Intrinsic TNFR2 signaling in T regulatory cells provides protection in CNS autoimmunity

Kamar-Sulu N Atretkhany, Ilgiz A Mufazalov, Josefine Dunst, Anna Kuchmiy, Violetta S Gogoleva, David Andruszewski, Marina S Drutskaya, Denise L Faustman, Marius Schwabenland, Marco Prinz, Andrey A Kruglov, Ari Waisman, Sergei A Nedospasov, Kamar-Sulu N Atretkhany, Ilgiz A Mufazalov, Josefine Dunst, Anna Kuchmiy, Violetta S Gogoleva, David Andruszewski, Marina S Drutskaya, Denise L Faustman, Marius Schwabenland, Marco Prinz, Andrey A Kruglov, Ari Waisman, Sergei A Nedospasov

Abstract

TNF is a multifunctional cytokine involved in autoimmune disease pathogenesis that exerts its effects through two distinct TNF receptors, TNFR1 and TNFR2. While TNF- and TNFR1-deficient (but not TNFR2-deficient) mice show very similar phenotypes, the significance of TNFR2 signaling in health and disease remains incompletely understood. Recent studies implicated the importance of the TNF/TNFR2 axis in T regulatory (Treg) cell functions. To definitively ascertain the significance of TNFR2 signaling, we generated and validated doubly humanized TNF/TNFR2 mice, with the option of conditional inactivation of TNFR2. These mice carry a functional human TNF-TNFR2 (hTNF-hTNFR2) signaling module and provide a useful tool for comparative evaluation of TNF-directed biologics. Conditional inactivation of TNFR2 in FoxP3+ cells in doubly humanized TNF/TNFR2 mice down-regulated the expression of Treg signature molecules (such as FoxP3, CD25, CTLA-4, and GITR) and diminished Treg suppressive function in vitro. Consequently, Treg-restricted TNFR2 deficiency led to significant exacerbation of experimental autoimmune encephalomyelitis (EAE), accompanied by reduced capacity to control Th17-mediated immune responses. Our findings expose the intrinsic and beneficial effects of TNFR2 signaling in Treg cells that could translate into protective functions in vivo, including treatment of autoimmunity.

Keywords: EAE; T regulatory cells; TNF/TNFR2; humanized mice; neuroinflammation.

Conflict of interest statement

The authors declare no conflict of interest.

Copyright © 2018 the Author(s). Published by PNAS.

Figures

Fig. 1.
Fig. 1.
TNFR2 humanization in hTNFKI mice restores frequency of Treg cells at steady state. (A and B) Analysis of Treg cells isolated from C57BL/6 mice (blue), hTNFKI (dark red), and hTNFKI × hTNFR2KI (gray) mice at steady state. (A) Representative FACS plots and frequencies of FoxP3+ cells among CD4+TCRb+ cells gated on live cells. (B) Representative FACS plots and frequencies of CD44hiCD62Llo cells among FoxP3+ cells gated on CD4+TCRb+ live cells. Data are representative of two independent experiments with three or more mice per group in each experiment. Each point in a diagram represents a single mouse; mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA test); NS, nonsignificant. (C–E) Humanization of TNFR2 in hTNFKI mice restores Treg cell proliferation in vitro. (C) Representative FACS histograms with Cell Trace Violet (CTV) dilution and summarized graphs of viable TCRb+CD4+FoxP3+ cells isolated from WT (C57BL/6, n = 5) and hTNFKI × hTNFR2KI mice (n = 6) and cultured under indicated conditions in the presence of aCD3, irradiated APC, and IL-2; repeated measures ANOVA with Bonferroni correction revealed: NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001. (D) Representative FACS histograms with CTV dilution of viable TCRb+CD4+FoxP3+ cells from hTNFKI × hTNFR2KI mice cultured (Left) with hTNF in the presence of IgG2b or anti-hTNFR2 blocking antibody or (Right) with IgG1 or anti-hTNFR2 activating antibody. (E) Summary of anti-hTNFR2 blocking antibody-mediated inhibition of hTNF-induced proliferation of regulatory T cells from hTNFKI × hTNFR2KI mice for n = 5 experiments (Left). Summary of anti-hTNFR2 activating antibody-mediated induction of regulatory T cell proliferation for n = 4 experiments (Right). Paired one-tailed t test revealed: *P < 0.05; ****P < 0.0001. FSC-A, forward-scatter area; LN, lymph nodes; Spl, spleen.
Fig. 2.
Fig. 2.
hTNFKI mice, but not doubly humanized hTNFKI × hTNFR2KI mice, develop exacerbated EAE. EAE analysis of C57BL/6 mice (blue), hTNFKI (dark red), and hTNFKI × hTNFR2KI (gray) mice. (A) Representative EAE clinical scores. (B–E) Analysis of immune response 2 wk postimmunization. (B) Total cell numbers of splenocytes and CNS-infiltrates. (C) Frequencies of FoxP3+ cells among CD4+TCRb+ cells gated on live cells. (D) Representative FACS histograms and mean fluorescence intensity (MFI) of FoxP3 in FoxP3+ cells gated on CD4+TCRb+ live cells. (E) Representative FACS plots and frequencies of IL-17A+ and GM-CSF+ cells among CD40L+ cells gated on CD4+TCRb+ live cells. Data are representative of three independent experiments with three or more mice per group in each experiment. Each point in a diagram represents a single mouse; mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001; NS, nonsignificant. Two-way ANOVA (A) or one-way ANOVA (B–E) tests were used.
Fig. 3.
Fig. 3.
TNFR2-mediated signaling is essential for the maintenance of Treg cells suppressive function. Analysis of hTNFKI x hTNFR2KI (gray) and hTNFKI x hTNFR2ΔTregs (red) mice at steady state. (A) Representative frequencies of CD44hiCD62Llow cells among CD4+TCRb+ cells in peripheral lymph nodes (LN) and spleen (Spl). (B) Representative frequencies of FoxP3+ cells among CD4+TCRb+ cells. (C) Representative FACS histograms and summary of FoxP3, CD25, CTLA-4, and GITR mean fluorescence intensity in FoxP3+ cells gated on CD4+TCRb+ live cells. FACS histograms are shown for spleen-derived Treg cells. Data are representative of three independent experiments with four mice per group in each experiment. Each point in a diagram represents a single mouse; mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed unpaired Student’s t test). (D) T cell suppression assay with TNFR2-sufficient and TNFR2-deficient Treg cells. Representative FACS histograms with Cell Trace Violet (CTV) dilution by viable TCRb+CD4+ responder T cells in the presence of Treg cells isolated from hTNFKI × hTNFR2KI (gray) and hTNFKI × hTNFR2ΔTregs (red) mice at ratio 1:1, without Treg cells (blue) or without activation (dark blue). The graph represents summary of responder T cell proliferation in the presence of Treg cells isolated from hTNFKI × hTNFR2KI (black) or hTNFKI × hTNFR2ΔTregs (red) mice, n = 6. Paired one-tailed t test revealed: ***P < 0.001.
Fig. 4.
Fig. 4.
Deletion of TNFR2 on Treg cells leads to exacerbated EAE. EAE analysis of hTNFKI x hTNFR2KI (gray) and hTNFKI x hTNFR2ΔTregs (red) mice. (A) Representative EAE clinical scores. (B–F) Analysis of CNS infiltration and peripheral immune response 2 wk postimmunization. (B) Total cell numbers in spleen and CNS-infiltrates. (C) Frequencies of FoxP3+ cells among CD4+TCRb+ cells in indicated organs and mean fluorescence intensity of FoxP3 in Treg cells. Mean fluorescence intensity of CD25 (D), GITR (E), and CTLA-4 (F) in Treg cells. (G) Frequencies of CCR6+ cells among FoxP3+ cells. (H) Frequencies of IL-17A+ cells among CD40L+CD4+T cells. Data are representative of three independent experiments with three or more mice per group in each experiment. Each point in a diagram represents a single mouse; mean ± SEM. *P < 0,05; **P < 0,01; ***P < 0,001; ****P < 0.0001; NS, nonsignificant. Two-way ANOVA (A) or two-tailed unpaired Student’s t tests (B–H) were used.

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