Tissue-restricted control of established central nervous system autoimmunity by TNF receptor 2-expressing Treg cells

Abstract

CD4⁺Foxp3⁺ regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell-mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients.

Keywords: TNF; Treg cells; autoimmune diseases.

Fig. 1.

Exacerbated EAE and impaired CNS Treg cell homeostasis in mice with constitutive ablation of TNFR2 in Treg cells. EAE was induced in Foxp3^CreTnfrsf1b^fl (cKO) and Foxp3^Cre control (Ctrl) mice. (A) EAE clinical score and disease survival. (B–D) FACS phenotyping at D10. Proportion of Treg cells (B) and of CTLA-4 (C) and Blimp-1 (D) expression among Treg cells is shown as mean ± SD from four independent experiments with each symbol representing a mouse. Upper panels show representative dot plots and histograms of CNS Treg cells. Two-tailed, unpaired Mann–Whitney U (A, for EAE scores, and B–D), and log-rank (Mantel–Cox, A, for survival curves) were used; **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., nonsignificant.

Fig. 2.

Ablation of TNFR2 in Treg cells after disease onset induced exacerbated EAE and impaired Treg cell activation in the CNS. Foxp3^Cre-ERT2Tnfrsf1b^fl (icKO) and Foxp3^Cre-ERT2 control (Ctrl) mice were immunized to induce EAE at day 0 and treated with tamoxifen from day 7 to 14. (A) EAE clinical score and disease survival. (B–D) FACS phenotyping at D14. Proportion of Treg cells (B) and of CTLA-4 (C) and Blimp-1 (D) expression among Treg cells is shown as mean ± SD from four independent experiments with each symbol representing a mouse. Upper panels show representative dot plots and histograms of CNS Treg cells. (E–G) Gene expression analysis of CNS-infiltrating Treg cells at day 14. (E) Volcano plot depicting differentially expressed genes. (F) Heatmap showing expression of selected genes. (G) GSEA plots of icKO Treg cells compared with genes up-regulated in 36-h culture in vitro activated vs. resting Treg cells (Left) or with genes up-regulated in CNS vs. dLN Treg cells (Right). Two-tailed, unpaired Mann–Whitney U (A, for EAE scores, and B–D) and log-rank (Mantel–Cox, A, for survival curve) were used; *P < 0.05, **P < 0.01, ****P < 0.0001; n.s., nonsignificant.

Fig. 3.

TNFR2 expression by Treg cells limits the activation and pathogenic function of Tconv cells in the CNS. EAE was induced in Foxp3^Cre-ERT2Tnfrsf1b^fl (icKO) and Foxp3^Cre-ERT2 control (Ctrl) mice that were treated with tamoxifen as in Fig. 2, and cells were analyzed at day 14. (A–D) Transcriptomic analyses on purified CD4⁺Foxp3⁻ Tconv cells, isolated from the CNS. (A) Volcano plot depicting differentially expressed genes. (B) Heatmaps showing expression of selected genes (FDR < 0.05) coding for cytokine, cytokine receptor, and T cell effector molecules (Left), transcription factors (Middle), and molecules involved in T cell migration (Right). (C) Heatmaps showing expression of selected genes (FDR < 0.05) coding enzymes of the glycolytic pathway (Left) and their position in the pathway (Right). (D) GSEA plots of icKO Tconv cells compared with genes up-regulated in effector memory versus naive CD4 T cells from the indicated GSE pathway. (E) Cytokines produced by CNS-infiltrating leukocytes restimulated ex vivo by the immunizing MOG antigen (10 or 100 µg/mL) or an anti-CD3 mAb were measured by ELISA in the supernatants. Graphs show the fold change concentration over control unstimulated cells (No Stim). Pool of two independent experiments with each symbol representing an individual mouse. Two-way unpaired ANOVA test with Sidak correction for multiple comparisons. *P < 0.05; n.s., nonsignificant.

Fig. 4.

TNF/TNFR2 signaling in Treg cells enables local suppression of EAE in the inflamed CNS. (A) Representative division profile (Left) and cumulative percentages of divided cells (Right) of MOG-specific CD90.1⁺ Tconv cells 3 d after transfer in Foxp3^CreTnfrsf1b^fl and Foxp3^Cre mice immunized to induce EAE. (B) Clinical score and disease survival of passive EAE induced in WT recipients transferred with pathogenic cells obtained from dLN of Foxp3^CreTnfrsf1b^fl (cKO) or Foxp3^Cre control (Ctrl) mice immunized 10 d earlier to induce EAE. (C) Clinical score and disease survival of passive EAE induced in cKO or control recipients transferred with pathogenic cells obtained from dLN of WT mice immunized 10 d earlier to induce EAE. (D) Clinical score and disease survival of passive EAE induced in WT recipients transferred with pathogenic cells obtained from dLN of WT mice immunized 10 d earlier to induce EAE. Recipients were treated with an anti-TNF or isotype control (Ctrl) mAb from day 0 to 8 after cell transfer. Data are from four (A) and two (B–D) independent experiments. In A, mean (±SD) from four experiments is shown; each dot represents a mouse. Unpaired t test was used. In B–D, mean (+SEM) from two experiments is shown. Two-tailed, unpaired Mann–Whitney U (for EAE scores and FACS analyses) and log-rank tests (Mantel–Cox, for survival curves) were used; **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., nonsignificant.

Fig. 5.

Systemic modulation of the TNF/TNFR2 axis affects EAE severity. (A–C) EAE clinical score in C57BL/6 mice immunized to induce EAE (day 0) and treated with anti-TNF, or isotype control mAbs or Etanercept for the indicated periods of time (gray boxes). Mean (+SEM) from two (B) or three (A and C) independent experiments. (D) Proportions of Treg cells and of dividing Treg cells in the CNS and dLN of mice treated with anti-TNF mAb as in A, and analyzed at days 29 to 45. Mean (±SD) from five experiments; each dot represents a mouse. (E) EAE clinical score in mice treated with a blocking anti-TNFR2 or isotype control mAbs from day 10 to 18. Mean from two independent experiments. (F) EAE clinical score in mice treated from day 4 to 18 with a TNFR2 agonist. Mean (+SEM) from two independent experiments. Two-tailed unpaired Mann–Whitney U test was used. *P < 0.05, **P < 0.01, ****P < 0.0001; n.s., nonsignificant.