incubated either alone or with TNFRII⁺ or TNFRII⁻ Tregs after uRBC or pRBC exposure at a 1∶1 ratio in 96 well plates pre-coated with 3 µg/mL anti-CD3 antibody (OKT-3) for 3 days. Sorted monocytes were used as APC. Bars show mean cpm+/−SD of triplicate wells.

PBMC were stained for the cell surface markers CD4, CD25, and CD127, followed by intracellular staining for Foxp3. (A) Lymphocytes were firstly gated on CD4 expression. All gated CD4 cells expressed CD3 (data not shown). CD4 T cells were then gated on cells expressing Foxp3 with low expression of CD127. Only cells that also expressed CD25 were considered Treg cells. (B) Treg cell frequency was significantly elevated in malaria patients (p = 0.007 by Kruskal-Wallis test, and in post-hoc pairwise comparison with controls, UM: p = 0.004, SM: p = 0.007). Treg cells are shown as the percentage of CD4 T cells. Horizontal lines depict the median with interquartile range. (C) Activated CD4 T cells were defined as CD25+CD4 T cells without co-expression of the Foxp3+CD127^lo phenotype. Ratios of activated CD4 T cells: Treg cells are shown for AC, UM, and SM (p = 0.007 by Kruskal-Wallis test, and in post-hoc pairwise comparison with controls, UM: p = 0.005, SM: p = 0.009). Horizontal lines depict the median with interquartile range. (D) Correlation of Treg cell frequency and PfHRP2 plasma levels in SM. (E) In patients with severe malaria, Treg cells were significantly elevated in subjects with hyperparasitemia (HP) compared to SM patients without hyperparasitemia (no HP); p = 0.02; Mann-Whitney U test. AC, malaria-exposed asymptomatic controls; UM, uncomplicated P. falciparum malaria; SM, severe P. falciparum malaria.