Treg phenotype in UM and SM.

<p>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 <i>P. falciparum</i> malaria; SM, severe <i>P. falciparum</i> malaria.</p

In vitro <i>P. falciparum</i> exposure induced Treg cell expansion, TNFRII expression, and enhanced Treg cell activity.

<p>PBMC from healthy malaria-unexposed blood donors (n = 4) were cultured overnight in the presence of <i>P. falciparum</i>–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 <i>P. falciparum</i>–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.</p