Plasmodium vivax: induction of CD4+CD25+FoxP3+ regulatory T cells during infection are directly associated with level of circulating parasites

Abstract

Circulation CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-beta) as well as pro-inflammatory (IFN-gamma, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4(+)CD25(+)FoxP3(+)GITR(+) or CD4(+)CD25(+)FoxP3(+)CTLA-4(+) and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.

Figure 1. Flow cytometric analysis of regulatory T cells. (A) CD25 and FoxP3 expression in gated CD4⁺CD3⁺ lymphocytes.

Dot plot show a representative data of 35 donors examined. (B) Absolute numbers of circulating CD4⁺CD25⁺FoxP3⁺ regulatory T cells in malaria-naïve and P. vivax-infected donors (n = 15 and 20, respectively). Absolute numbers (cells/mm³) are indicated on Y-axis and lines represent median. Statistical differences were detected using Mann-Whitney U test and are indicated on the graph with significant P values.

Figure 2. Flow cytometric analysis of surface markers (CTLA-4 and GITR) and cytokines (IFN-γ, IL-17, TGF-β, and IL-10) in CD4⁺CD25⁺FoxP3⁺ regulatory T cells in malaria-naïve and P. vivax-infected donors (n = 15 and 20, respectively).

Results were expressed as absolute numbers of cells expressing (A) GITR, (B) CTLA-4, (C) IFN-γ, (D) IL-17, (E) TGF-β, and (F) IL-10. Absolute numbers (cells/mm³) are indicated on Y-axis and lines represent median. Statistical differences were detected using Mann-Whitney U test and are indicated on the graphs with significant P values.

Figure 3. Expression of surface markers (CTLA-4 and GITR) and cytokines (IFN-γ, IL-17, TGF-β, and IL-10) on CD4⁺CD25⁺FoxP3⁺ regulatory T cells in malaria-naïve and P. vivax-infected donors (n = 15 and 20, respectively).

Results were expressed as median intensity of fluorescence (lines represent median) for (A) GITR, (B) CTLA-4, (C) IFN-γ, (D) IL-17, (E) TGF-β, and (F) IL-10 on CD4⁺CD25⁺FoxP3⁺ cells. Statistical differences were detected using Mann-Whitney U test and are indicated on the graphs with significant P values.

Figure 4. CD4⁺CD25⁺FoxP3⁺ regulatory T cells are directly correlated with parasite burden.

The relationship between absolute numbers of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and degree of parasitaemia among 20 patients with Plasmodium vivax malaria was examined using Spearman rank correlation.

Figure 5. Correlation of CD4⁺CD25⁺FoxP3⁺ regulatory T cell co-expressing (A) GITR, (B) CTLA-4, (C) IFN-γ, (D) TGF-β, (E) IL-10, and (F) IL-17 and degree of parasitaemia among 20 patients with Plasmodium vivax malaria.

Statistical significance was determined by Spearman rank correlation.

Figure 6. Indirect suppression elicited by CD4⁺CD25⁺ T regulatory cells from P. vivax-infected individuals.

(A) CFDA-SE Proliferation ratio of Pv-AMA-1- stimulated PBMCs (sPBMCs) and sPBMCs co-cultured with different proportions of autologous CD4⁺CD25⁺ lymphocytes (1∶2, 1∶5 and 1∶10, CD4⁺CD25⁺ cells: sPBMCs). Results are expressed for two malaria-infected donors who presented positive proliferative response after Pv-AMA-1 stimulation. (B) Representative FACS histogram plots for 1 out 2 donors with positive proliferative response after Pv-AMA-1 stimulation showing CFDA-SE staining after antigen stimulation and co-culturing with CD4⁺CD25⁺ T cells. CFDA-SE Proliferation ratio for sPBMCs was calculated by proliferative response observed in Pv-AMA-1-stimulated PBMCs (indicated by positivity for CFDA-SE) divided by basal proliferative response of non-stimulated cells (PBMCs only). CFDA-SE Proliferation ratio for co-cultured cells were calculated by proliferative response observed in Pv-AMA-1-stimulated PBMCs with CD4⁺CD25⁺ T cells divided by proliferative response observed in Pv-AMA-1-stimulated PBMCs only.