Leukemia
https://doi.org/10.1038/s41375-018-0292-9

ARTICLE
Immunotherapy

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2
antagonistic antibodies in advanced Sézary syndrome
H Torrey1 M Khodadoust2 L Tran1 D Baum1 A Defusco1 Y H Kim2 D L Faustman1
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Received: 25 April 2018 / Revised: 10 September 2018 / Accepted: 17 September 2018
© The Author(s) 2018. This article is published with open access

Abstract
Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphoma often refractory to treatment. SS is deﬁned as
adenopathy, erythroderma with high numbers of atypical T cells. This offers an opportunity for new interventions andperhaps antibody-based therapeutic by virtue of its high expression of the TNFR2 oncogene on the tumor cells and on Tregulatory cells (Tregs). Potent human-directed TNFR2 antagonistic antibodies have been created that preferentially target the
TNFR2 oncogene and tumor-inﬁltrating TNFR2+ Tregs. Here we test the therapeutic potential of TNFR2 antagonists on
freshly isolated lymphocytes from patients with Stage IVA SS and from healthy controls. SS patients were on a variety ofend-stage multi-drug therapies. Baseline burden Treg/T effector (Teff) ratios and the responsiveness of tumor and inﬁltrating
Tregs to TNFR2 antibody killing was studied. We show dose-escalating concentrations of a dominant TNFR2 antagonistic
antibody killed TNFR2+ SS tumor cells and thus restored CD26− subpopulations of lymphocyte cell numbers to normal.
The abundant TNFR2+ Tregs of SS subjects are also killed with TNFR2 antagonism. Beneﬁcial and rapid expansion of Teff
was observed. The combination of Treg inhibition and Teff expansion brought the high Treg/Teff ratio to normal. Our ﬁndings
suggest a marked responsiveness of SS tumor cells and Tregs, to targeting with TNFR2 antagonistic antibodies. These results
show TNFR2 antibodies are potent and efﬁcacious in vitro.

Introduction
Sezary Syndrome is a rare form of cutaneous T cell
lymphoma. SS is considered a late stage and aggressive
form lymphoma with a poor prognosis. SS subjects ave
signiﬁcant blood involvement with malignant T cells
known as Sézary cells (SC) [1, 2]. Effective treatments for
CTCL are limited and most forms of immunotherapy haveshown minimal effectiveness. SS offers a special opportunity for an antibody-based therapeutic intervention
because of its high expression of the newly characterizedElectronic supplementary material The online version of this article(https://doi.org/10.1038/s41375-018-0292-9) contains supplementarymaterial, which is available to authorized users.
* D L Faustman
Faustman@helix.mgh.harvard.edu
1

Massachusetts General Hospital and Harvard Medical School,
Building 149, 13th Street, Rm 3602, 02129 Boston, MA, USA

2

Stanford University School of Medicine/Cancer Institute, 94305
Palo Alto, CA, USA

oncogene tumor necrosis factor receptor 2 (TNFR2) on
tumor cells [3], as well as on Treg cells in the tumor
microenvironment [4–9]. In SS the signiﬁcant blood
involvement allows direct blood sampling of tumor cells
and the tumor microenvironment [3].
Recent research ﬁnds that TNFR2 is expressed directly
on tumor cells as an oncogene conferring preferential
growth [10]. This is particularly well-depicted in SS wherepoint mutations and genomic gains of TNFR2 (TNFRSF1B)
lead to enhanced NFκB signaling for cell expansion andgrowth [3]. In non-tumor settings TNFR2 is the bidirectional switch for either Treg expansion or Treg contraction with limited systemic expression [11]. In SS
alterations by point mutations or gain mutations suggest apotential role of oncogenic TNFR2 signaling and increased
TNFRSF1B transcript mRNA with expanded expression
onto the tumor cell themselves. Furthermore these SS
mutations correlate with worse outcomes. Relatedly, theTNFR2 oncogene expression on ovarian tumor cells renderthem susceptible to death by TNFR2 antagonism: TNFR2
antagonistic antibodies in vitro directly kill TNFR2expressing ovarian cancer cell lines, kill the tumorassociated TNFR2+ Tregs, and proliferate the beneﬁcial T

H. Torrey et al.

effectors (Teffs) [12, 13]. The TNFR2 oncogene is now
expressed on at least 25 tumor types [14].
Treg removal or inactivation in cancer is considered partof an essential strategy to remove or diminish hostgenerated immune suppression [15]. Tregs are a hostderived cell that suppresses the immune response andhampers host recognition of the cancer [16, 17]. Therefore,a targeted approach to remove or inactive host Tregs might
better control cancer. While Treg deﬁciency could put anindividual at risk of developing autoimmune disease [18] orgraft-versus-host disease (GvHD) [19], selective or transientdeletion of the most suppressive Tregs in only the tumor
microenvironment may provide optimal conditions for
antitumor immune response [20]. Finding restricted or
exclusive receptors speciﬁc to Tregs has been challenging[21–23]. Many surface receptor of Tregs are also expressed
diffusely in the immune system, with TNFR2 being a prominent exception with highest density in the tumor microenvironment [13]. The TNFR2 receptor is a member of the
TNF superfamily receptors that are composed of over 29
receptors. The expression of TNFR2 is limited: while most
TNF superfamily member receptors are expressed on all
lymphoid and sometimes all parenchymal cells, TNFR2
expression is restricted to a subpopulation of potent Tregs,
myeloid suppressor cells, and developing neurons [24, 25].
Research in primates ﬁnds that TNFR2-speciﬁc ligands
have minimal in vivo systemic toxicity [26], most likely
because of TNFR2’s restricted cellular distribution. Indeed
unlike many other broadly expressed Treg targets, TNFR2−/−
mice have no signs of autoimmunity. This combinedwith unique TNFR2 antagonistic antibodies that can only
kill rapidly proliferating TNFR2-expressing cells creates asetting where tumor microenviroment Treg targeting may bepossible [13]. This combined with the limited naturalexpression of TNFR2 makes it an ideal target for a possible
safer immunotherapy.
Naturally occurring Tregs have inducible TNFR2
expression with a tenfold higher density than TNFR1, theother most closely related receptor of the TNFR superfamily. TNFR2 receptor lacks an intracellular death domain
and thus is a cell growth pathway linked to NFκB and thus
cell growth through this known proliferative pathway.
TNFR2 is preferentially expressed on Tregs and is a functional receptor—indeed, the master switch—for Treg survival in humans and mice [11]. Tregs die with TNFR2
blockade or lack of stimulation in development or adulthood. Therefore, the TNFR2 surface protein is not merely
an identiﬁer of potent Tregs, but is the central control site forTreg survival.
Tregs in both mouse and humans that express the TNFR2
receptor are potently suppressive and are the predominant
inﬁltrating cells found in human and murine tumors [4–6, 8,
9, 27]. In some human cancers, the expression of TNFR2 on

inﬁltrating Tregs is estimated to be 100 times higher than on
circulating Tregs in control subjects. In other forms of humancancer, the overall abundance of TNFR2+ Tregs is higher
than in peripheral blood [9, 28]. In human cancers, the most
suppressive Tregs express excess TNFR2 receptors on theirsurface and exert very potent host immunosuppressive
effects [9, 28]. Agonism of the TNFR2 receptor results inmembrane cleavage, generating soluble TNFR2 (sTNFR2),
a serum marker diagnostic of poor cancer outcomes [29–33]. These features make TNFR2 an advantageous molecular target on Tregs and form the basis for Treg inactivation
approach using human-directed antibodies to TNFR2 forcancer therapies [10].
This in vitro study examines end-stage SS subjects forsusceptibility of their lymphocytes and cancer cells in culture to a TNFR2 antagonistic antibody. Subjects werereceiving a variety of treatments with advanced disease(Stage IVA). With the discovery of TNFR2 signaling
pathway polymorphisms and mutations in SS and long priorcourse of diverse therapeutic interventions, it is vital toshow that TNFR2 antagonistic antibodies have the capacity
to kill TNFR2+ Tregs and TNFR2 oncogene-expressing
tumor cells.

Results
End-stage Sézary syndrome patients express higher
TNFR2 in tumor-residing CD26− cells and Tregs
We initially characterized CD4+ cells from advanced SS
patients (Supplementary Table S1). Abnormally low or no
surface expression of CD26 on CD4+ T cells is a characteristic of the malignant cells of SS patients and a usefuldiagnostic marker of disease [34]. In all end-stage SS subjects, the proportion of CD26− CD4+ cells ranged from 40
to over 90% whereas the proportion in healthy controls was<20% (Fig. 1a). All subjects were in Stage IVA of theirdisease. The lack of CD7 on CD4+ cells is another indicatorof disease progression [35]. These same subjects were
found to exhibit varying degrees of CD7 expression, indicating varying disease progression (Fig. 1b). Adding to thediversity, each subject was on a unique treatment regimen
and samples were taken at various stages of treatment(Supplementary Tables S1 and S2).
Next, we assessed the level of TNFR2 expression. As
expected, we found a signiﬁcantly higher proportion of
TNFR2+ CD26− and TNFR2+ Tregs in SS patients thancontrols (Z test, 95% CI) (Fig. 1c). In addition to the greater
proportion of TNFR2+ cells, others have found higher
TNFR2 transcript levels in patient tumor samples [36].
Indeed, we found that the mean ﬂorescence intensity (MFI)of TNFR2 on CD26− and Tregs was also higher in patients,

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic. . .

indicating higher receptor density (Fig. 1c). In contrast, with
Teff, the proportion of TNFR2+ cells and the TNFR2 MFI
was signiﬁcantly lower in patients than healthy controls(Fig. 1c). In one patient where malignant clone-speciﬁc

TCR Vb was determinable (Subject E), CD26−SC wereenriched in the Vb-positive subset and the MFI of TNFR2
was higher (Supplementary File S2a). In another patient(Subject C), TNFR2+ CD26− SC of clone-speciﬁc

H. Torrey et al.
Fig. 1 Sézary syndrome patients exhibit characteristically high levelsof CD26− cells and Tregs with increased TNFR2 expression. a Percentage of CD26− cells from freshly isolated peripheral human CD4+
cells of Sézary syndrome patients and healthy controls (n = 1 Subjects
E, H, I; n = 2 Subjects A, D, C; n = 3 Subjects B, FG, and n = 10
Controls). b Proportion of CD26+/− and CD7+/− lymphocytes. c.
Proportion of lymphocyte populations (CD26−, Treg, and Teff) andTNFR2 expression of all patients except samples from subjects withtumor cells > 90% of CD4+ (Subjects E, I) and controls (n = 11
Patients, n = 11 Controls). d Individual histograms showing themassive amounts of TNFR2 expression on either tumor containing
CD26− cells or Treg, cells in a characteristic Sezary syndrome subject(pink) compared to a control subject (blue). Data are mean ± SEM,
underlined asterisks indicate signiﬁcant difference between patient andcontrols determined by Z test (95% CI)

Vb-positive cells were more susceptible to the effect of
TNFR2 antagonism than non-clonal cells (SupplementaryFile S2b). A set of representative ﬂow cytometry histogramof the MRI of TNFR2 on tumor cells and on Treg cells
compared to control cells shows on a log scale the massive
expression of TNFR2 oncogene on these two cells types inthis cancer during advanced disease (Fig. 1d). Taken together, these results support abnormally high CD4+ CD26−
phenotype, demonstrate variability in the CD7 proﬁle, andreveal signiﬁcant differences in level of TNFR2 expressionin SS patients compared to controls both with highexpression on the tumor cells themselves and on theassociated tumor-associated Tregs. They also suggest tumorspeciﬁc expression and possible merit for looking for sensitivity of the TNFR2 target to targeted immunotherapy.

A dominant TNFR2 antagonist antibody eliminates
TNFR2+ CD26− cells of Sézary syndrome patients
We previously reported the elimination of TNFR2expressing Tregs and TNFR2-expressing ovarian cancer
cells in a dose-dependent manner by dominant TNFR2
antagonistic antibodies [13]. Here we demonstrate thattumor-residing TNFR2+ CD26− are also susceptible to theinhibitory effects of one of the TNFR2 antagonists used in
the ovarian culture study. Even in short assays (48 to 72 h),
the proportion of TNFR2+ CD26− cells was signiﬁcantly
reduced (t test, p < 0.05 for TNFR2 antagonist ≥ 12.5 µg/ml)
in SS patient samples samples; Fig. 2a and SupplementaryFile S3a). In healthy controls, even though the baseline
proportion of CD26− and TNFR2+ CD26− cells was considerably lower than in patients (Fig. 1c; Z test, 95% CI), a
signiﬁcant reduction (t test, p < 0.05 for TNFR2 antagonist ≥ 50 µg/ml) in TNFR2+ CD26− was observed (Fig. 2b).
Importantly, after normalizing the data we found that therelative change in TNFR2+ CD26−cells was signiﬁcantly
greater (Z test, 95% CI) at a tenfold lower dose in patients(5 µg/ml) than controls (50 µg/ml; Fig. 2c and Supplementary File S3b). This suggests that tumor-residing CD26−

cells of SS patients are more sensitive to the action of the
TNFR2 antagonist than CD26− cells of healthy controls.
This may be due to faster turnover of the TNFR2 target on
proliferating cancer cells. Importantly, we conﬁrmed thatthe reduction in the proportion of CD26− cells, due to
TNFR2 antagonist treatment, equates to a reduction in total
CD26− cell number (Supplementary File S4a-d).
An important consideration of combination cancer therapy is the possibility that one type of therapy modulates the
efﬁcacy of another type of therapy. To assess whether SS
patients’ treatment regimens affect the in vitro efﬁcacy of
TNFR2 antagonist, we analyzed patient samples by treatment type. Interestingly, samples from treatment-naive
patients or those on Investigative Therapy A or B weresigniﬁcantly more susceptible (t test, p < 0.05) to the
TNFR2 antagonist (≥50 µg/ml) than samples from a methotrexate (MTX)-treated patient (Fig. 2d). While it is possible that the neoplastic lymphocytes obtained from the MTX
patient sample were less responsive to the TNFR2 antagonist than other patients, the ﬁndings are consistent with ourprevious observation that inhibition of DNA replication caninterfere with efﬁcacy of the antibody [13]. The mechanismof action of the TNFR2 antagonist antibodies, based on
structural biology observations, is the capture of newly
synthesized surface TNFR2 on rapidly proliferating cells.
This capture makes TNFR2 an anti-parallel dimers that caninhibit cell growth and kills the cells by preventing of signaling trimmers [12, 13].
In two patients (Subject C, E), clonal malignant T cells
were determined by TCR Vb subtyping. To assess the effectof the TNFR2 antagonistic antibody on the clonal population, we isolated these T cells for direct examination. In
both cases, treatment with the TNFR2 antagonist led toelimination of the clonal tumor cell populations (Fig. 3a, b
and Supplementary File S5 a-d). As expected, the inhibitory
effect of the antagonistic antibody was ampliﬁed for thesubset expressing high levels of TNFR2 (Fig. 3b, c). These
results demonstrate direct killing of tumor-residing cells by
the TNFR2 antagonist.

TNFR2 antibody inhibits Tregs and enables Teff
proliferation
TNFR2+ Tregs collected from the tumor microenvironment
are known to be highly immunosuppressive and reducing
their presence is a major objective in cancer immunotherapy. Therefore, in addition to direct killing of tumorresiding T cells, we sought to assess whether Tregs fromperipheral blood of SS patients would also be targeted by
the TNFR2 antagonist. Indeed, pooled data of Tregs from SS
patients demonstrated susceptibility to dose-dependent
killing by the TNFR2 antagonist (Fig. 4a). While all
patient samples were individually analyzed and responded

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic. . .

b 80

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Fig. 2 TNRF2 CD26 cells are reduced in response to treatment withTNFR2 antagonist. a Proportion of TNRF2+ CD26− cells from Sézary
syndrome patients (n = 15) after treatment with IL-2 (200 U/ml) andTNFR2 antagonist (0–125 µg/ml) for 48 to 72 h. b Proportion of
TNRF2+ CD26− cells from healthy controls (n = 11) treated as
described in a. c Relative change in TNFR2+ CD26− cells in patientsand controls as described in a. d Relative change in TNFR2+ CD26−

125

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cells in patients on various treatment regimens (Investigative Therapy
A (n = 4), Investigative Therapy B (n = 3), methotrexate (n = 2),
Control (n = 11). Data are mean ± SEM and asterisks indicates signiﬁcant difference from baseline (TNFR2 antagonist 0 µg/ml (a, b) or
0.1 µg/ml (c)) (t test p < 0.05), and underlined asterisks indicate signiﬁcant difference in patients versus controls (Z test 95% CI)

H. Torrey et al.

Vb18+ (Subject E)

Vb16+ (Subject C)

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Fig. 3 TCR Vbeta clonal CD4+ cells of Sézary syndrome patients are
inhibited in a dose-dependent manner by treatment with the TNFR2
antagonist antibody. a. Proportion of CD4+ TCR Vb-speciﬁc cells inpatient versus control for Subject C (left) and Subject E (right). b.

Relative change in the proportion of CD4+ Vb-speciﬁc clonal cells for
Subject C (left) and Subject E (right). c. Relative change in the proportion of TNFR2+ Vb-speciﬁc cells for Subject C (left) and Subject
E (Right) (bottom). Data are from a single representative experimentas expected (Supplementary File S6a), it is important tonote that Subjects E and I were removed from the pooleddata due to very low numbers of Tregs. The paucity of Tregs
in these subjects was likely due to very high tumor-residing
CD26− cell populations (Fig. 1a) or treatment regimens
targeting Tregs (Investigative Therapy B; Supplementary
Tables S1 and S2). We assessed Tregs from healthy controls
and observed the expected dose-dependent elimination by
the TNFR2 antagonist (Fig. 4b).

Previously we reported that Tregs from the ovariancancer tumor microenvironment were more susceptible tothe effect of the TNFR2 antagonist than Tregs from healthy
donors [13]. In this study, we predicted that Tregs fromperipheral blood of SS patients would also be more susceptible than those of healthy controls. Normalizedcomparison of pooled data showed that both patients andcontrols exhibited signiﬁcant reduction in Tregs frombaseline at TNFR2 concentration ≥ 12.5 µg/ml (Fig. 4c

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic. . .

b

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Patient

Fig. 4 TNFR2 antagonist inhibits Tregs in Sézary syndrome andhealthy control CD4+ cell culture. a. Proportion of Treg cells frompatients (n = 15) after incubation of freshly isolated CD4+ cells with
IL-2 and TNFR2 antagonist (0–125 µg/ml) for 48–72 hrs. b. Proportion of Treg cells from controls (n = 11) as described in (a). c. Relativechange in Treg of patients and controls after culture with TNFR2

antagonist as described in (a). d. Comparison of baseline proportionsof Treg from patients and controls treated with IL-2 alone or withTNFR2 agonist (12.5 µg/ml). Data are mean ± SEM, stars (*) indicate
signiﬁcant difference (T-test p<0.05) from baseline (TNFR2 antagonist
0 µg/ml (B) or 0.1 µg/ml (C)), and underlined stars (*) indicate signiﬁcant difference in patients versus controls (Z-test 95%CI)

and Supplementary File S6b). Thus, there was no signiﬁcant difference in the degree of elimination of Tregs.
However, we did ﬁnd that Tregs from peripheral blood of
SS patients had a signiﬁcantly greater (p < 0.05) responseto treatment with a TNFR2 agonist than healthy controls(Fig. 4d). This data supports our observations with Tregs
from ovarian cancer ascites ﬂuid and further suggests that
Tregs of the tumor microenvironment in SS are not only
expressing very high levels of TNFR2 but presumably arealso rapidly proliferating from the overabundance of thisH. Torrey et al.

b

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Fig. 5 TNFR2 antagonist enables continuous dose-dependent Teff
expansion in Sézary syndrome patient samples. a Proportion of Teff
cells from patients (n = 18 samples) after incubation of freshly isolated
CD4+ cells with IL-2 and TNFR2 antagonist (0–125 µg/ml) for 48–72
h. b Proportion of Teff cells from controls (n = 11) as described in a. c
Relative change in Teff of patients and controls after culture withTNFR2 antagonist as described in a. d Comparison of baseline proportions of Teff from patients and controls. Data are mean ± SEM,
asterisks indicate signiﬁcant difference from baseline (TNFR2
antagonist 0.1 µg/ml; t test p < 0.05), and underlined asterisks indicate
signiﬁcant difference in patients versus controls (Z test 95%CI)

growth receptor. Also it should be mentioned that theovarian cancer study used treatment-naive subjects justdiagnosed with ovarian cancer, and this study usedadvanced SS subjects on a range of salvage therapies
some of which might work to decrease in vivo Tregs cellnumbers thus accounting for the more dramatic Tregs effectof TNFR2 antagonism.
In the absence of suppressive Tregs, Teff cells proliferate
due to the removal of this cell mediated suppression. We
therefore predicted, with the reduction in Tregs by TNFR2
antagonist treatment, a corresponding expansion of Teff. As

expected, we observed a dose-dependent proliferation of
Teff at low concentrations of TNFR2 antagonist (0.1–12.5µg/ml) in both SS patients and controls (Fig. 5a, b andSupplementary File S7a). Teff proliferation continued toincrease with higher doses of the antibody (25–125 µg/ml)
in SS patients whereas it tapered off at high doses of theantibody in controls (Fig. 5c and Supplementary File S7b).
Interestingly, baseline levels of Teff were signiﬁcantly lower
in the patients (Fig. 5d) and were only restored to normal
levels (baseline of healthy control) in the presence of highdoses of the TNFR2 antagonist (50–125 µg/ml).

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic. . .

Ratio of Treg/Teff is corrected by TNFR2 antagonist
regardless of patient treatment history or treatment
stage
The ratio of Treg/Teff is an indicator of the suppressive
capacity of the immune system in the tumor microenvironment. With a dose-dependent decrease in the Tregs
and concurrent proliferation of Teff, we ﬁnd that the TNFR2
antagonist has the ability to correct the Treg/Teff ratio of SS
patient samples. Indeed, the Treg/Teff ratio of patients (n =15 samples) was brought down to the baseline level ofhealthy controls (n = 11 samples) after only 48 to 72 h
culture with the TNFR2 antagonist (125 µg/ml; Fig. 6a). To
investigate whether this antibody would work effectively at
various stages of clinical treatment, for each patient wetested two longitudinal samples which we assigned a labelof early or late (Table S2) depending on the date of sample
collection. In each case except one (Subject E), treatment ofeither sample with the TNFR2 antagonist (12.5 µg/ml)
resulted in an improvement of the Treg/Teff ratio (Fig. 6b).
The very low Treg/Teff ratio in Subject E is likely skewed
due to the very high proportion of CD26− in this patient(Fig. 1a). We also ﬁnd that in all cases except one (SubjectE), the proportion of tumor-residing CD26− decreases fromearly to late in the sample comparison (Fig. 6c). With
Subject E, the longitudinal increase in CD26− may be
explained by the fact that the patient was taken off, andremained off, therapy prior to sample collection. Importantly, for all patients receiving continuous therapy, there
was a notable decrease in the relative change in TNFR2+
tumor-residing CD26− cells in the longitudinal sample
analysis (Fig. 6d).

Discussion
Our population of SS patients had advanced disease(Stage IVA) and were on concurrent, multi-drug therapy.
This advanced disease state was evident by massive tumor
burdens within the CD26− lymphocytes in peripheral
blood. Our in vitro study reveals that TNFR2 antibody
antagonism, despite advanced disease and diverse treatment regimens was effective in directly killing TNFR2+
tumor cells, eliminating high numbers of TNFR2+ Tregs,
permitting the expansion of Teff, and restoring Tregs/Teffratios. This data suggests TNFR2 antagonism could
potentially offer a clinical beneﬁt once developed forin vivo therapy.
The mechanism by which TNFR2 antagonist kills
TNFR2+ tumor cells and TNFR2+ Tregs is likely byblocking activation of NFkB, a transcription factor necessary for cell survival. The mechanism by which TNFR2
enables Teff expansion is by lifting Treg suppression [11].

Designing a dominant TNFR2 antagonistic antibody
that can successfully exert effects in the presence of TNF
has been difﬁcult. This has also been true for antibody
antagonists to other receptors of the TNF receptor
superfamily. The past failures to create effective TNFR2
antagonistic antibodies have been largely due to the natural ligand, TNF, being a potent agonist that stabilizes the
TNFR2 into a trimer with tight associations not dislodged
by even high afﬁnity antibodies. Thus the natural ligand—trimeric TNF, or trimeric membrane TNF—is nearly
always dominant and when present result in agonism of
TNFR2 and cell expansion. The TNFR2 antagonistic
antibody used in this study is still effective in spite of highdoses of TNF [13]. This dominant antagonism is possibleby the selective capture of newly synthesized, nonassembled TNFR2 proteins on rapidly proliferating cells
prior to trimerization with TNF. The newly formed
structure, TNFR2 plus antagonistic antibody, is an
assembly of newly synthesized TNFR2 as an anti-parallel
dimer [10, 12]. This anti-parallel dimer blocks the TNF
binding site and may also form a potent inhibitory signaling lattice. Dominant TNFR2 antagonistic antibodies
also have speciﬁcity for the tumor-bearing cells andtumor-associated Tregs due to their rapid proliferation andthus surface supply of newly synthetized TNFR2 surface
protein.
This brings us to the therapy being used in SS and its
impact on TNFR2 antagonism effectiveness. This study
suggests that dominant TNFR2 antibody antagonism
effectiveness remains even in end-stage subjects. In
metastatic melanoma it is known that both anti-CTLA4
and anti-PDL1 treatment failures are driven by a tumor
microenvironment full of TNFR2+ Tregs, suggesting that
TNFR2 could be a similar escape pathway for the lost
effectiveness on current therapies in SS [37]. There is one
exception that will need to be repeated with further studies. As observed in Fig. 2d, if the SS subjects are on
methotrexate, the TNFR2 antagonist-induced killing of SStumor cells in culture appears to be blunted. It is possiblethat the neoplastic lymphocytes of this patient were
inherently less susceptible. However, further support isprovided by observations that TNFR2+ CD26− cells ofpatients were more susceptible than those healthy controls, and that those of naive patients were more susceptible than those of treated patients, since all treatments for
SS aim to slow down the proliferation of neoplastic
lymphocytes. This was also previously reported in ovariancancer subjects exposed to Mitomycin C, another antiproliferative agent [13]. A diminished TNFR2 antagonistic antibody killing response against ovarian cancer oragainst tumor-associated Tregs appears to occur if the celltarget is not rapidly proliferating. This clinical situation
with SS is consistent with dominant TNFR2 antagonistic

H. Torrey et al.

antibodies having the enhanced ability to target newly
produced surface TNFR2 if the cancer cells are rapidly
proliferating. If rapid proliferation is inhibited by

concurrent therapy, our TNFR2 antagonist is less effective. It is known that the dominant antibody antagonism
has such Treg speciﬁcity since the antibody captures a

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic. . .
Fig. 6 TNFR2 antagonism corrects Treg/Teff ratio and reduces TNFR2+
tumor cells in Sézary syndrome patient samples regardless of underlying therapy. a Ratio of Treg/Teff in patients (n = 15 samples) andcontrols (n = 11 samples) after incubation of freshly isolated CD4+
cells with IL-2 and TNFR2 antagonist (0–125 µg/ml) for 48–72 h. b
Ratio of Treg/Teff after TNFR2 antagonist treatment (0 and 12.5 µg/ml)
as describe in a of early and late longitudinal samples from patients on
various clinical treatment regimens. c Proportion of CD26− of earlyand late patient samples at baseline (TNFR2 0 µg/ml). d Relativechange in proportion of TNFR2+ CD26− in samples treated withTNFR2 antagonist (12.5 µg/ml). Data are mean ± SEM and ns indicates no signiﬁcant difference between patients and controls (t test p <0.05)

unique newly synthesized anti-parallel TNFR2 dimer on
the surface of rapidly proliferating cells [12, 38].
Genetic studies in Sezary Syndrome have identiﬁed
many alterations in the key T cell signaling pathways for T
cell expansion. Two genetic studies similarly identify
TNFR2 mutations and all studies have observed involvement of the downstream NFkB proteins to surface TNFR2cell signaling [39–41].
In summary, TNFR2 is an oncogene expressed on somecancers, such as SS, and a cell surface protein found inabundance on tumor-associated Tregs. In this study, we showthat dominant TNFR2 antagonistic antibody can kill completely the abundant Tregs of SS, directly kill the tumor cells
and this unleashes rapid expansion of Teff cell numbers tocorrected Treg/Teff ratios like controls. A recent limitation ofmoving TNFR2 antagonistic therapies into human trials has
been the lack of a mouse surrogate antibodies for murine
tumor studies. This issue has been recently solved with newdata using a mouse-directed TNFR2 antagonistic antibody
that shows efﬁcacy in treating two murine models of cancer[42]. Taken together, these ﬁndings support TNFR2
antagonism as a new multi-pronged approach in difﬁcult-totreat end-stage SS with implications for numerous other
TNFR2-expressing tumors. Indeed recent data similarlyshows TNF signaling in the malignant cell of myeloﬁbrosis
proliferate and expand preferentially through the TNFR2
receptor and linked pathway of clonal expansion [38]Materials and methods
Human subjects
Human blood samples were collected from SS patients (n =18 from 8 SS subjects) and healthy controls (n = 11 samples
from 11 subjects) according to a human studies protocol
approved by the Massachusetts General Hospital (MGH)
Human Studies Committee (MGH-2001P001379) andStanford University Human Studies Committee (IRB 5538
and IRB 13844). All the donors provided written informed
consent. We also stipulated with our request for patient

samples that the study drugs were not anti-mitotic drug
since we know from published data that this alone willinterfere with our effectiveness with TNFR2 antagonismthat require rapidly proliferating cells.
Blood was collected into BD Vacutainer EDTA Tubes(BD Diagnostics) and processed within 24 h of phlebotomy.
These human studies were approved by the MGH Human
Studies Committee (MGH-2015P002489).

Blood and cell culture
Fresh human blood was processed within 24 h of venipuncture. CD4+ cells were isolated using the Direct Human
CD4+ T Cell Isolation Kit (Stemcell Technologies). Isolated
CD4+ cells were re-suspended in RPMI GlutaMAX (LifeTechnologies) plus 10% FBS (Sigma-Aldrich) and 1%
penicillin streptomycin (Life Technologies). Because isolated and cultured human T cells die without IL-2 in the
media, all culture conditions in all experiments contained a
low amount of IL-2 (200 U/ml) to prevent IL-2 withdrawal
from inﬂuencing the data.

Cell culture assays
For cell culture assays, freshly isolated CD4+ cells wereseeded at a concentration of 0.2 × 106 cells per well in 96well round-bottom plates, treated with the TNFR2 antagonist or agonist, and incubated at 37 °C and 5% CO2 for 48 to
72 h. After incubation, cells were collected by centrifugation, washed once with 1× Hanks’ balanced salt solution(HBSS) (Invitrogen), and stained for FACS analysis.

Reagents and ﬂow cytometry
Monoclonal antibodies (mAbs) against human TNFR2 were
produced internally as previously described [11]. Recombinant human TNF was purchased from Sigma-Aldrich andrecombinant human IL-2 was purchased from Life Technologies. Cells were prepared for ﬂow cytometry using
Human Tregs Flow Kit (BioLegend) according to the manufacturer’s instructions. Fluorescently stained cells were resuspended in 1× HBSS (Invitrogen) and analyzed using a
BD FACS Calibur Flow Cytometer machine (Becton
Dickinson). Antibodies used for FACS analysis of Tregs
included AlexaFluor 488 anti-human FOXP3 (Clone 259D;
BioLegend) for intracellular staining of FOXP3, phycoerythrin (PE) human CD25 (Clone BC96; BioLegend), PE/
Cy7 human CD26 (Clone BA5b; BioLegend), and ﬂuorescein isothiocyanate (FITC) human CD7 (Clone CD7-6B7;
BioLegend) for cell surface staining of CD25, CD26, andCD7 respectively. Anti-human TNFR2/TNFRSF1B(MAB726; R&D Systems) was conjugated to allophycocyanin (APC) by Lightning-Link (Innova Biosciences) andH. Torrey et al.

used for assessing TNFR2 cell surface expression by FACS
with FL4 (far red). Treg populations were assessed by FACS
with FL2 (red) versus FL1 (green) and deﬁned as CD25hi
and FoxP3−positive, Teff populations were deﬁned as
CD25hi and FoxP3-negative, CD26− cells were deﬁned as
CD26low (Supplementary File S1). To be able to best discern Treg cells from Sezary cells that also express Foxp3, all
ﬂow cytometry gating utilized only CD4 T cells with highCD25, lacking CD127; this was done for identifying themost suppressive human Treg cells (TNFR2+) and minimizing the contamination of possible SS cells [43].
T cell receptor (TCR) Vbeta (Vb) 16 clone cells wereisolated from CD4+ cell suspensions by labeling with FITC
conjugated TCR Vbeta 16 mAb (Beckman-Coulter) andextracting the FITC-labeled cells with EasySepTM FITC
Positive Selection Kit (Stemcell Technologies) and assessedby FACS with FL1 (green). TCR Vbeta18 clone cells werelabeled with PE conjugated Vbeta 18 mAb (BeckmanCoulter) and assessed by FACS with FL2 (red). FACS data
were processed using FlowJo software (version 10.1).

Statistical analysis
Data analysis was performed by Student’s t test (unpaired,type 3) using Excel (Microsoft) or GraphPad Prism 5 software (GraphPad Software). Signiﬁcance was determined by
P < 0.05.
Acknowledgements We acknowledge the secretarial assistance of Ms.
Lynne Murphy and the editorial support by Miriam Davis, PhD. We
thank Ziba Rahbar for her coordination of samples and sample
retrieval. This work was supported by all non-proﬁt donations contributed to the Massachusetts General Hospital.
Author contributions HT carried out the experiments, analyzed data,and prepared the ﬁgures. MK and YHK both worked on patient
recruitment, deﬁne patient characteristics and treatments, performed
the human consents and patient care for the blood donations for thisstudy and reviewed the manuscript and provided critical feedback. LT,
DB, and AD all executed the ﬂow cytometric studies carried out in thispaper and processed patient samples for study. DLF designed andconceived the experiments, supervised the data on a daily and weekly
basis, and wrote the manuscript.

Compliance with ethical standards
Conﬂict of interest The authors declare that they have no conﬂict ofinterest.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, aslong as you give appropriate credit to the original author(s) and thesource, provide a link to the Creative Commons license, and indicate ifchanges were made. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is notincluded in the article’s Creative Commons license and your intended

use is not permitted by statutory regulation or exceeds the permitteduse, you will need to obtain permission directly from the copyrightholder. To view a copy of this license, visit http://creativecommons.
org/licenses/by/4.0/.

References
1. Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R,
Advani R et al. The 2016 revision of the World Health Organization classiﬁcation of lymphoid neoplasms. Blood. 2016;127:
2375–90.
2. Kim EJ, Hess S, Richardson SK, Newton S, Showe LC, Benoit
BM, et al. Immunopathogenesis and therapy of cutaneous T cell
lymphoma. J Clin Invest. 2005;115:798–812.
3. Ungewickell A, Bhaduri A, Rios E, Reuter J, Lee CS, Mah A,
et al. Genomic analysis of mycosis fungoides and Sezary syndrome identiﬁes recurrent alterations in TNFR2. Nat Genet.
2015;47:1056–60.
4. Byrne WL, Mills KH, Lederer JA, O’Sullivan GC. Targeting
regulatory T cells in cancer. Cancer Res. 2011;71:6915–20.
5. Teng MW, Ritchie DS, Neeson P, Smyth MJ. Biology and clinicalobservations of regulatory T cells in cancer immunology. Curr
Top Microbiol Immunol. 2011;344:61–95.
6. Facciabene A, Motz GT, Coukos G. T-regulatory cells: key
players in tumor immune escape and angiogenesis. Cancer Res.
2012;72:2162–71.
7. Chen X, Subleski JJ, Kopf H, Howard OM, Mannel DN,
Oppenheim JJ. Cutting edge: expression of TNFR2 deﬁnes a
maximally suppressive subset of mouse CD4 + CD25 + FoxP3 +T regulatory cells: applicability to tumor-inﬁltrating T regulatory
cells. J Immunol. 2008;180:6467–71.
8. Chen X, Subleski JJ, Hamano R, Howard OMZ, Wiltrout RH,
Oppenheim JJ. Co-expression of TNFR2 and CD25 identiﬁes
more of the functional CD4( + )FOXP3( + ) regulatory T cells inhuman peripheral blood. Eur J Immunol. 2010;40:1099–106.
9. Govindaraj C, Scalzo-Inguanti K, Madondo M, Hallo J, Flanagan
K, Quinn M, et al. Impaired Th1 immunity in ovarian cancer
patients is mediated by TNFR2 + Tregs within the tumor microenvironment. Clin Immunol. 2013;149:97–110.
10. Vanamee ES, Faustman DL. TNFR2: a novel target for cancer
immunotherapy. Trends Mol Med. 2017;23:1037–46.
11. Okubo Y, Mera T, Wang L, Faustman DL. Homogeneous
expansion of human T-regulatory cells via tumor necrosis factor
receptor 2. Sci Rep. 2013;3:3153.
12. Vanamee ES, Faustman DL. Structural principles of tumor
necrosis factor superfamily signaling. Sci Signal. 2018; 11:
eaao4910.
13. Torrey H, Butterworth J, Mera T, Okubo Y, Wang L, Baum D,
et al. Targeting TNFR2 with antagonistic antibodies inhibits
proliferation of ovarian cancer cells and tumor-associated Tregs.
Sci Signal. 2017;10:eaaf8608.
14. Uhlen M, Bjorling E, Agaton C, Szigyarto CA, Amini B,
Andersen E, et al. A human protein atlas for normal and cancer
tissues based on antibody proteomics. Mol Cell Proteom.
2005;4:1920–32.
15. Drake CG, Lipson EJ, Brahmer JR. Reply: Regulatory T cells-an
important target for cancer immunotherapy. Nat Rev Clin Oncol.
2014;11:307.
16. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat
Rev Cancer. 2012;12:278–87.
17. Wolf AM, Wolf D, Steurer M, Gastl G, Gunsilius E, GrubeckLoebenstein B. Increase of regulatory T cells in the peripheral
blood of cancer patients. Clin Cancer Res. 2003;9:606–12.
18. Zhang W, Sharma R, Ju ST, He XS, Tao Y, Tsuneyama K, et al.
Deﬁciency in regulatory T cells results in development ofTargeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic. . .
antimitochondrial antibodies and autoimmune cholangitis. Hepatology. 2009;49:545–52.
19. Li Q, Zhai Z, Xu X, Shen Y, Zhang A, Sun Z, et al. Decrease ofCD4( + )CD25( + ) regulatory T cells and TGF-beta at early
immune reconstitution is associated to the onset and severity ofgraft-versus-host disease following allogeneic haematogenesis
stem cell transplantation. Leuk Res. 2010;34:1158–68.
20. Klages K, Mayer CT, Lahl K, Loddenkemper C, Teng MW,
Ngiow SF, et al. Selective depletion of Foxp3 + regulatory T cells
improves effective therapeutic vaccination against establishedmelanoma. Cancer Res. 2010;70:7788–99.
21. Shimizu J, Yamazaki S, Sakaguchi S. Induction of tumor immunity by removing CD25 + CD4 + T cells: a common basis
between tumor immunity and autoimmunity. J Immunol.
1999;163:5211–8.
22. Sutmuller RP, van Duivenvoorde LM, van Elsas A, Schumacher
TN, Wildenberg ME, Allison JP, et al. Synergism of cytotoxic T
lymphocyte-associated antigen 4 blockade and depletion of CD25
( + ) regulatory T cells in antitumor therapy reveals alternative
pathways for suppression of autoreactive cytotoxic T lymphocyte
responses. J Exp Med. 2001;194:823–32.
23. Smyth MJ, Ngiow SF, Teng MW. Targeting regulatory T cells intumor immunotherapy. Immunol Cell Biol. 2014;92:473–4.
24. Heinrich M, Burger D, Wang L, Tahhan G, Reinhold P, Zhao M,
et al. TNFR1 and TNFR2 expression and induction on human Treg
cells from type 1 diabetic subjects. Antibodies. 2015;4:34–47.
25. Polz J, Remke A, Weber S, Schmidt D, Weber-Steffens D,
Pietryga-Krieger A, et al. Myeloid suppressor cells require
membrane TNFR2 expression for suppressive activity. Immun
Inﬂamm Dis. 2014;2:121–30.
26. Welborn MB III, Van Zee K, Edwards PD, Pruitt JH, Kaibara A,
Vauthey JN, et al. A human tumor necrosis factor p75 receptor
agonist stimulates in vitro T cell proliferation but does not produce
inﬂammation or shock in the baboon. J Exp Med. 1996;184:
165–71.
27. Chen X, Wu X, Zhou Q, Howard OM, Netea MG, Oppenheim JJ.
TNFR2 is critical for the stabilization of the CD4 + Foxp3 +regulatory T. cell phenotype in the inﬂammatory environment. J
Immunol. 2013;190:1076–84.
28. Govindaraj C, Tan P, Walker P, Wei A, Spencer A, Plebanski M.
Reducing TNF receptor 2 + regulatory T cells via the combined
action of azacitidine and the HDAC inhibitor, panobinostat forclinical beneﬁt in acute myeloid leukemia patients. Clin Cancer
Res. 2014;20:724–35.
29. Tarhini AA, Lin Y, Yeku O, LaFramboise WA, Ashraf M, SanderC, et al. A four-marker signature of TNF-RII, TGF-alpha, TIMP-1
and CRP is prognostic of worse survival in high-risk surgicallyresected melanoma. J Transl Med. 2014;12:19.
30. Heemann C, Kreuz M, Stoller I, Schoof N, von Bonin F, Ziepert
M, et al. Circulating levels of TNF receptor II are prognostic forpatients with peripheral T-cell non-Hodgkin lymphoma. Clin
Cancer Res. 2012;18:3637–47.
31. Babic A, Shah SM, Song M, Wu K, Meyerhardt JA, Ogino S,
et al. Soluble tumour necrosis factor receptor type II and survival
in colorectal cancer. Br J Cancer. 2016;114:995–1002.
32. Warzocha K, Bienvenu J, Ribeiro P, Moullet I, Dumontet C,
Neidhardt-Berard EM, et al. Plasma levels of tumour necrosis
factor and its soluble receptors correlate with clinical features andoutcome of Hodgkin’s disease patients. Br J Cancer.
1998;77:2357–62.
33. Dobrzycka B, Terlikowski SJ, Kowalczuk O, Kinalski M. Circulating levels of TNF-alpha and its soluble receptors in theplasma of patients with epithelial ovarian cancer. Eur Cytokine
Netw. 2009;20:131–4.
34. Jones D, Dang NH, Duvic M, Washington LT, Huh YO. Absence of
CD26 expression is a useful marker for diagnosis of T-cell lymphoma in peripheral blood. Am J Clin Pathol. 2001;115:885–92.
35. Rappl G, Abken H, Muche JM, Sterry W, Tilgen W, Andre S,
et al. CD4 + CD7- leukemic T cells from patients with Sezary
syndrome are protected from galectin-1-triggered T cell death.
Leukemia. 2002;16:840–5.
36. Lee CS, Ungewickell A, Bhaduri A, Qu K, Webster DE, Armstrong R, et al. Transcriptome sequencing in Sezary syndrome
identiﬁes Sezary cell and mycosis fungoides-associated lncRNAs
and novel transcripts. Blood. 2012;120:3288–97.
37. Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D,
Trombetta JJ, et al. Dissecting the multicellular ecosystem ofmetastatic melanoma by single-cell RNA-seq. Science.
2016;352:189–96.
38. Torrey H, Defusco A, Baum D, Rhabar Z, Khodadoust M, Kim
YH et al. Novel treatment of cutaneous T cell lymphoma: targetting TNFR2 an oncogene and marker of potent Treg. Cancer
Research 78 (13 Suppl):1790–1790.
39. Wang L, Xiao N, et al. Genomic proﬁling of Sezary Syndrome
identifeis alternations of key T-cell singaling and differentiation
genes. Nat Genet. 2015;47:1426–34.
40. da Silva Almeida A, Abate F, Khiabanian H, et al. The mutationallandscape of cutaneous T cell lymphoma and Sezary syndrome.
Nat Genet. 2015;47:1465–70.
41. Choi J, Goh G, Walradt T. et al. Genomic landscape of cutaneous
T cell lymphoma. Nat Genet. 2015;47:1011–9.
42. Ni X, Jorgensen JL, Goswami M, Challagundla P, Decker WK,
Kim YH, et al. Reduction of regulatory T cells by Mogamulizumab, a defucosylated anti-CC chemokine receptor 4 antibody, inpatients with aggressive/refractory mycosis fungoides and Sezary
syndrome. Clin Cancer Res. 2015;21:274–85.
43. Nie Y, He J, Shirota H, Trivett AL, Yang, Klinman DM, et al.
Blockade of TNFR2 signaling enhances the immunotherapeutic
effect of CpG ODN in a mouse model of colon cancer. Sci Signal.
2018;11:eaan0790.