Anti-IL-2 Treatment Impairs the Expansion of Treg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4+ T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (Treg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of Treg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4+ T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4+CD25+Foxp3+ cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4+ T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-γ production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-α and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4+ T cells from non-treated chronic mice, while it further increased the response of CD4+ T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of Treg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease

Loss- and Gain-of-Function Approaches Indicate a Dual Role Exerted by Regulatory T Cells in Pulmonary Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), is a pulmonary fungal disease whose severity depends on the adequate development of T cell immunity. Although regulatory T (Treg) cells were shown to control immunity against PCM, deleterious or protective effects were described in different experimental settings. To clarify the function of Treg cells in pulmonary PCM, loss-and gain-of-function approaches were performed with Foxp3GFP knock-in mice and immunodeficient Rag1-/- mice, respectively, which were intratracheally infected with 106 yeast cells. The activity of Foxp3-expressing Treg cells in pulmonary PCM was determined in Foxp3GFP transgenic mice. First, it was verified that natural Treg cells migrate to the lungs of infected mice, where they become activated. Depletion of Treg cells led to reduced fungal load, diminished pathogen dissemination and increased Th1/Th2/Th17 immunity. Further, adoptive transfer of diverse T cell subsets to Rag1-/- mice subsequently infected by the pulmonary route demonstrated that isolated CD4+Foxp3+ Treg cells were able to confer some degree of immunoprotection and that CD4+Foxp3- T cells alone reduced fungal growth and enhanced T cell immunity, but induced vigorous inflammatory reactions in the lungs. Nevertheless, transfer of Treg cells combined with CD4+Foxp3- T cells generated more efficient and balanced immune Th1/Th2/Th17 responses able to limit pathogen growth and excessive tissue inflammation, leading to regressive disease and increased survival rates. Altogether, these loss- and gain-of-function approaches allow us to clearly demonstrate the dual role of Treg cells in pulmonary PCM, their deleterious effects by impairing T cell immunity and pathogen eradication, and their protective role by suppressing exacerbated tissue inflammation

Adoptive transfer of Treg cells improves the protective effect of CD4⁺Foxp3^- T cells.

<p>(A) Survival curves of Rag1^-/- mice infected with Pb18 after adoptive transfer of 2×10⁵ Treg cells, 2×10⁶ CD4⁺Foxp3^- T cells, 2×10⁵ Treg cells + 2×10⁶ CD4⁺Foxp3^- T cells, or vehicle only (<i>p</i> < 0.05 between PBS and Treg; <i>p</i> < 0.001 between PBS and CD4⁺Foxp3^- T cells; <i>p</i> <0.001 between PBS and CD4⁺Foxp3^- + Treg; <i>p</i> < 0.05 between Treg and CD4⁺Foxp3^-; <i>p</i> < 0.001 between Treg and CD4⁺Foxp3^- + Treg; n ≥ 13). (B and C) Analysis of disease severity through recovery of CFU from lungs, livers and spleens from Rag1^-/- mice infected with Pb18 after adoptive transfer of 2×10⁵ Treg cells, 2×10⁶ CD4⁺Foxp3^- T cells, 2×10⁵ Treg cells + 2×10⁶ CD4⁺Foxp3^- T cells, or vehicle only. Horizontal bars indicate the mean value in each group (* = <i>p</i> < 0.05; ** = <i>p</i> < 0.005; *** = <i>p</i> < 0.001; n ≥ 10).</p

Treg cells display a natural-like phenotype and express typical surface and intracellular markers during infection.

<p>(A) Expression of surface and intracellular markers suggest a natural Treg-associated phenotype, as demonstrated by increased expression of Neuropilin-1, CD39, CD73 and Helios. Solid lines represent infected mice, whereas dashed lines indicate uninfected mice. (B) Treg cells in the lungs exhibit augmented expression of the chemokine receptor CCR5 and the suppressive cytokine IL-10 after infection with Pb18. Cells were stained for surface CCR5 or permeabilized and stained for intracellular IL-10. Histograms were gated on Foxp3^GFP+ cells and show infected (shaded area) and uninfected (black line) mice. Dot plots display the frequency of Foxp3^GFP+ cells expressing IL-10. Histograms and dot plots are representative of three independent experiments with at least five mice per group. (C) IL-10 mRNA levels in Treg cells increase after infection, as analyzed by RT-PCR. Treg cells from lungs of uninfected and infected mice (weeks 2 and 10 after infection) were isolated and total RNA was extracted. After cDNA synthesis, RT-PCR was performed using primers for IL-10. Bars represent mean ± SD from at least 5 mice per group. One representative experiment is shown (* <i>p</i> < 0.05; **<i>p</i> < 0.005).</p

Depletion of Treg cells induces balanced Th1/Th2/Th17 immunity in both stages of infection.

<p>(A) Quantification of activated/memory and naïve CD4⁺ and CD8⁺ T lymphocytes in lungs from mice treated with anti-CD25 or control IgG and analyzed after 2 (left) and 10 (right) weeks of infection. (B) mRNA relative expression of Tbet, GATA3, RORγC, Foxp3, PD-L1, CCR5, CCR6 and Granzyme B in lung cells of mice treated with anti-CD25 or control IgG, after 2 (top) and 10 (bottom) weeks of infection. <b>(C)</b> Cytokine quantitation by ELISA in lung homogenates from mice treated with anti-CD25 or control IgG. Bars show mean ± SD from at least five mice per group and are representative of three independent experiments (*<i>p</i>< 0.05, **<i>p</i> < 0.005, ***<i>p</i> <0.001).</p

Characterization of conventional CD4⁺ T cells as well as Treg cells during pulmonary PCM.

<p>(A) Representative FACS plots demonstrating the gating strategy for CD4⁺ T cells and T reg cells. (B) Expansion of CD4⁺ T lymphocytes during infection with <i>P</i>. <i>brasiliensis</i>. Percent (left) and total number (right) of CD4⁺Foxp3^- T cells and CD4⁺Foxp3⁺ T cells were analyzed by flow cytometry in the lungs of infected and uninfected C57BL/6 Foxp3^GFP mice. Numbers indicate mean ± SD for at least five mice per group and are representative of three independent experiments (* <i>p</i> < 0.05; ** <i>p</i> < 0.005; *** <i>p</i> < 0.001). (C, D) Flow cytometric characterization of CD4⁺Foxp3^- T cells (C) and CD4⁺Foxp3⁺ T cells (D) indicate an activated status at the site of infection of both cell subsets, as shown by CTLA-4, GITR, ICOS, and PD-1 expression. Solid lines represent infected mice, whereas dashed lines indicate uninfected mice. Median fluorescence intensity (MFI) values were calculated from all CD4⁺Foxp3^- T cells or CD4⁺Foxp3^GFP+ T cells. Bars reflect mean ± SD of three independent experiments with five mice per group (* <i>p</i> < 0.05; ** <i>p</i> < 0.005; *** <i>p</i> < 0.001).</p

In the absence of Tregs, mice show exacerbated inflammatory responses.

<p>(A) Photomicrographs of lungs, livers and spleens from Rag1^-/- mice infected with Pb18 after adoptive cell transfer of Treg, CD4⁺Foxp3^- T cells and Treg + CD4⁺Foxp3^- T cells. Organ sections were stained with H&E and Grocott and examined under a light microscope with a magnification of 100×. Morphometric analysis of lesions in lungs, livers and spleens from Rag1^-/- mice after 6 weeks of infection with Pb18. (B) Areas of lesions were quantified from photomicrographs of organ sections. Data represent the mean ± SD from three experiments (n ≥ 5; *<i>p</i> < 0.05; ** <i>p</i> < 0.005; ***<i>p</i> < 0.001; ND = not detectable).</p

Ablation of Treg cell leads to decreased fungal loads and diminished dissemination.

<p>(A) Groups of five C57BL/6Foxp3^GFP mice were given anti-CD25 or control IgG antibodies and inoculated with 10⁶<i>P</i>. <i>brasiliensis</i> yeast cells. The frequency of lung-derived CD4⁺CD25⁺ cells at weeks 2 and 10 post-infection is indicated. (B) Frequency and number of conventional CD4⁺ T cells as well as of Treg cells in the lungs at weeks 2 and 10 after infection following administration of control IgG or anti-CD25 antibody. Bars represent mean ± SD of three independent experiments with four to six mice per group (** <i>p</i> < 0.005; *** <i>p</i> < 0.001). (C) Fungal burden in organs of mice infected with Pb18 and treated with anti-CD25 or control IgG, after 2 (left) and 10 (right) weeks of infection. Horizontal bars indicate the mean value in each group (**<i>p</i> < 0.05, ***<i>p</i> < 0.005; n ≥ 15).</p

Treg cells limit inflammation and increase the differentiation of Th1, Th2 and Th17 cells.

<p>Rag1^-/- mice were reconstituted with different T cell subpopulations, infected with <i>P</i>. <i>brasiliensis</i> and sacrificed at the time points indicated. Lungs were excised and single cells were stained as described in Materials and Methods section. (A) Number of total leukocytes, lymphocytes, macrophages and granulocytes in the lungs of infected Rag1^-/- mice given Treg cells, CD4⁺Foxp3^- T cells or CD4⁺Foxp3^- T cells + Treg cells. (B) Frequency and number of CD4⁺CD69⁺Foxp3^- T lymphocytes and Treg cells in the lungs of infected Rag1^-/- mice previously reconstituted with Treg cells, CD4⁺Foxp3^- T cells or CD4⁺Foxp3^- T cells + Treg cells. (C-E) Numbers of CD4⁺ T lymphocytes producing IFN-γ, IL-17 and IL-4 in reconstituted and infected Rag1^-/- mice. (F) Quantitative PCR analysis of major transcription factors of T cells in the lungs of Rag1^-/- mice, as determined by RT-PCR after 2 and 6 weeks post-infection. Rag1^-/- mice were reconstituted with the different T cell subpopulations or vehicle only, infected with <i>P</i>. <i>brasiliensis</i> or left uninfected, and sacrificed after 2 and 6 weeks. Lungs were removed, RNA was extracted, reverse transcribed and the amplified cDNA was used for RT-PCR, as described previously. All bars indicate mean ± SD from at least 5 mice per group and are representative of three independent experiments (* <i>p</i> < 0.05; **<i>p</i> < 0.005; ***<i>p</i> < 0.001).</p

Effects of JES6-1 treatment on the early CD4⁺ T cell response to <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 7 p.i., splenic CD4⁺ T cells were analyzed for CD122, CD25, CD69 and CD44 expression and cell size (FSC). Data show gated CD4⁺ T cells expressing high levels of activation markers and large size (n = 3–4). (B) Non-stimulated (basal) and anti-CD3 mAb stimulated IFN-γ and IL-17 production was evaluated in CD4⁺ T cells from the same groups of mice. (C) Numbers of CD4⁺ T cells per spleen were determined in the same groups of mice. (D) On day 4 p.i., PRBC-stimulated CD4⁺ T cell proliferation was analyzed <i>in vitro</i> in the presence or absence of JES6-1 mAb. Histograms show CFSE fluorescence in gated CD4⁺ T cells. The means ± SD (n = 3–4) of the percentages of replicating (CFSE^LO) cells are shown. In A–D, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with cells from non-treated (NT) mice; and #p<0.05 with non-stimulated cells. Data are representative of three separate experiments.</p