Cellular Oncology
https://doi.org/10.1007/s13402-022-00742-0

ORIGINAL ARTICLETNFR2 antagonistic antibody induces the death of tumor infiltrating­CD4+Foxp3+ regulatory T cells
Tianzhen He1,2 · Yibo Chen1 · De Yang3 · Md Sahidul Islam1 · Chon‑Kit Chou1 · Jiarui Liu1 · Denise L. Faustman4 ·Joost J. Oppenheim3 · Xin Chen1,5,6
Accepted: 26 October 2022
© Springer Nature Switzerland AG 2022

Abstract
Background TNFR2 expression is a characteristic of highly potent immunosuppressive tumor infiltrating C­ D4+Foxp3+
regulatory T cells (Tregs). There is compelling evidence that TNF through TNFR2 preferentially stimulates the activationand expansion of Tregs. We and others, therefore, proposed that targeting TNFR2 may provide a novel strategy in cancer
immunotherapy. Several studies have shown the effect of TNFR2 antagonistic antibodies in different tumor models. However,
the exact action of the TNFR2 antibody on Tregs remained understood.
Method TY101, an anti-murine TNFR2 antibody, was used to examine the effect of TNFR2 blockade on Treg proliferationand viability in vitro. The role of TNFR2 on Treg viability was further validated by TNFR2 knockout mice and in the TY101
antagonistic antibody-treated mouse tumor model.
Results In this study, we found that an anti-mouse TNFR2 antibody TY101 could inhibit TNF-induced proliferative expansion of Tregs, indicative of an antagonistic property. To examine the effect of TY101 antagonistic antibody on Treg viability,
we treated unfractionated lymph node (L.N.) cells with Dexamethasone (Dex) which was known to induce T cell death. The
result showed that TY101 antagonistic antibody treatment further promoted Treg death in the presence of Dex. This led us
to find that TNFR2 expression was crucial for the survival of Tregs. In the mouse EG7 lymphoma model, treatment with
TY101 antagonistic antibody potently inhibited tumor growth, resulting in complete regression of the tumor in 60% of mice.
The treatment with TY101 antagonistic antibody elicited potent antitumor immune responses in this model, accompanied
by enhanced death of Tregs.
Conclusion This study, therefore, provides clear experimental evidence that TNFR2 antagonistic antibody, TY101, canpromote the death of Tregs, and this effect may be attributable to the antitumor effect of TNFR2 antagonistic antibody.
Keywords TNFR2 · TNFR2 antagonistic antibody · CD4+Foxp3+ regulatory T cells (Tregs) · Cell death · Lymphoma

Tianzhen He and Yibo Chen contributed equally to this work.
* Joost J. Oppenheim
oppenhej@mail.nih.gov

3

Cancer Innovation Laboratory, Center for Cancer Research,
National Cancer Institute, Frederick, MD 21702, USA

Xin Chenxchen@um.edu.mo

4

Immunobiology Laboratory, Massachusetts General Hospital,
Harvard Medical School, Boston, MA, USA

5

MoE Frontiers Science Center for Precision Oncology,
University of Macau, 999078 Macau, SAR, China

6

Guangdong-Hong Kong-Macau Joint Lab on Chinese
Medicine and Immune Disease Research, Guangzhou, China

1

2State Key Laboratory of Quality Research in Chinese
Medicine, Institute of Chinese Medical Sciences, Universityof Macau, Avenida da Universidade, Taipa, Macau,
SAR 999078, China
Institute of Special Environmental Medicine, Nantong
University, 226019 Nantong, China

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1 Introduction

2 Materials and methods

There is now compelling evidence that TNF-TNFR2 interaction plays a decisive role in the activation, expansion, phenotypic stability, and function of Tregs [1, 2]. Moreover, inmouse models and human patients, maximally immunosuppressive TNFR2-expressing Tregs accumulated in the tumor
environment [3, 4]. ­TNFR2+ Tregs in tumor tissue potently
inhibit the antitumor immune responses [5]. In humanpatients, TNFR2 expression levels on Tregs correlatedwith clinical pathology and disease progression [6–9]. The
presence of TNFR2-expressing Tregs represents a primarycellular mechanism underlying immune evasion of tumors.
Therefore, we and others proposed that targeting TNFR2 isa novel strategy in tumor immunotherapy [10, 11].
Currently, TNFR2 antagonists are investigated as potential tumor therapeutic agents[12]. For the first time, wereported that targeting TNFR2 with an antagonistic antibody (M861) in combination with CpG-ODN or anti-CD25
antibody markedly inhibited the growth of colon and mammary carcinoma in mice [13]. Furthermore, TY101, ananti-murine TNFR2 antibody, induced tumor regressionin CT26 and MC38 mouse colon carcinoma models [14].
Moreover, TY101, combined with anti-PD-1 antibodies or
R848 (TLR7/8 agonists, activating dendritic cells), potently
inhibited tumor growth [14, 15]. These results also indicated that eliminating tumor-infiltrating Tregs by TNFR2targeting antibody potently enhanced the antitumor immune
responses. Several studies also showed that anti-human
TNFR2 antagonistic antibodies also promoted the death ofTregs and ­TNFR2+ tumor cells [16, 17], indicating the therapeutic potential of anti-TNFR2 therapeutics in the treatmentof human cancer.
In this study, we examined the effect of TY101 antagonistic antibody on the Treg survival in different experimental system. The results showed that the treatment of TY101
antagonistic antibody inhibited TNF-induced Treg proliferation, but TY101 antagonistic antibody alone did not affectTreg viability. However, TY101 antagonistic antibody further impaired the Treg viability in the presence of Dex, aknown agent that could induce the death of T cells [18].
Additionally, TNFR2-deficient Tregs were more vulnerable
to Dex-induced cell death, while the activation of the TNFTNFR2 axis protected Tregs from Dex-induced cell death.
In vivo administration of TY101 antagonistic antibody into
EG7 lymphoma-bearing C57BL/6Jmice resulted in complete
tumor regression in 60% of mice. The antitumor effect was
attributable to decreased number of tumor-infiltrating Tregs
and increased number of infiltrating IFN-γ-expressing CD8cytotoxic T lymphocytes (CTLs). Our results indicate thatinduction of Treg cell death is a mechanism underlying theantitumor effect of the TNFR2 antagonistic antibody.

2.1 Mice and reagents

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Female wild-type (W.T.) C57BL/6J, B6.129S2Tnfrsf1btm1Mwm/J ­(TNFR2-/- mice) and BALB/c mice (8–12
weeks old) were provided by the Animal Facility of University of Macau. The animal study protocol was approved by
Animal Research Ethics Committee of University of Macau.
Anti-murine TNFR2 antibody (TY101) was a gift from Dr.
Denise Faustman’s group (Massachusetts General Hospital& Harvard Medical School) [14]. RPMI-1640 (1×), fetal
bovine serum (F.B.S.), trypsin-EDTA (0.25%) phosphatebuffered saline (1×), Pen Strep (100×), non-essential aminoacids solution (NEAA, 100×), HEPES (1 M) and Trypan
Blue Stain (0.4%), 2-mercaptoethanol (2-Me) and LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit and eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set
were purchased from ThermoFisher Scientific (U.S.A.).
Red blood cell lysis buffer was purchased from Beyotime
Biotechnology (C.N.). Mouse CD4 (L3T4) microbeads and
L.S. columns were purchased from Miltenyi Biotech. Antibodies were purchased from BD Pharmingen (San Diego,CA) consisted of PerCP-Cy5.5 anti-mouse TCR- β (H57597), PE anti-mouse CD120b/TNFR2 (TR75-89), APC antimouse CD8 (53 − 6.7) and PE anti-mouse IFN-γ (XMG1.2).
Antibodies purchased from eBioscience include eFluor 450
anti-mouse CD45 (30F11), APC anti-mouse CD4 (RM45), PE-Cy7 anti-mouse CD4 (GK1.5) and APC anti-mouse/
rat Foxp3 staining set (FJK-16s). Recombinant mouse IL-2
and TNF were obtained from BD Pharmingen. 1-monothioglycerol (M6135), dexamethasone (D4902) and dimethyl
sulfoxide (DMSO) were obtained from the Sigma-Aldrich(St. Louis, MO, U.S.A.).

2.2 Cell line and cell culture
EG7 and B16-F10 cell line were purchased from American
Type Culture Collection (Manassas, U.S.A.). EG7 cells were
cultured in Roswell Park Memorial Institute 1640 Medium(RPMI-1640) supplemented with 10% F.B.S., 1% Pen Strep,
1% L-glutamine and 50 µM monothioglycerol. B16-F10
cells were cultured in Dulbecco’s Modified Eagle’s Medium(DMEM) supplemented with 10% F.B.S. The incubation was
carried out at 37℃ in humidified air containing 5% ­CO2 ina tissue culture flask.

2.3 Cell purification and in vitro cell culture
Wild-type (W.T.) C57BL/6J or BALB/c mice were euthanized by ­CO2 inhalation, then the spleens and L.N.s in theaxillary, inguinal and mesenteric regions were harvested andTNFR2 antagonistic antibody induces the death of tumor infiltrating ­CD4+Foxp3+ regulatory…

pooled for the preparation of single-cell suspension. CD4 T
cells were purified with Mouse CD4 (L3T4) microbeads and
L.S. columns (Miltenyi Biotech).
Unfractionated lymph node cells or MACS-purified CD4 T cells isolated from W.T. C57BL/6J mice or­TNFR2−/− mice were cultured in RPMI-1640 supplemented
with 10% F.B.S., 1% Pen Strep, 5 × ­10− 5 M 2-Me, 1% nonessential amino acids with IL-2 (10 ng/ml), or with TNF(20 ng/ml), in the presence of TY101 (10 µg/ml) or Dexamethasone (10 nM). The cells were incubated at 37℃ in
humidified air containing 5% C
­ O2. About 5 × ­105 isolated
CD4 T cells or unfractionated lymph node cells were cultured in a 96-well U-type culture plates. The proportion andcell viability of ­CD4+Foxp3+ Tregs were analyzed by FCM(Flow cytometry).

2.4 Tumor cell inoculation and separation
2 × ­105 EG7 cells in 0.2 ml of PBS were subcutaneously
injected into the right flank of recipient mice. After indicatedtimes, the mice were euthanized by C
­ O2 inhalation. Mousetumors were excised, minced, and digested in RPMI 1640
supplemented with collagenase IV (1 mg/ml) and deoxyribonuclease I (0.1 mg/ml). The fragments were pushed througha 70-um pore size cell strainer to create a single-cell suspension. In some experiments, tumor-free mice after TY101
antagonistic antibody treatment were reinoculated with
2 × ­105 EG7 cells into the right flank, and the same numberof B16-F10 cells were injected subcutaneously into the left
flank. Tumor size was calculated by the following formula:(length × width ^ 2) / 2. “Survival” represented the time towhen a 4 ­cm3 tumor developed or mice became moribund.
A humane end point that triggered euthanasia. Mice weremonitored daily and were euthanized when signs defined ofmorbidity from metastatic disease burden became evident.

2.5 Flow cytometry (FCM)
eBioscience™ Foxp3 / Transcription Factor Staining Buffer
Set was used for cell fixation and permeabilization. About
1 × ­106 cells were stained with appropriately diluted antibodies in FACS buffer. Antibodies used in FCM analysis
included PerCP-Cy5.5 anti-mouse TCR β antibody (1:500
diluted), PE anti-mouse CD120b/TNFR2 (1:250 diluted),
APC anti-mouse CD8 (1:500 diluted). PE anti-mouse IFN-γ(1:250 diluted), eFluor 450 anti-mouse CD45 (1:500 diluted),
APC anti-mouse CD4 (1:500 diluted), PE-Cy7 anti-mouse
CD4 (1:500 diluted) and APC anti-mouse/rat Foxp3 staining
set (1:250 diluted). The acquisition was performed using a
Fortessa cytometer (B.D. Biosciences), and data analysis was
conducted using FlowJo software (Tree Star Inc.).

2.6 Statistical analysis
Two-tailed Student’s t test was used for the comparisonof two indicated groups. one-way analysis of variance(ANOVA) with Tukey’s post-test was used for the comparison of differences among groups as described in our previous study. Log-rank test was used for the comparison ofsurvival shown in Fig. 4D. All statistical analysis was performed with GraphPad Prism 7.0.

3 Results
3.1 TY101 antagonistic antibody abrogates
TNF‑induced proliferation of Tregs
Previously, we and others reported that, in the presence of
low levels of IL-2, TNF preferentially stimulates the proliferation of Tregs present in cultured CD4 cells [4, 19].
We thus used this assay to characterize the TY101 function in modulating TNFR2 function. To this end, MACSpurified mouse CD4 T cells isolated from C57BL/6J mice or
BALB/c mice were cultured with IL-2 and stimulated with
TNF. As expected, TNF treatment increased the proportionof Foxp3-expressing Tregs and the absolute number of Tregs
in the cultured CD4 cells by 51.7% and 150%, respectively(Fig. 1A-C, P < 0.01). However, treatment with TY101
antagonistic antibody (10 µg/ml) alone did not change theproportion of Tregs in CD4 cells cultured with IL-2 alone,while it completely blocked the expansion of Tregs inducedby TNF (Fig. 1A-C, P < 0.01). Therefore, just like M861
[13], TY101 is an antagonistic anti-mouse TNFR2 antibody
that inhibited TNF-induced proliferative expansion of Tregs.

3.2 TY101 treatment increases the death of Tregs
in the presence of Dex (Dexamethasone)
It was reported that anti-TNFR2 antibody was able to induce
the death of Tregs in purified CD4 T cells [17, 20]; we thus
used LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit
to assess the effect of TY101 antagonistic antibody on Treg
viability. However, TY101 alone did not affect Treg viability(Fig. S1A-B, P > 0.05). To further determine the role of the
TNF-TNFR2 axis on Treg viability, we used Dexamethasone(Dex), an anti-inflammatory agent that induces Treg cell death[18]. To this end, unfractionated lymph node cells from normal
C57BL/6J mice were cultured with Dex plus IL-2 (10 ng/ml),
Dex plus IL-2 and TNF (20 ng/ml), or Dex plus IL-2, TNF,
and TY101 antagonistic antibody (10 µg/ml) or IL-2 alone for
72 h. As shown in Fig. 2, IL-2 alone partially maintained thesurvival of Tregs, while the addition of TNF was able to almost
completely abrogate Treg death induced by Dex (Fig. 2C-D,

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T. He et al.

Fig. 1  TY101 antagonistic antibody abrogated TNF-induced Treg
proliferation. CD4 T cells were isolated from L.N. and spleen of W.T.
C57BL/6J or BALB/c mice with MACS. The cells were cultured with
IL-2, in the presence or absence of TNF, with or without TY101 for
72 h. The proportion of F
­ oxp3+ Tregs was analyzed by FCM. A Typical FCM plots. The number indicates the proportion of Tregs in CD4

T cells. B and C Summary of the proportion and the absolute numberof Tregs. Data (Mean ± S.E.M., n = 3) shown are representatives ofthree separate experiments with similar results. Comparison of indicated groups. *P < 0.05, **P < 0.01, ***P < 0.001. N.S., no significanceP < 0.01). Furthermore, TNF treatment resulted in a marked
increase in the proportion of F
­ oxp3+ Tregs in the cultured CD4
cells (Fig. 2A-B, P < 0.001), which was attributable to the proliferative expansion of Tregs even in the presence of Dex, as
we reported previously [18, 21]. These effects of TNF on the
expansion of Tregs and the survival of Tregs were inhibited by
the treatment of TY101 antagonistic antibody.

Dead Cell Stain Kit by gating on F
­ oxp3+ Tregs. The result
showed that, when unfractionated lymph node cells were
cultured in IL-2 alone, the viability of W.T. Tregs and
TNFR2-deficient Tregs were comparable (Fig. S2A-B,
P > 0.05). Thus, genetic ablation of TNFR2 did not enhancethe death of Tregs in the presence of IL-2 alone. Nevertheless, TNF-TNFR2 interaction markedly protected Tregs from
Dex-induced cell death, as evidenced by the fact that TNF
markedly enhanced the proportion and viability of Dextreated W.T. Tregs (Fig. 3A-D, P < 0.01), but not Dex-treated
TNFR2-deficient Tregs (Fig. 3A-D, P > 0.05). Interestingly,
TNF treatment did not enhance the viability of W.T. Teffs(Fig. S3A-B, P > 0.05), which could be attributable to therelatively lower TNFR2 expression on Teffs.
To verify the in vivo effect of TNFR2 expression on
Treg viability, Dex (5 mg/kg/d) was i.p. injected into the
W.T. C57BL/6J or T
­ NFR2−/− mice three times as described
previously [21]. One day after the last treatment, the mice
were sacrificed, and the proportion of ­CD4+Foxp3+ Tregs

3.3 TNFR2 signaling dampens death induction
by Dex on Tregs
To further clarify that TY101 antagonistic antibody inhibited Treg survival mediated by the blockade of TNFR2, we
examined whether TNFR2 deficiency impairs Treg viability
in vitro and in vivo. To this end, in the presence or absenceof Dex, unfractionated lymph node cells isolated from wildtype (W.T.) C57BL/6J mice and from ­TNFR2−/− mice were
cultured with IL-2, with or without TNF. The viability ofTregs was determined by LIVE/DEAD™ Fixable Near-IR

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TNFR2 antagonistic antibody induces the death of tumor infiltrating ­CD4+Foxp3+ regulatory…

Fig. 2  Effect of TY101 antagonistic antibody on Treg cell survival.
Unfractionated L.N. cells from W.T. C57BL/6J mice were treatedwith Dex, in the presence or absence of IL-2, or TNF, or TY101, for
72 h. The proportion of Tregs (A and B) and the proportion of living
Tregs (C and D) were analyzed by FCM, gating on ­Foxp3+ cells. A

and C Representative FCM plots. The number indicates the proportion of F
­ oxp3+ cells in CD4 T cells. B and D Summary of results.
Data (Mean ± S.E.M., n = 3) shown are representatives of three
separate experiments with similar results. Comparison of indicatedgroups. *P < 0.05, **P < 0.01, ***P < 0.001. N.S., no significancein the spleen and lymph nodes was analyzed by FCM, andthe number of Tregs were determined. The gating strategy
for ­CD4+Foxp3+ Tregs was shown in Fig. S4. The results
showed the proportion of Tregs decreased markedly in
spleens and lymph nodes in Dex-treated W.T. mice and­TNFR2−/− mice (Fig. 3F-G, P < 0.001 and Fig. S5). The
absolute number of splenic Tregs decreased by 50.3%(Fig. 3F) in the T­ NFR2−/− mice group. In contrast, thesplenic Treg percentage decreased by 30.7% in W.T. mice
(Fig. 3G, P < 0.01). The results indicated that TNFR2-deficient Tregs were more susceptible to Dex-induced cell deathin vivo (Fig. 3H). Therefore, the in vivo and in vitro data
supported that TNF promoted Treg viability in a TNFR2dependent manner.

the in vivo antitumor effect of TY101 in the mouse EG7
lymphoma model. As shown in the schematic diagram in
Fig. 4A, EG7 lymphoma cells were inoculated subcutaneously in W.T. C57BL/6J or BALB/c mice. Treatment
started on day 7 after tumor inoculation (tumor diameterreached about 10 mm). TY101, or isotype-matched IgG
control, was injected into the tumor-bearing mice (200 µg,i.p.) twice a week for up to five times. The results showedthat the administration of TY101 antagonistic antibody markedly inhibited the growth of EG7 lymphoma(Fig. 4B-C, P < 0.001), and complete regression of EG7
lymphoma was observed in 60% of the mice at the endof the experiment (50 days after tumor inoculation). In
contrast, the tumor grew rapidly in the mice treated with
control IgG, and all mice died from the tumor burden by
day 30 (Fig. 4D).
To investigate whether TY101 could induce long-term
tumor-specific immunity, treated mice with complete
tumor regression were reinoculated with EG7 lymphoma
into the right flank 8 weeks after becoming tumor-free. In

3.4 TY101 antagonistic antibody potently inhibits
the growth of EG7 lymphoma in mice
To investigate whether TY101 enhances antitumor
responses as previously reported [14, 15], we examined13

T. He et al.

Fig. 3  The effect of TNF on the viability of W.T. Tregs and TNFR2
deficient Tregs. A-D Unfractionated LN cells isolated from W.T.
C57BL/6J mice and ­TNFR2−/− mice were treated with Dex, in thepresence or absence of IL-2, or TNF, for 72 h. The proportion ofTregs (A and B) and the proportion of living Tregs (C and D) wereanalyzed by FCM, gating on F
­ oxp3+ cells. A and C Representative
FCM plots. The number indicates the proportion of F
­ oxp3+ cells in
CD4 T cells. B and D Summary of results. Data (Mean ± S.E.M.,
n = 3) shown are representatives of five separate experiments withsimilar results. E-H W.T. or TNFR2 KO mice were treated with

Dex once every two days for three times. One day after the lasttreatment, the mice were sacrificed. The proportion of Tregs wasanalyzed by FCM and the absolute number of Tregs in spleen were
determined. E The schematic diagram of the experiment procedure. F The representative FCM plots, the number indicate the proportion of ­CD4+Foxp3+ Treg in total CD4 T cells. G The absolute
number of Tregs in spleen. H The percent decrease of Tregs. Data
(Mean ± S.E.M., n = 6) shown are representatives of two separate
experiments with similar results. Comparison of indicated groups.
*P < 0.05, **P < 0.01, ***P < 0.001, N.S., no significancecomparison, irrelevant B16-F10 melanoma cells were inoculated into the left flank. As another control, normal mice werealso inoculated with EG7 lymphoma into the right flank andB16-F10 melanoma into the left flank in the same manner.

As a result, none of the mice with complete tumor regression
developed EG7 lymphoma (0/3), while all developed B16F10 melanoma (3/3). In contrast, all the normal control micedeveloped B16-F10 melanoma and EG7 lymphoma (3/3),

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TNFR2 antagonistic antibody induces the death of tumor infiltrating ­CD4+Foxp3+ regulatory…

Fig. 4  In vivo effect of TY101 antagonistic antibody on tumor growth
in mouse EG7 lymphoma model. A-D C57BL/6J or BALB/c Mice
were treated with TY101 or IgG control twice a week for five times,
starting on the day 7 after inoculation. A Schematic diagram of theexperimental procedure. B EG7 tumor growth curves for each individual EG7 tumor-bearing mouse treated with TY101 or IgG control.
C Mean growth curves of EG7 lymphoma, data are means ± S.E.M.
of 5 mice. D Survival curves of the EG-7 tumor-bearing mice treatedas described in A. E The mice with complete regression of tumor

were reinoculated with EG7 tumor cells into the right flank and B16F10 tumor cells into the left flank 8 weeks after the mice became
tumor-free. As a control, normal mice were inoculated with EG7
tumor cells into the right flank and B16-F10 tumor cells into the left
flank in the same manner. Data (n = 3 mice) show the percentage oftumor incidence of normal mice and surviving mice on day 26 after(re)challenge. *P < 0.05, **P < 0.01, ***P < 0.001, N.S., no significanceFig. 4E). Thus, this result indicated that TY101 treatment
induced long-term tumor-specific immune memory.

P < 0.01). Moreover, administration of TY101 decreased the
proportion of tumor-infiltrating C­ D4+ ­Foxp3+ Tregs by about
50% in mouse EG7 tumors (Fig. 5C-D, P < 0.05). The absolute number of tumor-infiltrating Tregs also decreased significantly in the TY101 treated mice group (Fig. 5E, P < 0.05).
In addition, the percentage of IFNγ-expressing CD8 T cells
was increased by about 50% compared to control (Fig. 5FG, P < 0.01). Furthermore, as we used same number of cells(1 × ­106 cells/tube) when analyzing the Treg and CD8 T cells
from tumor tissue, we thus calculated the ratio of CD8 T cells
to Tregs, an important indicator of clinical outcome of different cancer types [22, 23]. The results showed that TY101 significantly increase the ratio of CD8 T cells to Tregs (Fig. 5H,
P < 0.01). Therefore, TY101 could enhance the antitumor
immune responses by reducing intratumoral Treg viability.

3.5 TY101 antagonistic antibody treatment
promotes tumor‑infiltrating Treg cell deathand enhances the antitumor responses
To determine whether TNFR2 antagonistic antibody elicits antitumor immune responses by promoting intratumoral
Treg cell death, we started the treatment after the tumor
size reached about 10 mm in diameter in W.T. C57BL/6J or
BALB/C mice. The tumor-infiltrating Tregs were analyzed by
FCM. As shown in Fig. 5A-B, over 95% of Tregs were viablein the IgG-treated control group, while TY101 treatment significantly reduced the Treg viability in tumors (Fig. 5A-B,

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T. He et al.

Fig. 5  Effect of TY101 on tumor infiltrating Tregs and IFN-γ+ CD8
CTLs. A-H Treatment started when the tumor reached tumor size
reach about 10 mm in diameter, then the tumor tissue was cleaved
into single cell for flow cytometry (FCM). The Treg viability weredetected by LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit.
A-D The percentage of viable Tregs in total Tregs and the proportion of Tregs in CD4 T cells. A and C Representative FCM plots.
B and D The summary data. E The absolute number of Tregs pergram tumor tissue. F Representative FCM analysis of IFN-γ+ cells in­CD8+ cells from mice described in (A) and the summary data (G).
The number indicates the percentage of interferon–positive (IFN-γ+)
cells in total ­CD8+ cells. H The percentage of CD8 IFN-γ+ cells to
Tregs. Data (n = 4 mice) showed the percentage of Treg proportion
and Treg viability, as well as the proportion of IFN-γ+ CTLs and theratio of CD8/Tregs. *P < 0.05, **P < 0.01, ***P < 0.001, N.S., no significance4 Discussion

result, mobilized and activated antitumor immune responses,
resulting in the complete regression of EG7 lymphoma inthe majority of mice.
There is compelling evidence that TNF promotes activation, function, proliferation, and phenotypic stability ofTregs [1, 2, 19, 24]. Recent studies have shed light on themechanism of the role of TNFR2 in Treg activation andproliferation. It was shown that specific deletion of TNFR2
in Treg significantly reduced the expression of Myb [25],In this study, we showed a possible mechanism of the antitumor effect of TNFR2 antagonists. Genetic or pharmacological inhibition of TNFR2 promoted Treg cell death, while
activation of TNFR2 protected Tregs from cell death. We
further found that in vivo treatment of TY101antagonistic
antibody reduced the viability of the immunosuppressive
TNFR2-expressing Tregs in the tumor environment and, as a

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TNFR2 antagonistic antibody induces the death of tumor infiltrating ­CD4+Foxp3+ regulatory…

a cell cycle regulator important for controlling Treg differentiation and function [26]. Moreover, TNFR2 costimulation enhanced NF-κB signaling pathway in Tregs,
which could be associated with TNF-TNFR2-induced Treg
proliferation [27]. Upregulation of the pro-survival geneis also a characteristic of the transcriptomic profile of
TNFR2 agonist-treated Tregs [27]. Previous results fromDr. Faustmann and her colleagues showed antagonistic
anti-TNFR2 antibodies, in addition to inhibiting the proliferation of human Tregs, could induce the death of TNFR2expressing Tregs [14, 16, 17]. They showed that TNFR2
antagonistic antibody alone decreased Treg ratio or Treg
number in purified CD4 T cells, which was interpreted
that the blockade of TNFR2 induced Treg cell death.
However, our previous results showed M861, another antimurine TNFR2 antagonistic antibody, did not affect Treg
viability in spleen and lymph nodes [13]. In this study,
we confirmed that TNFR2 signaling was dispensable forTreg viability in normal culture conditions. Interestingly,
blockade of TNFR2 promoted Treg cell death in the presence of Dexamethasone, an anti-inflammatory agent thatinduces Treg cell death. A study further supported the
pro-survival effect of TNF and showed that TNF-treatedTregs are more resistant to oxidative stress-induced cell
death [28]. Therefore, activation of the TNF-TNFR2 signaling pathway could be essential for maintaining the Treg
viability in some conditions.
We further showed that TNFR2 antagonistic antibody
promoted the death of intratumoral Treg cells. Hypoxia,
increased R.O.S. (reactive oxidative species) and low-PH
value (high concentration of lactic acid) are the characteristics of the tumor microenvironment. These factors suppress
the function of CTLs but favor the accumulation of Treg
and other immunosuppressive subsets [29]. Several recent
studies showed that metabolic adaptation is vital for tumorinfiltrating Tregs’ immunosuppressive function and viability. Compared with effector T cells, tumor-infiltrating Tregs
are less dependent on glycolysis but use lactate to fuel theiractivities [30]. Moreover, upregulation of fat metabolism
was also found in mouse tumor-infiltrating Tregs [31]. It
was reported that the specific deletion of FASN (fatty acidsynthase) in Treg significantly inhibits mouse tumor growth[32]. The role of TNF-TNFRs signal in metabolic regulation
has been long investigated in different cell types, however,its role in T cells remains largely unknown [33]. Recently, it
was reported that TNFR2 stimulation promotes glycolysis
and glutamine metabolism in Tregs [34, 35]. However, thein vivo effect of TNF-TNFR2 on Treg metabolism remains
unknown. Therefore, whether TNFR2 blockade disrupts
intratumoral Treg metabolism, and induces tumor-infiltrating Treg cell death, should be further studied.
Interestingly, pharmaceutical companies are developing
TNFR2 antagonistic antibodies but also TNFR2 agonistic

antibodies for tumor treatment. Theoretically, as previously
reported, TNFR2 agonistic antibody would boost Treg proliferation [36], which may impair the antitumor immune
responses. However, one study found that TNFR2 agonistic
antibody promoted antitumor immune responses and inhibited tumor growth [37]. This study also found that TNFR2
agonistic antibody did not increase infiltration of Tregs
but promoted CD8 T cell infiltration in mouse tumors. In
comparison with Tregs, although Teffs and CTLs express alower level of TNFR2, Teffs’ activation and pro-inflammatory effect were also dependent TNF-TNFR2 axis [38, 39].
These studies suggested that a high dose of TNFR2 agonist
may also elicit the antitumor responses of Teffs and CTLs.
However, the role of TNFR2 expression by tumor-infiltrating
Teffs or CTLs has not been well studied. Mouse specificallylacking TNFR2 expression in Tregs has been developed andused to study autoimmune inflammatory diseases of thecentral nervous system [25]. This mouse strain will also be
useful in clarifying the role of TNF-TNFR2 in modulating
Teffs, CTLs, and other immune subsets in the tumor and,
consequently, advance the understanding of the mechanismof action of anti-TNFR2 therapeutics.
Taken together, in this study, we showed the activation of
TNF-TNFR2 promoted Treg viability, and induction of thedeath of Tregs represented an underlying mechanism of theantitumor effect of TNFR2 antagonistic antibodies.
Abbreviations TNF: Tumor necrosis factor; TNFR2: Tumor necrosis factor receptor 2; Treg: Regulatory T cells; Dex: Dexamethasone;
RPMI-1640: Roswell Park Memorial Institute Medium,; Pen: Penicillin; Strep: Streptomycin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; F.B.S.: Fetal bovine serum; 2-Me: 2-Mercaptoethanol; FcR: Fc receptor; MACS: Magnetic-activated cell sorting;Teffs: Effect T cells; FCM: Flow cytometry; FACS: Fluorescence-activated Cell Sorting; CTL: Cytotoxic T lymphocyte
Supplementary Information The online version contains supplementary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13402-0​ 22-0​ 0742-0.
Acknowledgements We appreciated Dr Denise Faustmann (Massachusetts General Hospital) for providing TY101 in this study.
Author contributions J.J.M, X.C, designed the study and reviewed
the manuscript. T.Z.H, Y.B.C, M.S.I, C.K.C, J.R.L, performed theexperiments and wrote the manuscript. J.J.M, X.C were involved in
the conceptualization, supervision, project administration and funding
acquisition. All authors have read and agreed to the published versionof the manuscript.
Funding This work was funded by grants from Macau Science andTechnology Development Fund (0099/2021/A2 and 0056/2019/A.F.J.),
University of Macau (CPG2022-00024-ICMS and MYRG201900169-ICMS), Guangdong-Hong Kong-Macau Joint Lab on Chinese
Medicine and Immune Disease Research (EF006/ICMS-CX/2021/
GDSTC), Applied Research Programs of Guangdong-Hong KongMacao Innovation Center sponsored by Guangzhou Development
District (EF032/ICMS-CX/2021/ RITH). It also been funded in partby the Intramural Research Program of the N.I.H., National Cancer
Institute, C.C.R., LCIM.

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T. He et al.
Data availability The original contributions presented in the study areincluded in the article/Supplementary Material. Further inquiries canbe directed to the corresponding authors.

Declarations
Consent for publication All authors have read and agreed to the published version of the manuscript.
Ethical approval and consent to participate The animal study wasreviewed and approved by Animal Research Ethics Committee of the
University of Macau.
Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.

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