Intrinsic TNFR2 signaling in T regulatory cells provides
protection in CNS autoimmunity
Kamar-Sulu N. Atretkhanya,b,c,1, Ilgiz A. Mufazalova,c,1, Josefine Dunstd, Anna Kuchmiya,d, Violetta S. Gogolevaa,b,David Andruszewskic, Marina S. Drutskayaa,b, Denise L. Faustmane, Marius Schwabenlandf, Marco Prinzf,g,h,Andrey A. Kruglovb,d,i, Ari Waismanc, and Sergei A. Nedospasova,b,d,i,2aLaboratory of Molecular Mechanisms of Immunity, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia;
Department of Immunology, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; cInstitute for Molecular Medicine, University
Medical Center of the Johannes Guttenberg University of Mainz, 55131 Mainz, Germany; dResearch Group Inflammation Biology, German Rheumatism
Research Center, Leibniz Institute, 10117 Berlin, Germany; eDepartment of Immunobiology, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02129; fInstitute of Neuropathology, Faculty of Medicine, University of Freiburg, D-79106 Freiburg, Germany; gBIOSS-Centre for BiologicalSignalling Studies, University of Freiburg, D-79104 Freiburg, Germany; hCIBSS-Centre for Integrative Biological Signalling Studies, University of Freiburg,
D-79104 Freiburg, Germany; and iBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russiab

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 significanceof 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 thesignificance 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 micedown-regulated the expression of Treg signature molecules (suchas FoxP3, CD25, CTLA-4, and GITR) and diminished Treg suppressivefunction in vitro. Consequently, Treg-restricted TNFR2 deficiency led
to significant exacerbation of experimental autoimmune encephalomyelitis (EAE), accompanied by reduced capacity to control Th17mediated immune responses. Our findings expose the intrinsic andbeneficial effects of TNFR2 signaling in Treg cells that could translate into protective functions in vivo, including treatment of
autoimmunity.
neuroinflammation
humanized micerole in experimental autoimmune encephalomyelitis (EAE), amouse model of MS (12–14). In contrast, the soluble form of
TNF through TNFR1 can trigger disease progression and demyelination (15, 16).
Emerging evidence indicates that TNFR2 is highly expressedon CD4+FoxP3+ Treg cells that are crucial for maintaining immune homeostasis and suppression of autoimmunity (17). Furthermore, TNFR2 is essential for the maintenance of FoxP3
expression by Treg cells and for their immunosuppressive functions in the colitis model (18). Nevertheless, the significance of
intrinsic TNFR2 signaling in T reg cells in vivo remains incompletely defined due to the lack of mouse models with conditional TNFR2 ablation. In humans, TNFR2 acts on CD4+
T cells as the master control switch for Treg expansion or Treg
contraction; therefore, TNFR2 agonists and antagonists may
have therapeutic potential (19, 20). However, one of the limitations in the development of drugs targeting TNF/TNFR2 signaling is the lack of preclinical animal models since many human
TNF-directed biologics do not interact with murine TNF or
Significance| EAE | TNF/TNFR2 | T regulatory cells |In spite of TNF involvement in the pathogenesis of multiple
sclerosis (MS), systemic TNF neutralization in MS patients was
not successful. One of the possible reasons is that TNF possesses both pathogenic and protective features that may berelated to TNFR1 versus TNFR2 receptor engagement. This
study uncovers one of such protective functions of TNF mediated by intrinsic TNFR2 signaling in Treg cells. In mice bearing
humanized TNF and TNFR2 genetic loci, TNFR2 ablation restricted
to Treg cells led to reduced capacity to control Th17 cell responses,
exacerbated experimental autoimmune encephalomyelitis (EAE)
development, and affected the maintenance of Treg cells. Thesefindings provide support for the emerging role of TNFR2 signaling in autoimmunity, as demonstrated here in mice with conditional inactivation of TNFR2.

T

NF is a cytokine with multiple functions in immune regulation, host defense, lymphoid tissue organogenesis, and control of inflammation. Dysregulation of TNF has been observed innumerous autoimmune diseases, including multiple sclerosis(MS). Strikingly, although anti-TNF therapy has shown remarkable efficacy in patients with rheumatoid arthritis (RA) (1)or inflammatory bowel disease (IBD) (2), this therapeutic approach was not successful in MS treatment (Lenercept study)(3). One possible reason is that TNF can acquire both pathogenic and protective features due to engagement with two distinct receptors, TNFR1 (CD120a or p55/p60) and TNFR2(CD120b or p75/p80), respectively. TNFR1 is a death domaincontaining receptor that can activate both cell death and expression of proinflammatory genes while TNFR2 is mostly linked
to the NF-κB–mediated proliferative pathway (4). Many featuresin TNFR1-deficient mice mirror the major defects observed in
TNF-deficient mice (5–7), suggesting that TNF signaling in vivo
may be predominantly mediated by TNFR1, and not by TNFR2.
However, recent studies revealed the involvement of TNFR2 in
the maintenance and functions of various cell types, such asneurons, oligodendrocytes, myeloid-derived suppressors, andTreg cells (8–11). Moreover, loss-of-function studies in micedemonstrated that TNFR2 and the membrane-bound form of
TNF, the preferred ligand for TNFR2, may have a protective
www.pnas.org/cgi/doi/10.1073/pnas.1807499115

Author contributions: K.-S.N.A., I.A.M., A.A.K., and S.A.N. designed research; K.-S.N.A.,
I.A.M., J.D., A.K., V.S.G., D.A., M.S., and A.A.K. performed research; D.L.F. contributed new
reagents/analytic tools; K.-S.N.A., I.A.M., J.D., M.S.D., M.P., A.A.K., and A.W. analyzed
data; and K.-S.N.A., I.A.M., M.S.D., D.L.F., A.A.K., A.W., and S.A.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution-NonCommercialNoDerivatives License 4.0 (CC BY-NC-ND).
1

K.-S.N.A. and I.A.M. contributed equally to this work.

2

To whom correspondence should be addressed. Email: sergei@nedos.net.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1807499115/-/DCSupplemental.

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Edited by Alexander Y. Rudensky, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved November 1, 2018 (received for review May 3, 2018)

TNFR2. For this purpose, we developed and validated a doubly
humanized TNF/TNFR2 mouse line with the option of conditional Cre-mediated deletion of exons 2 to 6 of the TNFR2 gene.
Such a mouse model allowed us to directly assess the consequences of Treg-restricted TNFR2 ablation at steady state andduring EAE. Furthermore, TNF/TNFR2 doubly humanized
mice have potential use for evaluation of agonistic and antagonistic antibodies against human TNF (hTNF) or human TNFR2(hTNFR2) in various models of autoimmunity.
Results
Generation and Evaluation of Mice with Functional Signaling of
Human TNF via both TNFR1 and TNFR2. To evaluate effects ofclinically utilized and novel hTNF inhibitors in vivo, we previously generated a knock-in mouse (hTNFKI), in which themouse Tnf gene was replaced by its human counterpart (21–23).
Earlier biochemical studies suggested that human TNF can bindand engage murine TNFR1, but not TNFR2 (24). Therefore, inthe current study, we aimed to generate mice with the additional
humanization of the extracellular portion of TNFR2 to ensure
functional TNF signaling through both receptors in vivo. In linewith this, we generated a hTNFR2KI mouse (see SI Appendix,
Fig. S1 for targeting strategy and technical details) and crossed
these mice to hTNFKI mice to produce doubly humanized homozygous hTNFKI × hTNFR2KI mice (SI Appendix, Fig. S2).
To directly address the role of TNFR2 signaling in distinct cell
types, including Treg cells, we additionally inserted two LoxP sites
within the human TNFR2 locus to allow for conditional Cremediated ablation of the extracellular portion of TNFR2.
Since TNFR2 signaling has been implicated in the proliferation of Treg cells (9), we first performed a comparative
analysis of these cells in hTNFKI and hTNFKI × hTNFR2KI
mice. We expected that hTNFKI mice might show defects in Treg
cell maintenance due to the inability of hTNF to signal via murine TNFR2 while doubly humanized hTNFKI × hTNFR2KI
mice would be comparable with WT C57BL/6 mice. Indeed, we
noted diminished frequencies of CD4+FoxP3+ cells in the secondary lymphoid organs from hTNFKI mice, compared with WT
controls, while additional humanization of TNFR2 resulted innormalized Treg frequencies (Fig. 1A), presumably due to restoration of hTNF signaling through hTNFR2. Additionally, frequencies of effector CD44hiCD62Llo Treg cells were increased in
hTNFKI mice compared with WT and hTNFKI × hTNFR2KI
mice (Fig. 1B). However, the total cell number and the expression levels of characteristic Treg markers, such as CD25, CTLA-4,
and GITR, were not affected in either singly or doubly humanized mice (SI Appendix, Fig. S3).
To directly assess the functionality of TNFR2 signaling in Treg
cells with humanized TNFR2, CD4+CD25+ Treg cells were sorted from spleens and lymph nodes of WT and hTNFKI ×
hTNFR2KI mice and stimulated in vitro with hTNF or mouse
TNF (mTNF) in the presence of IL-2. In line with previous
biochemical studies (24–26), T reg cells from hTNFKI ×
hTNFR2KI mice proliferated well in response to both mTNF
and hTNF while proliferation of Treg cells isolated from WT
mice was increased only in response to mTNF (Fig. 1C). Thesefindings are consistent with the notion that hTNF cannot efficiently signal through murine TNFR2 (24) and suggest that thisdefect can be rescued by humanization of the extracellular partof TNFR2. To further validate our model and to test whether
doubly humanized mice are suitable for evaluating TNFR2specific biologics, we performed similar assays with recently
characterized blocking or activating antibodies specific for
hTNFR2 (27). We found that addition of antagonistic hTNFR2
antibody impaired hTNF-induced Treg cell proliferation while
the agonistic hTNFR2 antibody alone induced a modest proliferative response (Fig. 1 D and E).
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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 ofCD44hiCD62Llo cells among FoxP3+ cells gated on CD4+TCRb+ live cells. Data arerepresentative of two independent experiments with three or more mice pergroup 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 cellproliferation 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, andIL-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 fromhTNFKI × 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-mediatedinhibition 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.

In summary, we validated doubly humanized hTNFKI ×
hTNFR2KI mice and demonstrated “normalization” of Treg cellcompartment, presumably due to functional TNF/TNFR2 signaling.
hTNFKI Mice, but Not Doubly Humanized hTNFKI × hTNFR2KI Mice,
Develop Exacerbated EAE. Since disrupted TNFR2 signaling in

hTNFKI mice resulted in decreased Treg cell numbers at steady
Atretkhany et al.

and SI Appendix, Fig. S4 D and E), whereas frequencies of IFNγexpressing T cells in the spleen were increased in hTNFKI mice
(SI Appendix, Fig. S4D).
Altogether, we concluded that TNFR2-mediated signaling isrequired to sustain Treg cell functionality, which, in turn, controls
pathogenic T cells during EAE.

Fig. 2. hTNFKI mice, but not doubly humanized hTNFKI × hTNFR2KI mice,develop exacerbated EAE. EAE analysis of C57BL/6 mice (blue), hTNFKI (darkred), and hTNFKI × hTNFR2KI (gray) mice. (A) Representative EAE clinicalscores. (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 arerepresentative of three independent experiments with three or more mice pergroup 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. Twoway ANOVA (A) or one-way ANOVA (B–E) tests were used.

state, we next tested the development of EAE in WT, hTNFKI,
and hTNFKI × hTNFR2KI mice. We observed a much higher
disease score and delayed EAE remission in hTNFKI mice (Fig.
2A), compared with WT mice and reminiscent of the phenotypeof TNFR2-deficient mice in EAE (14). Importantly, humanization of TNFR2 in hTNFKI × hTNFR2KI mice rescued thisphenotype and ameliorated disease to the level observed in WT
mice (Fig. 2A), further supporting the notion that TNFR2 indeed provides a protective signal in EAE.
The T cell compartment was next analyzed in three groups ofmice at the peak of disease. First, we found that total cellularity
was diminished in the spleen while CNS infiltration was increased in hTNFKI mice, compared with WT and hTNFKI ×
hTNFR2KI mice (Fig. 2B). Importantly, frequencies of Treg cells
in spleen and CNS upon immunization were lower in hTNFKI
mice, compared with both strains with functional TNF/TNFR2
axis (Fig. 2C). Moreover, FoxP3 expression levels in Treg cells
from hTNFKI mice, as judged by mean fluorescence intensity(MFI), were significantly reduced in spleen, but not in CNS, andwere restored back to normal in mice with double humanization(Fig. 2D). Expression levels of CD25, CTLA-4, and GITR, as
well as the frequencies of CD25+, CTLA-4+, GITR+, andCCR6+ Treg cells, were not affected in the spleen of hTNFKI
mice (SI Appendix, Fig. S4 A–C). In contrast, CD25 and CCR6
expression by Treg cells was compromised in the draining lymph
nodes of EAE-diseased hTNFKI mice, but restored in
hTNFKI × hTNFR2KI mice (SI Appendix, Fig. S4 A and C).
IL-17A–producing T cells represent the main pathogenic subset in CNS-directed autoimmunity (28). Analysis during EAE
revealed significantly higher frequencies of MOG-specific Th17
cells in the CNS, but not in the spleen, of hTNFKI mice among
all groups tested (Fig. 2E). Frequencies of GM-CSF and IFNγexpressing T cells, as well as hTNF levels in CNS, remained
unaffected, regardless of TNF/TNFR2 humanization (Fig. 2E
Atretkhany et al.

trinsic role of TNFR2 for Treg cell development and function, we
next generated mice with Treg cell-restricted deficiency in
TNFR2 by conditional deletion of exons 2 to 6 of the human
TNFR2 gene in doubly humanized mice after crossing hTNFKI ×
hTNFR2KI mice to FoxP3-Cre transgenic mouse (29) (SI Appendix, Fig. S2). The resultant mice were designated as hTNFKI ×
hTNFR2ΔTregs.
Evaluation of these mice did not reveal any obvious abnormalities in the cellularity of secondary lymphoid organs or frequencies of CD4+ T cells (SI Appendix, Fig. S5 A and B).
However, further evaluation showed increased frequency ofCD44hiCD62Llo effector T cells in the secondary lymphoid organs of hTNFKI × hTNFR2ΔTregs mice, compared with micewith sufficient TNFR2 signaling in Treg cells (Fig. 3A). Unexpectedly, the frequency of FoxP3+ cells among CD4+ T cells
also appeared higher in hTNFKI × hTNFR2ΔTregs mice (Fig. 3B)
while the percentage of CD44hiCD62Llo Treg cells was not affected (SI Appendix, Fig. S5C). Presumably, the shifted balance
between Treg cells and effector T cells could be due to compromised Treg functions in the absence of TNFR2. Indeed, we
found a significant reduction in the expression levels of Treg cellsignature molecules, such as FoxP3, CD25, CTLA-4, and GITR
in Treg cells from hTNFKI × hTNFR2ΔTregs mice (Fig. 3C). Interestingly, only the frequency of CD25+ cells among Treg cells
lacking TNFR2 signaling was reduced whereas percentages of
CTLA-4 and GITR positive cells among these Treg cells were notaffected (in contrast to the expression levels) (SI Appendix,
Fig. S5D). Additionally, hTNFKI × hTNFR2ΔTregs mice
showed diminished frequencies of CCR6+ Treg cells (SI Appendix,
Fig. S5E).
To directly address a possible impact of TNFR2 deletion on
Treg cell function, we evaluated suppressive capacity of Treg cells
on T cell proliferation in vitro. To achieve this, CD4+CD25+ Treg
cells were isolated from spleens and lymph nodes of hTNFKI ×
hTNFR2KI and hTNFKI × hTNFR2ΔTregs mice and cocultured
with responder T cells according to the standard protocol (30).
We observed that TNFR2-deficient Treg cells showed reduced
inhibitory capacity, compared with Treg cells with the functional
TNFR2 (Fig. 3D). Altogether, these findings demonstrated thesignificance of intrinsic TNFR2 signaling for suppressive capacityand maintenance of Treg cells at steady state.
TNFR2-Mediated Signaling in Treg Cells Is Protective in EAE. To assess
the functional significance of TNFR2 expression by Treg cells in
an autoimmunity model, we induced EAE in hTNFKI ×
hTNFR2 ΔTregs and control hTNFKI × hTNFR2 mice. Remarkably, hTNFKI × hTNFR2ΔTregs mice showed exacerbateddisease, compared with hTNFKI × hTNFR2KI mice (Fig. 4A),
resembling our earlier observation for hTNFKI mice (Fig. 2A).
The total number of cells and the frequency of CD4+ T cells inthe spleen at the peak of disease were comparable in hTNFKI ×
hTNFR2KI and hTNFKI × hTNFR2ΔTregs mice (SI Appendix,
Fig. S6A) whereas CNS infiltration was significantly increased in
hTNFKI × hTNFR2ΔTregs mice (Fig. 4B). Although frequencies
of FoxP3+ cells among CD4+ T cells were comparable in bothgroups, the expression level of FoxP3 was significantly diminished in Treg cells from hTNFKI × hTNFR2ΔTregs mice (Fig.
4C). We also observed a significant reduction in the expression
levels of several signature Treg cell markers, such as CD25,
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Intrinsic TNFR2-Mediated Signaling Is Essential for the Suppressive
Capacity and Maintenance of Treg Cells. To demonstrate the in-mice was characterized by increased levels of hTNF in CNS (SIAppendix, Fig. S6D) and severe demyelination, as well as elevated axonal damage (SI Appendix, Fig. S7).
Thus, TNFR2 expression and its intrinsic signaling in Treg cells
are essential for the functional status of Treg cells and provide an
overall protective signal in EAE via control of autoreactive
Th17 cells.
In summary, in this study, we developed and validated a humanized mouse with the conditional deletion of TNFR2 in Treg
cells that allowed us to directly demonstrate the critical protective role of intrinsic TNFR2 signaling in EAE, a murine
model of MS.
Discussion
TNF is a pleiotropic cytokine and a critical target for therapeutic
neutralization in several autoimmune diseases. Nevertheless, due
to the complexity of TNF signaling through two different receptors, it appears important to define cellular sources of pathogenic and protective TNF, as well as the involvement of aparticular TNF receptor expressed by distinct cell type in a given
disease setting. Cell type-restricted targeting in autoimmune
diseases may prevent side effects and increase efficiency of thetreatment. However, the progress in the development of new
biologics targeting hTNF is largely hampered by lack of adequate
in vivo model systems.
In the present study, we describe doubly humanized mice withthe functional human TNF-TNFR2 signaling axis and with theadditional option for conditional TNFR2 inactivation in desired
cell types.
Previous studies employing cell type-specific gene targetingsuggested that myeloid-derived TNF is required for EAE initiation while TNF produced by T cells protected animals from

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 ofCD44hiCD62Llow 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 asingle 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-sufficientand TNFR2-deficient Treg cells. Representative FACS histograms with Cell
Trace Violet (CTV) dilution by viable TCRb+CD4+ responder T cells in thepresence 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 cellproliferation 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.

CTLA-4, and GITR, in spleen while, in the CNS, only CD25
expression was compromised (Fig. 4 D–F), suggesting functional
insufficiency of Treg cells lacking TNFR2. Moreover, percentagesof CD25 and GITR positive Treg cells were diminished in theabsence of TNFR2 on Treg cells in the spleen (SI Appendix, Fig.
S6B). Additionally, hTNFKI × hTNFR2ΔTregs mice demonstrated lower percentage of CCR6+ Treg cells, both in CNS andin the spleen (Fig. 4G), and increased frequency of MOGspecific Th17 cells (Fig. 4H), but not of GM-CSF–producing
T cells or Th1 cells (SI Appendix, Fig. S6C). Impaired CCR6
expression on Treg cells can affect their recruitment to the CNS
during inflammation (31, 32), where Treg cells suppress inflammation. Exacerbated EAE in hTNFKI × hTNFR2ΔTregs
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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 andperipheral immune response 2 wk postimmunization. (B) Total cell numbersin 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), andCTLA-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 groupin 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.

Atretkhany et al.

Atretkhany et al.

of autoimmunity, due to compromised control of pathogenic
T cells by Treg cells.
In summary, we have generated and validated a humanizedmouse model with conditional ablation of TNFR2 in Treg cells
that allowed us to unambiguously demonstrate the critical protective role of TNFR2 intrinsic signaling in EAE, a murine
model of MS. Additionally, this mouse system can be used forevaluation of biologics that target hTNF or hTNFR2 signaling
pathways.
Materials and Methods
Mice. hTNFKI mice and Foxp3-Cre mice have been described elsewhere (22, 23,29). Generation of hTNFKI × hTNFR2KI and hTNFKI × hTNFR2ΔTregs mice is
described in SI Appendix, Figs. S1 and S2. All mice were bred and housed
under specific pathogen-free (SPF) conditions at the Institute for Molecular
Medicine (Mainz), at the German Rheumatism Research Center (DRFZ)(Berlin), and at the Pushchino Animal Breeding Facility (Branch of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academyof Sciences). Cohoused, age, and gender-matched animals (7 to 12 wk old) ofdifferent genotypes were utilized throughout the study. Experiments were
performed in accordance with the local guidelines of the corresponding
animal facility and the institution [Translational Animal Research Center(TARC), University of Mainz, Landesamt für Gesundheit und Soziales(LaGeSo), German Rheumatism Research Center and Scientific Council,
Engelhardt Institute of Molecular Biology].
Organ Preparation and Cell Isolation. Single cell suspensions were prepared bymechanical dissociation of spleen and lymph nodes in PBS supplemented with
2% FCS. To obtain CNS infiltrating lymphocytes, brain and spinal cord wereisolated from transcardially perfused mice, pooled, enzymatically digested,
and centrifuged in a 30/37/70 Percoll gradient (47).
In Vitro Treg Cultures. Treg cells were sorted from spleen and lymph nodes of
C57BL/6 and hTNFKI × hTNFR2KI mice. Treg cells were labeled with CellTrace
Violet (CTV) (ThermoFisher) at a concentration of 2.5 μM according to the
manufacturer’s instructions. Then, 50,000 CD4+CD25+ cells were cultured
together with 200,000 irradiated antigen-presenting cells (APCs) in complete
RPMI 1640 in the presence of either plate-bound (10 μg/mL) or soluble(0.5 μg/mL) anti-CD3 (145-2C11; in-house/DRFZ) and 10 to 100 ng/mL IL-2
(R&D systems) in a humidified atmosphere for 72 h at 37 °C and 5% CO2.
Where indicated, murine TNF (ThermoFisher) or human TNF (Dr. MadausGmbH) were added at 10 ng/mL. Anti-human TNFR2 blocking antibody (TY877),
or anti-human TNFR2 activating antibody (TY010), or the respective isotype
controls, rat IgG2b (RTK4530; Biolegend) or rat IgG1 (19E1; in-house/DRFZ),
were added at 12.5 μg/mL (27).
In Vitro Treg Suppression Assay. Treg cell suppression assay was performed aspreviously described (30). Briefly, naive CD4+ T cells from spleen and lymph
nodes of C57BL/6 mice were magnetically sorted (Miltenyi) and labeled with
CellTrace Violet (CTV) at a concentration of 5 μM according to the manufacturer’s instructions. CD4+CD25+ Treg cells were isolated from spleens andlymph nodes of hTNFKI × hTNFR2KI and hTNFKI × hTNFR2ΔTregs mice using amagnetic bead separation kit (Miltenyi). CTV-labeled T cells were cocultured
with Treg cells in complete RPMI 1640 medium in the presence of 1 μg/mL
anti-CD3 (145-2C11; in-house/DRFZ) and 6 ng/mL of anti-CD28 (37.51; inhouse/DRFZ) in a humidified atmosphere for 80 h at 37 °C and 5% CO2.
T cell proliferation was analyzed by flow cytometry by measuring the
CTV label.
MOG-Induced EAE. Mice were s.c. immunized with 50 μg of MOG35-55 peptide(Gene Script) emulsified in complete Freund’s adjuvant (CFA) supplemented
with 5 mg/mL Mycobacterium tuberculosis (Difco), followed by 150 ng of
Pertussis toxin (List Biological Laboratories) administration on day 0 and 2.
Mice were scored daily, and clinical signs were assessed according to standard protocol. Briefly, the following scores were used: 0, no disease; 0.5,
partial tail paralysis; 1, complete tail paralysis; 1.5, partially impaired righting
reflex; 2, impaired righting reflex; 2.5, impaired gait with limping; 3, hindlimbs paresis; 3.5, complete paralysis of hind limbs; 4, forelimbs paresis; 4.5,
complete paralysis of forelimbs; 5, inability to move; 5.5, moribund.
ELISA Analysis. For hTNF measurement, brain and spinal cord homogenates
were incubated in complete radioimmunoprecipitation assay (RIPA) buffer(Sigma Aldrich) with Protease Inhibitor Mixture (Roche) and centrifuged at

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pathogenic T cell responses and subsequent CNS demyelination (33). Interestingly, TNFR2 signaling on myeloid cells may
play contrasting roles, since TNFR2-expressing microglial
cells appeared to be protective while TNFR2+ macrophages
contribute to the severity of EAE (10). However, the impact of
TNFR2 signaling in T cells, especially in Treg cells, to neuroinflammation remains incompletely understood. Despite theevidence that TNFR2 is highly expressed on Treg cells, the role of
TNFR2 in differentiation and maintenance of Treg cells in vivo
remains controversial since most reported studies were based on
the adoptive transfer of Treg cells. Some studies indicated that
TNF has a beneficial effect for Treg cells through increased
proliferation and maintenance of suppressive phenotype (9, 27,34) while others reported the opposite role or no role for TNFR2(14, 35, 36).
Using humanized mice with the Cre-mediated TNFR2 deletion specifically in FoxP3+ cells, we directly demonstrated theloss of suppressive functions of Treg cells and increased EAEseverity in the absence of TNFR2. Selective ablation of TNFR2
on Treg cells resulted in increased pathogenic T cell responses
during EAE. Our data indicate that TNF-TNFR2 signaling in
Treg cells is not essential for disease initiation but is critical forthe control of autoreactive Th17 responses and disease severity.
The downstream effects of TNFR2 agonism are mediated byboth canonical and noncanonical NF-κB pathways (37). Throughdistinct combinations of NF-κB subunits, TNFR2 may regulatedistinct Treg features, including the initial burst in FoxP3 expression and its stabilization during Treg cell proliferation anddifferentiation (18, 38). Indeed, we found that TNFR2 deficiency
in Treg cells leads to reduced expression of its master regulator
FoxP3 and Treg-associated molecules, such as CD25, CTLA-4,
and GITR, that are known to be critical for dampening the inflammatory responses (39, 40). Interestingly, TNFR2 is highly
expressed on most suppressive Treg cells (17, 41), and one possible role of this receptor could be its competition with othercells for TNF. Since TNFR2 has higher affinity for transmembrane TNF than TNFR1, Treg cells may bind this form of
TNF more efficiently and, thus, acquire a proliferative or survival
advantage. Another possible implication of TNFR2 signaling for
Treg cell functions is the prevention of caspase-8–mediateddegradation of FoxP3 during inflammation (35). Additionally,in the absence of TNFR2 signaling, expansion of CCR6+ Treg
cells is reduced. This subpopulation can migrate following
CCL20 chemokine gradient and regulate Th17 immune responses in target organs, including the CNS (32). It would be
beneficial to conduct further experiments using inducible Tregspecific ablation of TNFR2 to shed light on the role of TNFR2 in
the priming phase of T cell response and in EAE progression.
Dysfunctional Treg cells are a characteristic trait of autoimmunity. In some human autoimmune diseases, the total numbersof Treg cells are not altered, but the potent TNFR2+ Treg cell
subpopulation is underrepresented (42). Growing evidence suggests that TNFR2 may define a major central switch for Treg cell
expansion or Treg cell contraction (19, 43). Recently, a TNFR2
agonist was used to expand a stable population of human Treg
cells ex vivo that highly expressed FoxP3 and Helios, which waslater confirmed by studies in mice (19, 44). The same TNFR2
agonist showed therapeutic potential in murine models of acutegraft-versus-host disease (GvHD) (43), and in collagen-induced
arthritis (45), although the exact cellular targets were not identified. Selective stimulation of TNFR2 and inhibition of TNFR1
by receptor-specific antibodies was beneficial in NMDA-induced
acute neurodegeneration (46) while employing TNFR2 antagonist inhibited proliferation of tumor-associated Treg cells, suggesting TNFR2 as an important target also in cancer treatment(27). However, similarly to the checkpoint therapy paradigm,
blockade of suppressive mechanisms may, on one hand, promote
antitumor immunity but, at the same time, may lead to exacerbation

20,000 × g for 30 min at 4 °C. Total protein concentration was measured
with a Bradford Protein Assay (Bio-Rad). hTNF concentration in supernatants
was measured using ELISA Ready-Set-Go kits (eBioscience) and normalized
to total protein level.
Histology. A detailed procedure of histology analysis is provided in SI Appendix, Supplementary Materials. Briefly, spinal cords were isolated on day
17 after EAE immunization and fixed in 4% buffered paraformaldehyde(PFA). Specimens were dehydrated and embedded in paraffin and cut andstained with luxol fast blue-periodic acid–Schiff (LFB-PAS) to assess the degree of demyelination and with antibody to amyloid precursor protein (APP)
to determine axonal damage. Histological images were taken with a BZ9000 Microscope (BioRevo; Keyence) and quantified as described (48).

the presence of monensin or brefeldin A, harvested, washed, and stained with
an eBioscience FoxP3 Fixation Permeabilization kit. Gating strategies are
summarized in SI Appendix, Figs. S8 and S9.
Statistical Analyses. All statistical analyses were done using Prism software(GraphPad). Unpaired or paired t tests and one-way or two-way ANOVA
tests were used. Differences were considered significant when P values
were <0.05.

Flow Cytometry. Single cell suspensions prepared from lymph nodes, spleen,and central nervous system were stained with fluorescently labeled antibodies
purchased from BD Biosciences, BioLegend, or eBioscience (SI Appendix, Table
S2) and acquired with BD FACSCanto II or LSR II flow cytometers. Flowcytometry data were analyzed using FlowJo software. For intracellular staining of cytokines, cells were stimulated with MOG-peptide (20 μg/mL) for 6 h in

ACKNOWLEDGMENTS. We thank Drs. S. Kozlov and S. Woertge for helping
us to generate hTNFKI and hTNFR2KI mice, respectively; and M. Blanfeld forassistance with mouse colony maintenance. We thank Drs. D. Kuprash andG. Efimov for critical reading of the manuscript; and Dr. T. Bopp for providing
FoxP3-Cre mice on C57BL/6 background (originally from Prof. S. Sakaguchi).
This work was supported by Russian Science Foundation Grant 14-50-00060
and by Deutsche Forschungsgemeinschaft (DFG) Grant NE 1466/2. A.W. isa member of the Research Center Immunology (FZI) Mainz and was
supported by DFG Grant CRC/TR 128. K.-S.N.A and I.A.M. were partially
supported by independent European Federation of Immunological SocietiesImmunology Letters (EFIS-IL) fellowships.

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