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J. Exp. Med. 2012 Vol. 209 No. 11 2113-2126
Current advances in T cell biology have chal
lenged the notion that differentiated CD4+
T cells are irreversibly hardwired to a particular
lineage as defined by the expression of specific
transcription factors and cytokines. It is now
clear that cellular microenvironments can pheno
typically and functionally redirect a T cell pop
ulation to different lineages (O’Shea and Paul,
2010). For example, recent evidence demon
strated that Foxp3+ regulatory T (T reg) cells
and Th17 cells are interconvertible (Zhou et al.,
2009). Even the more canonical Th1 and Th2
lineages can display unstable phenotypes (Hegazy
et al., 2010). Furthermore, contrary to their
traditionally defined roles, helper subsets have
been shown to attain direct cytolytic properties
(Brown, 2010). An important consequence of
the inherent plasticity of CD4+ T cells is that it
can be exploited to elicit more potent immuno
therapeutic effects such as in the context of
adoptive T cell transfer to treat malignancies.
An important challenge in mobilizing an
antitumor immune response is that the pre
cursor frequency of T cells recognizing tumor
antigens is very low (Moon et al., 2007; Rizzuto
et al., 2009). Therefore, supplementing the host
with tumorspecific T cells represents a logical
approach (Grupp and June, 2011). Although
extensive focus has been devoted to the study
of CD8+ T cells in adoptive transfer protocols
Jedd D. Wolchok:
Abbreviations used: CTX,
eomesodermin; GrzB, granzyme
B; TDLN, tumordraining LN;
ViD, viability dye.
T. Merghoub and J.D. Wolchok contributed equally
to this paper.
Induction of tumoricidal function in CD4+
T cells is associated with concomitant memory
and terminally differentiated phenotype
Daniel Hirschhorn-Cymerman,1 Sadna Budhu,1 Shigehisa Kitano,5
Cailian Liu,1 Feng Zhao,1 Hong Zhong,1 Alexander M. Lesokhin,1,2
Francesca Avogadri-Connors,1 Jianda Yuan,5 Yanyun Li,1
Alan N. Houghton,1,2,3 Taha Merghoub,1 and Jedd D. Wolchok1,2,4,5
1Swim Across America Laboratory, Immunology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065
2Weill Cornell Medical College and 3Graduate School of Medical Sciences of Cornell University, New York, NY 10065
4Ludwig Institute for Cancer Research, New York Branch, New York, NY 10065
5Ludwig Center for Cancer Immunotherapy at Memorial Sloan-Kettering Cancer Center, New York, NY 10065
Harnessing the adaptive immune response to treat malignancy is now a clinical reality.
Several strategies are used to treat melanoma; however, very few result in a complete
response. CD4+ T cells are important and potent mediators of anti-tumor immunity and
adoptive transfer of specific CD4+ T cells can promote tumor regression in mice and
patients. OX40, a costimulatory molecule expressed primarily on activated CD4+ T cells,
promotes and enhances anti-tumor immunity with limited success on large tumors in mice.
We show that OX40 engagement, in the context of chemotherapy-induced lymphopenia,
induces a novel CD4+ T cell population characterized by the expression of the master regu-
lator eomesodermin that leads to both terminal differentiation and central memory pheno-
type, with concomitant secretion of Th1 and Th2 cytokines. This subpopulation of CD4+
T cells eradicates very advanced melanomas in mice, and an analogous population of human
tumor-specific CD4+ T cells can kill melanoma in an in vitro system. The potency of the
therapy extends to support a bystander killing effect of antigen loss variants. Our results
show that these uniquely programmed effector CD4+ T cells have a distinctive phenotype
with increased tumoricidal capability and support the use of immune modulation in repro-
gramming the phenotype of CD4+ T cells.
© 2012 Hirschhorn-Cymerman et al. This article is distributed under the
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for the first six months after the publication date (see http://www.rupress
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The Journal of Experimental Medicine
Potent tumoricidal CD4+ T cells with novel phenotype | Hirschhorn-Cymerman et al.
in combination with CTX can promote transferred CD4+
T cells to durably regress very large tumors containing antigen
loss variants. Closer examination revealed that OX40 ligation
promoted the generation of highly cytolytic CD4+ T cells
with a unique phenotype characterized by expression of mark
ers of both terminal differentiation and memory, capable of
secreting both Th1 and Th2 cytokines. In sum, the linear im
pact of this work extends from demonstration of the potency
of optimally activated CD4+ T cells to the description of the
phenotype of this novel population of effector cells.
CTX and OX86 synergize with anti-tumor CD4+ T cells
to eradicate large established tumors
Previously, we showed that CTX in combination with OX40
engagement synergizes to regress established tumors (0.2–
0.4 cm in diameter; HirschhornCymerman et al., 2009).
Given that OX40 preferentially provides costimulation to
CD4+ T cells and CTX promotes lymphopenia, we hypothe
sized that the addition of tumorspecific CD4+ T cells would
induce regression of more advanced tumors. To this end, mice
bearing 3wkold B16 tumors (1.0–1.5 cm in diameter) were
injected with CTX. The next day, purified CD4+ T cells from
Trp1 transgenic mice (thereafter, Trp1 cells; Muranski et al.,
2008) were transferred along with the antiOX40 agonist
antibody OX86 or IgG as a control. We found that all mice
treated with the triple combination therapy (CTX + OX86 +
Trp1 cells) durably eradicated tumors 3 wk after treatment
(Fig. 1 A). Mice that received Trp1 cells, CTX, and IgG
showed only temporary tumor regression. Mice treated with
CTX and OX86 without Trp1 cells showed activity only in
the subset of mice bearing small tumors. Mice that received
CTX with IgG did not show any regression. The efficacy of
the therapy was largely dependent on the number of Trp1
cells infused (unpublished data). Moreover, the triple combi
nation therapy was effective in eliminating 3wkold JBRH
melanoma and radically decreasing the tumor burden of
TG3 and Grm1 spontaneous melanoma (unpublished data).
To further assess the potential clinical relevance of OX40
engagement in adoptive transfer protocols, we investigated
whether other forms of immune modulation could enhance
the antitumor potential of Trp1 cells. For that, we compared
OX86 with anti–CTLA4 (9D9), GITR (DTA1), CD40
(FGK45), and PDL1 (10F.9G2). Although anti–CTLA4 and
antiCD40 provided higher antitumor activity than controls,
use of OX86 yielded significantly higher efficacy by regress
ing tumors in all recipients. Overall, these results show that
adoptive transfer of antitumor CD4+ T cells synergizes with
CTX and OX40 engagement to provide lasting antitumor
immunity (Fig. 1 B).
Most adoptive transfer studies have focused on CD8+
T cells (Overwijk et al., 2003; June, 2007; Stromnes et al.,
2010). For this reason, we compared the antitumor activity of
CD8+ and CD4+ T cells in combination with CTX and OX86.
Groups of mice bearing 3wkold tumors were pretreated
with CTX and received equivalent numbers of Trp1 cells or
(Dudley et al., 2008; Rosenberg et al., 2008), CD4+ T cells
have several potential advantages. CD4+ T cells can help
orchestrate a global antitumor immune response by mobiliz
ing numerous components of the immune system (Hunder
et al., 2008; Muranski and Restifo, 2009). Furthermore, CD4+
T cells can acquire direct cytolytic activity under certain
conditions, such as lymphopenia (Quezada et al., 2010; Xie
et al., 2010).
It is well established that lymphopenia can enhance the
potency of adoptive T cell therapies (Wrzesinski and Restifo,
2005). Cytotoxic agents, such as cyclophosphamide (CTX),
induce lymphopenia and provide multiple immunomodula
tory effects beneficial for adoptive T cell transfer (North,
1982; Bracci et al., 2007). CTX can remove suppressive cell
populations (Awwad and North, 1988), sensitize tumor cells
for immune destruction (van der Most et al., 2009), release
tumor antigens and TLR agonists (Nowak et al., 2003;
Apetoh et al., 2007), and promote homeostatic proliferation of
transferred cells (Brode and Cooke, 2008).
Recent advances in immunotherapy have shown that
checkpoint blockade with CTLA4 and PD1 blocking anti
bodies have resulted in significant clinical benefit in a variety
of different malignancies (Brahmer et al., 2010; Wolchok et al.,
2010). CTLA4 blockade with ipilimumab produces an over
all survival benefit in patients with metastatic melanoma,
yet only 20–30% of patients seem to be sensitive to this inter
vention (Hodi et al., 2010; Robert et al., 2011). These recent
advances in immune modulation, particularly checkpoint block
ade with monoclonal antibodies, advocate for the incorpora
tion of novel strategies that target T cell costimulation.
OX40 is a costimulatory molecule belonging to the
TNFR family expressed primarily on activated effector T
(T eff) cells and naive T reg cells (Croft, 2010). Ligation of
OX40, primarily on CD4+ T cells, activates NFB and
upregulates antiapoptotic molecules from the Bcl2 family,
leading to T cell expansion, memory, activation, and cyto
kine secretion (Gramaglia et al., 2000; Rogers et al., 2001;
Redmond et al., 2009). Furthermore, OX40 engagement
on CD4+ Foxp3+ T reg cells leads to expansion, deactivation,
or cell death depending on the local milieu (Colombo and
Piconese, 2007; Vu et al., 2007; HirschhornCymerman et al.,
2009; Ruby et al., 2009). Given that OX40 engagement can
potently stimulate T cells and potentially inhibit/eliminate
T reg cells, OX40 agonists have been investigated in multiple
preclinical tumor models (Weinberg et al., 2000; Piconese
et al., 2008; Houot and Levy, 2009) and an anti–human
OX40 monoclonal antibody is currently being evaluated in
clinical trials (Clinical trial registration numbers NCT01303705
and NCT01416844; Weinberg et al., 2011). Despite this
potential, targeting OX40 alone or in combination with other
approaches has only shown effectiveness in preclinical models
with low tumor burden.
We hypothesized that programming tumorspecific CD4+
T cells with an agonist OX40 antibody in the context of
chemotherapyinduced lymphopenia would effectively treat
more advanced tumors. Here, we show that OX40 engagement
JEM Vol. 209, No. 11
Figure 1. CTX and OX86 synergize with Trp1 CD4 T cells to eradicate large established tumors. (A) C57BL/6 mice (6–10/group) were inoculated
intradermally in the flank with B16 cells. After 3 wk, mice were injected with CTX. The next day, mice were injected with OX86 (or IgG as control) with or
without Trp1 cells as indicated. Tumors were measured periodically. Top: graphs represent tumor area of individual mice over time for each treatment.
Middle: Kaplan-Meir overall survival curves. Bottom: representative photographs of mice treated with the triple combination therapy at day 0 or 21 after
treatment. (B) C57BL/6 mice (7–11/group) inoculated intradermally in the flank with B16 cells. After 3 wk, mice were injected with CTX. The next day, Trp1
cells were transferred to all mice and groups were injected with 1 dose of agonists Abs (OX40, GITR, or CD40) or three doses of antagonist Abs (CTLA-4 or
PD-L1) given 3 d apart. Tumor size was measured periodically. Graphs represent tumor area of individual mice over time for each treatment. (C) C57BL/6
mice (7–11/group) were inoculated intradermally in the flank with B16 cells. After 3 wk, mice were injected with CTX. The next day, Trp1 cells or Pmel-1
CD8+ T cells were injected and mice received OX86 (or IgG as control). Tumor size was measured periodically. Graphs represent tumor area of individual
mice over time for each treatment. (D) C57BL/6 mice (10/group) were inoculated intradermally in the flank with Hep-55.1C or Hep-55.1C-Trp1 cells.
On day 10, mice were injected with CTX. On day 11, mice received Trp1 cells and OX86 or IgG. Plots represent tumor area over time for each individual
mouse. **, P < 0.005. All experiments were repeated at least twice with similar results.
Potent tumoricidal CD4+ T cells with novel phenotype | Hirschhorn-Cymerman et al.
Trp1 T reg cells is partially FcR dependent. Levels of Trp1
T reg cells are significantly higher in tumors and TDLNs of
FcRdeficient hosts (unpublished data).
OX40 engagement promotes enhanced Trp1 cell lytic
function by up-regulation of eomesodermin (Eomes)
Several studies have shown that tumorspecific CD4+ T cells
can acquire cytotoxic properties (Quezada et al., 2010; Xie
et al., 2010). To assess if CTX and OX86 promote direct
lytic potential of Trp1 cells, we applied the combination
therapy to mice lacking mature T and B cells (Rag1/)
bearing B16 tumors. As shown in Fig. 3 A, the triple com
bination shows comparable efficacy in Rag1/ and wild
type hosts, suggesting that the transferred Trp1 cells directly
kill B16 without the need for induction of an additional
adaptive immune response. In addition, purified Trp1 cells
from tumors, TDLNs, and spleens of mice treated with the
combination therapy showed high ex vivo B16 cytotoxicity
(Fig. 3 B and not depicted). These results indicate that the
combination of CTX and OX86 enables Trp1 cells with
direct tumoricidal activity.
Cytotoxic effector cells typically kill through FAS, TRAIL,
or granzymeperforin–dependent mechanisms (Trapani and
Smyth, 2002). We sought to elucidate the primary mechanism
responsible for the lytic activity of the Trp1 cells after CTX
and OX86 combination therapy. For this, we measured surface
expression of FAS and TRAIL on Trp1 cells ex vivo and did
not find differences between OX86 treatment and control (un
published data). However, Trp1 cells showed higher levels of
CD107a and granzyme B (GrzB) in TDLNs, spleens, and
tumors when exposed to CTX and OX86 (Fig. 3, C and D; and
not depicted). Of interest, the endogenous CD4+ T cell popu
lation (CD45.1) showed elevated levels of GrzB expression
as well (Fig. 3 D). These results suggest that OX86 and CTX
promote the cytotoxic potential of not only adoptively trans
ferred CD4+ T cells but also endogenous CD4+ T cells.
The expression of GrzB and other lytic granules is under
the control of the effector master regulator Eomes (Pearce
et al., 2003). Recently, it has been reported that OX40 and
41BB engagement can induce the expression of Eomes on
CD4+ T cells as well as differentiation to a Th1 phenotype
(Qui et al., 2011). We therefore analyzed Eomes expression in
CD4+ T eff cells after OX86 and CTX treatment. As shown
in Fig. 3 E, a larger proportion of the Trp1 cells exposed to
CTX and OX86 expressed both GrzB and Eomes compared
with control. Interestingly, most GrzB+ cells were also Eomes+
suggesting that OX40mediated GrzB expression is under the
control of Eomes.
We then tested if similar induction of Eomes+ Trp1 cells
results from treatment with other clinically relevant immune
modulating antibodies. We treated mice with anti–CTLA4
(9D9), antiGITR (DTA1), antiCD40 (FGK45), and anti–
PDL1 (10F.9G2) in addition to OX86 or control IgG after
Trp1 cell transfer in tumorbearing hosts pretreated with
CTX. As shown in Fig. 3 F, only OX86 showed a substantial
increase of Eomes+ Trp1 cells.
CD8+ T cells from a TCR transgenic mouse specific for the
melanoma antigen gp100 (Pmel1 cells) followed by OX86
or rat IgG (Overwijk et al., 2003). Although OX86 improved
the antitumor efficacy of Pmel1 CD8+ T cells after CTX
administration, the combination with Trp1 cells showed much
higher antitumor activity (Fig. 1 C).
Given the potency of the combination therapy, we asked
whether antigen expression in tumor cells is necessary be
cause Trp1 cells can be activated by Trp1 protein expressed
in melanocytes. We applied the combination therapy to cohorts
of mice bearing established tumors from a hepatoma cell line
expressing Trp1 (Hep55.1CTrp1) or the parental cell line
(Hep55.1C). Fig. 1 D shows that the combination therapy
was only effective in mice bearing Hep55.1CTrp1, con
firming that antigen expression by the tumor is necessary for
Combination therapy increases Trp1 T eff/T reg cell ratio
by expanding T eff and reducing T reg cells via activation-
induced cell death
Tumors may also evade immune elimination by recruiting
and expanding CD4+ Foxp3+ T reg cells (Curiel, 2007). Strat
egies that eliminate T reg cells and, at the same time, increase
T eff cells are therefore highly desirable as they improve the
T eff/T reg cell ratio. Because a significant fraction of the
transferred Trp1specific transgenic cells express Foxp3 (Xie
et al., 2010), we measured how CTX and OX86 therapy
affects the Trp1 T eff/T reg cell ratio. Tumorbearing mice
were treated with CTX or PBS and, the next day, Trp1 cells
were injected along with OX86 or IgG. On day 14 after treat
ment (when tumors show signs of regression), we found ele
vated T eff/T reg cell ratios that were several hundred–fold
higher in both the tumordraining LNs (TDLNs) and tumors
in mice treated with CTX and OX86 compared with indi
vidual components of the combination therapy or control
(Fig. 2 A). This dramatic increase in T eff/T reg cell ratio was
caused by both profoundly decreasing the number of Trp1
Foxp3+ T reg cells (Fig. 2 D) and increasing Trp1 Foxp3 T eff
cells (Fig. 2 B).
Given our prior observation that the combination of
CTX and OX86 promotes T reg cell–specific activation
induced cell death (HirschhornCymerman et al., 2009), we
explored whether this mechanism is responsible for the
decrease of Trp1 Foxp3+ T reg cells. Fig. 2 E shows that a
larger percentage of Trp1derived T reg cells stained positive
for a viability dye (ViD) in both the tumors and TDLNs from
mice treated with the CTX and OX86 compared with con
trol. A majority of ViD+ Trp1 T reg cells costained with the
proliferation marker Ki67 (Fig. 2 F), indicating that Trp1 T reg
cells underwent activationinduced cell death. Although lev
els of Ki67 were high in Trp1 T eff cells treated with the
combination therapy (Fig. 2 C), we did not detect higher
apoptosis levels (not depicted), implying that activation
induced cell death was relatively specific for the T reg cell
subpopulation. Furthermore, we found that elimination of
JEM Vol. 209, No. 11
Figure 2. Combination therapy increases Trp1 T eff to T reg cell ratio by expanding T eff cells and reducing T reg cells via activation-
induced cell death. C57BL/6 mice (4–5/group) were injected subcutaneously in the flank with B16 cells in Matrigel. 3 wk after tumor challenge, CTX
or PBS was injected. The next day, Trp1 cells were transferred followed by injection of OX86 (or IgG as control). On day 14 after treatment, single cell
suspensions were prepared from TDLNs and tumors. Cells were stained and analyzed by flow cytometry. (A) Graph represents T eff/T reg cell ratio ± SEM
in tumors and TDLNs. *, P < 0.05, ^, P = 0.0599. (B) Representative plots showing CD45.1 (Trp-1 cells) versus CD4 for each group pregated on
ViD Foxp3. ViD is a viability dye (ViD: live cells). (C) Graph represents Ki67 MFIs ± SEM of CD45.1+ CD4+ population in TDLNs. **, P < 0.01.
(D) Representative plots of Foxp3 versus CD4 depicting the percentage of Trp1+ Foxp3+ in tumors and TDLNs. Events were pregated on ViD CD45.1+.
(E) The graph represents the percentage of dead Trp1 T reg cells (ViD+ in the Foxp3+ CD45.1+ gate) in tumors and TDLNs. *, P < 0.05; **, P < 0.01. Error
bars represent SEM. (F) Representative plots of activated dead Trp1 T reg cells ViD+ versus Ki67. Events were pregated on CD45.1+ Foxp3+. The experi-
ments were repeated at least five times with equivalent results.
To test if Eomes upregulation in Trp1 cells is necessary
for the antitumor efficacy of the combination therapy, we
knocked down Eomes expression in Trp1 cells by delivery of
an Eomes shRNAGFP encoding retrovirus. After 5 d in culture,
cells were FACS sorted for GFPhigh expression and analyzed by
flow cytometry. We found a profound reduction of Eomes and
GrzB, but not the related gene Tbet, in Eomes shRNA trans
duced cells compared with control retrovirus transduced cells
(Fig. 3 G). Functionally, Eomes shRNA transduced cells show
reduced B16 killing in an in vitro cytotoxicity assay (Fig. 3 H).
Potent tumoricidal CD4+ T cells with novel phenotype | Hirschhorn-Cymerman et al.
Figure 3. Direct cytotoxicity by Trp1 cells as a result of OX40 engagement during CTX-induced lymphopenia is Eomes dependent.
(A) C57BL/6 wild-type or Rag1/ mice (6–10/group) were inoculated intradermally in the flank with B16 cells. After 3 wk, mice were injected with
CTX, followed the next day by OX86 (or IgG as control) and Trp1 cells. Tumors were measured periodically. Graphs represent tumor area of individual
mice over time for each treatment. (B) C57BL/6 mice (10 mice/group) were injected subcutaneously with B16 in Matrigel. 3 wk after tumor challenge,
CTX was injected. The next day, mice were injected with Trp1 cells and OX86 (or IgG). On day 14 after treatment, Trp1 cells were purified by FACS from
splenocytes and were used for in vitro killing assays using B16 cells as targets at a 10:1 effector to target ratio. Means of three individual wells and
SEM are shown. *, P < 0.05. (C) Single cell suspensions of TDLNs and spleens from mice that were treated, as in B (8 mice/group), were stimulated
overnight with Trp1 peptide in the presence of monensin and anti–CD107a-FITC. The next day, the samples were stained for CD45.1 (Trp1 cells) and
other phenotypic markers and analyzed by flow cytometry. Plots show CD107a MFIs of individual mice gated on ViD CD45.1+ CD4+ Foxp3. *, P < 0.005;
**, P < 0.05. Error bars represent SEM. (D) Single cell suspensions of TDLNs and spleens from mice that were treated as in B (4–5 mice/group)
were stained and analyzed by flow cytometry for GrzB and other phenotypic markers on day 14 after treatment. Representative plots are shown for
GrzB versus CD45.1. Events were pregated on ViD, CD4+, and Foxp3. (E) C57BL/6 mice (7–10/group) were treated as in B and bled on day 14 after
treatment. PBMCs were stained for GrzB and Eomes and analyzed by flow cytometry. Events were pregated on ViD, CD45.1+, CD4+, and Foxp3.
JEM Vol. 209, No. 11
the tumor microenvironment. To this end, we incubated puri
fied Trp1 cells from tumors and TDLNs of treated mice in the
presence of APCs and Trp1 peptide. After 4 d in culture, the
supernatants were tested for the secretion of Th1, Th2, and
Th17 cytokines. Surprisingly, we found that Trp1 cells ex
posed to OX86 secreted higher levels of both Th1 (IL2,
IFN, and TNF) and Th2 (IL4, IL5, and IL13) cytokines
but produced insignificant levels IL17 (Fig. 4 B). A possible
explanation is that purified Trp1 cells could include sepa
rate populations of Th1 and Th2 cells. We therefore tested
if Trp1 cells can concomitantly secrete both sets of cytokines
by intracellular cytokine staining. We found that a large
proportion of Trp1 T eff cells secreted IFN and TNF
(Fig. 4 C–E) and the majority of cells secrete both cyto
kines simultaneously, consistent with a polyfunctional Th1
phenotype. Moreover, Trp1 T eff cells exposed to the combi
nation therapy also secreted IL4 (Fig. 4 F), with the majority
of cells secreting IL4 also secreting IFN (Fig. 4 G). Further
more, OX86 treatment reduced the levels of IL17–secreting
Trp1 T eff cells (Fig. 4 H). Overall, these results suggest
that CTX and OX86 promote Trp1 CD4+ T eff cells to se
crete both Th1 and Th2 cytokines while modestly inhibiting
a Th17 lineage.
Triple combination therapy promotes bystander killing
of antigen loss variants
Tumors may be populated by cells that lose expression of anti
genic proteins (Dunn et al., 2002; Spiotto et al., 2004). This
heterogeneity not only is a predominant mechanism to avoid
immune destruction but also renders immunotherapies that
target a single antigen eventually ineffective. For this reason, we
tested whether the combination of CTX and OX86 induces
Trp1 cells to eliminate tumors containing cells which do not
consistently express the cognate antigen (Trp1). We injected
groups of mice with a mixture of B16 melanoma cells and
B78H1, a B16 clone which does not express the Trp1 protein
(Graf et al., 1984). As controls, we injected mice with B16 or
B78H1 alone or both cell lines in different flanks, and the triple
combination therapy was applied (Fig. 5, scheme). We found
that the triple combination therapy eradicated pure B16
tumors, as expected, but was ineffective in treating B78H1
tumors (Fig. 5). Moreover, when B16 and B78H1 were injected
in different flanks on the same mouse, only B16 regressed
To establish if Eomes is necessary for the potency of the com
bination therapy, we injected OX86 and transferred Trp1 cells
transduced with Eomes shRNA or empty virus into Rag1/
mice bearing large established B16 tumors pretreated with
CTX. As shown in Fig. 3 I, mice treated with Trp1 cells
transduced with empty retrovirus showed significant tumor
regression, whereas mice treated with Eomes shRNA trans
duced Trp1 cells exhibited lower antitumor activity. Overall,
these results show that the combination of CTX and OX86
enable CD4+ T eff cells to acquire a cytotoxic phenotype, at
least in part, via Eomes.
CTX and OX86 promote the expression of terminal
differentiation and memory markers and the secretion
of both Th1 and Th2 cytokines in Trp1 cells
Cytotoxic CD4+ T cells are typically associated with markers
of terminally differentiated shortlived effectors (Brown,
2010). However, Eomes expression has recently been linked
to the formation of memory in CD8+ T cells (Banerjee et al.,
2010; Zhou et al., 2010). Moreover OX40 engagement on
CD4+ T cells can promote clonal expansion and memory
(Croft, 2010). To reconcile these potential disparities, we pheno
typed single cell suspensions from TDLNs of treated mice by
staining for markers associated with terminal differentiation
and memory. As shown in Fig. 4 A, Trp1 Foxp3 T eff cell
exposed to the combination therapy expressed higher levels
of Eomes (as expected) but also higher levels of Klrg1 and
lower levels of Bcl6, markers typically associated with
cytolytic potential/terminal differentiation (Belz and Kallies,
2010; Crotty et al., 2010; Fig. 4 A). Interestingly, the com
bination therapy also modulated the expression of markers
consistent with increased memory and less exhausted phe
notypes on Trp1 CD4+ Foxp3 T eff cell: CD127high,
Tbetlow, CD62Lhigh, and PD1low (Belz and Kallies, 2010;
Wherry, 2011; Fig. 4 A). A similar expression pattern was
observed in the endogenous CD4+ Foxp3 T eff cell pop
ulation (unpublished data). These results indicate that OX86
engagement can induce a unique CD4+ T cell population
to concomitantly acquire both cytolytic effector and mem
Because CD4+ T eff cells differentiate into distinct lin
eages with unique cytokine profiles, we examined how CTX
and OX86 influenced Trp1 T eff cytokine expression within
Representative plots are shown of GrzB versus Eomes. (F) C57BL/6 mice (4–5/group) were injected subcutaneously with B16 in Matrigel. 3 wk after tumor
challenge, CTX was injected. The next day, mice were injected with Trp1 cells and OX86, DTA-1, FGK45, 9D9, 10F.9G2, or IgG. Two additional doses of
9D9 and 10F.9G2 were given every 3 d. On day 14 after treatment, single cell suspensions were stained for Eomes and other phenotypic markers
and analyzed by flow cytometry. Events were pregated on ViD, CD45.1+, CD4+, and Foxp3. Representative plots are shown of GrzB versus Eomes.
(G) In vitro activated Trp1 cells were transduced with Eomes shRNA-GFP retrovirus or GFP retrovirus as control. Transduced cells were FACS sorted on
GFPhigh gate 5 d later. Cells were stained intracellularly for Eomes, GrzB, and T-bet and analyzed by flow cytometry. Representative histograms are
shown. (H) In vitro cytotoxicity assay with transduced Trp1 cells sorted on GFPhigh gate. B16 cells were used as targets at a 10:1 effector to target
ratio. Bars represent the means of duplicate wells and the SEM is shown. (I) C57BL/6 Rag1/ mice (6/group) were inoculated intradermally in the
flank with B16 cells. 3 wk later, CTX was injected followed the next day by the transfer of 70,000 transduced Trp1 cells (sorted on GFPhigh) and injec-
tion of OX86. Tumor area was periodically monitored. Each point represents the mean tumor area and error bars are SEM. *, P < 0.05. All experiments
were repeated at least twice with similar results.
Potent tumoricidal CD4+ T cells with novel phenotype | Hirschhorn-Cymerman et al.
Figure 4. CTX and OX86 promotes Trp1 cells to acquire terminal differentiation and memory phenotype with mixed Th1/Th2 cytokine
secretion. (A) C57BL/6 mice (4–5 mice/group) were injected subcutaneously in the flank with B16 in Matrigel. 3 wk after tumor challenge, CTX was
injected. The next day, Trp1 cells were transferred followed by injection of OX86 or IgG. On day 14 after treatments, single cell suspensions from
TDLNs were stained and analyzed by flow cytometry for Eomes, Klrg1, Bcl-6, T-bet, PD-1, CD127, and CD62L. Events were pregated on ViD CD4+
CD45.1+ Foxp3. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ^, P = 0.0632. Experiment was repeated at least three times with similar results. Error bars
represent SEM. (B) C57BL/6 mice (10 mice/group) were injected subcutaneously in the flank with B16 in Matrigel. 3 wk after tumor challenge, CTX was
injected. The next day, Trp1 cells were transferred followed by injection of OX86 or IgG. On day 14 after treatments, tumors from each group were
pooled and single cell suspensions were prepared. Trp1 cells were purified by MACS and co-cultured with irradiated APCs (pulsed with Trp1 peptide).
After 4 d, cytokines levels were measured in supernatants by cytokine bead arrays. Graphs show the means ± SEM of three individual wells per condi-
tion. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C–H) C57BL/6 mice (4–5 mice/group) were injected subcutaneously in the flank with B16 in Matrigel.
3 wk after tumor challenge, CTX was injected. The next day, Trp1 cells were transferred and injected with OX86 (or IgG). On day 14 after treatment,
single cell suspensions were prepared from tumors and incubated for 8 h in the presence of APCs pulsed with Trp1 peptide. The cells were stained and
analyzed by intracellular flow cytometric analysis. (C) Representative plot of IFN- versus CD45.1. Events were pregated on ViD CD4+ Foxp3.
(D) Representative plot of TNF versus CD45.1. Events were pregated on ViD CD4+ Foxp3. (E) Representative plot of TNF versus IFN-. Events were
pregated on ViD CD4+ Foxp3 CD45.1+. (F) Representative plot of IL-4 versus CD45.1. Events were pregated on ViD CD4+ Foxp3. (G) Representative
plot of IL-4 versus IFN- pregated on ViD CD4+ Foxp3 CD45.1+. (H) Representative plot of IL-17 versus CD45.1 pregated on ViD CD4+ Foxp3.
Experiments were repeated at least three times with similar results.
whereas B78H1 was unaffected. Interestingly, the majority of
B16:B78H1 chimeric tumors (7/8) regressed, indicating that
the combination therapy can eliminate tumors by triggering a
local bystander killing effect on tumor cells not expressing cog
nate antigen. This observation is of potential clinical impor
tance given tumor heterogeneity.
JEM Vol. 209, No. 11
of Tbet and PD1 (Fig. 6 C). These discrepancies could be a
result of differences in human OX40 signaling or in vitro cul
ture conditions. Overall, these results highlight the transla
tional potential of OX40 engagement in promoting and or
enhancing antitumor cytotoxic CD4+ T cells.
We have demonstrated that in vivo engagement of OX40
in the context of adoptive transfer of antigenspecific CD4+
T cells is a potent approach that can completely and durably
regress advanced B16 melanoma tumors. Our results show that
in this context, the OX40 agonist antibody provides superior
antitumor efficacy compared with other immunomodulatory
antibodies when combined with CTX and antigenspecific
CD4+ T cells. Under the same conditions, antigenspecific CD4+
T cells achieve higher potency than antigenspecific CD8+
T cells. The efficacy of this therapy was accompanied
by increasing Trp1 CD4+ T eff cells and reducing T reg
cells, resulting in a favorable T eff/T reg cell ratio within the
Several attempts have been made to engineer T cells from
patients in vitro to increase tumoricidal activity. Our results
imply that in vivo modulation with antibodies provides an
alternative in reprogramming transferred cells to improve
clinical outcomes. The availability of immunomodulatory
antibodies enhances the feasibility of this approach.
To confirm the clinical applicability of our findings, we
established an in vitro system in which the ability of OX40
engagement to increase the tumoricidal capability of human
antigenspecific T cells was tested. NYESO1–specific CD4+
T cell lines exposed to a human OX40 agonist increased their
ability to lyse autologous melanoma cells, supporting the
cytolytic phenotype observed in mouse models.
The selective pressure that the adaptive immune system
exerts on tumors leads to antigen downregulation (Dunn
et al., 2002; Spiotto et al., 2004). This important escape mech
anism poses a challenge when targeting a single antigen. We
found, however, that local destruction of tumor cells lacking
the target antigen (Trp1) is possible when potent immuno
therapies mediate sufficient bystander killing. Although the
precise mechanisms leading to the observed effect are cur
rently elusive, it is unlikely that systemic epitope spreading is
responsible for the regression of mixed B16:B78H1 tumors
because B78H1 tumors were not eliminated when injected
concomitantly with B16 at a distant site. We hypothesize
that Trp1 cells exposed to OX86 and CTX infiltrate tumors,
directly killing antigenexpressing cells and, at the same
time, promote a favorable local inflammatory milieu that
recruits innate cells eliminating antigen escape variants and/
or stromal cells.
Our results indicate that, under CTXinduced lympho
penic conditions, OX40 engagement promotes Trp1 cells as
well as endogenous CD4+ T cells to acquire a highly cyto
toxic phenotype by inducing Eomes. However, the endoge
nous CD4+ T cell population is not necessary for eliminating
tumors because regression could be recapitulated in mice
Human NY-ESO-1–specific CD4+ T cell lines exhibit
enhanced killing of melanoma cell lines when exposed
to OX40 agonist in vitro
We further asked if OX40 engagement could enhance the
tumoricidal activity of human CD4+ T cells. First, we set up
in an in vitro system to assess whether OX40 ligation is suffi
cient to enhance direct tumor cell killing by Trp1 cells. Trp1
cells activated with peptidepulsed APCs in the presence of
OX86 were subjected to an in vitro cytotoxicity assay and the
killing potential of cells treated with OX86 was increased
threefold (Fig. 6 A). With this system in place, CD4+ T cell
lines specific for NYESO1 derived from three patients with
advanced melanoma (unpublished data) were expanded with
platebound antiCD3 in the presence or absence of human
agonist OX40LFc protein. After 10 or 14 d in culture, the
CD4+ T cell lines were tested for cytotoxic potential using
patientmatched melanoma cell lines as targets. As shown in
Fig. 6 B, OX40 engagement significantly enhanced the cyto
toxicity of all NYESO1–specific CD4+ T cell lines to levels
equivalent to the Trp1 cells. Moreover, RTPCR analysis re
vealed a phenotype comparable to Trp1 cells, with the exception
Figure 5. Triple combination therapy promotes bystander tumor
killing of antigen loss variants. C57BL/6 mice (8/group) were inocu-
lated intradermally in the flank with B16 cells, B78H1, B16, and B78H1
in opposite flanks, or a B16:B78H1 mixture. 3 wk later, CTX was injected
followed the next day by treatment with Trp1 cells and OX86 as de-
scribed in the top diagram. Graphs represent tumor area of individual
mice over time for each treatment. Similar results were obtained in two
Potent tumoricidal CD4+ T cells with novel phenotype | Hirschhorn-Cymerman et al.
where OX40 and 41BB engagement were shown to drive
cytotoxic Th1 differentiation, here we show that secretion of
both Th1 and Th2 cytokines is a hallmark of our newly de
scribed CD4+ T cell population. This difference in CD4+
T cell phenotype may explain the superior antitumor efficacy
with the conditions described in our manuscript. Moreover,
we found that OX86 diminished Th17 and TFh (T follicular
helper) lineages, shown by lower levels of IL17 and down
regulation of Bcl6 (Johnston et al., 2009). Although Eomes
can inhibit RoRt, which could account for IL17 down
regulation (Ichiyama et al., 2011), the observation that Trp1
cells secrete both Th1 and Th2 cytokines under these condi
tions is perplexing. Given that Tbet represses Th2 cytokines
such as IL4 to promote Th1 polarization (Zhu et al., 2010),
it is feasible that Tbet downregulation overcomes IL4 sup
pression and high Eomes expression promotes IFN secre
tion. Moreover, several experiments in viral models where
CD4+ T cells were polarized into different helper lineages
(Th0, Th1, Th2, and Th17) show that cells with the Th0
phenotype exhibit higher cytotoxic potential (Brown, 2010).
We speculate that OX40 engagement drives Trp1 cells to
acquire a highly cytotoxic Th0like phenotype in vivo, char
acterized by Eomes expression. This suggests that OX40
engagement may induce a new effector Th lineage which
warrants further characterization.
lacking the capacity to generate adaptive immunity (Rag1/).
Moreover, the observation that CD4+ T cells with the newly
described phenotype have potent direct antitumor proper
ties suggests that this approach could be a means to potentiate
engineered T cells.
Given that Trp1 cells—in the context of OX40 engage
ment and lymphopenia—durably control advanced tumors,
we further characterized and defined the phenotypic “finger
print” of these uniquely potent cells. Cytotoxic CD4+ T cells
can develop under conditions of chronic antigen exposure
leading to a terminally differentiated phenotype (Brown,
2010). However, upon OX40 stimulation, Trp1 cells not only
acquire a terminally differentiated phenotype (Klrg1high
and Bcl6low) but also exhibit markers correlated with memory/
low exhaustion (CD62Lhigh, Tbet low, CD127high, and PD1low;
Belz and Kallies, 2010; Wherry, 2011). A recent study showed
that Eomes expression enables CD8+ T cells to acquire both
effector and memory/selfrenewal functions (Banerjee et al.,
2010). An intriguing hypothesis is that the inherent plastic
ity in both CD4+ and CD8+ T cells allows for the possibility
of developing even more potent cells for clinical use when
The cytokine profile of antitumor CD4+ T cells exposed
to the combination therapy reveals both Th1 and Th2 cyto
kine secretion. As opposed to other studies (Qui et al., 2011)
Figure 6. Human and mouse tumor-specific CD4+ T cells showed enhanced anti-tumor lytic activity with OX40 ligation in vitro. (A) Purified
Trp1 cells were incubated with APCs and Trp1 peptide and, 48 h after incubation, 10 µg/ml OX86 (or IgG as control) was added to the cultures. After 5 d,
the cells were subjected to in vitro cytotoxicity assay using B16 cells as targets at 10:1 effector to target ratio. Graphs show the mean percent killing of
duplicate wells. Error bars represent SEM. (B) NY-ESO-1–specific CD4+ T cell lines from three different patients were expanded with plate-bound anti-CD3
and IL-2 in the presence or absence of 10 ng/ml of recombinant human OX40L-Fc. After 10 or 14 d in culture (ptn 1 or 7 and 10, respectively), the CD4+
T cell lines were subjected to an in vitro cytotoxicity assays using patient-matched melanoma cell lines (SKMEL-ptn 1, 7, and 10) pulsed with NY-ESO-1
peptide pools as targets. Bars depict the means of percentage of targets killed of duplicate or triplicate wells/treatment. Errors bars represent SEM.
***, P = 0.0004. Experiments were repeated at least three times with similar results. (C) NY-ESO-1–specific CD4+ T cell lines (patient 7) were expanded with
plate-bound anti-CD3/CD28 and IL-2 in the presence or absence of 5 ng/ml of recombinant human OX40L-Fc. After 7 d, RNA was extracted and subjected
to RT-PCR for EOMES (Eomes), KLRG1 (KLRG1), TBX21 (T-bet), BCL6 (Bcl-6), PDCD1 (PD-1), IL7R (CD127), and SELL (CD62L). Graphs represent means of
triplicate wells/treatment. Error bars represent SEM. *, P < 0.05; ^, P = 0.056. Experiments were repeated at least twice with similar results in cell lines
derived from two patients.
JEM Vol. 209, No. 11
Percoll (GE Healthcare) gradient centrifugation as described in Hirschhorn
Cymerman et al. (2009). Cells from TDLNs and tumors were prepared
by mechanical dissociation in 40 µM filters and red blood cells were removed
by incubation in ACK Lysing Buffer (Lonza). For CD107a mobilization assay,
lymphocytes from TDLNs and spleens were incubated at 37°C with 2.5 µg/ml
of Trp1 peptide in HTM medium (RPMI 1640 supplemented with 10%
FCS, 1× nonessential amino acids, 1 mM sodium pyruvate, 2 mM lglutamine,
and 50 µM mercaptoethanol) overnight with 10 µg/ml of monensin (Sigma
Aldrich) and 1 µl of CD107a FITC. For intracellular cytokine staining, lym
phocytes from tumors were incubated at 37°C with 2.5 µg/ml of Trp1
peptide in HTM medium for 8 h with 10 µg/ml monensin and 1X GolgiPlug
(BD). Before staining, cells were treated with saturating antiCD16/CD32
(BD) in staining buffer (2% bovine serum albumin and 2 mM EDTA in PBS)
on ice for 15 min. Staining of surface antigens was performed in staining
buffer on ice for 40 min. All intracellular staining was conducted using the
Foxp3 fixation/permeabilization buffer (eBioscience) according to the man
ufacturer’s instructions. Flow cytometry was performed on an LSRII (BD).
FlowJo software (version 9.4.10; Tree Star) was used for all flow cytometry
analysis. FACS sorting was conducted on a FACSAria II cell sorter (BD).
Peptides. Trp1 peptide (Muranski et al., 2008) was synthesized by Genemed
Synthesis, Inc. Overlapping NYESO1 peptides (20mers overlapping by
10 peptides), were purchased from JPT Peptide Technologies.
Retrovirus construction and transduction. Four candidate Eomes
shRNA sequences and control psHIVNH1 lentivirus backbone were ob
tained from GeneCopoeia with the following specific sequence: Eomes1,
5ACAAAGCGTCCAAGAAGTT3; Eomes2, 5CAAAGCGGACAAT
AACATG3; Eomes3, 5GCACTCTCTGCACAAATAC3; Eomes4,
5CTGTGACCAACAAGCTAGA3; or scrambled shRNA. The psHIVNH1
lentivirus plasmids were cotransfected with Fugene 6 (Roche) with full
length Eomes in MigR1 backbone (gift of S. Reiner, University of Pennsyl
vania, Philadelphia, PA) into HEK 293 cells in 6well plates. After 48 h, the
cells were fixed and permeabilized with Cytofix/Cytoperm (BD), stained for
Eomes, and analyzed by flow cytometry. shRNA Eomes4 sequence almost
entirely inhibited Eomes expression and was further used for subsequent
experiments. Because of our inability to effectively transduce activated CD4+
T cells with a lentivirus, the shRNA Eomes4 sequence was subcloned into
MigR1 retrovirus backbone and was called Eomes shRNAGFP thereafter.
Transduction of Trp1 cells was performed as described by Lee et al. (2009).
In brief, purified CD4+ T cells from TDLNs and spleens of Trp1 mice were
activated with T cell–depleted, irradiated splenocytes (APCs) pulsed with
2.5 µg/ml of Trp1 peptide at 1:3 ratio in HTM media supplemented
with 50 U/ml of recombinant human IL2 (Roche). After 2 d in culture,
the activated CD4+ T cells were inoculated with high titer virus solution of
Eomes shRNAGFP or empty MigR1 by centrifugation (3,500 RPM, 24°C,
60 min) in sterile non–tissue culture plates treated with 20 µg/ml of plate
bound RetroNectin (Takara Bio Inc.). The next day, the cells were subjected
to a second inoculation. After 4 d, the cells were FACS sorted on GFPhigh
DAPIlow population and either analyzed by flow cytometry, used as effectors
in a cytotoxicity assay, or used in adoptive transferred experiments.
In vitro expansion of Trp1 cells and NY-ESO-1 CD4+ T cell lines.
NYESO1 CD4+ T cell lines developed from three patients diagnosed with
Stage IV melanoma were provided by S. Kitano. Approximately 1 × 105 cells
were expanded with 1 µg/ml of platebound antiCD3 with or without
antiCD28 at 1 µg/ml (BD) in Xvivo 15 Media (BioWhittaker) supple
mented with 10% heat inactivated human serum (Gemini Bioproducts) and
30 U/ml IL2 (Roche). Some wells were treated with 10 ng/ml of recombi
nant human OX40LFc (PeproTech). After 10 or 14 d, the expanded cells
were subjected to in vitro cytotoxicity assays.
Trp1 cells were purified as described and 1 × 105 cells were incubated
with 3 × 105 irradiated T cell–depleted splenocytes pulsed with 2.5 µg/ml of
Trp1 peptide in HTM media. After 48 h, 10 µg/ml OX86 or IgG was added to
the cultures. After 4 d, the cells were subjected to in vitro cytotoxicity assays.
Overall, our observations highlight the clinical potential
of OX40 engagement in promoting a cytotoxic CD4+ T cell
population with unique phenotypic markers. As adoptive
transfer of designer or engineered cells becomes more feasible,
optimizing their antitumor potential is of clear importance.
The ability to modulate cells with sufficient lineage plasticity
in vivo with a single monoclonal antibody opens a new ave
nue and overcomes many of the challenges encountered by
adoptive T cell therapy protocols.
MATERIALS AND METHODS
Mice and tumor cell lines. All mouse procedures were performed in
accordance with institutional protocol guidelines at Memorial SloanKettering
Cancer Center (MSKCC) under an approved protocol. C57BL/6J or
C57BL/6J Rag1/ (8–10wkold males) were obtained from The Jackson
Laboratory. Pmel1 TCR transgenic mice (Overwijk et al., 2003) and Trp1
CD4+ TCR transgenic mice (Muranski et al., 2008) were obtained from the
N. Restifo laboratory (National Institutes of Health, Bethesda, MD). Trp1
CD4+ TCR transgenic were crossed to Rag1/ Trp1/ CD45.1 back
ground. All mice were bred at MSKCC. The B16F10 mouse melanoma line
was originally obtained from I. Fidler (M.D. Anderson Cancer Center, Hous
ton, TX). B78H1 was obtained from A. Albino (SloanKettering Institute).
Inoculation with 105 B16F10 or 106 B78H1 cells was determined to be
a lethal dose when injected intradermally in the flank (50 µl/injection).
In experiments where B16F10 and B78H1 were coinjected, the lethal dose
for each tumor was mixed in the same volume. For experiments where isolation
of tumor lymphocytes was performed, B16Matrigel was subcutaneously
injected (105 B16F10 cells in 0.2 ml of Matrigel Matrix Growth Factor
Reduced; BD). Parental Hep 55.1C (Kress et al., 1992) was a gift from
M. Schwarz (University of Tübingen, Tübingen, Germany). Hep 55.1CTrp1
were prepared by transfection with pCRANmgp75 and stable Trp1
expressing clones were selected after treatment with 1 mg/ml G418 (Gibco).
Trp1 expression was confirmed by Western blotting. 1 × 105 cells were
titrated to be a lethal dose.
Monoclonal antibodies, drug treatment, and adoptive transfer
experiments. OX86 (antiOX40; alShamkhani et al., 1996), FGK45 (anti
CD40; Rolink et al., 1996), and DTA1 (antiGITR; Ko et al., 2005) were
produced and purified by the Monoclonal Antibody Core Facility at
MSKCC. 9D9 (anti–CTLA4; Quezada et al., 2006) and 10F.9G2 (anti–
PDL1; Eppihimer et al., 2002) were purchased from BioXcell. Anti–rat IgG
(SigmaAldrich) was reconstituted in sterile PBS before injection. For all
treatments, a single dose of 0.5 mg OX86, 0.5 mg rat IgG, 0.25 mg FGK45,
and 1 mg DTA1 was injected. Three doses of 0.1 mg 9D9 and 0.2 mg
10F.9G2 were administered every 3 d. CTX monohydrate (SigmaAldrich)
mixed in sterile PBS was administered as a single dose at 250 mg/kg. Both
agents were administered intraperitoneally. Trp1 CD4+ T cells (Trp1 cells) or
Pmel1 CD8+ T cells were purified from LN and spleen by positive selection
magnetic cell sorting using CD4 beads (L3T4) or CD8 (Lyt2; Miltenyi
Biotech) according to the manufacturer’s instructions. Unless stated, 1 × 105
purified Trp1 T cells or Pmel1 CD8+ T cells were tail vein injected.
FACS analysis and cell sorting. The following antibodies were used for
flow cytometry analysis: CD4 Pacific blue, Ki67 FITC, CD107a FITC, IL17
PE, TNF APC, and PE–Texas red Streptavidin were obtained from BD. PD1
Biotin, Eomes PE, Tbet PercpCy5.5, CD45.1 APC–eFluor 780, CD62L
PerCPCy5.5, Foxp3 APC, Foxp3 FITC, Klrg1 PECy7, CD127 PECy7,
Bcl6 PE, IFN PerCPCy5.5, and IL4 PECy7 were obtained from
eBioscience. GrzB PETX and ViD (LIVE/DEAD Fixable Aqua Dead Cell
Stain kit) were obtained from Invitrogen. All antibodies were used according
to the manufacturer’s instructions with the recommended buffers.
Lymphocytes from tumors were prepared by digestion in 1× Liberase/
DNase (Roche) solution in plain RMPI 1640 for 30 min at 37°C. After pass
ing the solution through a 40 µM filter, live lymphocytes were isolated using
Potent tumoricidal CD4+ T cells with novel phenotype | Hirschhorn-Cymerman et al.
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Measurement of cytokines produced by Trp1 cells from treated
mice ex vivo. Mice were treated with the combination therapy as described
previously. Single cell suspensions from tumors were incubated on ice with
CD45.1 PE antibody (BD) for 15 min. Trp1 cells (CD45.1+) were further
purified by with antiPE MicroBeads (Miltenyi Biotec) according to the
manufacturer’s instructions. Purified Trp1 cells (5 × 104) were incubated with
1.5 × 105 irradiated T cell depleted splenocytes with 2.5 µg/ml of Trp1 pep
tide in 200 µl HTM media. After 4 d, supernatants were assayed for Th1, Th2,
and Th17 cytokines using the mouse Th1/Th2 10plex FlowCytomix Multi
plex (eBioscience) according to the manufacturer’s instructions.
Collagen I–fibrin gel in vitro cytotoxicity assay. Using a clonogenic assay
to asses melanoma cell killing, Budhu et al. (2010) showed that collagenfibrin
gels were 5,000× more sensitive in detecting cytotoxicity then conventional
CTL killing assays. Therefore, we used collagenfibrin gels along with the clo
nogenic assay to examine killing of melanoma cells by Trp1 cells and human
NYESO1 CD4+ T cell. Collagenfibrin gels were formed, incubated, and
lysed, and their contents of melanoma cells assayed exactly as previously de
scribed (Budhu et al., 2010). Target melanoma cells (with or without CD4+
T cells) were coincubated in PBS containing 1 mg/ml human fibrinogen,
1 mg/ml rat tail collagen I, 10% FBS, and 0.1 U human thrombin. The gels
were formed by incubating this mixture at 37°C for 20 min and then were
overlaid with HTM media. 24 h later, the gels were lysed by sequential collage
nase (2.5 mg/ml) and trypsin (2.5 mg/ml; SigmaAldrich) digestion. The lysed
gels were then diluted and the recovered melanoma cells were plated in 6well
plates for colony formation. After 7–21 d in culture, the plates were fixed with
formaldehyde, stained with 2% methylene blue, washed, dried, and the colonies
were counted manually. A 10:1 effector to target ratio was used in all experi
ments. For the preparation of targets, B16 cells were incubated overnight with
10 ng/ml of recombinant IFN (PeproTech) and single cell suspension were
prepared using Cellstriper (Cellgro) before the assay. Human melanoma cell
lines derived from three patients were incubated overnight with recombinant
10 ng/m IFN and single cell suspension was prepared with Cellstriper.
To increase sensitivity the melanoma cells were pulsed with 10 µg/ml of
NYESO1 peptides for 40 min at 37°C. The percentage of target cells killed was
calculated using the equation: 1 [melanoma + T cells]/[melanoma alone].
RT-PCR. Total RNA was extracted from CD4 T cell lines with the RNeasy 96
kit (QIAGEN) and cDNA synthesized using High Capacity cDNA Reverse
Transcription kit (Applied Biosystems) according to manufacturer’s instructions.
All primers and probes were from TaqMan Gene Expression Assays (Applied
Biosystems). Realtime PCR reactions were prepared with 3 µl cDNA according
to the manufacturer’s instructions. All amplifications were done using the ABI
7500 Real Time PCR system (Applied Biosystems). Each gene was amplified in
triplicate and cDNA concentration differences were normalized to GAPDH.
Relative gene expression of the target genes were shown by 2 Ct (Ct =
Ct(target gene) Ct(GAPDH)) using mean Ct (threshold cycle) of triplicates.
Statistical analysis. Statistical differences between experimental groups
were determined by the twotailed Student’s t test and Logrank test using
Prism software (GraphPad Software).
The authors wish to thank S. Terzulli for assistance in the creation of this
manuscript and S.L. Reiner, G.A. Rizzuto, J.C. Sun, and A.M. Beaulieu for their helpful
critical comments. We would like to thank A. Burey, X. Yang, and the members of
the immunomonitoring facility at MSKCC for technical support. We also would like
to thank the members of the Wolchok\Houghton Laboratory at MSKCC.
This work was supported by grants from the National Cancer Institute (R01
CA56821, P01 CA33049, and P01 CA59350), Swim Across America, the Lita
Annenberg Hazen Foundation, the T.J. Martell Foundation, the Mr. William
H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Cancer Foundation for
Research, and the Experimental Therapeutics Center of MSKCC.
The authors have no conflicting interests.
Submitted: 8 March 2012
Accepted: 27 August 2012
JEM Vol. 209, No. 11
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