SCIENCE SIGNALING | RESEARCH RESOURCE
BIOCHEMISTRY

A novel TNFR2 agonist antibody expands
highly potent regulatory T cells
Heather Torrey*, Willem M. Kühtreiber*, Yoshiaki Okubo, Lisa Tran, Katherine Case,
Hui Zheng, Eva Vanamee, Denise L. Faustman†

Copyright © 2020
The Authors, somerights reserved;
exclusive licensee
American Association
for the Advancementof Science. No claim
to original U.S.
Government WorksINTRODUCTION

Traditional therapies for inflammatory disorders have depended on
immunosuppressive medications that globally diminish immune
responses. Although these agents are highly effective for many
patients, their common and sometimes serious adverse effects, including life-threatening opportunistic infections, combined with
the need for high-dose, long-term use have given impetus for thedevelopment of more specific and safer immunotherapies.
One relatively recent approach is to enhance the function of regulatory T cells (Treg cells), a small subset of T lymphocytes that express CD4, high-density interleukin-2 (IL-2) receptor (CD25), andinducible levels of intracellular transcription factor forkhead box P3(Foxp3) (1). Treg cells regulate the immune response in health andin diseases such as autoimmunity versus cancer. Enhancement ofTreg cell function is considered beneficial for many disease states (2–9),
including autoimmune diseases ranging from type 1 diabetes (T1D),
Crohn’s disease, ankylosing spondylitis, and arthritis to colitis (2–9).
Greater Treg cell activity may prevent transplant rejection and graft-­versus-host disease (3, 10). Last, Treg cell enhancement may play a specialrole in neurodegenerative disease, including multiple sclerosis,
Parkinson’s disease, and dementia. The benefit of a Treg cell-targeted
therapy to the central nervous system (CNS) may be derived not
only from simple removal of the disease process but also by CNS
repair and regeneration, as demonstrated also for pancreatic islet
regeneration (8–13).
Tumor necrosis factor receptor 2 (TNFR2) is abundantly expressedon the surface of the most potent subtype of Treg cell (14–15).
TNFR2 is not merely a cell surface marker; rather, it appears to act asa master control switch for potent Treg cell expansion or Treg cell inhibiImmunobiology Laboratory, Massachusetts General Hospital and Harvard Medical
School, Boston, MA 02129, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: faustman@helix.mgh.harvard.edu
Torrey et al., Sci. Signal. 13, eaba9600 (2020)

8 December 2020tion (16). Agonism of the TNFR2 expands Treg cells, whereas antagonism kills them. Agonism culminates in nuclear factor B (NF-B)–­
dependent gene transcription through the DNA binding activity of
RelA (p65), RelB, c-Rel, NFkB1 (p50), and NFkB2 (p53) (p52), as
well as through the activity of signaling complexes such as TNFR-­
associated factor (TRAF) and cIAPs (cellular inhibitors of apoptosis
proteins), which are associated with cell proliferation (17). TNFR2
is also an attractive candidate for controlling the right balance ofTreg cells to T effector cells (Teff cells), which are Foxp3-­expressing
CD4+ or CD8+ T cells. The right balance of Treg cells to Teff cells isnecessary for health. Cancer typically is characterized by too many
Treg cells and too few Teff cells, whereas autoimmunity is associatedwith either too few Treg cells or Treg cells of insufficient potency andtoo many Teff cells (15, 18). Although TNFR2 is underexpressed on
Treg cells in diverse autoimmune diseases, the pathogenic cytotoxic
T cells (a subpopulation of CD8+ Teff cells) that cause autoimmunity overexpress TNFR2 and are also targeted by agonism thatkills these high–TNFR2 density cells (19). TNFR2 agonism expands
Treg cells but specifically kills pathogenic cytotoxic T cells in diverse
human autoimmune diseases (19).
TNFR2 targeting carries some additional advantages. Unlike
many other Treg cell markers that are also expressed broadly on diverse
lymphoid populations, TNFR2 has a far more restricted lymphoid
distribution (20). A pivotal study in baboons has shown that TNFR2
agonism, in the form of TNF trimeric muteins with only TNFR2
binding, has minimal toxicity (21). This stands in contrast to
TNFR1, a receptor with ubiquitous bodily expression and severe
toxicity with agonism (22).
The creation of a novel agonistic antibody to TNFR2 might be,at first glance, straightforward. However, a closer look reveals that
progress has been elusive. Little efficacy has been achieved in early
results of ongoing cancer trials using agonistic antibodies to members of the TNF superfamily such as OX40, 4-1BB, and GITR (23).
There is much speculation on why agonistic antibodies are difficult
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Regulatory T cells (Treg cells) restrict immune system activity, such as in response to self-antigens, and are switchedon by tumor necrosis factor receptor 2 (TNFR2). Therapeutic activation of TNFR2, thereby expanding Treg cells andsuppressing immune activity, may be beneficial to patients with various inflammatory diseases. Here, we characterized a new human TNFR2-directed antibody agonist isolated from mice. We found that the antibody agonist
expanded the number of Treg cells within cultures of primary human CD4+ T cells from healthy donors and patientswith type 1 diabetes or Sézary syndrome. These Treg cells had increased metabolic gene expression and intracellular itaconate concentrations, characteristics associated with maximally suppressive, anti-inflammatory Treg
cells. Furthermore, antibody-expanded Treg cells repressed the activity of primary human CD8+ effector T cells (Teffcells). Epitope mapping suggested that the antibody bound to TNFR2 through a natural cross-linking surface andthat Treg cell expansion was independent of the antibody Fc region. In addition, Treg cell expansion was not increased byadding either supplemental TNF ligand or a cross-linking reagent, suggesting that the antibody agonist by itself
can elicit maximal activity, a notion that was confirmed by increased secretion of soluble TNFR2. Pending in vivo
tests, these features indicate that this TNFR2 antibody agonist has the potential to safely and effectively treat
various inflammatory disorders.

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RESULTSTNFR2 density on Treg and Teff cells varies by disease
TNFR2 expression on human T cell populations, Treg and Teff cells,
isolated from human participants with cancer (a lymphoma subtype known as Sezary syndrome) or autoimmune disease (T1D)
demonstrated substantial variation compared to those isolated fromhealthy controls. According to mean fluorescent intensity (MFI) of
the receptor detected with flow cytometry, a significantly higher
proportion of Treg cells from cancer participants expressed TNFR2
compared to those from either healthy controls or type 1 diabetics(Fig. 1A). Treg cells from type 1 diabetics expressed a lesser densityof TNFR2 compared to those from normal participants or patientswith cancer (Fig. 1A), whereas their Teff cells expressed a significantly greater amount (Fig. 1B).
TNFR2 agonist induces proliferation of Treg cellswith maximal agonism
As described in Materials and Methods, TNFR2 antibodies were
produced in BALB/c mice with peptides and whole extracellular
portions of the human TNFR2 protein. Screening for agonistic activity occurred after confirming selective TNFR2 binding with no
TNFR1 binding. Agonistic activity was identified by examining
fresh human CD4+ T cells for selective Foxp3+ and CD25+ expression using flow methods, typically after a 2- to 4-day incubation
period. An increase in cell numbers of Treg cells and an increase in
the density (MFI) of both Treg cell markers allowed the selection of theclone with the greatest potential for further functional studies.
When CD4+ T cells from normal blood donors were treated withTNFR2 agonist antibody, dose-dependent proliferation of Treg cells
occurred (Fig. 1C and fig. S1A). We previously reported that TNFR2
agonism (using a different antibody) yields expansion of highly potent and homogeneous Treg cells instead of a mixed population of
CD4+ T cells in controls and autoimmune participants (16, 26). A
mixed population poses safety problems for clinical trials (owing tocytokine secretion, among other factors) and thus faces more regulatory hurdles. When autoimmune CD4+ T cells from type 1 diabetic participants were tested, the TNFR2 agonist antibody was able
to proliferate the Treg cells in a dose-responsive manner (Fig. 1D
and fig. S1B).
Torrey et al., Sci. Signal. 13, eaba9600 (2020)

8 December 2020

Agonist (µg/ml)

Agonist (µg/ml)

Agonist (µg/ml)+ TNF (20 ng/ml)

Fig. 1. TNFR2 expression in cancer and autoimmunity and the expansion ofTreg cells by the TNFR2 agonist antibody. (A) Mean fluorescent intensity (MFI) of
TNFR2 abundance on Treg cells isolated from the peripheral blood of cancer (lymphoma, n = 10), normal (n = 10), and T1D participants (n = 24). (B) As described in(A) on Teff cells, each n = 10. (C) Percentage of Treg cells in a culture of freshly isolated
peripheral human CD4+ cells from nondiabetic control patients in response to
TNFR2 agonist antibody for 48 hours (n = 13). Analysis of P values for trend in thelinear mixed-effects model (LMEM) up to 2.5 µg/ml, P = 0.0018 and greater than
2.5 µg/ml, P = 0.80. A representative set of FACS plots is shown in fig. S1A. (D) Percentage of Treg cells in a culture of freshly isolated peripheral human CD4+ cells frompatients with T1D in response to TNFR2 agonist antibody for 48 hours (n = 11). Representative FACS plots are in fig. S1B. (E) As described in (C), with added TNF(20 ng/ml; n = 3). LMEM analysis, P = 0.07. Representative FACS plots are in fig. S1C.
In all panels, data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
by Student’ t test (unequal variance, two tailed).

When the TNFR2 agonist was combined with TNF at high concentrations (20 ng/ml; Fig. 1E and fig. S1C), expansion of humanTreg cell occurred, but it was no greater than that with TNFR2 agonist
alone (Fig. 1C and fig. S1A). This demonstrated that supplemental
TNF is not obligatory for Treg cell expansion using this form of TNFR2
agonistic antibody. These data suggested that maximal agonism by
the TNFR2 antibody may already be present with the antibody
without added TNF ligand. This also suggested lower toxicity in human trials. Supplementation with the natural ligand risks high toxicity and has impeded clinical trials with cancer drugs. Although thedata are represented as a percentage of cells, for each experiment,
total cell number counts were also performed to rule out cell conversion without the total number of cells increasing as well (fig. S2).
One common way to induce Treg cell proliferation is with IL-2 or
TNF, although both IL-2 and TNF are difficult treatments in vivo
because of their toxicity (26). A dose comparison was performed for
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Downloaded from http://stke.sciencemag.org/ on December 10, 2020to produce (23). In some cases, agonistic antibodies under development were produced with full immunoglobulin G1 (IgG1) function
and have required Fc receptor activation through dependence on
antibody-dependent cellular cytotoxicity (ADCC). In the clinic, TNF
superfamily agonism with ADCC antibodies has, in some cases, resulted in liver toxicity. Also, signaling of TNF superfamily receptors
requires a strategic clustering of receptors with the right geometry
for sufficient signal strength and efficient intracellular signal transduction for intracellular pathway activation (24, 25).
Here, we first examined the differential expression of TNFR2 on
Treg cells and Teff cells in cancer and autoimmunity and then, using
these cells, characterized in detail a new human-directed agonistic
antibody. We found that this antibody induces Treg cell proliferation,
specifically of highly potent Treg cells, and efficient downstream signaling, all in a manner that is independent of the Fc region of theantibody, indicating the likelihood of more limited toxicity in vivo
than that which to date has hampered the use of human-directedagonists in the clinic.

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from patients with T1D. In coculture, we found that the proliferative capacity of the responders—CD8+ T cells (or Tresp cells), stainedwith carboxyfluorescein succinimidyl ester (CFSE)—was inhibitedby either a greater proportion of Treg cells compared to responders
or by Treg cells expanded in the presence of TNFR2 agonist antibodies. As expected, suppression of responder cell proliferation increased as more TNFR2 agonist–expanded Treg cells were added, as
shown in both representative and pooled data from seven donors(Fig. 2, B and C). The TNFR2 agonist antibody was thus effective
at generating functional Treg cells in vitro from patients with T1D,
known to be inherently defective in generating activated Treg cells(27). Similar potency was observed with normal donors (Fig. 1, C
and E).

AC

TNFR2 agonist

Fig. 2. Comparison of IL-2 and TNF on Treg cell proliferation and the suppressive
ability of TNFR2 agonist–expanded Treg cells. (A) Expansion of Treg cells (as apercentage) within nondiabetic human CD4+ T cell samples in response to increasing doses of exogenously added human IL-2 (left, n = 7 samples) and TNF (right,
n = 8). (B) Functional evaluation, in terms of capacity to suppress CD8+/Tresp cellproliferation, of exogenously TNFR2 agonist antibody-expanded Treg cells added inincreasing ratio to CD8+ T cell populations from patients with T1D. Cocultures were
stimulated with soluble anti-CD3 (clone Hit3a) and IL-2 (50 U/ml) in 10% FBS RPMI
for 4 days. CD8+-gated proliferation in a representative of seven experiments isshown. (C) Quantification of the data described in (B), as means ± SEM from sevennormal blood donors. *P < 0.05, **P < 0.01, and ****P < 0.0001 by Student’s t test
(unequal variance, two tailed).

the impact of treatment with IL-2 or TNF alone (Fig. 2A) versus
treatment with TNFR2 agonistic antibody (Fig. 1C), which showed
the superiority of the latter in expanding human CD4+ Treg cells. It
has previously been reported that TNFR2 expansion of Treg cells in
culture also generates a more potent Treg cell, not just more Treg cells(14). Additional experiments confirmed that the absolute numberof Treg cells, not just the percentage of cells, was higher with TNFR2
agonistic antibody (fig. S2). This result rules out the possibility thatthe percentage increase in Treg cells was solely due to conversion ofTreg cells to Teff cells rather than actual Treg cell expansion.
Functional assay reveals that expanded Treg cells suppress
CD8+ T cells
To determine the functional capacity of TNFR2 agonist–expanded
Treg cells, we tested their ability to suppress autologous CD8+ T cells
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TNFR2 agonist–driven Treg cell expansion does not dependon the Fc region or on a cross-linking agent
Evaluation of the F(ab′)2 fragment of TNFR2 agonist antibody
demonstrated efficacy at Treg cell proliferation, and therefore, functionality of the antibody is independent of the Fc region (Fig. 3A).
Functional activity of the TNFR2 agonist antibody is also independentof receptor cross-linking as demonstrated by coculturing of rodent
anti-IgG antibody with TNFR2 agonist, an experiment that did
not show greater dose-­dependent Treg cell proliferation (Fig. 3B).
This finding suggests that the mechanism of action is through anactivation pathway that overrides the effect of obligatory cross-linking
or Fc binding. It also suggests that treatment with a TNFR2 agonist
antibody is likely to be less toxic because it does not require the
ADCC mechanism and does not need to be combined with across-linking agent. In addition, the lack of reliance on a cross-linking
agent suggests that antibody-­receptor binding is sufficiently clustered for maximal agonism.
We next studied the TNFR2 antibody agonist for an agonism
biomarker, soluble TNFR2 (sTNFR2), in the media. sTNFR2 is abiomarker of maximal agonism developed by the cancer field (28–31).
When there is high-TNFR2 agonism, the membrane receptor iscleaved by TNF- converting enzyme; this action releases sTNFR2
into the extracellular space and can be used as a biomarker of agonism. We found minimal sTNFR2 in the media with IL-2 alone,some sTNFR2 with TNF, but the most sTNFR2 was seen withTNFR2 agonism (Fig. 3C). This finding is important for translation
into the clinic because it suggested that sTNFR2 has potential as aserum-based biomarker of binding efficacy for diseases that do notreadily lend themselves to tissue sampling.
TNFR2 agonism induces gene signature indicative ofpotent Treg cells
Although we have shown that the agonist antibody increases Treg
cell numbers and percentages, we do not know from these expansion experiments whether the increases were occurring in the subpopulation of highly potent Treg cells. We therefore selected 33 Treg
cell signature genes that characterize potent Treg cells for gene
expression after brief and low-dose exposure of CD4+ T cells to
TNFR2 agonistic antibody (32). This selection of Treg cell-associatedgenes represents well-described surface proteins and three potent
transcription factors that directly control Foxp3 expression. T cell
receptor activation resulted in the binding of CREB (cyclic adenosine monophosphate response element–binding protein) to an intronicenhancer element (hereafter referred to as CNS2) in the FOXP3 gene(33). Also, critical is ETS1 binding to the CNS2 region, supporting a
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% CD8+ proliferation

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20,000Fig. 3. Expansion of Treg cells in response to F(ab)′ fragments and cross-linking
antibody and the impact of TNFR2 agonism on sTNFR2 secretion. (A) Treatment of peripheral human CD4+ cells (n = 5 samples) with F(ab′)2 fragments of
TNFR2 agonist antibody with the evaluation of the proliferation of Treg cells relative
to treatment with IL-2 alone. (B) Incubation of human CD4+ cells (n = 3 samples)with cross-linking antibody (ab9165 at 2.5 g/ml) and examination for augmented
Treg cell proliferation capacity of TNFR2 agonist antibody. Similar results were obtained
at 24-, 48-, and 72-hour incubation. (C) Soluble TNFR2 (sTNFR2), as a measure of
TNFR2 shed from brisk agonism, in culture medium from human CD4+ cells (n = 10
samples) was evaluated in response to IL-2 alone (200 U/ml), TNF alone (20 ng/ml),or the TNFR2 agonist (2.5 g/ml). Data are means ± SEM. *P < 0.05, **P < 0.01, and***P < 0.001 by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.

direct effect of FOXP3 transcription as well as a role in epigenetic
changes required for stable FOXP3 expression. Histone modification and DNA demethylation are also associated with stable expression of FOXP3 (34, 35).
Potent Treg CD4+ T cells are known to express panels of proteins
related to active and potent suppressor functions. We investigated,using RNA sequencing (RNA-seq) methods, the expression of these
Treg cell signature genes in normal CD4+ T cells exposed to the TNFR2
agonist for 10 hours. The panel of known Treg cell signature genes were
those that encode nuclear transcription factors that directly stimuTorrey et al., Sci. Signal. 13, eaba9600 (2020)

8 December 2020

Fig. 4. Induction of Treg cell signature genes after TNFR2 agonism. (A) RNA-seq
gene expression analysis of key mRNA-encoding proteins known to be importantfor potent Treg cell function. The graph shows %change in gene expression after treatment with TNFR2 agonist antibody (2.5 g/ml, 10 hours) as compared to untreatedcontrol, performed on purified human fresh CD4+ T cells from nondiabetic controls(n = 12). Blue bars represent increases in mRNA after treatment with agonist antibody. (B) Effect of the TNFR2 agonist antibody or a TNFR2-antagonist antibody(each 2.5 g/ml for 10 hours) compared to IL-2 alone on the expression of the
FOXP3-inducing genes ETS1, EP300, and CREB in fresh human CD4+ T cells isolated
from nondiabetic controls. Data are means ± SEM from n = 12 samples. ***P < 0.001
by Student’s t test (paired, two tailed).

late FOXP3 expression at the important CNS2 promoter site (ETS,
EP300, and CREB) and those that encode common cell surface proteins and secreted cytokines that identify the most potent Treg cells:
CTLA4, Helios (IKZF2), Eos (IKZF4), Il2RA (CD25), TNFRSF18(GITR), TNFR2, PDL2, IL-2, IL-10, IL-4, CD274, MYC, CD3D,
CD3G, CDKN1C, MAPRE2, RUNX1, TNFRSF25, TOLLIP, ITGAL,
EST1, FOXP3, TNFRSF18, TNF, CTLA4, IRF8, and PDCD1. A 10-hour
exposure to TNFR2 agonist induced many Treg cell signature genes(Fig. 4A, blue bars). For statistical significance, an induction of atleast 1200 normalized reads was considered significant compared to
CD4+ T cells cultured with IL-2 without antibody for the samelength of time. Those genes induced rapidly by the agonist included
IL7R, IL1B, IL2RA, CD274 (which encodes PDL1), MAPRE2, RUNX1,
PDCD1LG2 (encodes PDL2), TNFRSF9 (encodes 4-1BB), ITGAL,IKZF2 (encodes IKAROS), ETX1, EP300 (which encodes histone
acetyltransferase p300, also known as E1A), CREBBP, and FOXP3.
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B

SCIENCE SIGNALING | RESEARCH RESOURCETNFR2 agonism, through induced adenosine
monophosphate kinase and carnitine palmitoyl transferase
1A, augments FOXP3 expression and may augment
the Krebs cycle with fatty acid supply
Treg cells are dependent on mitochondrial metabolism, with the useof oxidized lipid in preference to glucose, whereas other T cells
commonly use glucose through glycolysis and do not use fatty acids(37–39). Treg cells express high levels of carnitine palmitoyl transferase 1a (CPT1a), the rate-limiting enzyme of fatty acid oxidationthat allows the entry of acyl groups into the mitochondria. This supports the possibility that Treg cells can use multiple fuel sources.
This, too, was observed in TNFR2 agonist–expanded Treg cells; gene
expression-level data suggest that TNFR2 agonist–induced Treg
cells switched to augmented fatty acids for energy metabolism, given
the up-regulation of CPT1A, CPT1B, and adenosine monophosphate kinase (AMPK) expression (Fig. 5B and fig. S4B). CPT1A also
directly induced Foxp3 expression, another indication of a potent
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8 December 2020

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Fig. 5. TNFR2 agonist antibody changes metabolic gene expression in potent
Treg cells. (A) Human blood derived CD4+ T cells from nondiabetic controls wascultured with and without TNFR2 agonist antibody (2.5 g/ml) for 10 hours andevaluated for glycolysis- and glutaminolysis-associated mRNA expression. Data arenormalized reads of mRNA (%change) compared to untreated control shown asmeans ± SEM from 12 samples. Heat plots are summaries of the pooled data.
(B) Expression of fatty acid utilization–associated genes in CD4+ T cells from nondiabetic control patients (n = 12) stimulated with TNFR2 antibody agonist (2.5 g/ml)
or IL-2 for 1 hour. Data are means ± SEM. ***P < 0.001 by paired Student’s t test.

Treg cell signature in response to TNFR2 agonism. These mRNA-seq
data are suggestive of increased fatty acid supply and Foxp3 induction, but this remains to be validated at a functional level.
TNFR2 agonism induces early Krebs steps including
induction of a possible suppressive metabolite pathway,
itaconate
Last, we explored the potential metabolic status of Treg cells upon
TNFR2 stimulation in relation to the Krebs cycle. TNFR2 agonism
of CD4+ T cells induced gene expression profiles indicative of glutaminolysis and augmented glutamate and fatty acid supply into the
Krebs cycle (Fig. 6 and fig. S4C). For example, this is illustrated by
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TNFR2 agonism may induce targeted metabolic changes
characteristic of potent Treg cells
Advances have been made in understanding of the metabolism ofTreg cells compared to other lymphoid cell populations. Metabolic
data combined with Treg cell surface markers allow the most potent Treg
cells to be identified and characterized (16). To understand this in
more detail, in relation to the efficient expansion of human Treg
cells with TNFR2 agonism, fresh human T cells were incubated withTNFR2 agonistic antibodies and were studied for changes in metabolism consistent with potent Treg cell induction (10 hours, 2.5 µg/ml ofTNFR2 agonist). Differentiated and potent Treg cells display low
glycolytic flux and oxidize lipids at higher rates than other T cellsubsets (Fig. 5 and fig. S4, A and B) (36–39).
We then assessed glycolysis and glutaminolysis, at the gene expression level, in response to the TNFR2 agonist antibody. On thebasis of down-regulated gene expression in cells from pooled donors, we inferred that various steps of glycolysis were suppressed in
CD4+ T cells compared to baseline values, including the associatedsteps in the pentose phosphate pathway (Fig. 5A and fig. S4A). In
contrast, also inferred from gene expression [specifically mRNA sequencing (mRNA-seq) analysis], TNFR2 agonist appeared to
up-regulate glutaminolysis in Treg cells (Fig. 5A and fig. S4A). The
up-regulation of glutaminolysis would result in augmented amountsof glutamate that can be used in the Krebs (also known as Szent-­
Györgyi Krebs) cycle; however, this extrapolation from the gene
expression-level data remains to be validated at the protein andfunctional levels.

A

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ETS1, CREB and p300 are all known to interact and directly facilitate FOXP3 transcription. This pattern of rapid induction of Treg
cell signature genes contrasted greatly from the mRNA response ofa TNFR2 antagonist (25) similarly applied to the same CD4+ isolated cells, also for 10 hours (Fig. 4B). A TNFR2 antagonistic antibody
had no impact on the expression of transcripts encoding ETS1,
EP300, and CREB and rather decreased that of transcripts encoding
ETS1 and EP300 (Fig. 4B). Culturing human CD4+ T cells in media
with only IL-2 did not lead to a substantial response. Together,
these data are consistent with TNFR2 antagonism suppressing andeventually killing Treg cells (25, 36), as well as inducing highly
potent Treg cells. Validating in vivo functional data, however, remains to be obtained.

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Epitope mapping shows that TNFR2 agonist binds
to the exterior and cross-links adjacent TNFR2
trimeric complexes
We sought to understand the receptor binding characteristics of potent TNFR2 agonists versus the binding characteristics of manyother TNFR2-generated antibodies with no enhanced TNFR2 agonistic functions. During the generation of these antibodies, we gradually observed that immunizations of BALB/c mice with various
fragments of the TNFR2 protein led to the more successful generation of agonistic antibody to predominantly, but not exclusively to,
only one region of the receptor (Fig. 7). With the use of both en-

zyme-linked immunosorbent assay (ELISA) peptide mapping of thereceptor regions with tightest binding to potent Treg agonistic cell antibody and the use of Pepscan three-dimensional (3D) modelingdata, the repeat receptor-binding site of the potent TNFR2 agonists
was mostly concentrated within the CRD1 and CRD2 region, as
described in Materials and Methods. We propose that, on the cellsurface, the ligand-occupied or unoccupied receptor as a trimer isenhanced by single monomeric TNFR2 agonistic antibodies thatbind and stabilized adjacent trimeric structures. This is a predictive
structure and is consistent with functional data that showed that
Fab agonistic antibodies lose their agonistic function but F(ab′)2
antibodies retain their function. These stabilized complexes, withenhanced clustering, can then form the cell surface lattice to induce
sufficient agonistic stimuli downstream of TNFR2.
TNFR2 agonistic antibody activation likely involves scenario of
the pulling together of two adjacent fully trimerized TNFR2 structures. This sort of network then resulted in recruitment of TRAF2
homotrimers inside of the cell (Fig. 7A). Of note is the required
separation between TRAF2 trimers to accommodate its zinc-finger
and RING domains. This necessitates the separation of the TNFR2
trimers at a sufficient distance and can be bridged by the antibodies
to magnify a hexagonal lattice, a structure referred to as a “beehive”
from the top down view (Fig. 7A). The orientation of the Fc portionof the antibody may not be exact.
On a functional level, this clustering of surface TNFR2 canmagnify NF-B signaling. This was verified by quantitative polymerase
chain reaction (qPCR) and the use of TaqMan Array Human NF-B
pathway mapping plates (Fig. 7B). The panel of TNFR2-regulated
genes in this plate included the study of direct target genes of Rel/NFKB
family members, namely, NFKB1, NFKB2, RELA, RELB, and REL,
and pathway-associated genes, including those encoding inhibitorof B (IB) kinase, IBs, Toll-like receptor (TLR), TNF, TNFR, andTRAF. Additional genes associated with NF-B function in apoptosis, immune and inflammation responses, as well as chemokines andcytokines were also included. The representative, pooled data that
we present from this analysis show the induction of BIRC3 and RELB
gene expression with TNFR2 agonism compared to the same cells
incubated with IL-2 alone (Fig. 7B).
DISCUSSION

Fig. 6. TNFR2 agonism induces gene expression associated with early steps in
the Krebs cycle. Expression of mRNAs associated with Krebs cycle steps in humanCD4+ T cells isolated from 12 nondiabetic donors and cultured either with IL-2(“control”) or the TNFR2 agonist antibody (2.5 µg/ml) for 10 hours. Data are shownas means ± SEM (left) %change and as a heatmap averages (right). Genes highlighted in red show up-regulation.
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In this study, we profiled an agonistic antibody to the TNFR2 andfound that it has potential properties for optimized activity. Antibody agonist–induced Treg cell expansion was independent of exterior cross-linking with secondary cross-linking antibodies and was
independent of the Fc receptor binding to adjacent cells, meaning
an ADCC mechanism. Removal of the Fc region of the antibody
demonstrated Fc receptor independence, which is suggestive oflower toxicity for clinical applications. As also shown by previously
reported studies on TNFR superfamily antibodies, the agonistic
antibody likely creates adjacent dimeric linkages between two fully
formed TNFR2 trimeric structures (24, 25). We further propose that
this creates stabilizing hexagonal networks for strong intracellularsignaling through lattices that appear to look like beehives on the cellsurface (24, 25). With only a 10-hour exposure of cultured CD4+
T cells to TNFR2 agonistic antibodies, the induction of well-known
Treg cell signature genes was demonstrated. The surface network clustering for maximal agonism was functionally verified by the resulting
appearance of sTNFR2 receptors proteins in the media, indicative of
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increased expression of CS, ACO1, and AC02 mRNA after CD4+
cells were exposed to TNFR2 agonism (Fig. 6), which may correspond to augmented abundance of citrate, oxaloacetate, isocitrate,
and acetyl–coenzyme A (fig. S4C). In contrast, gene expression ofproteins associated with late steps of the Krebs cycle were not augmented but were rather down-regulated, such as all steps after IDH2
(Fig. 6 and fig. S4C). Further proof of this shunting to suppressive
metabolites from early use of the Krebs cycle was an examination of
the mRNA levels of IL12B and KEAP1 that should be down-regulatedwith this pathway activation. At the mRNA level, both IL12B and
KEAP1 were down-regulated within 10 hours of CD4+ TNFR2 agonism exposure (fig. S3), consistent with aconitase pathway usage(fig. S4C). The mechanism proposed remains to be tested further,such as by analyzing Treg cell expansion in the presence of the TNFR2
agonist with AMPK inhibitors or enhancers. Using a low-dose, short-­
term exposure to the TNFR2 agonist antibody (0.1 to 2.5 g/ml for6 hours), we also measured the generation of the suppressive metabolite itaconate. Itaconate is of interest because it has direct
immunesuppressive abilities; it has been proposed that the aconitase/itaconate pathway is an immunosuppressive pathway that
operates in Treg cell-mediated immunosuppression (40). Culture supernatant levels of itaconate increased in a dose-specific response to
the TNFR2 agonistic antibody (fig. S3). This was assessed only witha 6-hour, very low-dose exposure to the TNFR2 agonist. Thus, thesemRNA data of early utilizations of the Krebs cycle buttressed bydirect measurements of the metabolite itaconate support a functional significance of the proposed trends.

SCIENCE SIGNALING | RESEARCH RESOURCE
glutaminolysis, inhibition of glycolysis,
augmented fatty acid use, and preferential use of early Krebs cycle steps withaugmentation of aconitase signaling. The
suppressive itaconate metabolite was
induced as well, supporting a functional
metabolic effect by the TNFR2 agonist.
Future mechanistic studies will be needed
to validate these conclusions and should
include the reapplication of these methods on sorted and purified Treg cells. It
is not known at this time if TNFR2 agonism only expands and increases thepotency of existing Treg cells or also recruits CD4+ T cells to become Treg cells
as an additional mechanism of mediated suppression.
Early on, it was thought that all agonistic antibodies to the TNF superfamilyof receptors required Fc receptor bindB
ing (25). Our screening of antibodies
was independent of Fc receptor bindingby performing cellular screening assays
on freshly isolated CD4+ T cells, unlike
the many Fc receptors on monocytes.
In all of these cases, the antibodies presumably stabilize the receptor or receptor-­
ligand complexes in the membrane witha geometry allowing for efficient TRAF
recruitment and receptor activation evenwhen FcRs are absent (41–46).
Deeper mechanistic insights haveemerged as more successful antibody
agonists have been made to the TNF superfamily receptors. In the case of CD40
for autoimmunity, the receptor needs
to be antagonized; for OX40 in cancer,
the receptor needs to be agonized (44).
Therefore, antibody agonism or antagonism of the TNF receptor superfamily
is dependent on linkage of these recepFig. 7. A structural model of TNFR2 and antibody agonist and analysis of downstream TNFR2 signaling–
tors to death or proliferative pathways.
associated gene expression. (A) Schematic representation of the proposed agonist antibody predictive model binding
Similar to our TNFR2 agonistic antito the TNF-TNFR2 complex. TNF is shown in magenta and TNFR2 in blue in surface representation. Two TNF-TNFR2
bodies, CD40-specific agonistic antibodies
complexes are linked and stabilized by an agonist antibody. The antibody heavy chain is shown in purple and thebind for optimized activity on membrane-­light chain in light purple. Each TNFR2 trimer is bound in the intracellular region to a TRAF2 homotrimer (green-yellow).
distal regions of the CD40 receptor (45).
Two TRAF2 homotrimers are linked via their N-terminal zinc-finger and RING dimerization domains (cyan). The top
Also, some but not all CD40 agonists
view of multiple TNF-TNFR2 agonist antibody complexes in the proposed hexagonal beehive lattice arrangement isrequire Fc receptor function. By conshown. The TNF-TNFR2 complexes sit at each vertex of the hexagon, and the antibodies (purple) connect two
trast, our agonist was largely indepenTNF-TNFR2 complexes forming the sides of the hexagon. The structural models were generated with PyMOL (69).
dent of Fc receptor function. This is(B) A NF-B activation assay assessing downstream TNFR2 signaling–associated gene expression in response to
TNFR2 agonism and IL-2 in human donor CD4+ T cells. Red bars denote genes with significant expression over backimportant, because Fc receptor requireground controls (black bars, cells treated with IL-2 alone), two of which are featured on the right. White bars arements of antibodies in vivo may cause
TNFR2 genes with background or negative signals. Data are pooled from seven samples.
heightened liver toxicity or platelet aggregation (46). Avoidance of the ADCC
optimized and tight membrane networks for efficient membrane dependence is highly desirable to minimize toxicity. Antagonisticcleavage of the trimerized receptor. Broad RNA-seq studies of antibodies prefer a dimeric form of TNF superfamily members, a
metabolism-related genes in the expanded human Treg cells through form that blocks ligand binding (24, 25). Treg cell proliferation is often
TNFR2 agonistic antibody suggested a metabolic switch to a highly accomplished with IL-2, but, as shown, TNFR2 agonism is superior
potent Treg cells with well-described metabolic activity of induced because it relates to total cell numbers and the potency of the expanded
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literature suggests that lower agonistic dosing, like shown in thesein vitro studies, may be more efficacious than high dosing (23).
MATERIALS AND METHODS

Research study participants
Human studies that involved blood drawing for autoimmune participants and control participants had full Massachusetts General
Hospital (MGH) institutional approval through Massachusetts General
Hospital and Partners Health Care (study nos. 2007P001347,
2012P002243, and 2013P002633). Informed consent was obtained
from all participants, and the experiments conformed to the principles set out in the World Medical Association Declaration of Helsinki
and the Department of Health and Human Services Belmont Report.
Whole blood was collected from research study participants in BD
Vacutainer EDTA tubes (BD Diagnostics) and processed within
2 hours of phlebotomy. Human blood samples were also received
from cancer participants with advanced lymphoma (Sezary syndrome)from the Stanford University Human Studies Committee [IRB (Institutional Review Board) 5538 and IRB 13844].
The donors of all blood samples were adults and spanned in age
from 25 to 52 years. Gender information was not part of the informed consent process for all these fresh blood draws. Lymphoma
participants were all stage II/IV. At the time of the fresh blooddraws, some of these lymphoma participants were receiving therapeutic intervention; however, we found no correlation between
TNFR2 MFI and the treatment medication. All donors were provided written informed consent.
Isolation of human CD4+ T cells
For purple top (K2-EDTA anticoagulant) collection, fresh humanwhole blood was washed twice with 1× Hanks’ balanced salt solution (HBSS) (Invitrogen) plus 2% fetal bovine serum (FBS) (Sigma-­
Aldrich). CD4+ T cells were isolated using magnetic EasySep Direct
Human CD4+ T Cell isolation kits (STEMCELL Technologies,
Vancouver, BC, Canada), following the instructions of the manufacturer. Briefly, 1200 l of Isolation Cocktail and 1200 l of RapidSpheres were mixed with 24 ml of whole blood in a 50-ml centrifuge
tube and incubated for 5 min at room temperature. Then, 24 ml of
Ca2+- and Mg2+-free phosphate-buffered saline (PBS) was added,
and the tube placed into an Easy 50 EasySep magnet. This immobilized the unwanted cells at the side of the tube. After 10 min, theCD4+ T cell enriched suspension was transferred into another tube,
and the magnetic separation process was repeated for 5 min withfresh RapidSpheres. The resulting highly enriched CD4+ T cell suspension was transferred into another tube and purified for a thirdtime using the EasySep magnet. The resulting purity of the final
CD4+ T cell preparation was >95%. Isolated CD4+ cells were cultured in RPMI GlutaMAX (Life Technologies) with 10% FBS (Sigma-­
Aldrich) and 1× penicillin-streptomycin (Life Technologies). Cells
were seeded in 96-well round-bottom plates at a concentration of
0.2 to 1 × 106 cells per well, treated with various reagents, and incubated for up to 72 hours at 37°C with 5% CO2. All cell culture experiments included low-level IL-2 (200 U/ml) in the media.
Antibody production, reagents, and flow cytometry
Functional TNFR2 agonistic antibody production and characterizations were conducted over a 7-year time span through screening of
hybridomas after BALB/c mice were repeatedly immunized using
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cells. IL-2 alone expansion is weak, and TNF itself can expand thenumber of Treg cells, but those Treg cells are less potent as we show
here and as published in the past (16). TNF binds to TNFR1 better
than TNFR2; TNFR1 is known in baboons to solely carry the wellknown toxicity of TNF (22). Treg cell proliferation is also accomplishedby the addition of rapamycin; as demonstrated with an earlier
TNFR2 agonistic antibody, TNFR2 agonism is superior and addition of rapamycin to a TNFR2 agonism culture is not additive (16).
A recent review of optimized agonistic antibody properties also
highlights the complex monomeric, dimeric binding of antibodies
and the need for caution with dosing so that the correct stoichiometry is achieved, typically at low dose, for current cross-linking activities and the avoidance of saturation conditions. It is believedthat saturation conditions will culminate in only monomeric interactions that could prevent the proper receptor membrane clustering
for intracellular signaling (23). On this point, it is important to notethat preformed trimeric TNFR2 already in complex with three TNF
ligands is already present on the cell surface such that a baseline amountof TNF is sufficient. In this study, we showed that, for optimized
TNFR2 agonists, additional TNF ligand is not obligatory, distal
binding is preferred, and Fc receptor cross-linking is not necessary.
The literature on antibody development yields some rules about
the region of TNF superfamily receptors guiding the function of
antibodies that successfully bind. It should be mentioned that thesetargeted agonists are very different from “blocking” antibodies.
Therapeutic blocking antibodies to PD1 and CTLA4 appear to haveminimal target receptor region specificity, and most regions ofthese target proteins with bound antibody have diminished function. This does not seem to be the case for any of the agonistic antibodies to the TNF superfamily receptors that have been reported todate. As future studies progress, it may not only be the receptor sitesof TNF superfamily antibodies determining antibody function but
agonism may also be determined by the framework structures of theantibodies, with IgG1, IgG2, and IgG4 having different properties
influenced by the hinge of the antibody. They feature changes in theantigen-binding arms of antibodies.
The need for TNFR2 agonistic antibodies for diverse human diseases has been strongly documented in animal models and tissue
culture models of human disease. Many animal and human in vivo
studies in Sjogren’s syndrome, rheumatoid arthritis, multiple sclerosis, lupus, ulcerative colitis, T1D, and Crohn’s disease show that
TNFR2 agonism protects or reverses autoimmunity and may also
supplement end organ healing (4–6, 11, 13, 19, 47–54). It also appears that the “sluggish” or not fully activated Treg cells of humanautoimmunity, at least in culture, fully respond to TNF2 agonism,
suggesting that the defect might be able to be corrected with thisantibody approach (27). The literature also shows the value of
TNFR2 agonism in reversing graft-versus-host disease and thwartingtransplant rejection (10, 54–59). The CNS effects of TNFR2 agonism
include CNS healing, and this might have applications to Alzheimer’s
disease, spinal cord injury, and multiple sclerosis (8, 9, 12, 60–66).
The availability of a new TNFR2 agonist that induces highly potent
Treg cells with potentially minimal toxicity has wide-ranging potential applications for treatment of inflammatory diseases or diseases
with inflammatory components.
A key limitation of this study is that in vivo data are still forthcoming. It is also important to point out that in the clinic, agonistic
antibodies to the TNF superfamily have been harder to dose correctly than blocking or antagonistic antibodies. A summary of this

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Torrey et al., Sci. Signal. 13, eaba9600 (2020)

8 December 2020

Measurement of secreted TNFR2
Secreted sTNFR2 was measured from cell culture supernatants using Quantikine ELISA kits (R&D Systems) with some modifications.
Briefly, culture supernatants were collected after 24 to 42 hours of
incubation of CD4+ cells with IL-2 (200 U/ml) alone or with TNF(20 ng/ml) or TNFR2 mAbs (12.5 µg/ml) and incubated on either
the commercial plates or custom plates coated with 2 µg per well of
TNFR2-directed antibodies. ELISA was performed according to the
manufacturer’s instructions. Absorbance at 450 nm was measured
using the SpectraMax 190 Absorbance Plate Reader and analyzed
with SoftMax Pro 6.3 (Molecular Devices).
Cell proliferation and suppression assays
For peripheral blood leukocytes (PBLs) proliferation experiments,
PBLs were stained with 1 M CFSE. Cells were plated at a density of
2 × 105 cells per well in a 96-well plate precoated with anti-CD3
mAb (5 g/ml) (OKT3, eBiosciences). Four days later, cells werecollected and analyzed by flow cytometry. The proliferation rate
was calculated by the percentage of cells undergoing division. For
Treg cell suppression assays, autologous peripheral blood mononuclear
cells (PBMCs) were used as responder cells. PBMCs were collected
at the day of venipuncture by density gradient separation using
Ficoll-Hypaque Plus (GE Healthcare, Piscataway, NJ, USA), cryopreserved at −80°C, and thawed at the day before mixed with Treg cells
and rested overnight in RPMI 1640 medium supplemented with 1%
FBS and IL-2 (10 U/ml). On the following day, responder cells were
stained with CFSE (1 M). Responder cells (5 × 104 cells) and expanded Treg cells were mixed at the ratio of 0:1, 1:1, 2:1, and 4:1 in
culture media and stimulated with anti-CD3 mAb (HIT3a, BD Biosciences) and IL-2 (50 U/ml). After 4 days, cells were collected andanalyzed by flow cytometry. Suppression index was calculated by
the percentage of CD8+ T cells responder cells that underwent division. The suppression index was calculated using the following
equation: (Treg cell proliferation without Treg − Tresp proliferation
with Treg) / Tresp proliferation without Treg cell.
Epitope mapping
ELISA was used for linear epitope mapping of TNFR2 agonists on
the TNFR2 external membrane protein sequence. Peptides werepurchased from GenScript, diluted in coating buffer, and placed on
Immulon 4HBX Flat Bottom Microtiter Plates (Thermo Fisher Scientific) at a concentration of 1 mg per well. Primary TNFR2 mAbs
(0.1 mg per well) were incubated with substrates. Secondary antibodies against rodent IgGs were used to label the primary mAbs.
Absorbance was measured using the SpectraMax 190 Absorbance
Plate Reader and analyzed with SoftMax Pro 6.3 (Molecular Devices).
3D peptide mapping was performed using Chemically Linked
Peptides on Scaffolds (CLIPS) technology (Pepscan). Briefly, thetarget protein was converted into a library of up to 10,000 overlapping linear peptides, which were then bound to a solid support andshaped into a matrix of CLIPS constructs. The affinity of the antibody to the various peptide conformations was used to determine
the precise discontinuous epitopes. This antibody had a KD (dissociation
constant) of 1.2(2) nM. The CRD1 and CRD2 discontinuous TNFR2
binding sites were QMCCSKCSPGQH, CSSDQVET, and NRICTCRP.
Transcription profiling analysis (RNA-seq)
CD4+ T cells were isolated from peripheral blood using EasySep Direct Human CD4+ T Cell isolation kits (STEMCELL Technologies),
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fragments of the human (h) TNFR2 protein, specifically a fragment
containing amino acids 50 to 122. Mice were also immunized withthe whole exterior TNFR2 protein to increase the affinity of thegenerated clones. All mice were immunized both intravenously andsubcutaneously at least five times before the splenic harvest for hybridoma generation. Tail bleed titer checks by ELISA confirmed
responding mice. A desired titer of 1:30,000 to 1:100,000 was used
for continuing the project with that animal’s spleen. Screening ofantibody candidates was carried out on fresh human CD4+ T cells
in functional assays looking for specific proliferation of Treg cellswith methods described within this paper. Supernatant testing was
first performed, and then any clones with subcloning that showedremaining activity were then purified. All antibodies of the IgM isotype were discarded; the antibody under study was an IgG1 isotype.
Antibody candidates were purified using affinity chromatography
followed by PBS solution. The purity of the antibody was >95%.
Bacterial endotoxin levels were determined using the Endosafe-PTS
system and Endosafe PTS cartridges (Charles River Laboratories),and endotoxin levels were <1 EU/mg before any in vitro antibody
testing. Antibody concentration was determined by measuring absorbance at 280 nm and calculated using the standard extinction
coefficient 205,500 M−1 cm−1 (or 1.0 mg/ml = A280). Additional
screening also involved confirmed binding to TNFR2, no binding to
TNFR1, and binding to TNFR2 amino acids 50 to 122 using ELISAs.
Antibody secondary selection was chosen on the basis of candidates
that had the most reliable human Treg cell expansion and the least
TNFR1 binding (TY101-10) on greater than 25 random human donors. Murine hybridoma immunizations were performed at theDana Farber Hybridoma Facility or through contract work. Before
all experiments, animal care institutional review boards confirmed
the safe and humane treatment of mice. Fluorescence-activated cellsorting (FACS) data were processed using FlowJo software (version
10.0.8) and analyzed on Prism (GraphPad Software, La Jolla, CA).
Other reagents and flow cytometry monoclonal antibodies (mAbs)
against human TNFR2 and sTNFR2 were produced internally or
obtained from external commercial vendors: Antibody MAB2261(R&D Systems) was used for measuring TNFR2 cell surface expression. The TNFR2 antagonistic antibody used in this paper was produced by our laboratory and is described elsewhere (25). Recombinanthuman TNF and IL-2 were purchased from Sigma-Aldrich. F(ab′)2
fragments of mAbs were prepared using Pierce F(ab′)2 Preparation
Kit (Life Technologies). Cross-linking antibody against rodent IgG(ab9165) was purchased from Abcam. Cells were prepared for flow
cytometry using Human Treg cell Flow Kit (BioLegend) according to the
manufacturer’s instructions. The mAbs or F(ab′)2 fragments wereconjugated with fluorescent allophycocyanin (APC) using theLightning-Link APC conjugation kit from Innova Biosciences using
the manufacturer’s instructions. Fluorescently stained cells were resuspended in 1× HBSS (Invitrogen) and analyzed using a BD FACSCalibur flow cytometer machine (Becton Dickinson). Antibodies
used for FACS analysis of Treg cells included Alexa Fluor 488 anti-human FoxP3 (Clone 259D; BioLegend) for intracellular stainingof FoxP3 and phycoerythrin anti-human CD25 (Clone BC96; BioLegend) for cell surface staining of CD25. Treg cell populations wereassessed by FACS with FL2 (red) versus FL1 (green) and defined as
CD25hi and FoxP3+, whereas Teff populations were defined as CD25hi
and FoxP3−. For cell suppression assays, responders were stainedwith CFSE (BioLegend) and CD8 APC (Clone SK1; BD Biosciences)
and analyzed by FACS with FL4 (far red) versus FL1 (green).

SCIENCE SIGNALING | RESEARCH RESOURCE

Measurement of itaconate
Culture supernatants were harvested from fresh human CD4 +T cells cultured with TNFR2 (0, 0.1, 0.5, and 2.5 g/ml) for 6 hours in
96-well plates (100,000 cells per well). Each well in each 96-wellplate yielded 50 µl of culture sample. The harvesting of the culture
supernatants was from numerous normal blood donors treated in
the dose-specific manner (n = 6 donors) with TNFR2 agonistic antibody. The culture supernatants were sent to Metabolon (Research
Triangle Park, NC) for the analysis of itaconate levels.
Measurement of NF-B pathway induction
Similar to the CD4 + T cell methods above [fresh human CD4 +T cells cultured with TNFR2 agonistic antibody (2.5 µg/ml) for
6 hours], the Applied Biosystems TaqMan Array Human NF-B pathway plates were used per manufacturer’s instructions to assess theability of this antibody to activate downstream NF-B signaling(Thermo Fisher Scientific). The panel of assays in this plate targets
genes encoding the proteins of the Rel/NF-B family signaling pathways (NFKB1, NFKB2, RELA, RELB, and REL) associated with regulatory proteins [including IB, IB kinase, TLR, TNF and TNFRs,
and TRAF], and those associated with NF-B functions in apoptosis,
immune and inflammation responses, as well as chemokines andcytokines were also studied.
Torrey et al., Sci. Signal. 13, eaba9600 (2020)

8 December 2020

Binding affinity measurement
The affinity of antibody binding to recombinant human TNFR2
was measured by Biacore Analysis Services (Precision Antibody).
The KD was 1.2(2) nM. Briefly, the antibody was biotinylated at 5:1
stoichiometry with biotinyl-LC-LC-NOSE (Thermo Fisher Scientific) in PBS. Excess biotinylating reagent was removed by centrifugation chromatography, and the biotinylated antibody was captured
on 3000 RU of streptavidin surface to a level of 100 RU. Theoreticalmaximum of signal with TNFR2 with that level of antibody capture
was 26 RU, and that signal was reached with a preliminary experiment using 500 nM TNFR2 in the running buffer. Analysis of the
kinetics of antigen binding was performed at a flow rate of 60 l/min
with 2-min injections. Antibodies were injected at a concentrationof 1 ng/ml to the final capture of 100 RU. The instrument used was
Biacore 3000 with the BioCap chip (GE Healthcare). The double
reference method was used for analysis. The reference channel contained the identical level of streptavidin.
Statistical analysis
Statistical significance was determined using Student’s t test (twotailed and unequal variance) in Excel (Microsoft) or GraphPad
Prism 8 software (GraphPad Software). The significance of dose-­
response slopes was determined using linear mixed-effects modeling (LMEM) with participant-level random effects. Computations
were performed using SAS version 9.4 (SAS Institute Inc.). Confidence levels were set to 0.05. The level of significance is indicated in
the figures by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001.
SUPPLEMENTARY MATERIALS

stke.sciencemag.org/cgi/content/full/13/661/eaba9600/DC1
Fig. S1. Representative FACS data.
Fig. S2. TNFR2 agonism results in Treg cell expansion.
Fig. S3. KEAP1, IL-12B, and itaconate analysis.
Fig. S4. Reference cell metabolism schematics.
Data file S1. RNA-seq data.
View/request a protocol for this paper from Bio-protocol.

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Acknowledgments: We thank M. Davis for the critical reading of this paper. Funding: This
work was supported by the Iacocca Foundation. Author contributions: H.T. performed anddesigned the experiments in Figs. 1 to 3, W.M.K. performed and designed the experiments in
Figs. 4 to 6, Y.O. performed and designed the experiments in Fig. 2B, and E.V. prepared Fig. 7A.
L.T. isolated the CD4+ cells from normal blood donors and rescreened the antibody clones,
H.Z. checked the statistics and analyzed trends using LMEM, and K.C. performed and assistedwith data processing. D.L.F. oversaw and designed the experiments, reviewed all the data, andwrote the paper. Competing interests: The authors declare that they have no competinginterests. H.T. is currently an employee of IMV Inc. but was employed at MGH for the durationof this study. Data and materials availability: Reagents presented in this paper are available
from MGH through a material transfer agreement. The RNA-seq data have been deposited at
datadryad.org and are available from https://doi.org/10.5061/dryad.wh70rxwkj. All other dataneeded to evaluate the conclusions in the paper are presented in the paper and/or the
Supplementary Materials.
Submitted 20 January 2020
Accepted 3 November 2020
Published 8 December 2020
10.1126/scisignal.aba9600
Citation: H. Torrey, W. M. Kühtreiber, Y. Okubo, L. Tran, K. Case, H. Zheng, E. Vanamee,
D. L. Faustman, A novel TNFR2 agonist antibody expands highly potent regulatory T cells. Sci. Signal.
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Heather Torrey, Willem M. Kühtreiber, Yoshiaki Okubo, Lisa Tran, Katherine Case, Hui Zheng, Eva Vanamee and Denise L.
FaustmanSci. Signal. 13 (661), eaba9600.
DOI: 10.1126/scisignal.aba9600

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An antibody to trigger Treg cells
For patients with autoimmune and other inflammatory diseases, increasing the number and activity of regulatory T
cells (T reg cells) may be therapeutically beneficial. Torrey et al. identified an antibody that activated the receptor TNFR2,
resulting in T reg cell expansion, and repressed the activity of effector T cells (Teff cells) within cultures of patient-derived
T cells. Various physical and functional features of the antibody suggested it may be more potent and less toxic thancurrent antibody or ligand-based methods of activating Treg cells. The findings suggest that this antibody has thepotential to restore the balance between T reg cells and Teff cells to treat inflammatory disease.