Plasmodium falciparum induces Foxp3hi CD4 T cells independent of surface PfEMP1 expression via small soluble parasite components

Abstract

Elevated levels of regulatory T cells following Plasmodium infection are a well-reported phenomenon that can influence both protective and pathological anti-parasite responses, and might additionally impact on vaccine responses in acutely malaria infected individuals. The mechanisms underlying their induction or expansion by the parasite, however, are incompletely understood. In a previous study, Plasmodium falciparum infected red blood cells (iRBCs) were shown to induce effector-cytokine producing Foxp3int CD4+ T cells, as well as regulatory Foxp3hi CD4+ T cells in vitro. The aim of the present study was to determine the contribution of parasite components to the induction of Foxp3 expression in human CD4+ T cells. Using the surface PfEMP1-deficient parasite line 1G8, we demonstrate that induction of Foxp3hi and Foxp3int CD4+ T cells is independent of PfEMP1 expression on iRBCs. We further demonstrate that integrity of iRBCs is no requirement for the induction of Foxp3 expression. Finally, transwell experiments showed that induction of Foxp3 expression, and specifically the generation of Foxp3hi as opposed to Foxp3int CD4 T cells, can be mediated by soluble parasite components smaller than 20 nm and thus likely distinct from the malaria pigment hemozoin. These results suggest that the induction of Foxp3hi T cells by P. falciparum is largely independent of two key immune modulatory parasite components, and warrant future studies into the nature of the Foxp3hi inducing parasite components to potentially allow their exclusion from vaccine formulations.

Keywords: Foxp3; Malaria; PfEMP-1; Plasmodium falciparum; hemozoin; regulatory T cell.

FIGURE 1

Infected red blood cell (iRBC)-mediated Foxp3 expression is independent of surface PfEMP-1 expression. PBMC were cultured alone or in the presence of normal (nRBC) or infected RBCs (iRBC) at an RBC:PBMC ratio of 2:1. RBCs were infected with either the parental strain 3D7 or the SBP-1 knock-out parasite line 1G8. On day 6, cells were harvested and analyzed by flow cytometry. (A) Lymphocytes were selected by FSC/SSC gating and further gated based on CD3 and CD4 staining. CD4+ T cells were then analyzed based on CD25 and Foxp3 expression. (B) Representative dots plots for PBMC from one donor stimulated with 3D7 and 1G8 parasites. (C) Proportions of Foxp3hi and Foxp3int CD4+CD25+ T cells determined as a percentage of CD3+CD4+ T cells for eight donors in six independent experiments. (D) Values of all conditions were normalized for each individual donor on proportions induced by 3D7 iRBCs. Vertical bars represent median values.

FIGURE 2

iRBC-mediated Foxp3 expression does not require intact iRBCs. PBMC were cultured alone or in the presence of normal (nRBC) or infected RBCs (iRBC) at an RBC:PBMC ratio of 2:1. PBMC were co-cultured with equal amounts of either intact 3D7 iRBCs, or 3D7 iRBC lysate resulting from five rounds of freezing and thawing. (A) Representative dots plots for PBMCs from one donor stimulated with intact iRBCs versus iRBC lysate. (B) Proportions of Foxp3hi and Foxp3int CD4+CD25+ T cells were determined on day 6 as a percentage of CD3+CD4+ T cells for five donors in two independent experiments. (C) Values of all conditions were normalized for each individual donor on proportions induced by intact iRBCs. Vertical bars represent median values. Dotted lines show the upper 95% confidence interval of the mean of uRBC-stimulated control cultures.

FIGURE 3

Foxp3hi and to a lesser degree Foxp3int induction is mediated by soluble iRBC components smaller than 20 nm. To exclude large membrane fragments and intact hemozoin crystals and to examine the contribution of small soluble molecules derived from iRBCs to Foxp3 induction, intact iRBC were separated from PBMC through a transwell membrane (pore size 20 nm). (A) Representative dots plots for PBMCs from one donor cultured in direct contact with iRBCs compared to transwell-separated iRBCs. (B) Proportions of Foxp3hi and Foxp3int CD4+CD25+ T cells were determined on day 6 as a percentage of CD4+ T cells for five donors in two independent experiments. (C) Values of all conditions were normalized for proportions induced by direct co-culture with iRBCs for each donor. Vertical bars represent median values. (D) The ratio of Foxp3hi:Foxp3int cells within the CD4+CD25+ T cell population was calculated for each individual donor in direct or transwell separated co-cultures. Vertical bars represent median values. Individual donors are identified by unique symbols. Dotted lines show the upper 95% confidence interval of the mean of uRBC-stimulated control cultures.