Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4(+) Foxp3(+) regulatory T cells

Abstract

Recently, we demonstrated elevated numbers of CD4(+) Foxp3(+) regulatory T (Treg) cells in Plasmodium yoelii-infected mice contributing to the regulation of anti-malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus-derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3(+) Treg cells expressing neuropilin-1 (Nrp-1) decreased at early time-points during P. yoelii infection, whereas percentages of Helios(+) Foxp3(+) Treg cells remained unchanged. Both Foxp3(+) Nrp-1(+) and Foxp3(+) Nrp-1(-) Treg cells from P. yoelii-infected mice exhibited a similar T-cell receptor Vβ chain usage and methylation pattern in the Treg-specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3(-) T cells adoptively transferred to P. yoelii-infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3(+) Treg cells.

Keywords: parasitic protozoan; regulatory T cells; rodent.

Figure 1

Plasmodium yoelii infection of BALB/c mice resulted in reduced percentages of neuropilin‐1 (Nrp‐1)‐expressing Foxp3⁺ regulatory T (Treg) cells, with unaffected Helios expression. Foxp3/eGFP reporter mice were infected with 1 × 10⁵ infected red blood cells (iRBC) intravenously. Non‐infected [0 days post‐infection (d p.i.)] and P. yoelii‐infected Foxp3/eGFP mice were killed at 3, 5 and 7 d p.i. (a) The frequency (left panel) and absolute number (right panel) of Foxp3⁺ (eGFP⁺) Treg cells and (b) the mean fluorescence intensity (MFI) of CD25 expression on Foxp3⁺ Treg cells were determined on gated CD4⁺ T cells and CD4⁺ Foxp3⁺ Treg cells, respectively by flow cytometry. Representative dot plots are shown in the upper panel. (c) Helios and neuropilin‐1 (Nrp‐1) expression were analysed on CD4⁺ Foxp3⁺ Treg cells by flow cytometry. Representative dot plots are shown in the upper panel. Percentages and absolute numbers of Helios and Nrp‐1‐expressing Treg cells are summarized. (d) The frequencies of Helios⁺ Nrp‐1⁻, Helios⁻ Nrp‐1⁺ and Helios⁺ Nrp‐1⁺ co‐expressing Foxp3⁺ Treg cells were determined by flow cytometry. Representative dot plots are shown in the left panel. Results from two independent experiments with n = 6 mice are summarized as mean ± SEM. Each dot represents one animal. One‐way analysis of variance with Dunett's post test was used for statistical analysis. *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

T‐cell receptor‐Vβ (TCR‐Vβ) chain usage of neuropilin‐1‐positive (Nrp‐1⁺) and Nrp‐1⁻ Foxp3⁺ regulatory T (Treg) cells at day 3 and day 7 after Plasmodium yoelii infection. (a) Representative dot plots illustrating the gating strategy for analysis of TCR‐Vβ chain repertoire on different CD4⁺ T‐cell subsets by flow cytometry. Percentages of (b) Nrp‐1⁺ Foxp3⁺ Treg cells, (c) Nrp‐1⁻ Foxp3⁺ Treg cells and (d) Foxp3⁻ T cells using TCR‐Vβ2, TCR‐Vβ5, TCR‐Vβ6, TCR‐Vβ8.1/8.2, TCR‐Vβ8.3, TCR‐Vβ9, TCR‐Vβ11, TCR‐Vβ12 and TCR‐Vβ13 isolated from spleen of non‐infected [0 days post‐infection (d p.i.), white bars) and P. yoelii‐infected Foxp3/eGFP mice 3 days (grey bars) and 7 days p.i. (black bars). Results from two or three independent experiments with n = 6 to n = 10 mice in total are summarized as mean ± SEM. One‐way analysis of variance with Dunett's post test was used for statistical analysis. *P < 0·05, ***P < 0·001.

Figure 3

The majority of neuropilin‐1‐positive (Nrp‐1⁺) and Nrp‐1⁻ Foxp3⁺ regulatory T (Treg) cells from Plasmodium yoelii‐infected mice are highly demethylated in their Treg‐specific demethylation region (TSDR). (a) Nrp‐1⁺ Foxp3⁻ and Nrp‐1⁻ Foxp3⁻ T cells as well as (b) Nrp‐1⁺ Foxp3⁺ and Nrp‐1⁻ Foxp3⁺ Treg cells were isolated from spleen of non‐infected Foxp3/eGFP reporter mice and P. yoelii‐infected Foxp3/eGFP mice at days 3 and 5 post‐infection (d p.i.). DNA was isolated and bisulphate treated before methylation analysis by real‐time PCR. Results from three or four independent experiments with n = 4 to n = 6 mice each were summarized as mean ± SEM. One‐way analysis of variance with Dunett's post test was used for statistical analysis. *P < 0·05, **P < 0·01.

Figure 4

Adoptively transferred CD4⁺ Foxp3⁻ T cells do not acquire Foxp3 expression in Plasmodium yoelii‐infected mice. From 3 × 10⁶ to 5 × 10⁶ sorted Thy1.2⁺ CD4⁺ Foxp3⁻ T cells or Thy1.2⁺ CD4⁺ Foxp3⁺ T cells from naive Foxp3/eGFP mice were injected intravenously into Thy1.1⁺ BALB/c mice before infection with 1 × 10⁵ infected red blood cells (iRBC) or in naive, non‐infected mice. At 3 and 5 days post‐infection (d p.i.) mice were killed and Foxp3 expression was analysed in gated CD4⁺ Thy1.2⁺ and CD4⁺ Thy1.1⁺ T cells, respectively by flow cytometry. (a) Purity of sorted CD4⁺ Foxp3/eGFP⁻ T cells and CD4⁺ Foxp3/eGFP⁺ T cells is shown in representative dot plots. (b) Representative dot plots for either non‐infected or P. yoelii‐infected Thy1.1 mice receiving either Thy1.2⁺ CD4⁺Foxp3⁻ T cells or Thy1.2⁺ CD4⁺ Foxp3⁺ T cells 3 d p.i. / cell transfer. (c) Data from two or three independent experiments with n = 4 to n = 11 mice in total are summarized as mean ± SEM. Student's t‐test was used for statistical analysis. ***P < 0·001.