cells
Review

The Roles of TNFR2 Signaling in Cancer Cells and the Tumor
Microenvironment and the Potency of TNFR2 Targeted Therapy
Hiroyuki Takahashi 1,2 , Gumpei Yoshimatsu 1 and Denise Louise Faustman 2, *1

2*

Citation: Takahashi, H.; Yoshimatsu,

Department of Gastroenterological Surgery, Fukuoka University Hospital, Fukuoka 814-0180, Japan;
htakahashi3@mgh.harvard.edu (H.T.); gumpei.ysohimatsu@fukuokauniversityhospital.org (G.Y.)
Immunobiology Department, Massachusetts General Hospital and Harvard Medical School,
Charlestown, MA 02129, USA
Correspondence: dfaustman@mgh.harvard.edu; Tel.: +1-617-726-4084; Fax: +1-617-726-4095

Abstract: The appreciation that cancer growth is promoted by a dynamic tumor microenvironment(TME) has spawned novel approaches to cancer treatment. New therapies include agents that activate
quiescent T effector cells and agents that interfere with abnormal neovascularity. Although promising,many experimental therapies targeted at the TME have systemic toxicity. Another approach is totarget the TME with greater specificity by taking aim at the tumor necrosis factor receptor 2 (TNFR2)
signaling pathway. TNFR2 is an attractive molecular target because it is rarely expressed in normal
tissues (thus, has low potential for systemic toxicity) and because it is overexpressed on many typesof cancer cells as well as on associated TME components, such as T regulatory cells (Tregs), tumorassociated macrophages, and other cells that facilitate tumor progression and spread. Novel therapies
that block TNFR2 signaling show promise in cell culture studies, animal models, and human studies.
Novel antibodies have been developed that expressly kill only rapidly proliferating cells expressing
newly synthesized TNFR2 protein. This review traces the origins of our understanding of TNFR2’s
multifaceted roles in the TME and discusses the therapeutic potential of agents designed to block
TNFR2 as the cornerstone of a TME-specific strategy.

G.; Faustman, D.L. The Roles of
TNFR2 Signaling in Cancer Cells andthe Tumor Microenvironment and
the Potency of TNFR2 Targeted

Keywords: TNFR2; tumor microenvironment; immune checkpoint inhibitor; Tregs; immunotherapy;
anti-TNFR2 antibody

Therapy. Cells 2022, 11, 1952.
https://doi.org/10.3390/
cells11121952
Academic Editor: Michael Morgan
Received: 24 April 2022
Accepted: 14 June 2022
Published: 17 June 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

1. Introduction
Since the identification of nitrogen mustard as an anti-cancer drug in 1940s, pharmaceutical options for malignant disease have remarkably progressed to increasingly targeted
therapies [1]. Cancer immunotherapy using immune checkpoint inhibitors (ICIs) suchas anti-programmed cell death protein 1 (PD-1) drugs or anti-cytotoxic T lymphocytesassociated protein 4 (CTLA-4) drugs have become common in the clinical setting [2]. ICIs
harness the human immune system to attack tumors, predominantly by enhancing theanti-tumor activity of cytotoxic T lymphocytes (i.e., effector T cells, Teffs) [3]. While ICIs
may prolong survival, tumors often acquire primary or secondary resistance to them [4].
Further, ICIs have unavoidable adverse events because they act broadly on the immune
system. Adverse events include autoimmune disease, cardiotoxicity, and cytokine release
syndrome [5,6]. Recent attention has focused on targeting the tumor microenvironment(TME), which largely consists of infiltrating immune cells, notably T-regulatory cells (Tregs),
neo-vascular cells, stromal cells, and tumor cells [3,7]. Interactions between cancer cells
and other cells of the TME affect overall tumor growth, homeostasis, and progression [3].
Uncovering TME’s detailed roles in tumor progression has led to a shift in anti-tumor
therapies to include TME’s supportive molecules, such as protein kinases, endothelial
growth factors, and superficial receptors [8]. Drugs directed at these molecular targets arethought to exhibit less systemic toxicity vs. conventional chemotherapy but they still exert

4.0/).

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2 of 22systemic effects [9,10]. Thus, the ideal anti-cancer strategy for the next era may require
targeting molecules more specifically expressed in the TME.
One component of the TME is tumor necrosis factor (TNF) and its receptors. TNF isa unique cytokine that exerts two distinct actions depending on its two receptors; one istumor necrosis factor receptor 1 (TNFR1), which initiates inflammation and tissue apoptosis
and/or necrosis depending on the cell type affected, and the other is TNFR2, which
modulates the immune system and tissue regeneration [7,11]. Although both receptors
are part of the human TNF superfamily, they are linked to separate intracellular signaling
pathways. The TNFR1 surface receptor is linked to a cell death pathway, while TNFR2 islinked to a cell proliferation pathway. Consequently, TNF-related signaling may exert both
tumor promotion and suppression [12]. While TNFR1 has been widely studied with respect
to the TME, TNFR2 has received scant attention. One possible reason is that TNFR1 isdiffusely expressed on mammalian cells, so it was identified much earlier than TNFR2 [13].
Yet, as more knowledge emerges, it has become apparent that TNFR2 is overexpressed inmany cancers and in other cells of the TME, while it is rarely expressed in normal tissue.
Therefore, a greater understanding of this axis may yield clinical benefits for cancer patients.
In this review, we outline recent developments in the pathophysiology of TNFR2 signaling
in the TME and the benefits of targeted therapies that block TNFR2 signaling.
2. The Structure and Function of TNF/TNFR
In 1975, Carswell and his colleagues initially identified TNF as a cytokine that leads tothe necrosis of tumor tissue [14]. Indeed, TNF stands for tumor necrosis factor. However,
TNF has become recognized as a multi-functional molecule that controls tissue homeostasis
in various and paradoxical ways, including proinflammation, regeneration, tissue necrosis,
and cell proliferation [15,16]. This 34 kDa cytokine can be both a cytokine and found in theserum. TNF is also a type II transmembrane trimeric protein and a representative memberof the TNF superfamily (TNFSF), consisting of 19 ligands and 29 receptors in humans [17].
TNF is composed of trimetric molecules and works by being membrane-attached (mTNF)
or a solvable form (sTNF). The latter is cleaved from the stalk region on cellular surfaces by
the metalloprotease TNFα-converting enzyme (TACE) (Figure 1) [18]. Activated signaling
through TNFR2 by mTNF binding is usually more effective than stimulation by sTNF,
which can be triggered only by an intact trimetric ligand [17]. Additionally, while mTNF
activates both TNFR1 and TNFR2, sTNF activates only TNFR1 because sTNF binding
to TNFR2 lacks secondary clustering of initial trimetric TNF-TNFR2 complex [19]. Tight
trimerization is required for downstream signaling. TNF is produced by various cells,
including activated macrophages, Teffs, fibroblasts, and keratinocytes, while production
is suppressed by some immunomodulatory cells, such as Tregs and mesenchymal stem
cells [20,21]. Lymphotoxin alpha (LTα or TNFβ), another soluble ligand belonging to
TNFSF, and progranulin (PGRN), may also interact with these two receptors [22,23].
TNF receptor super family (TNFRSF), a member of type 1 transmembrane receptors, has co-evolved together with TNFSF, acquiring diversity by genetic duplication ortransposition [24]. The TNFRSF consists of the ectodomain, transmembrane domain, andintracellular domain, usually from either a parallel or antiparallel dimer [25]. While the parallel dimer exposes the ligand binding site that activates the downstream signal pathway,
the antiparallel dimer buries the ligand binding site to block activation when stimulated byan antagonistic antibody [17].

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Figure 1. The roles of TNFR2 signaling within cancer cells. Many types of cancer cells express
tumor necrosis factor receptor 2 (TNFR2) on their surface as well as trimeric membrane TNF, whose
expression is also upregulated via TNFR2 signaling. After TNF binding, TRAF1/2/3 and clAP1/2
located on the intracellular domain of TNFR2 are clustered, activating further downstream pathways
consisting of phosphatidyl inositol 3 kinase (PI3K)/AKT, mitogen-activated protein kinase (e.g.,
extracellular signal-regulated kinase, ERK; c-jun N-terminal kinase, JNK), nuclear factor-κ B (NFκB),
and signal transducer and activator of transcription 3 (STAT3). The activated TNFR2 signal facilitates
proliferation and migration of the cancer cell as well as autocrine signaling by macrophage chemotaxis
protein-1 (MCP-1) via the C-C chemokine receptor type 2 (CCR2), which drives the cancer cell to
invade or migrate. Malignant transformation and tumorigenesis are induced via the signal; TNF
or progranulin (PGRN) binding to TNFR2 activates IκBα and NFκB or PI3K/AKT/mammaliantarget of rapamycin (mTOR) and cMyc. The colitis-associated carcinogenesis is also developed by
NFκB activation. In addition, TNF and TNFR2 are cleaved in their stem regions by TNFα-converting
enzyme (TACE), resulting in their soluble forms, sTNF and sTNFR2.

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TNFR1 (TNFRSF1A or CD120a) is classified as a death receptor group in the TNFRSF,
which includes the death domain (DD) in their intracellular portion [17]. Activated TNFR1
signaling can aggregate DD and lead to recruitment of TNFR1-associated death domain
protein, Fas-associated death domain, receptor-interacting protein and mitogen-activatedprotein kinase (MAPK) activating death domain. This results in the downstream caspase-8
activation and typically cell death [26]. Simultaneously, the activated signaling can induce
the enlargement of cell death-independent proinflammatory pathways activating nuclear
factor-κ B (NFκB) and MAPK [27]. Thus, TNF signaling via TNFR1 plays crucial roles in
the cellular apoptotic signaling mechanism. Additionally, the TNFR1 receptor is expressedon all cells in both the human immune lymphoid cells and parenchymal cells.
It is important to distinguish TNFR1 from the TNFR2 receptor—although their names
are very similar and they both can bind TNF, their functions are very different. TNFR2 (TNFRSF1B or CD120b) is a representative of the TNFR-associated factor (TRAF)-interacting
receptor subgroup of the TNFRSF and is an inducible receptor and lymphoid-only expressing receptor. Additionally, TNFR2 is inducible unlike the near constant expression of
TNFR1 on all mammalian cells [17]. Stimulation via mTNF can suppress inflammation by
downregulating the NFκB pathway, resulting in cell survival and proliferation [17]. After
TNF binding, TNFR2 recruits adaptor proteins such as TRAF and clAP, which translocate
activated NFκB and c-Jun N-terminal kinase (JNK) into the nucleus [28,29]. In addition,
when TNFR2 trimerizes by mTNF binding, this tightly clustered complex becomes susceptible to TACE and cleaved into the soluble form (sTNFR2) [30]. Soluble forms of TNFRSF
usually work similar to the decoy receptor, which lack an intracellular domain and act as
TNFSF ligand inhibitors [17]. Importantly, the evaluation of serum sTNFR2 level has been
attractive in various cancer studies, owing to the long-term stability and sensitivity and is asign of the marked agonism of this pathway [31].
The most determinative differences between TNFR1 and TNFR2 are: (1) the absenceof a DD in TNFR2, (2) TNFR2’s linkage to the proliferative NFkB pathway, (3) ubiquitous
expression of TNFR1 on all tissues, and (4) ineducability of TNFR2 at sites in inflammation and overall restricted cellular expression. Accordingly, we may roughly understand
TNFR1-associated signaling as cellular death, while TNFR2-associated signaling as cellular
proliferation/regeneration. Furthermore, the bodily distributions of these receptors arevery different. While TNFR1 is normally expressed by almost all mammalian cells, TNFR2’s
expression is limited to lymphocytes, myeloid, and endothelial cells [32]. The greater localization of TNFR2 expression is expected to be a therapeutic advantage in terms of fewer
adverse events. Additionally, TNFR1 typically has constitutive expression; whereas TNFR2
expression is inducible by various cytokines. This also explains the differential expression
and abuse of this receptor in the TME.
3. TNFR2 Expression Is Upregulated in Cancer Cells; TNFR2 Signaling
Accelerates Proliferation
Human and murine TME features aberrantly high TNFR2 receptor expression (Table 1).
The reason for overexpression of the TNFR2 protein for preferential cell growth is obvious
but the cause(s) and mechanisms behind upregulation are diverse. For example, in humancolorectal cancer, the overexpression of TNFR2 is driven via activated STAT3 through IL-6
and TNF induction (Figure 1) [33]. Activated TNFR2 signaling in malignant cells includes
breast, lung, colorectal, renal, cervical, skin cancer, melanoma, and malignant lymphoma.
The activation of phosphatidyl inositol 3 kinase (PI3K)/AKT, MAPK, STAT3, and NFκB
promote the downstream signaling pathway in these cancer cells [22,23,33–39]. As anotherexample, in vitro overexpression of TNFR2 in colorectal cancer cells increases their proliferation by 2-fold; conversely, knockdown of TNFR2 leads to significant downregulation of
proliferation [33]. Similarly, TNFR2/p75 knockout mice in vivo exhibit more than 2-folddecrease in growth of Lewis lung carcinoma compared with wild type mice [40]. Thus,
TNFR2 is overexpressed in a variety of cancers and its activation promotes tumor growthand progression.

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Table 1. Summary of TNFR2 Signaling-Related Mechanisms in the Tumor Microenvironment.
Malignant Disease

Human (C, Cancer Cell Line; P, Primary
Cancer Cell)

Thyroid(P) Non-coding RNA H19 inhibits lymphatic
metastasis via TNFR2 downregulation [41].

Breast(P) TNFR2 is more highly expressed on
TAMs [42].
(C) TNFR2 signaling contributes toAdriamycin resistance by repairing DNA
damage via AKT signaling [43].

(M) TNFR2 signaling promotes cancer cellproliferation via activated p42/p44 MAPK [34].
(M) TNFR2+ Tregs have greater suppressivefunction over TNFR2+ Teffs [44].
(M) Tumor growth and angiogenesis are driven
via TNFR2 signaling [36].
(M) TNFR2 signaling facilitates
MDSCs-mediated immunosuppression andmetastasis [45].
(M) TNFR2+ Tregs have greater suppressivefunction, which overcomes TNFR2+ Teffs [44].

Lung

StomachAnimal (M, Mouse; R, Rat)

(P) CAFs-derived IL-33 released via
TNF/TNFR2 signaling promotes gastriccancer metastasis [46].
(R) TNFR2 signaling promotes recurrence of
HCC via STAT3 activation [47].

Liver

Gallbladder(C) MMP secretion and increased
invasiveness are driven via TNF/TNFR2
signaling [48].

Colorectal(C) IL-6 and TNF induce TNFR2 expression
via STAT3 activation [33].
(C) TNFR2 signaling promotes tumor growth
via PI3K/AKT pathway [35].
(C) PGRN promotes cancer cell proliferation
and angiogenesis via TNFR2/AKT pathway[23].
(C) PGRN/TNFR2 drives proliferation andmigration of CAFs via AKT or ERK pathway[49].

Kidney(P) and (C) TNFR2 signaling increases tumor
progression via Etk-VEGFR2 cross talk [37].
(P) TNFR2 signaling drives CSC’s
proliferation and increases their sensitivity tocytotoxicity [51].

Ovary(P) TNFR2+ Tregs in malignant ascites are
more suppressive than those in blood [52].

Uterus(C) PGRN/TNFR2 signaling is needed for
malignant transformation via mTOR
signaling [38].

(M) TNFR2 signaling facilitates
MDSC-mediated immunosuppression andliver metastasis [45].
(M) TNFR2 signaling enhances Treg’s
suppressive function [50].

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Table 1. Cont.
Malignant Disease

Human (C, Cancer Cell Line; P, Primary
Cancer Cell)

Animal (M, Mouse; R, Rat)

Skin/Lymphoma(C) LTα/TNFR2 signaling promotes tumor
growth and angiogenesis in cutaneous
lymphoma [22].

(M) Both TNFR1 and TNFR2 are necessary for
optimal TNF signaling during skin cancer
development [39].
(M) Tumor growth and angiogenesis are driven
via TNFR2 signaling [36].
(M) TNFR2 signaling expands Tregs, driving
lung metastasis [53].

Melanoma

Leukemia/Myeloma(C) Transendothelial migration is driven via
TNFR2 signaling and upregulated MCP-1
secretion [54].
(P) Compared to TNFR- Tregs, TNFR2+ Tregs
express higher CXCL4, which facilitates
migration in AML [55].
TNFR2, tumor necrosis factor receptor 2; TAMs, tumor-associated macrophages; CAFs, cancer-associated fibroblasts; EMT, endothelial mesenchymal transition; MMP, matrix metalloprotease; STAT3, signal transducer
and activator of transcription 3; PI3K, phosphatidyl inositol 3 kinase; PGRN, progranulin; ERK, extracellularsignal-regulated kinase; VEGFR2, vascular endothelial growth factor 2; CSCs, cancer stem cells; Tregs, regulatory
T cells; mTOR, mammalian target of rapamycin; LTα, lymphotoxin α; MCP-1, macrophage chemotaxis protein-1;
CXCL4, C-X-C chemokine receptor type 4; AML, acute myeloid leukemia; MAPK, mitogen-activated protein
kinase; Teffs, effector T cells; MDSCs, myeloid-derived suppressor cells; HCC, hepatocellular carcinoma.

4. Activated TNF/TNFR2 Signaling Is Associated with Proliferation and Recruitmentof Stromal Cells in the TME
Stromal cells in the TME such as macrophages and fibroblasts can also express TNFR2
on their surface [56]. These cells undergo “malignant education”, which is a cellular
activating process resulting in a switch from their normal into malignant phenotypes:
tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), respectively (Figure 2) [49,57]. TAMs and CAFs are implicated in tumor progression by directly
driving cancer cell proliferation and/or by enhancing angiogenesis and modulating immune cells [58]. Although the decoy receptor 3 is common as the TAMs promoter among
the TNFRSF [59], TNFR2 signaling also seems to play an important role in the TME [49,60].
TAMs harvested from patients with triple negative breast cancer (TNBC) expressed higher
TNFR2 as compared with those from non-TNBC patients [42]. Interestingly, the degree of
TNFR2 expression in TAMs is strongly correlated with the chemokine C-C motif ligand
5 (CCL5) expression, which facilitates the recruitment of TAMs in the TME [42]. TNFR2 isdominantly expressed in CAFs as well, and the proliferation and migration can be accelerated through activated signaling [49,61]. Thus, these cancer-associated stromal cells arepivotal modulators of tumorigenesis.

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Figure 2. The interaction between cancer cells and stromal cells of the TME via TNFR2 signaling.
TNFR2+ macrophages and fibroblasts are influenced by receptor binding of TNF, resulting in a transformation to tumor-associated macrophages (TAMs) and cancer-associated fibroblast (CAFs). Thesecells can accelerate tumor proliferation via phosphatidyl inositol 3 kinase (PI3K)/AKT activationand migration by autocrine or paracrine of chemokine C-C motif ligand 5 (CCL5)/CCR5. Malignant
stromal cells are promoters of tumor burden and metastasis through angiogenesis and immunomodulation. In addition, sTNFR2 released from TAMs directly drives cancer invasion. TNFR2+ regulatory
T cells (Tregs), which are a minor subpopulation in the normal state, accelerate proliferation andmigration of malignant tissue via TNFR2 signaling and contribute to tumor growth by strongly suppressing effector T cells (Teffs). The degree of TNFR2 expression is associated with the suppressing
function, expression of C-X-C motif chemokine receptor 4 (CXCR4), migration of Tregs, and predicts
the prognosis of patients with malignant disease. Meanwhile, TNFR2- Tregs, which usually express
programmed cell death protein 1 (PD-1) and cytotoxic T lymphocytes-associated protein 4 (CTLA-4),
are considered to play a critical role in immunomodulation in the normal state.

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5. Cancer Invasion and Metastasis Are Facilitated by TNFR2 Signaling
For widely metastatic cancers, TNFR2 may play a role in promoting the metastatic
process and its progression. In metastatic disease, cancer cells from the primary lesion
invade the local parenchymal tissue and then intravasate into either blood or lymphatic
vessels to enter the circulation [62]. Following this dissemination step, the cancer cells aretrapped in the microvessels at distant organs, where they extravasate from the circulation
to enter the parenchymal tissue, resulting in the colonization at the site [62]. To date,some studies have shown the association of TNFR2 signaling with the metastatic process(Figure 3). In murine colon and lung cancer models, hepatic colonization and growth ofthe metastatic lesion are promoted via TNFR2-activated signaling [45]. Conversely, theinduced genetic loss of TNFR2 in the mice decreases metastasis of melanoma within thelung [53].
There is support for activated TNFR2 signaling driving the invasion and migrationof cancer cells. In multiple myeloma patients, for example, transendothelial migration
through the bone marrow endothelial cell monolayer is accelerated by the autocrine action of monocyte chemoattract protein-1 (MCP-1) following activated TNFR2 signaling(Figure 2) [54]. In addition, sTNFR2 derived from TAMs directly promotes the invasivenessof TNBC [42].
The neogenesis of blood or lymphatic vessels in the TME can be facilitated by activated TNFR2 signaling. In renal cell carcinoma, greater TNFR2 expression on vascularendothelial cells (VECs) is observed with higher malignant grades [37]. The activated signaling promotes angiogenesis by upregulating vascular endothelial growth factor (VEGF),
hepatocyte growth factor (HGF) and platelet derived growth factor (PDGF) [22,23,36,63].
Conversely, vascular regeneration is impaired in TNFR2−/− mice [36]. The neovascularity
may promote metastatic disease but also perhaps tumor nutrient resources [64]. TNFR2
downregulation in human papillary thyroid carcinoma occurs using H19 long non-coding
RNAs [41]. H19 inhibits TNFR2 expression and may be used as a potential diagnostic tooland therapeutic target for this cancer.
TNFR2 signaling also may play a role in driving epithelial mesenchymal transformation (EMT) and releasing matrix metalloprotease (MMP) during the tumor infiltration
process. EMT is an acquired mesenchymal phenotypic alteration and essential biologicalprocess for cancer cells to migrate or intravasate [65]. It is known that EMT is promoted
via IL-1β, transforming growth factor β, and TNFα signaling [65,66]. Of great interest,
CAFs-derived IL-33 can induce EMT of gastric cancer cells, which is facilitated via activated
TNFR2 signaling [46]. On the other hand, MMP is an important tool for tumor cells to
invade into the extracellular matrix by destroying connective tissue. TNF released by cancer
cells or TAMs is related to MMP [67]. Tanimura and colleagues showed MMP secretion via
activated TNFR2 signaling following cancer invasion with the cholangiocarcinoma cell line
which displays only TNFR2 [48].
TNFR2 signaling exerts an important regulatory role in distant metastatic sites both
before and after colonization. Ham and colleagues demonstrated that signal activation
could recruit myeloid-derived suppressor cells (MDSCs) into the distant liver in colorectal
cancer. This action suppresses anti-cancer immunity and stabilizes the colonization [45].
Curiously, the recruitment occurred even before the colonization of the metastatic cancer cells [45]. Several tumor-derived cytokines and molecules play a necessary role inmobilizing tumor-supportive host cells from the distant anatomical sites, known as thepremetastatic niche [62]. These host cells are rendered pro-tumorigenic even prior to theirmobilization, indicating that the MDSCs induced via TNFR2 signaling may contribute to
the establishment of the premetastatic niche.

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Figure 3. Emerging mechanisms of cancer metastasis via TNFR2 signaling. Cancer cells can leavemalignant tissue and migrate into the extracellular matrix (ECM) by the endothelial mesenchymaltransition (EMT), a process driven via IL-33 released from cancer-associated fibroblast (CAF) activated
via TNF/TNFR2 signaling. By autocrine action of macrophage chemotaxis protein-1 (MCP-1) andsecretion of matrix metalloprotease (MMP), the cancer cell invades into the blood capillary newly
generated by angiogenesis. The proliferation of vascular endothelial cell (VEC) is also accelerated via
TNFR2 signaling in addition to the interaction of vascular endothelial growth factor (VEGF)/VEGFR2,
platelet derived growth factor (PDGF)/PDGFR, and hepatocyte growth factor (HGF)/cMet. Before
and after the metastasis, TNFR2+ regulatory T cells (Tregs) and myeloid-derived suppressor cells(MDSCs) are recruited to the site from bone marrow with upregulated C-X-C motif chemokine
receptor 4 (CXCR4) expression after the binding of TNF. These immunosuppressor cells can establish
premetastatic niches by downregulating effector T cells (Teffs).

6. TNFR Signaling Controls Immunomodulating Cells, Tregs, and MDSCs
Limiting host anti-cancer immunity is important for malignant progression. The
immunomodulating cells with this potential are also essential components of the TME.
Both TME Tregs and MDSCs rely on the TNFR2 signaling pathway.

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Increased numbers of Tregs are found in the TME, even in the peripheral tissue ofcancer patients, and Tregs play a master role in the suppression of anti-tumor immunity(Figure 2) [50,68]. Owing to the highly suppressive potential of Tregs, Teffs are not potent
enough for killing the tumor. TNFR2 signaling propels the differentiation of Tregs in thethymus, and their expansion and stabilization through epigenetic mechanisms [69–71].
Herein, significant TNFR2 expression is found in the tumor-infiltrating Tregs, and the activated TNFR2 signaling facilitates the proliferation and suppressive function via PI3K/AKT
and/or NFκB activation in various cancers. Tregs are known to promote diverse cancers
such as breast cancer, lung cancer, colorectal cancer, ovarian cancer, malignant lymphoma,melanoma, and leukemia [25,44,50,52,53,55,72–75]. Although Tregs expressing TNFR2 areonly a subpopulation of total Tregs in the human TME, the degree of TNFR2 expression is
tenfold higher than that of TNFR1 in human blood, and the TNFR2+ Tregs are equipped tobe the most suppressive subset against Teffs [52,70,76]. In TNFR2-deficient mice, more Teff
numbers are found, and thus, tumor control is potentiated [77]. In humans, TNFR2+ Tregs
in acute myeloid leukemia (AML) patients also display higher levels of C-X-C chemokine
receptor type 4 (CXCR4) expression, which is positively correlated with TNFR2 expression
levels on Tregs [55]. This suggests that the CXCR4/CXCL12 axis may play a role in theaccumulation of TNFR2+ Tregs in the TME. Although TNFR2 is expressed on Teffs as well,much higher levels are found in the Tregs of the TME [44,52].
Activated TNFR2 signaling controls the proliferation and suppressive activity of
MDSCs [78]. MDSCs are often found in peripheral tissues and result in the tumor growthboth at primary and distant metastatic sites [45,79]. A recent finding is that MDSCs
accumulate and survive in the TME via activated TNFR2 signaling [80]. For instance, whilemTNF-expressed cancer cells recruit these MDSCs into the TME, TNFR2 deficiency on
MDSCs also impairs CXCR4 expression [81]. Thus, TNFR2 is dominantly expressed onthese immunomodulatory cells in the TME.
Other immune cells such as dendritic cells and natural killer cells could be also affectedby the TNF/TNFR2 signaling pathway [82]. However, it is still unclear how they work in
TME; thus, future investigations are desired in this field.
7. Oncogenesis Can Be Driven by TNFR2 Signaling
Over the past decades, evidence of oncogenesis induced by TNFR2 signaling has
accumulated. This should perhaps not be surprising since any receptor that preferentiallypromotes growth would directly favor oncogenesis. Initially, Onizawa and his grouprevealed that specific upregulation of TNFR2 was observed in inflamed intestinal epithelial
cells and that TNFR2-mediated epithelial NFκB activation correlated with colitis-associatedcarcinogenesis (Figure 1) [83]. Additionally, the investigators extended their observation
by demonstrating that the myosin light chain kinase expression was induced via TNFR2signaling-mediated tumorigenesis [84].
Upregulated TNFR2 signaling in colonic epithelial cells leads to colitis-associatedcarcinogenesis [83]. TNFR2-induced activation of transcription factor-associated protein
induced the malignant transformation of hepatic progenitor cells and liver tumorigenesis,
and the recurrence of hepatocellular carcinoma via STAT3 activation [47,85]. Other studies
reveal another mechanism using murine brain cancer models: activated mammalian target of rapamycin (mTOR) signaling pathway followed by PGRN binding to TNFR2 was
identified in malignant cervical cancer cells, whereas the inhibition of mTOR suppressedmalignant transformation and tumor growth [38]. He and colleagues reported the tumorigenesis by the loss of TNFR2 allele using a murine breast cancer model [86]. These studies
suggest that the TNFR signaling pathway may promote oncogenesis.
8. TNFR2 Signaling in Cancer Stem Cells: Still Controversial
Cancer stem cells (CSCs) seed many if not most tumors, reigning on the top of thecancer hierarchy [87]. CSCs also can play a role in anti-cancer drug resistance [88]. Some

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survival, and differentiation in non-malignant tissues [89,90].
TNFR2 signaling may negatively modulate CSCs in acute myeloid leukemia. Some
research reveals that TNFR2 gene expression was significantly decreased in the leukemia
samples and correlated with the inactivity of CSCs as compared with those from thenon-leukemia control samples [91]. Conversely, Al-Lamki and colleagues showed thatthe proliferation of CD133+ CSCs in renal clear cell carcinoma was promoted by TNFR2
signaling [51,92]. Importantly, while most types of CSCs strictly limit the self-proliferation
and bear the resistance to various chemotherapies, activated CD133+ CSCs via TNFR2
signaling could drive cellular division and sensitivity to chemotherapy drugs such as
cyclophosphamide [51]. The relationship between the signaling and CSCs is only beginningto emerge.
9. TNFR2 Signaling Can Contribute to Chemotherapy ResistanceSome investigators have proposed that chemotherapy drug resistance is associatedwith TNFR2 signaling by cancer cells. The initial report was a negative correlation. Zhang
and colleagues reported that apoptotic response of colorectal cancer cells to 5-fluorouracil
was negatively mediated by induced TNFR2 [93]. In contrast, later reports indicated apositive association with TNFR2 signaling. Sprowl and coworkers showed that TNFR2
expression was significantly upregulated in Adriamycin-resistant human breast cancer
cells [94]. In addition, Yang and colleagues also demonstrated the underlying mechanism
by repairing DNA damage in cancer cells via an activated TNFR2/AKT pathway [43].
In metastatic melanoma, accumulating evidence suggests a TNFR2 escape mechanism
after failed checkpoint inhibitor therapy. A detailed single cell mRNA sequencing study forboth failed CTLA-4 and PD1 human cancer identified signature mRNAs indicative of Tregs(Foxp3) with upregulation. Additionally, this study found upregulated TNFR2, also known
as the TNFRSF1B gene [95]. Similar data confirmed this is the setting of anti-PD1 therapy
with pembrolizumab, even after one cycle of therapy. Tregs were abundant in the TME ofhumans with metastatic melanoma and retained their immunosuppressive phenotype andfunctionality following anti-PD-1 [96]. Epigenetic, transcriptomic, and proteomic analysis
of Tregs after immune checkpoint therapies implicated TNFR2 signaling as a possible
driver of CD8+ T cell suppression. TNFR2 was preferentially expressed by Tregs in the
TME of humans with advanced disease. In parallel mouse studies, dual blockade of TNFR2
and PD-1 led to potent CD8+ T cell expansion in two mouse tumor models, and restored
sensitivity to immune checkpoint inhibitors [97]. This was buttressed by a study showingthat TNFR2 antagonistic antibodies administered to mice prior to checkpoint inhibitors
led to effective Treg depletion and enhanced tumor survival rates in two models of coloncancer [98].
10. Anti-Cancer Therapy Targeting TNFR2; TNFR2+ Treg Depletion Leads to
Beneficial Outcomes
Anti-TNFR2-based therapy holds promise for cancer treatment. TNFR2 is an attractive target because it is directly upregulated as an oncoprotein on cancer cells andassociated components including TAMs, CAFs, Tregs, and MDSCs that facilitate tumor
dissemination [25,99]. While several drugs modulating TNFR signaling are available for a
few autoimmune diseases, TNFR2-specific targeted therapies are still challenging in thefield of oncology [25]. However, reducing TNFR2 expression in malignant disease seems
to show promise in the clinical setting, according to two recent trials. One clinical trial
targeting TNFR2+ Tregs was performed on AML patients who were ineligible for intensive
chemotherapy. Fourteen patients received combined therapy with azacytidine, the DNA
methyltransferase inhibitor, and panobinostat, the histone deacetylase inhibitor [55]. The
patients, prior to treatment, showed a significantly higher number of TNFR2+ Tregs, which
also possessed high migration potential as compared with those from healthy donors [55].
After treatment, patients showed a significantly decreased number of TNFR2+ Tregs both

Cells 2022, 11, 1952

12 of 22in peripheral blood and bone marrow, while TNFR2- Tregs were not lowered. A reductionin TNFR2+ Tregs was accompanied by increased interferon-γ and IL-2 released from Teffs
in the bone marrow, and prolonged disease free survival [55]. In another clinical trial, thesame group reported that combined therapy with azacytidine and lenalidomide, the immunomodulatory drug, also contributed to a reduced number of TNFR2+ Tregs and TNFR2
expression on T cells, resulting in the durable clinical remission in AML patients [100]. Curiously, this study found that lenalidomide can independently reduce TNFR2+ Tregs, which
was enhanced by adding azacytidine in vitro [100]. The epigenetics-modulating drugs
have already been tried in myelodysplastic syndrome and multiple myeloma treatment;
however, the adverse effects, such as a bone marrow suppression, limit therapy [101].
The expression of TNFR2 on Tregs is, thus, a mechanism for hampered cancer elimination. Many early mouse and human studies confirmed Tregs with this protein marker as
the most functionally potent subtype of Treg cells [52,76,102]. It was in cutaneous T cell
lymphoma that data started to accumulate that TNFR2 could also act as an oncogene andbe directly expressed on tumor cells for preferential growth [103].
11. Clinical Evaluation of TNFR2: Higher Levels of TNFR2 Expression Reflect Poor
Prognosis and Risk of Cancer Development
Abundant and aberrant expression of TNFR2 on cancer cells and Tregs of the TME has
led to the concept that this receptor could be used for diagnosis, prognosis, and deciding atreatment plan. Human studies have focused on sTNFR2 in peripheral blood, in TNFR2 incancer cells and Tregs, and in metastatic tissue (Table 2).
Table 2. Summary of Human Studies of TNFR2 in Malignant Diseases.
Malignant Disease

Peripheral Blood

Brain

Plasma sTNFR2 levels are significantly
higher in patients with recurrent GBM
than healthy donors [104].

Head and Neck

Decreased plasma sTNFR2 levels
reflect treatment efficacy at 3 months
post-IMRT [105].
TNFR2 expression is positively
correlated with increased tumor size,
advanced clinical stage, poordifferentiation, shorter OS, DFS [106],
and tumor growth andangiogenesis [107].
TNFR2 expression on TAMs is
upregulated in TNBC compared to
non-TNBC [42].

BreastLung

Primary Region

Tregs express higher levels of TNFR2
than Teffs, and higher TNFR2+ Tregs
are related to poor prognosis [75].
TNFR2 expression is positively related
to an advanced clinical stage, poordifferentiation, and poor OS [109].

Esophagus

Pancreas

Higher plasma sTNFR2 levels are
marginally associated with a higher
risk of pancreatic cancer [110].

Colorectal

A higher plasma TNFR2 level isrelated to shorter OS in patients withmetastasis after second-line
chemotherapy [111].
Pre-diagnosis higher plasma sTNFR2
levels are associated with shorter
OS [112].

Metastatic Region

Increased TNFR2+ B cells in metastatic
LN are related to good prognosis [108].

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13 of 22

Table 2. Cont.
Malignant Disease

Peripheral Blood

Ovary

Uterus

Peripheral TNFR2+ Tregs andcirculating sTNFR2 are increased, andthe percentage of TNFR2+ Tregs isinversely correlated with clinicalstage [114].
Higher plasma sTNFR2 levels areassociated with advanced clinicalstage [115] and cancer risk [116].

Metastatic Region
TNFR2+ Tregs are abundantly present
in malignant ascites and show more
suppressive characteristics than thosein the peripheral blood [52].

Tumor-infiltrating TNFR2+ Tregs are
significantly increased [114].

Both total and TNFR2+ Treg
populations are significantly higher in
AML patients as compared withhealthy donors [55].
TNFR2+ Tregs have higher levels of
CTLA-4, Ki67 and CXCR4 as
compared with TNFR2- Tregs in AML
patients [55].
Higher TNFR2+ Tregs and lower
TNFR2+ Teffs are observed in AML
patients as compared with healthy
donors, and increased TNFR2+ Tregs
are related to cancer relapse [100].

Leukemia/Melanoma

Lymphoma

Primary Region
TNFR2 expression is higher in cancer
cells than in benign ovarian cells, andthe elevated expression reflects cancer
progression [113].

Higher plasma sTNFR2 levels are
related to higher NHL risk [117,118].
sTNFR2, soluble tissue necrosis factor receptor 2; GBM, glioblastoma; IMRT, intensity-modulated radiation
therapy; Tregs, suppressive T cells; Teffs, effector T cells; OS, overall survival; NHL, non-Hodgkin lymphoma;
DSF, disease free survival; TAMs, tumor-associated macrophage; TNBC, triple negative breast cancer; CTAL-4,
anti-cytotoxic T lymphocytes-associated protein 4; CXCR4, C-X-C chemokine receptor type 4; AML, acute myeloid
leukemia; LN, lymph node.

Two overarching points emerge from Table 2. First, the degree of TNFR2 expression in malignant cells is positively associated with disease progression and prognosis.
Higher TNFR2 expression is correlated with the highest risk of tumor growth in ovarian,esophageal, and breast cancer; moreover, TNFR2 gene expression in breast cancer cells
also indicates a higher grade of malignancy [42,106,107,109,113]. Although higher levelsof a protein are hard to quantify, two studies—one in human lung cell carcinoma and one
in cutaneous T cell lymphoma—show this overexpression was actually logs of overexpression as measured by flow cytometry, an impressive signal [74]. Similarly, the numberof TNFR2+ Tregs in malignant tissue indicates tumor progression. Compared to othercommonly studied tumor markers such as OX40 and 4-1BB, TNFR2 overexpression was
still 10× greater. The increased TNFR2 expression on Tregs is associated with lymphatic
invasion, distant metastasis, and more advanced clinical stage in lung cancer. Furthermore,
the increased degree of the TNFR2+ T cell populations among CD4+ T cells reflects relapse
in AML [55,75,100].
Second, measuring sTNFR2 level in the serum is also useful. When there is augmented
TNFR2 agonism, the receptor is shed. Elevated levels of sTNFR2 are a surrogate for extreme
TNFR2 agonism [69]. For example, higher levels of sTNFR2 imply poor prognosis in colorectal, cervical cancer, and non-Hodgkin lymphoma [111,112,114,117]. A stage-dependent
release of sTNFR2 into the bloodstream occurs in the endometrial cancer and an elevatedsTNFR2 level indicates recurrence in glioblastoma [104,115]. Meanwhile, it reflects thetherapeutic effect of radiotherapy in the head and neck cancer [105]. Importantly, thehigher circulating sTNFR2 level can be a risk factor for cancer generation. Prude andhis group analyzed the baseline serum levels of 67 immune and inflammation markers
from 301 patients with non-Hodgkin lymphoma diagnosed over 5 years after the bloodcollection and 301 control patients within another disease screening trial, revealing thatthe higher sTNFR2 levels were strongly associated with lymphoma risk [118]. The relationship between elevated sTNFR2 level and cancer development also has been reported

Cells 2022, 11, 1952

14 of 22in endometrial and pancreatic cancer [110,116]. Thus, the serum sTNFR2 level serves as anovel biomarker that reflects TNFR2 activity, further the state of malignant disease, andalso the drive of severe agonism of this receptor for preferential tumor growth.
12. Discussion
Specifically targeting TNFR2 is believed to be an ideal cancer therapy because TNFR2
expression is limited to minor subpopulations of normal cells, while its expression is enriched in the TME, including Tregs. The creation of TNFR2 antibodies with the ability to killrapidly proliferating cells presents a rare example of a TME-specific cancer therapy [73,119].
Therefore, the targeting of TNFR2+ malignant cells with TNFR2-specific antagonistic antibodies may not only control cancer growth but also minimize adverse effects [25]. Here,
we describe the current challenges to creating such potential drugs and discuss future
perspectives on targeting TNFR2 therapy for malignant diseases. This discussion is basedon Table 3, which is a summary of cell culture studies, animal models, and human studies
of TNFR2-targeted antibody therapy for malignant diseases.
Table 3. Summary of studies using TNFR2-targeted therapy for malignant diseases.
Author(Year) [Ref.]

Drug

Specimen/Tissue

Mechanism/Outcomes••
Govindaraj, C. et al.
(2014) [55]Combination ofazacytidine andpanobinostat

NFR2+ Tregs in AML
patients•

Govindaraj, C. et al.
(2014) [100]Torrey, H. et al.
(2017) [73]Combination ofazacytidine andlenalidomide

Anti-TNFR2 antibody•

Lenalidomide reduced TNFR2+ Tregs andaugmented Teffs, which was enhanced byazacytidine, resulting in prolonged
clinical remission.

•

The antibody bound to identical TNFR2
regions and locks in receptor resting state
as antiparallel dimer form, which blocks
downstream signaling pathway.
Proliferation of Tregs was inhibited, while
Teffs were expanded.
In addition to TNFR2+ Tregs, TNFR2+
cancer cells were directly killed.

NFR2+ Tregs in clinically
remitted AML patients

Human Tregs in ovariancancer ascites
Human ovarian cancer
cell line•••

Nie, Y. et al.
(2018) [72]Torrey, H. et al.
(2019) [74]Combination ofanti-TNFR2 antibody
and anti-CD25 antibody

Anti-TNFR2 antibody

TNFR2+ Tregs in murine
colorectal and breast
cancer model
TNFR2+ cancer cells andTregs from patients withstage IV SS

The combination therapy might inhibit
DNA methyltransferase 1 and histone
deacetylase in AML patients.
The level of TNFR2+ Tregs in peripheral
blood and bone marrow of patients wasdecreased, while the population of
TNFR2- Tregs was not reduced.
Positive clinical response corresponded todecreased TNFR2+ Tregs and increased
IFN-γ+ Teffs in bone marrow before vs.
after treatment.

••

Remarkably decreased TNFR2+ Tregs andincreased IFN-γ+ Teffs infiltrated in thetumor.
Long term tumor-free survival was
improved in murine cancer models.
TNFR2+ SS tumor cells and Tregs weredose-dependently decreased by the
antibody, while beneficial and rapid
expansion of Teffs was observed.

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Table 3. Cont.
Author(Year) [Ref.]

Drug

Specimen/Tissue

Mechanism/Outcomes•

Tam, E. M. et al.
(2019) [120]Anti-TNFR2 antibody

Murine colorectal, breast,
fibroblast fibrosarcoma,
and B cell lymphoma
cell lines•

Murine TNFR2+ Tregs••
Human colorectal cancer,
lymphoma andleukemia cell line
Yang, M. et al.
(2020) [119]•Anti-TNFR2 antibody•
Human Tregs and Teffs
in the blood•

Case, K. et al. (2020) [98]Combination ofanti-TNFR2 antibody
and anti-PD-1 antibody

Murine colorectal cancer•

The antibody bound to outside of the
TNF-binding region in TNFR2 andshowed anti-tumor activity as
Fc-dependent agonism of Teffs.
Anti-TNFR2 treatment is mediated byTeffs and NK cells, downregulates TNFR2
on T cells, which leads to Teffs expansion
and improved functionality.
The antibody did not deplete Tregs andnever caused spontaneous immune cell
activation.
The anti-TNFR2 antibody showed
specific killing of TNFR2-expressing
tumor cells and Tregs, but sparing Teffs,
which proliferated.
The IgG2 isotype of the antagonists
functioned better than the IgG1 isotype.
The mutations to its amino acid sequencestabilized the natural variability of the
IgG2 isotype’s hinge and improved
function.
The combination therapy had the greatest
efficacy by complete tumor regression
and elimination, and the next mosteffective therapy was anti-TNFR2 alone,whereas the least effective was anti-PD-1
alone.
The mode of action was by killing Tregs
and increasing Teffs.

TNFR2, tissue necrosis factor receptor 2; AML, acute myeloid leukemia; SS, Sézary syndrome; Tregs, regulatory T
cells; IFN-γ, interferon-gamma; Teffs, effector T cells; NK cells, natural killer cells.

Early development of antibodies against TNFR2 was fraught with limitations. The
antibodies worked in culture but did not work well in animal models. This was in largepart driven by the fact that in vitro they blocked apparent signaling, but in vivo a tiny bit of
TNF superfamily ligand uniformly caused their failure. After nearly 15 years of research, anovel type of true antagonistic antibodies was created [73,119]. Newly synthesized TNFR2
on rapidly proliferating cells was frozen as TNFR2 receptors into an anti-parallel dimer,
thus, blocking the TNF binding site. Additionally, these antibodies only killed rapidly
proliferating cells and left alone preformed TNFR2 timers, suggesting a more favorable
structural biology to prevent off-target effects on circulating non-proliferating Tregs [73].
These antibodies have been called “dominant” antagonistic antibodies due to their ability
to diminish human ovarian cancer cells in the ascites and cutaneous T cell lymphoma, evenunder TNF induction. The other type of antibody is called “recessive,” because it cannot
overcome a tumor burden with the additional TNF [73,74]. The dominant antibody also
inhibits Treg proliferation, especially strongly suppressive Tregs, in the TME only, resulting
in activated Teffs [25]. There are some mechanistic clues supporting their usefulness:
(1)

(2)

The dominant antibody targets a different site on the TNFR2 protein from the recessive antibody, resulting in stabilized TNFR2 in an antiparallel dimer formation andhexagonal lattice on the cellular surface, which blocks the activation of the signaling
pathway [17,73].
The dominant antibody does not require Fcγ receptor-mediated antibody binding [18,73].

Cells 2022, 11, 1952

16 of 22(3)
(4)

The dominant antibody inhibits the cleavage of the receptor from the cellular surface [73].
We also expect that this dominant anti-TNFR2 antibody may overcome the drug resistanceof CSCs with the expectation of achieving a complete cure in some malignant diseases.

For example, renal CSCs are not sensitive to chemotherapy until they proliferate
through activated TNF/TNFR2 signaling [51]. This indicates that the dominant anti-TNFR2
antibody may effectively remove such drug-resistant CSCs.
Anti-TNFR2 antibody dominance may optimize therapy in additional ways. Using thechimeric mutant constructs and a structural biological technique, the IgG2 isoform of theantibody effectively kills rapidly growing cancer cells and immunosuppressive Tregs withhyperexpressed TNFR2 [119]. This accomplishment demonstrates that the stabilization of
the antibody’s hinge and expansion of its arms may achieve the greatest efficacy as a cancer
immunotherapy [17]. Furthermore, our anti-TNFR2 antibody, when combined with an ICI,
effectively eliminates murine colorectal cancer [98]. Mice treated with the combination
therapy displayed significantly decreased Treg and increased Teff number in their TME [98].
Importantly, the anti-TNFR2 antibody alone also displayed significantly longer survivalin the treated mice as compared with the non-treated mice, suggesting that the antagonist
may be an alternative Treg inhibitor for patients with tolerance to the existing ICIs.
Other research groups have attempted to identify specific anti-TNFR2 antibodies
for cancer treatment. Nie and the coworkers combined their antagonistic anti-TNFR2
antibody, TR75-54.7, with an anti-CD25 antibody as therapy in murine colorectal and breast
cancer models; they found significantly fewer TNFR2+ Tregs and more infiltrating IFN-γ+
Teffs, and remarkably decreased tumors [72]. The rationale for the combined therapy was
to achieve synergy between the anti-TNFR2 antibody and an immune stimulant. Morerecently, Tam and colleagues developed another anti-TNFR2 antibody, Y2, which binds
to the receptor outside of the TNF-binding site and accelerates the anti-tumor activity as
Fc-dependent agonism of conventional T cells [120]. While Y2 facilitated the expansion
and function of Teffs and downregulated the TNFR2 expression on T cells, Tregs were notdepleted. Spontaneous immune cell activation never occurred in the healthy or tumorbearing mice [120]. These results indicate less toxicity in the cancer therapy using theanti-TNFR2 antibody. The reason of less autoimmune effects by targeting TNFR2 therapy
relative to ICIs may be intelligible by our proposed mechanism. Tregs expressing PD-1 andCTLA-4 act as critical immunomodulators, hence, severe side effects can be expected whentargeting these immune checkpoint molecules. Meanwhile, TNFR2+ Tregs belong to theminor subpopulation of potent Tregs in the normal condition but can highly infiltrate in
TME (Figure 2) [25].
As described above, a universal demethylation inhibitory drug such as azacytidine
contributes to improved outcomes in some blood cancers by downregulating TNFR2+
Tregs [55]. Thus, drugs in this class, which regulate by an epigenetic mechanism, may be apotential treatment for malignant disease. However, to our knowledge, no studies have thus
far directly investigated DNA methylation/demethylation of TNFR2 in malignant disease;it has not been uncovered whether the gene duplication and/or activating mutations occur
in the malignant tissue, and it is not known whether gene duplication and/or activation
occurs in the malignant tissue [121,122]. The epigenetic axis has shed light on the current
cancer research and the detailed mechanism including PD-1 and CTLA-4 [121].
In conclusion, the TNFR2 signaling pathway plays pivotal roles in cancer progression.
Targeted anti-TNFR2 therapy is known to deplete malignant cells and immunosuppressive Tregs, conferring both tumoricidal capacity. The evidence base developed thus far
suggests that that more detailed investigations of TNFR2 signaling in the TME are desirable and that specific targeting of TNFR2 in the TME is an exciting new strategy forcancer immunotherapy.

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Author Contributions: H.T.; writing—original draft preparation, H.T.; writing—review and editing,
G.Y.; supervision, D.L.F.; supervision, editing and writing. All authors have read and agreed to the
published version of the manuscript.
Funding: This study was supported by The Iacocca Foundation.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank to Lynne Murphy and Sheila A. Williams for supporting our researchand Miriam Davis for editing the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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