Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection

Abstract

Using a model of lethal oral infection with Toxoplasma gondii, we examined the fate of both induced and natural regulatory T (Treg) cells in the face of strong inflammatory responses occurring in a tolerogenic-prone environment. We found that during highly T helper 1 (Th1) cell-polarized mucosal immune responses, Treg cell numbers collapsed via multiple pathways, including blockade of Treg cell induction and disruption of endogenous Treg cell homeostasis. In particular, shutdown of interleukin 2 (IL-2) in the highly Th1 cell-polarized environment triggered by infection directly contributes to Treg cell incapacity to suppress effector responses and eventually leads to immunopathogenesis. Furthermore, we found that environmental cues provided by both local dendritic cells and effector T cells can induce the expression of T-bet transcription factor and IFN-gamma by Treg cells. These data reveal a mechanism for Th1 cell pathogenicity that extends beyond their proinflammatory program to limit Treg cell survival.

Figure 1. Treg cell number decreases during acute toxoplasmosis

C57/BL6 mice were infected orally with 40 cysts of ME-49. (A) Shows parasite burden in Lamina propria (LP) of small intestine at day 4, 6, 8 and 10 after infection. (B) Histological assessment of ileum from naïve or day 10 post infection (hematoxylin and eosin stain; magnification × 200). (C) Amounts of hepatic Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) were assessed in sera from naïve or day 9 infected mice. (D) Survival curve of infected mice, (n=6). (E) Comparative assessment of CD4⁺Foxp3⁺ Treg cell frequencies in LP compartments in naïve or day 10 infected mice. Cells were stained for TCRβ, CD4, and Foxp3 and TCR-β⁺CD4⁺-gated cells analyzed for expression of Foxp3 by flow cytometry. Numbers in quadrants refer to the percentage of CD4⁺ T cells expressing Foxp3. (F) Percentages of TCR-β⁺CD4⁺ cells expressing Foxp3 between day 0 and 10 after infection in spleen, MLN and LP compartments. (G) Foxp3^eGFP mice were infected with 40 cysts of ME-49 expressing RFP. Day 10 after infection small intestine sections were stained for CD4 and infected sites analyzed by confocal microscopy (CD4: purple; Foxp3^eGFP green; T. gondii : red). (H) Absolute numbers of TCR-β⁺CD4⁺Foxp3⁺ cells in various tissues between day 0 and 12 post-infection. Histograms represent the mean number of Treg cell (n=3) ± SDs. All experiments shown were performed at least 3 times, with similar results. Statistical comparisons were performed using the Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 2. Failure to sustain Treg cell conversion after T. gondii infection

CD45.1⁺ mice were orally infected or not with ME-49. 4 days after infection, recipient mice received CFSE labeled CD45.2⁺eGFP⁻OT-II T cells (1.5×10⁶) and fed with OVA antigen in drinking water for 5 days. On day 9 after infection, TCR-β⁺CD4⁺CD45.2⁺-gated T cells were assessed for intracellular Foxp3 expression and CFSE dilution. (A) Detection of Foxp3⁺ cells is illustrated for small intestinal LP in naïve or day 9 infected mice; (B) Proliferation of transferred cells from the LP was assesses by CFSE dilution (C) Percentage of CD45.2⁺eGFP⁻OT-II T cells expressing Foxp3 in LP or MLN from naïve (open circle) or day 9 (closed circle) infected mice. Each dot represents an individual mouse (n=3). (D) LP cells were briefly restimulated with PMA and ionomycin and stained for TCRβ, CD4, CD45.2, T-bet and IFN-γ. Histograms summarize the percentage of TCR-β⁺CD4⁺CD45.2⁺ cells expressing T-bet and IFN-γ. Error bars represent the SDs of the means of 3 individual mice. Experiment shown was performed 3 times, with similar results. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3. Reduced Treg cell proliferation parallels reduced IL-2 production by effector T cells during acute infection by T. gondii

Mice were inoculated orally with 40 cysts of ME-49. Spleen and MLN cells were isolated from naïve or infected mice and stained for TCRβ, CD4, Foxp3 and Ki-67 (A) Comparative analysis of Ki-67 expression in CD4⁺TCR-β⁺Foxp3⁻ (open circle) or CD4⁺TCR-β⁺Foxp3⁺ (closed circle) cells during infection by flow cytometry. Experiment shown was performed two times, with similar results (n=3). (B) At day 0, 4, 6, 8, 10 after infection, LP cells were shortly restimulated in vitro with PMA and ionomycin in the presence of Brefeldin A and stained for TCR-β, CD4, Foxp3, IL-2 and IFN-γ. Dot plots illustrate IL-2 and IFN-γ staining profiles of TCR-β⁺CD4⁺Foxp3⁻ cells. (C) At day 9 after infection, mRNA was extracted from splenic or LP cells and quantitative RT-PCR of IL-2 mRNA was performed. Histograms show the means of 3 individuals mice +/− SDs (AU = Arbitrary Unit) (D) Spleen cells were extracted from naïve, day 6 or day 10 infected mice, stimulated with α-CD3, and IL-2 secretion evaluated by ELISA 12 hours after stimulation. (E) Histograms and dot plots represent IL-2 production by CD4⁺Foxp3⁻ and Treg cell frequencies between day 0, 4, 6, 8 and 10 after infection in LP compartments. Experiments shown in panels (B), (C) and (D) were performed 3 times, with similar results (n=3; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 4. Acquisition of effector phenotype by Treg cell during T. gondii infection

CD4⁺TCR-β⁺ cells from the spleen, MLN, LP and IEL compartments were analyzed for expression of Foxp3, IL-10 and IFN-γ by flow cytometry 8 days after oral infection with 40 ME-49 cysts. (A) Dot plots illustrate IL-10 and IFN-γ intracellular staining profiles of Foxp3⁻ or Foxp3⁺ cells from naïve or day 8 infected spleen and LP compartments. Numbers in quadrants refer to the percentage of each subset. (B) CD4⁺TCRβ⁺ cells from naïve or day 9 infected LP compartment were incubated with splenic DC in presence of STAg for 15 hours and Brefeldin A. Dot plots illustrate Foxp3 and IFN-γ intracellular staining profiles of TCR-β⁺CD4⁺ gated cells. (C) Percentages of Foxp3⁻ or Foxp3⁺ CD4⁺TCR-β⁺ cells producing IFN-γ during the infection in spleen, MLN, LP and IEL compartments (cells were restimulted as in A) (D) T-bet and IFN-γ expression were analyzed by flow cytometry in naïve or day 8 infected MLN compartment on CD4⁺TCR-β⁺Foxp3⁻ and CD4⁺TCR-β⁺Foxp3⁺ cells. (D) CD45.2⁺CD4⁺eGFP⁻ T cells (2×10⁶) isolated from Foxp3^eGFP mice were transferred into CD45.1⁺ recipient mice. At day 8 after infection, CD4⁺TCR-β⁺CD45.1⁺ or CD45.2⁺ cells from the spleen were analyzed by flow cytometry for Foxp3 and IFN-γ expression. The results shown are representative of 3 panels C) independent experiments. Each dot plot represents one mouse and error bars represent the SD of the means of 3 individual mice.

Figure 5. DCs from infected mice imprint T-bet expression and IFN-γ production on both endogenous and induced Treg cells

(A) CD4⁺eGFP⁺ cells isolated from peripheral lymphoid tissue (spleen, MLN) or the thymus of Foxp3^eGFP mice were cultivated with purified LpDC from naïve or day 6 infected mice in presence the of IL-2 and α-CD3. Isotype controls or αIL-12p40 and αIFN-γ were added at the beginning of cocultures of peripheral Treg cell and LpDCs. After 5 days of culture, cells were stained for TCR-β, CD4, Foxp3 and T-bet and analyzed by flow cytometry. Histogram represents the mean of triplicate wells ± SDs. (B) Spleen and MLN CD4⁺CD45RB^loCD25^hi cells isolated from WT or Tbx21^−/− mice were cultivated with purified LpDCs from day 6 infected mice in presence of IL-2 and α-CD3, with or without addition of IL-12. After 5 days of culture, cells were shortly restimulated in vitro by PMA and ionomycin in the presence of Brefeldin A and stained for TCR-β, CD4 and Foxp3. Dot plots are representative staining for T-bet and IFN-γ on TCR-β⁺CD4⁺Foxp3⁺ cells. Experiments shown in panel A and B were performed 3 times, with similar results. (C) At day 8 after oral infection with 40 ME-49 cysts, MLN cells were briefly restimulated in vitro with IL-12, stained for TCR-β, CD4, Foxp3 and pSTAT-4, and analyzed by flow cytometry. Graphs show the percentage of TCR-β⁺CD4⁺Foxp3⁺ expressing pSTAT-4 from naïve (open circle) or day 8 infected (closed circle). Each dot represents one mouse. (D) and (E) CD4⁺CD25⁻CD44^loFoxp3⁻ T cells isolated from Foxp3^eGFP were cultured with exogenous TGF-β and α-CD3 in presence of LpDC purified from naïve or day 6 infected mice with indicated blocking mAb (αIL-12p40, αIFN-γ) or isotype control or with indicated cytokines (IL-12p70, IFN-γ). After 5 days of culture, cells were shortly restimulated in vitro and T-bet (D) or IFN-γ (E) expression was analyzed on TCR-β⁺CD4⁺Foxp3⁺ cells by flow cytometry. Histograms show summary of results obtained for triplicate wells ± SDs. Experiments shown in panel C, D and E were performed two times, with similar results (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 6. Enhancement of Treg cell survival and stability protects mice from lethal outcome

Mice inoculated orally with 40 ME-49 cysts were treated with IL-2-anti-IL-2 complexes or not for 5 consecutive days. At day 9 after infection, TCR-β⁺CD4⁺cells were analyzed for expression of Foxp3, Bcl2 and T-bet by flow cytometry. (A) Treg cell percentages in spleen and LP compartments from naïve (grey circle), infected and untreated (closed circle) or infected and treated (open circle) mice. Each dot represents an individual mouse. (B) Percentages of Treg cell expressing Bcl-2 in spleen or MLN compartments. (C) Representative staining for Foxp3 and T-bet expression by TCR-β⁺CD4⁺ gated LP cells. (D) Histograms summarize the percentage of TCR-β⁺CD4⁺Foxp3⁺ cells producing IFN-γ in the spleen and LP compartments from day 9 infected mice treated or not with IL-2-anti IL-2 complexes. Error bars represent the SDs of the means of 3 individual mice. (E) Mice were inoculated orally with 5 ME-49 cysts and the number of cysts was counted in the brain at 40 days after infection. Each dot represents an individual mouse (n=5). (F) The amount of hepatic Alanine Aminotransferase (ALT) was assessed in the serum at day 9 after infection (n=4). (G) Histological assessment of liver. (I) Comparative assessment of CD4⁺Foxp3⁺ Treg cells in spleen and colon compartments in age-matched Il2^+/− or Il2^−/− naïve mice. (H) Dot plots illustrate the T-bet and IFN-γ expression of TCR-β⁺CD4⁺ Foxp3⁺ or Foxp3⁻ cells in the spleen of naïve Il2^+/− or Il2^−/− mice. The results shown are representative 3 independent experiments with similar results (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 7. Treg cell collapse correlates with parasite virulence

(A) Mice were inoculated orally with 10 or 60 ME-49 cysts. At day 9 after infection, pathology in the small intestine duodenum (D), jejunum (J), ileum (I) and Peyer’s patches (PP) was compared (n=3). (B) Mice were inoculated orally with 10 or 40 cysts of ME-49. Comparative assessment of CD4⁺Foxp3⁺ Treg cells frequencies in MLN and LP compartments in naïve or day 9 infected C57BL/6 mice. Cells were stained for TCR-β, CD4, and Foxp3. TCR-β⁺CD4⁺-gated cells were analyzed for Foxp3 expression by flow cytometry. Each dot represents one mouse (open circle: naïve, grey circle: 10 cysts, closed circle: 40 cysts). The results shown are representative of two (panel A) or three (panel B) independent experiments with similar results. Statistical comparisons were performed using the Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Mice were inoculated orally with 40 cysts of ME-49 clone F1 or clone C1. (C) The survival curves of infected mice were compared (n=5; ***, p < 0.001). (D) At day 9 after infection, LP cells were analyzed by flow cytometry for TCR-β, CD4 and Foxp3 expression or for TCR-β, CD4, IL-2, IFN-γ, and Foxp3 expression after in vitro restimulation with PMA and ionomycin in the presence of Brefeldin A. Each dot plot represents one mouse among 3 used per condition. Numbers in quadrants represent the percentage of each subset. The results shown in panel C and D are representative of two independent experiments.