International Immunopharmacology 101 (2021) 108345

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International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

A TNFR2 antibody by countering immunosuppression cooperates with
HMGN1 and R848 immune stimulants to inhibit murine colon cancer
Mengmeng Jiang a, 1, Jia Liu b, c, 1, De Yang b, 1, Debra Tross b, Ping Li a, Fengyang Chen a,
Md Masud Alam b, Denise L. Faustman d, Joost J. Oppenheim b, *, Xin Chen a, e, f, g, *aInstitute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macau SAR 999078, China
Laboratory of Cancer Immunometabolism, Center for Cancer Research, National Cancer Institute at Frederick, NIH, Frederick, MD, USA
Department of Pharmacy, Changchun University of Chinese Medicine, Changchun, Jilin 130117, Chinad
Immunobiology Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USAe
Department of Pharmaceutical Science, Faculty of Health Sciences, University of Macau, Macau SAR 999078, Chinaf
MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR 999078, Chinag
Guangdong-Hong Kong-Macau Joint Lab on Chinese Medicine and Immune Disease Research, Chinabc

A R T I C L E I N F O

A B S T R A C T

Keywords:
CD4+Foxp3+ regulatory T cells (Tregs)
TNFR2
HMGN1 and R848
Antitumor
Immunotherapy

Immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs) promote tumor immune evasion and thus targetingof Tregs has become an strategy in cancer immunotherapy. Tumor necrosis factor receptor 2 (TNFR2) is highly
expressed and important for the immunosuppressive function of Tregs in humans and mice. Thus, the benefit oftargeting TNFR2 in cancer immunotherapy merits more investigation. A previous report identified a new murine
monoclonal anti-TNFR2 antibody (designated TY101), which showed therapeutic efficacy in murine cancer
models, but its mechanism of action was less understood. In this study, the capacity of a combination of
immunostimulants to enhance the effect of this inhibitor of Tregs was investigated. We examined the efficacy ofTY101 as an anti-tumor immune reagent combined with HMGN1 (N1, a dendritic cell activating TLR4 agonist)
and R848 (a synthetic TLR7/8 agonist). This immunotherapeutic combination exerted synergistic antitumor
effects as compared with any single treatment. The antitumor response was mainly mediated by the depletion ofTregs and stimulation of cytotoxic CD8 T cell activation. The result also suggested that the effect of TY101 wassimilar to that of anti-PD-L1 when used in combination with these immunostimulants. Therefore, we propose thattreatment strategies of antagonizing TNFR2 on Tregs would behave as potent checkpoint inhibitors and canpotentially be utilized to develop a novel antitumor immunotherapy.

1. Introduction
There is accumulating evidence that CD4+Foxp3+ regulatory T cells(Tregs) are highly enriched in the tumor microenvironment (TME) andplay crucial roles in promoting tumor progression [1,2]. To date, a large
increase in intratumoral Tregs has been demonstrated in melanomas,
colorectal cancers and gastric cancers. Furthermore, the accumulation oftumor-infiltrating Tregs was also widely associated with poor prognosis[3–5]. Tregs inhibit not only effector T cells, but also innate immune

dendritic cells (DCs) and natural killer cells [6,7]. Consequently, targeting of Tregs has become a successful strategy in the treatment of
human cancers [8–10].
We were the first group to report that tumor necrosis factor (TNF)
could promote activation and expansion of Tregs by interacting with
TNF receptor type 2 (TNFR2) [11]. Moreover, our research and subsequent studies have confirmed that TNFR2 was preferentially expressedon Treg cells, and high expression of TNFR2 identified the maximallyimmunosuppressive subset of Tregs in humans and mice [12–14]. In

Abbreviations: CFSE, Carboxyfluorescein succinimidyl ester; CTL, Cytotoxic T lymphocyte; DCs, Dendritic cells; dLNs, draining lymph nodes; MSCs, Mesenchymal
stem cells; MDSCs, Myeloid-derived suppressor cells; Tregs, CD4+Foxp3+ regulatory T cells; TME, Tumor microenvironment; TNF, Tumor necrosis factor; TNFR2,
Tumor necrosis factor receptor type 2; TLRs, Toll-like receptors.
* Corresponding authors at: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR
999078, China (X. Chen). Laboratory of Cancer Immunometabolism, National Cancer Institute, NIH, USA (J.J. Oppenheim).
E-mail addresses: Oppenhej@mail.nih.gov (J.J. Oppenheim), xchen@umac.mo (X. Chen).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.intimp.2021.108345
Received 10 July 2021; Received in revised form 2 November 2021; Accepted 2 November 2021
Available online 15 November 2021
1567-5769/© 2021 Elsevier B.V. All rights reserved.

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International Immunopharmacology 101 (2021) 108345

contrast, as a result of TNFR2 knock-down or knock-out, Treg cells only
showed minimal or no suppressive activity by disturbing Foxp3
expression [15]. Overall, the evidence clearly demonstrates that TNFR2
plays a key role in maintaining and promoting the immunosuppressivefunction of Tregs. Thus, targeting of TNFR2 may provide a therapeutic
approach in cancer immunotherapy.
Tumor infiltrating dendritic cells express a tolerogenic immaturephenotype in the immunosuppressive tumor microenvironment and failto promote anti-tumor response. Stimulant of Toll-like receptors (TLRs)
expressed by DCs plays crucial roles in inducing their maturation andactivation [16]. Currently, HMGN1 (N1), a TLR4 ligand which was
identified as an alarmin that activates DCs [17], and R848, a synthetic
TLR7/8 agonist that also activates DCs [18], together induce therapeutic
antitumor responses. The combination of N1 and R848 have been reported to synergistically promote the activation of dendritic cells andmobilize antitumor immune responses in mice [19].
We have used a murine anti-TNFR2 antibody (TY101, isotype is Rat
IgG), which has the capacity to block the binding of TNF to CRD3 and
CRD4 regions of TNFR2 receptor, with the ability to inhibit Treg activities and promote anti-tumor immune responses [20]. In order toenhance its efficacy in promoting anti-tumor immune responses, TY101
was given together with N1 and R848 to promote the generation of
antitumor immune responses by triggering the maturation of tumorinfiltrating dendritic cells and their subsequent homing to draining
lymph nodes [21]. Given the effective therapy of CT26-bearing mice by
the combination of N1 + R848 + anti-PD-L1 or N1 + R848 + TY101(present study), it is presumed that tumor-infiltrating dendritic cells andmacrophages express TLR4, TLR7 and PD-L1. In fact, it has been documented since 2003 that antigen presenting cells (including DCs andmacrophages) in the TME of both humans and mice expressing functional TLR4 and TLR7 receptors [22].
Furthermore, we also compared the effect of TY101 with anti-PD-L1
treatment given together with N1 and R848. This study demonstrates
that the combination of TY101 with N1 and R848 can exert robust
antitumor effects based on depletion of TNFR2-expressing tumor infiltrating Tregs and increased activity of cytotoxic CD8 T cells. The antitumor effect of TY101 equaled the effect of anti-PD-L1 antibody.
Therefore, combination therapy of TY101 with other immunostimulants
can be considered a promising cancer treatment strategy.

CD4+FoxP3+ Tregs. The purified cells were cultured in 96-well roundbottom plates in medium of RPMI-1640 (Corning, VA, USA) containing
10% heat inactivated fetal bovine serum (FBS, BioWhittaker, USA), 2mM L-glutamine, 10 mM HEPES buffer, 0.1 mM nonessential aminoacids, 1 mM sodium pyruvate, 1% penicillin (100 U/ml)/streptomycin(100 mg/ml), and 50 µM 2-Methylmercaptoethanol, at 37℃ with 5%
CO2 incubator.
2.3. Reagents and flow cytometry
Antibodies purchased from BioLegend included Percp/cy5.5 antimouse CD45 (Clone I3/23), PE anti-mouse CD120b (TNFR Type II /p75) (clone TR75-89); antibodies purchased from BD Pharmingen
included FITC Rat anti-mouse CD4 (clone RM4-5); antibodies purchasedfrom eBioscience (Invitrogen) included eFlour 450 anti-Hu/Mo CD44
(clone IM7), APC anti-mouse CD62L (clone MEL-14), eFlour 450 antimouse CD8a (clone 53-6.7), eFlour 660 anti-mouse CD107a (clone
eBiol D4B), and Percp/Cyanine 5.5 anti-mouse CD4 (clone RM4-5).
Besides, APC anti-Mo/Rt Foxp3 (clone FJK-16s) for intracellular staining of Foxp3 was purchased from Invitrogen. Cells were resuspended in
FACS buffer (PBS + 2% FBS + 0.05% NaN3), and cell surface staining
was conducted at room temperature for 30 min. For intracellular
staining of Foxp3, cells were fixed and permeabilized using Transcription Factor Staining Buffer Kit (Cat# TND-0607-KIT, TONBO biosciences), following the manufacturer’s instructions. After blocking FcR,
cells were incubated with appropriately diluted antibodies. Flowcytometry was performed on LSR II (BD FACSCalibur), FACS analysis
was gated on the live cells only by using a LIVE/DEAD Fixable Dead Cell
Stain kit, then analyzed by FlowJo software (version 10.6.1).
2.4. Effects of anti-TNFR2 antibody (TY101) on Tregs
CD4+CD25+ T cells were purified and seeded in a round bottom 96well plate with 1 × 105 cells/well. Cells were cultured with IL-2 (10 ng/ml) and/or TNF (20 ng/ml) to maintain and activate their survival andexpansion. To examine the effect of anti-TNFR2 antibody (TY101) on
Tregs, the cells were treated with increasing concentrations of TY101 (0,0.625, 1.25, 2.5 μg/ml) in the culture medium containing IL2 and TNF
for 72 h. After the cells were collected and fixed/ permeabilized, thenstained with specific antibodies of PercP/cy5.5 anti-mouse CD4, PE antimouse CD120b (TNFR2) and APC anti-mouse FoxP3, the relative isotype
was used as an internal control, for FACS analysis. The CellTrace CFSE
(carboxyfluorescein succinimidyl ester) Cell Proliferation Kit was used
for labeling of cells to detect cell proliferation.

2. Materials and methods
2.1. Mice and therapeutic drugs
Female wild-type Balb/c or C57BL/6 mice, 6–8 weeks old werepurchased from The Jackson Laboratory. The NCI as authorized byAmerican association for the accreditation of laboratory animal care
(AAALAC) follows the public health service policy for the care and use oflaboratory animals. Animal care was provided according to the procedures and guidelines for care and use of laboratory animals (National
research council, Washington, DC). All animal studies were approved by
the Institutional Animal Care and Use Committee (IACUC) of NCI.
Anti-TNFR2 antibody (TY101, 10.90 mg/ml) was generated andpurified as described previously [20]. Anti-mouse PD-L1 (B7H1) antibody (Clone 10F.9G2, cat# BE0101) was purchased from Bio X Cell.
Recombinant HMGN1 (N1) was generated and purified as previously
reported [17]. R848 (Cat# 144875-48-9) was purchased from InvivoGen. Recombinant murine IL2 (Cat# 212-12) and TNF-α (Cat# 31501A) were purchased from PeproTech, USA.

2.5. In vivo effect of TY101 on murine colon cancer models
Thirty BALB/c or C57BL/6 mice were randomly divided into six
groups. All of the mice were subcutaneously (s.c.) injected with 2 × 105
CT26 or MC38 tumor cells suspended in PBS on the right flank. Once thetumors reached 50 to 100 mm3 (usually 7 to 10 days after tumor inoculation), the tumor-bearing mice were intratumorally (i.t.) injected witheither hamster IgG (10 µg), or anti-TNFR2 antibody (TY101) (10 µg), or
i.t. injections of (N1 (10 µg) + R848 (10 µg)), or with TY101 (10 µg) +(N1 (10 µg) + R848 (10 µg)) or anti-PD-L1 (10 µg) + (N1 (10 µg) + R848
(10 µg)) twice per week for a total of 5 doses. The survival, tumor sizeand body weight of tumor bearing mice were monitored twice weekly.
The calculating formula to determine tumor volume was V = length ×
width × width/2. Animals reached an endpoint and were euthanizedwhen tumor volume reached 2000 mm3, exhibited large necrotic areas
on the skin or the mice had a 20% weight loss.

2.2. Cell isolation and culture
CD4+CD25+ T cells were purified from spleen and lymph nodes(including inguinal, axillary and mesenteric regions) of C57BL/6 or
Balb/c mice, using the mouse CD4+CD25+ Regulatory T Cell Isolation
Kit (Cat# 130091041, Miltenyi Biotec), yielding a purity of 90% for2.6. Isolation of tumor-infiltrating lymphocytes and immunostaining
Following treatments, the tumors and draining lymph nodes (dLNs)
of mice were surgically dissected and dissociated using Miltenyi2

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International Immunopharmacology 101 (2021) 108345

Dissociation Kit (Miltenyi, MA, USA). After dissociation, 1 × ACK lysing
buffer (Invitrogen, ThermoFisher, USA) was used to further lyse thecontaminating red blood cells (RBCs). Cells were washed twice using
FACS buffer and centrifuged at 300g for 5 min. Finally, cell suspensions
were stained with anti-CD45/PerCP-Cy5.5, anti-CD4/FITC, anti-CD8/
APC-Cy7 for the detection of CD4 and CD8 T cells; anti-CD62L/APC
and anti-CD44/Pacific Blue for the detection of effector T cells; antiTNFR2/PE and anti-FoxP3/APC for Tregs. Flow cytometry analysis
was performed on LSRII (BD FACSCalibur), and data analyzed by FlowJo
software (version 10.6.1).

(dLNs: CT26). After 24 h incubation, cells were harvested and stainedwith anti-CD8-eFluor450 and anti-CD107a-eFluor660. Flow cytometry
was conducted using BD LSRII, and the result was analyzed by FlowJo
software.
2.8. Statistical analysis
The statistical significance between two groups were analyzed by
two tailed student’s t-test, and the multiple groups comparison wereanalyzed by one-way ANOVA and Tukey’s post hoc test using GraphPad
Prism 8.3 (GraphPad, San Diego, CA, USA). The results data were represented as mean ± standard deviation (SD) of multiple experiments.
Statistical significance was determined by * p < 0.05, ** p < 0.01, *** p< 0.001, **** p < 0.0001.

2.7. Cytotoxic T lymphocyte (CTL) assay for immune function
A monolayer of CT26 cells was prepared using a 48-well plate byseeding 5 × 104 cells/well and incubating at 37℃ with 5% CO2 incubator overnight. The dLNs cells were added to the plate at a ratio of 10:1

Fig. 1. Effect of in vitro treatment with TY101 on the proportion and proliferation of Tregs and their TNFR2 expression. CD4+CD25+ T cells were purified and treatedwith different concentrations of anti-TNFR2 antibody TY101 (0, 0.625, 1.25, 2.5 μg/ml) in the presence of 10 ng/ml IL-2 with or without 20 ng/ml TNF in RPMI1640
complete medium for 3 days. A, Summary of percentage of Foxp3-expressing cells and mean fluorescence intensity (MFI) of Foxp3 expression present in the
CD4+CD25+ T cell subsets (N = 3, means ± SEM); B, Flow cytometric plot of TNFR2-expressing cells in Tregs. Number shows the proportion of gated cells. C,
summary of the proportion of TNFR2-expressing cells present in CD4+FoxP3+ Tregs (N = 3, means ± SEM); D, CellTrace CFSE stained CD4+CD25+ T cells weretreated with 2.5 μg/ml TY101 in the presence of 10 ng/ml IL-2 with or without 20 ng/ml TNF were cultured for 3 days, the proportion of proliferating cells is
indicated in the histogram; E, the summarized statistical data of proliferating Treg cells are shown (N = 3, means ± SEM). F, The mean fluorescence intensity (MFI) of
CTLA4 and PD-L1 expression by Tregs. All data shown are representative of three independent experiments with similar results. The statistical significance wasanalyzed using one-way ANOVA by comparison with IL-2 plus TNF group, * p < 0.05, ** p < 0.01, *** p < 0.001.
3

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3. Results and discussion

increase the proportion of Foxp3+ Tregs and TNFR2 expression by Tregs(~1.2-fold and ~1.4-fold, respectively), which may reflect the effect of
TNF on the maintenance of Foxp3 expression by in vitro cultured Tregs.
However, treatment with TY101 reduced the proportion of Foxp3+
Tregs (from 72.3% to 61.4%) as well as Foxp3 expression (Fig. 1A) anddown-regulated TNFR2 expression by Tregs (~0.4-fold) (Fig. 1B and C)
compared to TNF treatment Tregs, in a dose-dependent manner (p <0.05). Based on cell viability assays, the proportion of live cells in
CD4+CD25+ T cells treated with TY101 was equal to those in the control
cells (data not shown), indicating TY101 was not cytotoxic for Tregs.
To determine the effect of TY101 on the proliferation of Tregs, Tregs
stained with CFSE were cultured in the absence or presence of TNF and
TY101. After 3 days of incubation with IL-2, 20.1% cells were proliferative (Fig. 1D left panels). In the presence of IL-2, TNF promoted the3.1. Effect of TY101 on the proportion, proliferation and TNFR2
expression of Tregs in vitro
Mouse CD4+CD25+ T cell subsets purified using a CD4+CD25+
Regulatory T Cell Isolation Kit, yielded greater than 90% pure of
CD4+FoxP3+ Tregs as previously reported [23]. TNF could activate
Tregs and promote their proliferation and expansion by interacting withTNFR2. To confirm the effect of anti-TNFR2 antibody (TY101) on Tregs,
purified CD4+CD25+ T cells were cultured in a medium containing IL-2
(10 ng/ml) and TNF (20 ng/ml). The cells were treated with increasing
concentrations of TY101 (0.625–2.5 μg/ml). The results showed that in
the presence of IL-2 (required for in vitro survival of Tregs), TNF could

Fig. 2. Therapeutic effect of TY101 therapy alone or in combination with N1 and R848 on mouse colon cancer models. 2 × 105 CT26 or MC38 colon cancer cells
were subcutaneously (s.c.) injected into Balb/c or C57BL/6J mice on day 0, respectively. At day 8, when tumor size reached 50–100 mm3, TY101, anti-PD-L1, or
HMGN1 plus R848, alone or in combinations of TY101 or anti-PD-L1 together with HMGN1 plus R848 were injected intratumorally (i.t.) for 5 doses biweekly. A,
shows the overall therapeutic procedural design scheme. Blue arrow represents TY101 or anti-PD-L1 injection, green arrow represents HMGN1 plus R848 injection; B,
CT26 tumor growth in individual mice treated with TY101 or anti-PD-L1 (n = 10, respectively) or control (PBS) group (n = 10) is shown (means ± SEM), and thesurvival curves for each group are also indicated; C, Effect on CT26 tumor growth in individual mice being treated with TY101 or anti-PD-L1 combined with N1 andR848 group (n = 10, each group) is shown (means ± SEM), and the survival curves for each group are also indicated; D, Effect on MC38 tumor growth in individual
mice of TY101 treatment alone (n = 5) or combined with N1 and R848 (n = 5), the tumor growth for each mouse is shown and the survival curves for each group arealso indicated; E: Tumor free mice in TY101 combination group (n = 5) and anti-PD-L1 combination group (n = 4) were subcutaneously injected with 2 × 105/mouseof CT26 cells in the right flank and 4T1 cells in left flank and the tumor growth curves were represented, data are summarized as means ± SEM. Red line represents
CT26, Blue line represents 4T1. Statistically significant difference was analyzed using one-way ANOVA in comparison with PBS group. * p < 0.05, ** p < 0.01, *** p< 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)4M. Jiang et al.

International Immunopharmacology 101 (2021) 108345

proliferation of Tregs up to 39.9%, however, when treated with TY101,
the proportion of replicating cells decreased back to 21.6% (Fig. 1D rightpanels). Therefore, TY101 was an antagonistic antibody to TNFR2 andcompletely inhibited the proliferation of Tregs induced by TNF (p <0.01, Fig. 1E). Moreover, to explore whether TY101 interferes with otherinhibitory markers of Tregs by blocking TNF-TNFR2 axis, we assessed
the effect on the expression of CTLA4 and PD-L1. The expression oftheses inhibitory markers was decreased markedly (Fig. 1F), whichindicated the important role of TY101 in interfering the suppressive
phenotype of Tregs.

or without N1 and R848 for up to 5 doses (twice a week), starting fromday 8 after tumor reached 50–100 mm3 in size, as schematically shownin Fig. 2A. Mice receiving a single treatment of TY101 showed partial
inhibition of tumor growth, and prolonged the survival of tumor bearing
mice, the potency of TY101 was comparable to anti-PD-L1 treatment(Fig. 2B). However, none of the CT26 tumor bearing mice was cured bytheir treatment. We previously documented that N1 and R848 synergistically activated DCs and promoted antitumor responses. As a result,intratumoral delivery of N1 plus R848 with therapeutics capable ofcountering tumor immunosuppression (e.g., Cytoxan, anti-PD-L1), themouse tumors could be cooperatively suppressed to a much great extent[19]. We therefore examined whether N1 and R848 could enhance theantitumor effect of anti-TNFR2 antibody. Although treatment of
HMGN1 and R848 could not induce complete regression of tumors, thecombination of TY101 with N1 plus R848 resulted in a cure rate of 50%(5/10). Moreover, to compare the effect of this combination with anti-

3.2. Effect of TY101 in combination with N1 and R848 on the growth ofmurine colon tumors
To determine the in vivo effect of TY101, CT26 or MC38 tumorbearing mice were treated with TY101 or anti-PD-L1, combined with

Fig. 3. Effect of TY101 combined with N1 and R848 on the tumor infiltration by Tregs and TNFR2 expression, and on effector CD4 and CD8 T cells and CTL. After
treatment by TY101, or anti-PD-L1, or N1 plus R848, and their respective combinations for 3 doses, the immune cells in tumors and draining lymph nodes (dLNs)
were detected by flow cytometry. To examine the cytotoxic CD8 T lymphocytes (CTL) function, the draining lymph nodes (dLNs) from each treatment group were
cocultured with CT26 tumor cells at a ratio of 100:1, then flow cytometry was performed to detect the proportion of CTL. A, The gating strategies of CD4 T, CD8 T,
Tregs and TNFR2+ Tregs in tumor tissue by flow cytometry; B, summary of the ratio of CD8+ versus CD4+ T cells (n = 5, means ± SEM) in individual mouse fromeach group treated with Ctrl Ab, TY101, anti-PD-L1, N1 plus R848, or a combination of TY101 or anti-PD-L1 with N1 plus R848; C, summary of the proportion of
CD4+FoxP3+ Tregs in CD4+ T cells in tumor tissues from each group (n = 5), data shown are means ± SEM; D, summary of the percentage of TNFR2-expressing cells
in Treg cells from each group (n = 5), data shown are means ± SEM; E, summary of the percentage of effector CD4 T (CD4+CD44high/CD62L-) cells and CD8 T(CD8+CD44high/CD62L-) cells in dLNs from each group (n = 5), data shown are means ± SEM; F, summary of the percentage of CD8+ T cells and CT26-specificcytotoxic CD8+ T (CD8+CD107a+) cells in dLNs from each group (n = 5), data shown are means ± SEM. Statistically significant difference was analyzed using
one-way ANOVA by compared to Ctrl Ab group. * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001.
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PD-L1 inhibitor, we found the combination of anti-PD-L1 with N1 andR848 also resulted in a cure rate of 40% (4/10). The overall survival ofmice in both combination groups was also markedly prolonged (Fig. 2C).
Similarly, in MC38 tumor bearing mice, TY101 treatment alone could
induce a cure rate of 20%, whereas the combination of TY101 with N1and R848 resulted in 60% of mice being cured. Consequently, the longterm survival of tumor bearing mice was also markedly improved(Fig. 2D).
To investigate whether tumor free mice could generate persistenttumor-specific immune responses, CT26 and murine mammary carcinoma 4T1 cells were injected into tumor-free mice cured by TY101
combined with N1 and R848 (n = 5) or by anti-PD-L1 combined with N1and R848 (n = 4) treatment, on their contralateral flanks three weeks
later after the completion of first treatment. We then monitor the tumor
growth for another month. All of the cured mice in both groups developed 4T1 tumors, whereas none of the cured mice formed CT26 tumors(Fig. 2E). This indicates that the cured mice have developed tumorspecific immunity.

expressing cells may be attributed collaboratively to the overall antitumor effect of TY101. However, most likely that the inhibition ofTregs, may play a key role. To further identify additional relevant
cellular targets of TNFR2 blocking antibodies merits further
investigation.
As previously reported, a number of tumor cells also express TNFR2,
and it was shown that TY101 had a moderate cytotoxic effect on TNFR2expressing CT26 and MC38 tumor cells, as determined by trypan blue
staining [21]. To further confirm its effects on tumor cells, we examined
the proliferation of tumor cells in the presence of different concentrations of TY101 with a cell counting kit-8 (CCK-8) assay. However, we did
not detect any significant inhibitory effect of TY101 on the proliferationof tumor cells (data not shown). Although the discrepant results may becaused by the different assays, we favor the idea that the major antitumor effect of TY101 was mediated by reduction of TNFR2expressing Tregs, rather than any direct inhibition on tumor cells.
CD8 T cells also express TNFR2, however, the proportion of TNFR2expressing cells in CD8 subset was ~12 folds lower than that of Tregs(data not shown). The role of TNFR2 expressed by CD8 T cells is highly
controversial and there are plenty of publications showing that TNFR2
negatively regulates CD8 cell function. For example, it was reported that
TNFR2-expressing CD8 T cells are exhausted cells or immunosuppressive CD8+ Treg cells [27]. Intriguingly, it was shown that TNFR2deficient memory CD8 T cells provided superior protection against
tumor cell growth [28], presumably due to the fact that CD8 cells
deficient in TNFR2 were more resistant to activation-induced cell death(AICD) [29]. Therefore, it is possible that blockade of TNFR2 by TY101
could inhibit the activation of CD4 Tregs, while reversing the exhaustive
phenotype of CD8 T cells, promoting the survival of CD8 CTLs andenhancing their anti-tumor activity. This possibility should be experimentally studied in the future.
It should be noted that a previous report showed intraperitoneal (i.p)
treatment by TY101 alone of CT26 tumor bearing mice resulted in a
~55% cure rate, while the combination of TY101 with anti-PD-1 therapy could induce a cure rate of up to ~63% [20]. However, in ourreport, none of the CT26 tumor bearing mice was cured by the treatment
with TY101 by itself and had only a ~20% cure rate in MC38 tumor
bearing mice. The reason for this discrepancy may be attributable to themethod of administration and dosage of TY101, because the intratumorally administration in our study did not result in a more potent
immune responses in comparison with the reported intraperitoneal injection of higher doses of TY101. However, the comparatively lower
doses used in our study would be advantageous. Furthermore, the highexpression of TNFR2 on tumor cells and/or abundance of tumor infiltrated TNFR2+ Tregs in tumors would be more susceptible to antiTNFR2 therapy.
Unlike in previous studies, our approach endeavored to reduce or
eliminate the immunosuppressive environment to improve the effector T
cells activation. In this study, TY101 by selective blocking TNFR2, might
have prevented downstream signaling by NF-kB and PI3K/Akt [30],which is necessary for Tregs survival and expansion. TY101 exhibited
potent in vitro and in vivo efficacy in decreasing the numbers of TNFR2expressing Tregs and reducing their suppressive function. Though N1
plus R848 did not have any direct antitumor effects, they could markedly activate the maturation of DCs in the tumor microenvironment.
Stimulation by this combination resulted in the production of proinflammatory cytokines and antigen-presenting capacities of DCs.
Therefore, the combination of anti-TNFR2 antibody with N1 and R848
by dual-targeting of immunosuppressive Tregs and immature DCs,
resulted in greater promotion of presentation of tumor-specific antigens
to CTL effectors, thus promoting greater antitumor immune responses.
Furthermore, combination therapy of TY101 with N1 plus R848 equaled
the anti-tumor effects of anti-PD-L1 together with N1 and R848, indicating that anti-TNFR2 antibody could be developed as a novel treatment option for cancer therapy.
In conclusion, our study clearly shows that combination therapy by

3.3. Effect of TNFR2 antagonism in combination with N1 and R848 on
the levels of CD4 and CD8 T cells intratumorally and Tregs and in draining
lymph nodes
To demonstrate the effects of immunotherapy on immune cell content in CT26 tumor bearing mice, we profiled the tumor infiltrating
leukocytes and immune cells present in tumor tissue and in draining
lymph nodes by flow cytometry. The flow cytometry gating strategies asshown in Fig. 3A. TY101 treatment alone or in combination with N1 andR848 increased the ratio of intratumoral CD8/CD4 T cells equally as dida single treatment of anti-PD-L1 or in combination with N1 and R848
(Fig. 3B). Moreover, after treatment with TY101 or anti-PD-L1 in combination with N1 plus R848, the numbers of CD4+FoxP3+ Tregs in thetumor microenvironment were markedly decreased (Fig. 3C). Since
TY101 acted as an TNFR2 antagonist, we further examined the expression of TNFR2 by Tregs, which revealed that treatment with TY101
alone or when combined with N1 and R848 markedly decreased theexpression of TNFR2 by Tregs in tumor tissue (Fig. 3D). Therefore,
TY101 has the capacity to reduce Tregs in tumor bearing mice.
Furthermore, the combination of TY101 or anti-PD-L1 together with
N1 and R848 resulted in greater increases in effector CD4(CD4+CD44high/CD62L-) and effector CD8 (CD8+CD44high/CD62L-) T
cells (Fig. 3E) as compared with control groups. Moreover, based on
coculture of dLNs with CT26 tumor cells to examine the cytotoxic
function of CD8 T cells, combination immunotherapy also resulted in thehighest levels of CD8 T cells as well as tumor-specific effector CD8(CD8+CD107a+) T cells present in draining lymph nodes (Fig. 3F).
Overall, these data indicate that combination therapy by TY101 with N1and R848 exerted its antitumor effects based on depletion of Tregs andresultant stimulation of cytotoxic CD8 T cells.
In this study, monotherapy with TY101 moderately reduced the
proportion of Tregs and more profoundly decreased the expression of
TNFR2 by tumor infiltrating Tregs. The combination therapy with N1and R848 more dramatically reduced the proportion of Tregs in tumor,
presumably resulting from the restriction of Tregs, and from the
expansion of Teffs though the activated DCs induced by N1 and R848
treatment. The infiltration of CD8 T cells in tumor microenvironment by
combination therapy was also markedly increased.
In addition to Tregs, recent studies have shown that TNFR2 is also
expressed by other immunosuppressive cells, such as mesenchymal stem
cells (MSCs) and myeloid-derived suppressor cells (MDSCs) [24,25].
Thus, the immune suppressive effect of these immunosuppressive cells
was likely inhibited by TY101 treatment as well. Moreover, endothelial
progenitor cells (EPCs) also express TNFR2 [26]. Since EPCs play
important roles in the formation of new blood vessels to support tumor
growth [26], the inhibition of TNFR2 may mitigate the tumorpromoting effect of EPCs. Global inhibition of all lineages of TNFR26

M. Jiang et al.

International Immunopharmacology 101 (2021) 108345

anti-TNFR2 antibody with N1 and R848 may represent a novel treatment for solid tumors. The mechanism of TNFR2 antagonism is presumably based on blocking TNF-TNFR2 signaling of Tregs in the tumor
microenvironment. Recently, multiple humanized anti-TNFR2 antibodies have been developed for cancer immunotherapy, and our results
suggest the efficacy and safety of anti-TNFR2 merit clinicalinvestigation.

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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This project has been funded by The Science and Technology
Development Fund, Macau SAR (FDCT, File No. 0056/2019/AFJ),
University of Macau (File No. MYRG2019-00169-ICMS, CPG-CPG202100007-ICMS and SKL-QRCM(UM)-2020-2022), 2020 Guangdong Provincial Science and Technology Innovation Strategy Special Fund
(Guangdong-Hong Kong-Macau Joint Lab), No: 2020B1212030006; andsupported by the Applied Research Programs of Guangdong-Hong KongMacao Innovation Center sponsored by Guangzhou Development District. It also been funded in part by the Intramural Research Program of
the NIH, National Cancer Institute, CCR, LCIM.
Contributions
Mengmeng Jiang and Jia Liu performed and completed the experiments. De Yang, Xin Chen and Joost J. Oppenheim designed the experiments. Mengmeng Jiang drafted the manuscript. Debra Tross, Ping
Li, Fengyang Chen and Md Masud Alam helped to review and editing themanuscript. Denise L. Faustman contributed to provide drug analysis.
Joost J. Oppenheim, De Yang and Xin Chen revised the manuscript. All
authors agreed to submit the manuscript.
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