Download Chimeric NKG2D–Modified T Cells Inhibit Systemic T

Research Article
Chimeric NKG2D–Modified T Cells Inhibit Systemic T-Cell
Lymphoma Growth in a Manner Involving Multiple
Cytokines and Cytotoxic Pathways
Tong Zhang, Amorette Barber, and Charles L. Sentman
Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire
Abstract
In this study, the efficacy and mechanisms of chimeric NKG2D
receptor (chNKG2D)–modified T cells in eliminating NKG2D
ligand–positive RMA/Rae1 lymphoma cells were evaluated.
Intravenous injection of RMA/Rae1 cells led to significant
tumor formation in spleens and lymph nodes within 2 weeks.
Adoptive transfer of chNKG2D-modified T cells after tumor
injection significantly reduced tumor burdens in both spleens
and lymph nodes, and prolonged the survival of tumorbearing mice. Multiple treatments with chNKG2D T cells
resulted in long-term tumor-free survival. Moreover, these
long-term survivors were resistant to rechallenge with RMA
tumor cells (NKG2D ligand–negative), and their spleen and
lymph node cells produced IFN-; in response to RMA but not
to other tumors in vitro, indicating immunity against RMA
tumor antigens. ChNKG2D T cell–derived IFN-; and granulocyte-macrophage colony–stimulating factor, but not perforin
(Pfp), tumor necrosis factor–related apoptosis-inducing ligand, or Fas ligand (FasL) alone were critical for in vivo
efficacy. T cells deficient in both Pfp and FasL did not kill
NKG2D ligand–positive RMA cells in vitro. Adoptive transfer of
Pfp/FasL/ chNKG2D T cells had reduced in vivo efficacy,
indicating that chNKG2D T cells used both mechanisms to
attack RMA/Rae1 cells. Taken together, these results indicate
that chNKG2D T-cell–mediated therapeutic effects are mediated by both cytokine-dependent and cytotoxic mechanisms
in vivo. [Cancer Res 2007;67(22):11029–36]
Introduction
Immunotherapy has the potential to provide a highly selective
means to control cancer in patients. Adoptive transfer of tumor
antigen–specific T cells has shown promise in the treatment of
cancers (1, 2). One of the major hurdles to successful T cell
adoptive therapy is the difficulty in isolation and expansion of large
numbers (>109) of antigen-specific T cells (2, 3). To overcome this
problem, alternative strategies to obtain a large number of tumorspecific T cells by genetic modification of T cells with immunoreceptors [such as T cell receptor (TCR) or single-chain antibody
(‘‘T body’’)] have been developed (4–8).
A recent clinical trial showed that adoptive transfer of genetically engineered T cells with tumor-specific TCRs could lead to
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Charles L. Sentman, Department of Microbiology and
Immunology, Dartmouth Medical School, 6W Borwell Building, One Medical Center
Drive, Lebanon, NH 03756. Phone: 603-650-8007; Fax: 603-650-6223; E-mail:
[email protected].
I2007 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-2251
www.aacrjournals.org
cancer regression in some patients (8). However, several factors,
such as incorrect pairing between endogenous and exogenous TCR
molecules, variable signaling capacity, MHC polymorphism, and
down-regulation of MHC on target cells might limit the use of
TCRs as immunotherapy (4–6). We previously described a strategy
to genetically modify T cells with a chimeric natural killer (NK)
receptor that comprises of a NK-activating receptor, NKG2D, linked
to a gene encoding signaling domains of CD3~ (9, 10). Murine
or human T cells expressing such a chimeric receptor (chNKG2D)
can recognize NKG2D ligand–positive tumor cells, become directly
activated, secrete cytokines, and kill tumor cells.
A variety of effector mechanisms have been shown to be
involved in immune responses against tumors. Direct cytotoxicity
of tumors by NK cells or CTLs using perforin and/or FasL are
important mechanisms for tumor elimination (11–13). In studies
using spontaneous or transplantable tumors in perforin or FasLdeficient mice, there were higher incidences of tumors suggesting
important roles for these host-derived molecules in the rejection of
tumors. The requirement for donor T cell–derived perforin or FasL
for therapeutic efficacy in cell therapy models is still not wellestablished. CTL-derived cytokines, especially Th1 cytokines, can
also contribute to their antitumor effects by either stimulating
the host immune system or directly inhibiting tumor cell growth
(14–17). IFN-g production by type 1 CD8+ T cells (Tc1) have been
shown to be critical for Tc1-mediated therapeutic effects (18, 19).
Thus, both cytotoxic effector pathways and cytokines may be
involved in mediating protective effects against tumor growth.
In a subcutaneous RMA lymphoma model, we showed that
adoptive transfer of chNKG2D-bearing T cells at the tumor site
inhibited NKG2D ligand–bearing RMA (RMA/Rae1) tumor growth
(9). Moreover, mice that had remained tumor-free were resistant to
subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor antigens (9, 20).
In this study, we examined the therapeutic effects of chNKG2D
T cells in a systemic lymphoma model using RMA/Rae1 cells given to
mice i.v. The cytotoxic pathways and effector cytokine mechanisms
responsible for therapeutic efficacy were also determined using
T cells with specific deficiencies in perforin, FasL, tumor necrosis
factor–related apoptosis-inducing ligand (TRAIL), IFN-g, or granulocyte-macrophage colony–stimulating factor (GM-CSF).
Materials and Methods
Mice. C57BL/6 (B6, wild-type, wt) mice were purchased from the
National Cancer Institute. IFN-g–deficient mice B6.129S7-Ifngtm1Ts /J
(IFN-g/), Fas ligand–deficient mice B6Smn.C3-Faslgld /J (FasL/),
and perforin-deficient mice C57BL/6-Prf1tm1Sdz /J (Pfp/) were obtained
from the Jackson Laboratory. GM-CSF knockout mice (GM-CSF/) were
obtained from Dr. Jeff Whitsett of the University of Cincinnati. TRAIL
knockout mice (TRAIL/) were obtained from Dr. Jacques Peschon
(Amgen, Seattle, WA). A mouse strain deficient for perforin and FasL
11029
Cancer Res 2007; 67: (22). November 15, 2007
Cancer Research
(Pfp/FasL/) was generated by crossing Pfp/ mice with FasL/
mice. The genotypes of the mice were determined by PCR of tail DNA. The
primers for genotyping Pfp/ mice are p1, 5¶-GCTATCAGGACATAGCGTTGG-3¶, 5¶-GGAGGCTCTGAG ACAGGCTA-3¶; and p3, 5¶-TACCACCAAATGGGCCAAG-3¶. The PCR products for Pfp/ and Pfp+/+ mice are 250 and
187 bp, respectively. The primers for genotyping FasL mutation (FasL/)
mice are f1, 5¶-TGATCAATTTTGAGGAATCTAAGGCC-3¶ and f2, 5¶-ATAGGTCTTAAGAAGACTCTCATTCAAG-3¶. The reactions for both PCRs were
as follows: 95jC for 3 min, followed by 35 cycles of 95jC for 30 s, 56jC for
30 s, and 72jC for 30 s, ending with 72jC for 3 min. To identify the presence
or absence of the FasL mutation in FasL/ mice, 9 AL of the PCR product
was digested with 10 units of StuI (NEB) for 3 h at 37jC. The resulting
product was run on a 3% agarose gel, and the presence or absence of bands
at 112 and 136 bp indicated the presence of a wild-type FasL allele and a gld
mutant FasL allele, respectively. All experiments were conducted according
to protocols approved by Dartmouth College’s Institutional Animal Care
and Use Committee.
Tumor cell lines. A RMA subline RMA/RG that coexpresses a NKG2D
ligand, Rae1h and green fluorescent protein (GFP), was generated by
retroviral transduction using dualtropic retroviral vectors containing the
rae1b and gfp genes according to our previous protocol (9). All cells were
grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine
serum (Hyclone), 100 units/mL of penicillin, 100 Ag/mL of streptomycin,
1 mmol/L of pyruvate, 10 mmol/L of Hepes, 0.1 mmol/L of nonessential
amino acids, and 50 Amol/L of 2-mercaptoethanol.
Flow cytometry. For fluorescence-activated cell sorting analysis of Rae1
expression, tumor cells were stained with APC-conjugated anti-Rae1 (R&D
Systems). Cell surface phenotyping of transduced primary T cells was
determined by staining with APC-conjugated anti-CD3q (clone 145-2C11;
PharMingen), PE-conjugated anti-NKG2D (clone A10; eBioscience) and
FITC-conjugated anti-CD8 (clone CT-CD8a; Invitrogen) monoclonal antibodies. To detect intracellular IFN-g production by T cells, spleen cells
(3 106) from naı̈ve B6 or tumor-surviving mice (125 days after tumor
injection) were cultured with irradiated (120 Gy) RMA or ID8 cells (20:1) for
24 h. Intracellular IFN-g staining was done as in our previous study using
PE-conjugated anti–IFN-g (clone XMG12; eBioscience) or PE-conjugated rat
IgG1 isotype control (eBioscience; ref. 21). Surface markers of T cells were
analyzed using FITC-conjugated anti-CD8b (clone CT-CD8b; eBioscience)
and APC-conjugated anti-CD4 (clone RM4-5; BD Biosciences). All samples
were preincubated with FcR block antibody (antimouse CD16/CD32, clone
93; eBioscience) to reduce nonspecific staining. Cell fluorescence was
monitored using a FACSCalibur cytometer (Becton Dickinson).
Cytotoxicity assays. Cytotoxicity of chNKG2D-bearing T cells against
target cells was determined by 5-h 51Cr release assays as previously
described (9). To block perforin-mediated cytotoxicity, T cells were
pretreated with concanamycin A (CMA, 20 nmol/L; Sigma) for 2 h before
an equal volume of target cells were added to cocultures.
Treatment of mice with genetically modified T cells. Retroviral
transduction of murine primary T cells was done using ecotropic viruses, as
we have previously described (9). C57BL/6 mice were injected with various
numbers (2–5 106) of RMA/RG cells via tail veins in 400 AL of PBS. For
treatment with T cells, mice were administered i.v. with 7.5 106 of
wtNKG2D- or chNKG2D-modified T cells 2 days after tumor injection.
Genetically modified T cells (6–7 days posttransduction) were harvested,
washed, and resuspended in cold PBS before injection. Adoptively
transferred T cells were a mixture of CD8+ (70–90%) and CD4+ (10–30%)
T cells. For treatment with three doses of T cells, 5 106 T cells were given
i.v. on days 2, 6, and 10 after tumor injection. For determination of tumor
burdens in tumor-bearing mice, spleens and lymph nodes (axillary, brachial,
and inguinal) were collected 10 or 13 days after tumor injection. The
lymphoid tissues were mechanically teased and mashed. RBC were lysed
with ACK lysis buffer [0.15 mol/L NH4Cl, 1 mmol/L KHCO3, 0.1 mmol/L
EDTA (pH 7.3)]. The number of cells was counted, and the percentage of
GFP+ cells was determined by flow cytometry. The absolute number of
tumor cells in these tissues was determined by multiplying the percentage
of GFP+ cells by the number of total cells. For survival studies, mice were
monitored closely and sacrificed when moribund signs were observed.
Cancer Res 2007; 67: (22). November 15, 2007
Tumor rechallenge. Mice that survived for 75 days without signs of
sickness were regarded as tumor-free and then were inoculated s.c. with 104
wild-type RMA cells on the shaved right flank. Naı̈ve B6 mice were used as
controls. Tumor size was monitored every 2 days, and mice were sacrificed
when tumor burden became excessive.
IFN-; production in secondary stimulation cultures. Coculture of 106
lymph node or spleen cells from naı̈ve or tumor-surviving mice with 105
RMA or ID8 tumor cells was done in 48-well plates in 0.75 mL of complete
medium. Tumor cells were irradiated (120 Gy) before use. Cell-free
conditioned media were collected after 5 days and assayed for IFN-g by
ELISA using mouse Duoset ELISA kits (R&D Systems) according to the
instructions of the manufacturer.
Statistical analysis. Differences between groups were analyzed using
Student’s t test or ANOVA. P < 0.05 were considered significant. KaplanMeier survival curves were plotted and analyzed using Prism software
(GraphPad Software).
Results
Intravenous injection of RMA cells leads to systemic
lymphoma. Lymphoma is characterized by the infiltration of
tumor cells in lymph nodes, spleens, and other lymphoid tissue. We
Figure 1. Intravenous injection of RMA/RG cells leads to tumors in lymphoid
organs. Ten and 13 d after i.v. injection of either 2 or 5 106 of RMA/RG cells,
GFP+ tumor cells in spleens (A) and lymph nodes (LN, B ) were determined
by flow cytometry as described in Materials and Methods (n = 4). C, GFP+ tumor
cells in bone marrow (BM ) and thymus were also determined at day 15 from
mice given 2.5 106 of RMA/RG tumor cells (n = 3). Numbers shown are
mean F SE. Bars represent GFP positive cells.
11030
www.aacrjournals.org
Mechanisms of ChNKG2D Receptors against Lymphoma
Figure 2. Treatment with chNKG2D T cells
reduces tumor burden in a dose-dependent
manner. Mice that were inoculated i.v. with
2.5 106 RMA/RG cells at day 0 were treated
with either wtNKG2D (wt ) or chNKG2D (ch ) T cells
via the i.v. route for a single dose (7.5 106 on
day 2) or three doses (5 106 on days 2, 6,
and 10). Both percentages (A and C ) and absolute
numbers (B and D) of GFP+ cells in spleens
(A and B ) and lymph nodes (C and D) were
determined at day 13 as described in Materials and
Methods. Significance was determined by ANOVA.
used a systemic lymphoma model to mimic the clinical disease
with widespread distribution of lymphoma cells into these
lymphoid organs. To achieve this goal, we used a lymphoma cell
line RMA/RG which expressed both a NKG2D ligand Rae1 and GFP,
and injected the tumor cells i.v. into mice. Flow cytometry was
done to determine the percentage of GFP+ tumor cells as well as
the absolute numbers of tumor cells in various organs. As shown in
Fig. 1A and B, 10 days after i.v. injection of 2 or 5 106 RMA/RG
cells, GFP+ tumor cells could be readily detected in the spleen. Mice
that were injected with 5 106 RMA/RG cells had larger tumor
burdens than those which were injected with 2 106 tumor cells.
The numbers of tumor cells increased rapidly thereafter. Thirteen
days after injection of 5 106 RMA/RG cells, the percentages of
tumor cells increased by >20-fold in spleens ( from 1.2% to 27.8%)
and 70-fold ( from 0.025% to 1.95%) in lymph nodes, compared with
that at day 10. A similar trend was observed in mice that were
injected with 2 106 RMA/RG cells. Besides spleens and lymph
nodes, RMA/RG cells also infiltrated bone marrow, thymus
(Fig. 1C), and liver (data not shown).
ChNKG2D-modified T cells significantly reduce NKG2D
ligand–positive tumor burden and promote long-term survival of mice in a systemic lymphoma model. To determine the
efficacy of chNKG2D-bearing T cells in eliminating established
tumors in a systemic lymphoma model, 2.5 106 RMA/RG cells
were injected i.v. into B6 mice. Two days after tumor inoculation, 7.5 106 T cells (transduced with chNKG2D or wtNKG2D
receptors) were administered i.v. As shown in Fig. 2, treatment
with a single dose of chNKG2D-modified T cells significantly
reduced both the percentages and absolute numbers of RMA/RG
cells in the spleens and lymph nodes of tumor-bearing mice by 90%
and 60%, respectively, compared with the treatment with wtNKG2D
T cells. Besides reducing tumor burdens, the same treatment
regimen using chNKG2D T cells also doubled the median survival
from 15 to 30 days (Fig. 3A). Two of 16 (12.5%) mice became
long-term survivors after one treatment with chNKG2D T cells.
We observed even lower tumor burdens after three injections of
5 106 T cells (on days 2, 6, and 10; Fig. 2B and D). All mice that
www.aacrjournals.org
were treated with three doses of chNKG2D T cells remained alive
>120 days after tumor injection (Fig. 3B). This result indicates that
multiple treatments with fewer chNKG2D T cells was a better
treatment regimen than a single dose. To determine whether a
small tumor burden still existed in the lymphoid organs of longterm survivors, a nested-PCR analysis for a tumor-specific gene,
gfp, was done on DNA samples extracted from the organs
(Supplemental Fig. S1A). The sensitivity of this PCR is at least
one tumor cell per reaction. All long-term survivor DNA samples
from spleen, lymph node, or bone marrow were negative 125 days
post–tumor inoculation for tumor DNA. The NKG2D ligand Rae1
expression on RMA/RG cells in vivo was also determined after
treatment with either wtNKG2D or chNKG2D T cells. RMA/RG cells
expressed lower levels of Rae1 in chNKG2D T cell–treated mice
than in wtNKG2D-treated mice (Fig. 3C and D), suggesting that
treatment with chNKG2D T cells might generate immune selection
pressure and lead to survival of tumor ‘‘variants’’ that have downregulated or lost Rae1 expression. However, multiple treatments
with chNKG2D T cells resulted in long-term tumor-free survival.
Treatment with chNKG2D T cells induces the generation of
memory responses against RMA cells that do not express
ligands for NKG2D. Because NKG2D ligand–negative tumor cells
may preferentially grow out, it would be beneficial if treatment with
chNKG2D T cells induced host immunity against other tumor
antigens. Eight of the mice that remained tumor-free after 75 days
(Fig. 3B) were challenged s.c. with RMA tumor cells. These tumorfree mice were resistant to a subsequent challenge of wild-type
RMA cells, whereas all control naı̈ve mice had aggressive tumors
(tumor area, f120 mm2) after 18 days (Fig. 4A). To test whether
long-term tumor survivors generated specific memory responses
against RMA cells, four tumor-free mice (Fig. 3B) were sacrificed
120 days post–tumor inoculation. Lymph node and spleen cells
were cocultured with either RMA cells or ID8 tumor cells. ID8
is a B6-derived ovarian cancer tumor cell line (22). As shown in
Fig. 4B–D, compared with the spleen and lymph node cells from
naı̈ve mice, tumor-free survivor-derived spleen and lymph node
cells produced IFN-g in response to stimulation by RMA but not
11031
Cancer Res 2007; 67: (22). November 15, 2007
Cancer Research
ID8 cells. Both CD8+ and CD4+ T cells produced IFN-g when cultured with RMA cells, but not with ID8 cells, as shown by intracellular staining (Fig. 4D). This memory T cell response was
mediated by host cells, not the adoptively transferred chNKG2D
T cells, because there were no chNKG2D T cells present 120 days
post–tumor inoculation based on a nested-PCR assay for the chimeric receptor gene (Supplemental Fig. S1B). These data indicate
that adoptive transfer of chNKG2D T cells allowed hosts to
generate immunologic memory against RMA tumor antigens.
Both perforin and Fas ligand, but not TRAIL, contribute to
the cytotoxicity mediated by chNKG2D-bearing T cells in vitro.
It is well known that perforin granule exocytosis and FasL-Fas
interactions are two major mechanisms that CTLs use to kill target
cells. RMA cells are Fas positive (23). To determine the mechanisms
Figure 3. Adoptive transfer of chNKG2D T cells promotes the survival of
RMA/RG tumor-bearing mice. Treatment of RMA/RG (2.5 106, i.v., day 0)
tumor-bearing mice with chNKG2D T cells (x) at a single dose (7.5 106 cells)
on day 2 (A ) or three doses (5 106 cells/treatment) on days 2, 6, and 10 (B)
significantly enhanced survival compared with wtNKG2D T cells (n). Data is
presented in Kaplan-Meier survival curves (*, P < 0.001). Rae1 expression on
the injected RMA/RG cells (from spleens) after treatment with either wtNKG2D
(C) or chNKG2D (D ) T cells was determined by flow cytometry from days 15
to 30 d post–tumor inoculation (shaded histograms ) along with isotype
controls (open histograms ). Histograms are representative of four independent
mice. MFI, mean fluorescence intensity.
Cancer Res 2007; 67: (22). November 15, 2007
of cytotoxicity of RMA/Rae1 cells, we used T cells derived from
Pfp/, FasL/ or TRAIL/ mice as effector cells. There was a
5-fold reduction in the cytotoxicity against RMA/Rae1 cells by
Pfp/ or FasL/-derived T cells (Fig. 5B). However, significant
cytotoxicity remained in spite of the absence of perforin or FasL.
TRAIL deficiency did not affect the cytotoxicity of chNKG2D T
cells. In addition, T cells that lacked IFN-g or GM-CSF did not
have reduced cytotoxicity against RMA/Rae1 cells (Supplemental
Fig. S2). The killing of target cells by chNKG2D T cells is NKG2D
ligand–dependent because no specific killing of RMA cells was
achieved by chNKG2D T cells (Fig. 5A and C). We have previously
shown that blocking the NKG2D receptor with anti-NKG2D
antibodies prevented chNKG2D-mediated in vitro cytotoxicity
(9, 10).
To pinpoint the relative contribution of each pathway to
chNKG2D T cell cytotoxicity, we generated B6 mice deficient in
both perforin and FasL. Pfp/FasL/ chNKG2D T cells lack
cytotoxic activity against RMA/Rae1 cells (Fig. 5D), indicating that
the combination of perforin and FasL accounted for all of the
chNKG2D-bearing T cell–mediated cytotoxicity against RMA/Rae1
cells. This result was further confirmed by combining CMA, which
is an inhibitor of the perforin-dependent pathway (24), with the use
of FasL/ T cells. Similar to the observation obtained using
perforin-deficient T cells, the addition of CMA alone led to a
reduction but not total abrogation of cytotoxicity of tumor cells
(Supplemental Fig. S3). However, CMA-treated FasL-deficient
chNKG2D T cells had no cytotoxic activity against RMA/Rae1 cells
(Supplemental Fig. S3). These results indicate that chNKG2Dbearing T cells use both FasL and perforin to kill RMA/Rae1 tumor
cells.
Direct killing of RMA/Rae1 cells by chNKG2D T cells is
involved in their in vivo efficacy. Considering the important
roles of perforin and FasL in chNKG2D T cell–mediated in vitro
cytotoxicity, we tested whether these two effector molecules were
also involved in chNKG2D T cell–mediated therapeutic efficacy
in vivo. ChNKG2D-bearing T cells derived from either Pfp/,
FasL/, TRAIL/, or B6 mice were adoptively transferred into
RMA/RG-bearing B6 mice. ChNKG2D T cells derived from either
Pfp/ or FasL/ mice displayed an equivalent capacity as T cells
from B6 mice to reduce tumor burden (Fig. 6A), indicating that
either perforin or FasL alone was not required for in vivo
therapeutic effects. TRAIL-deficient chNKG2D T cells did not show
any reduced functionality in therapeutic effects in vivo, which
was in line with the fact that TRAIL did not contribute to the
in vitro cytotoxic activity. As shown in Supplemental Fig. S4A
and C, Pfp/ or Pfp/FasL/ chNKG2D T cells produced
similar amounts of IFN-g in response to RMA/Rae1 cells as B6
chNKG2D T cells. Thus, deficiency in these cytotoxic pathways
did not inhibit other effector functions. However, T cells deficient
in both perforin and FasL showed reduced therapeutic efficacy
in vivo. The data indicate that chNKG2D T cell–mediated direct
cytotoxicity of tumor cells is involved in the in vivo effects,
although either perforin or FasL alone are not required for
chNKG2D T cells to be effective.
Donor cell–derived IFN-; and GM-CSF are critical for
chNKG2D T-cell–mediated therapeutic effects. Cytokines may
activate host immune cells and alter the local tumor microenvironment. To determine whether cytokines from chNKG2D T
cells were involved in their in vivo antitumor effects, we used T
cells derived from mice with defects in ifn-c or gm-csf. We
have previously shown that chNKG2D T cells produce IFN-g and
11032
www.aacrjournals.org
Mechanisms of ChNKG2D Receptors against Lymphoma
Figure 4. Adoptive transfer of chNKG2D T cells
induces memory responses. A, tumor-free mice
(o) in the chNKG2D-treated group (shown in
Fig. 3B) and naı̈ve mice (E) were challenged
with wild-type RMA cells (104) s.c. into the right
flank. Points, mean of tumor areas; bars, SE
(P < 0.05 on days 6–18). Lymph node (B ) and
spleen cells (C ) from tumor survivors (Fig. 3B)
produced significantly higher (P < 0.05) IFN-g
than cells from naı̈ve mice after stimulation with
irradiated RMA cells (filled columns ). Only
background amounts of IFN-g production were
observed in lymph nodes or spleen cells when
cultured with ID8 cells (hatched columns ) or
media alone (open columns ). D, intracellular
production of IFN-g by CD8+ or CD4+ T cells
upon culture with RMA (filled columns ),
ID8 (hatched columns ), or media only
(open columns ) for 24 h (*, P < 0.05).
GM-CSF upon culture with tumor cells that express NKG2D ligands
(9, 10). Although IFN-g is not directly linked to chNKG2D T cell–
mediated cytotoxicity as shown in in vitro cytotoxic assays
(Supplemental Fig. S2A), in vivo it may activate immune cells,
such as macrophages, dendritic cells, and/or lymphocytes to
achieve therapeutic effects. As shown in Fig. 6B, administration of
IFN-g–deficient chNKG2D T cells failed to reduce tumor burden,
indicating the critical role for IFN-g production by these
transferred cells in vivo. GM-CSF–deficient chNKG2D T cells had
an impaired ability to reduce tumor burdens compared with
chNKG2D T cells, although these T cell–treated mice had lower
tumor burdens than the mice treated with IFN-g–deficient
chNKG2D T cells. This impaired in vivo activity was not due to a
lack of IFN-g production because GM-CSF deficiency did not affect
the T cell production of IFN-g (Supplemental Fig. S4B). In addition,
a lack of GM-CSF did not affect T cell cytotoxicity (Supplemental
Fig. S2B). Thus, the production of cytokines was critical for the
antitumor responses mediated by chNKG2D T cells in vivo.
Discussion
T cells have a highly restrictive antigen specificity through their
TCR, which limits their antitumor activity to cells that express the
particular antigen. In addition, enough MHC expression of the
proper allele on tumor cells is required to ensure significant killing
by T cells. Therefore, down-regulation of MHC on tumor cells helps
tumor cells evade T cell recognition (25). NKG2D ligands are
primarily expressed on tumor cells but not normal cells (26). Thus,
the chNKG2D receptor–NKG2D ligand system provides a relatively
specific system for T cells to recognize tumor cells and become
fully activated irrespective of tumor cell MHC/tumor antigen
expression. Many human leukemia and lymphoma cells have been
found to express NKG2D ligands (27). It has been shown that the
antileukemia effect of NK cells in leukemia involves NKG2D-target
cell interactions (28). Therefore, the generation of systemic
lymphoma by i.v. administration of RMA/Rae1 tumor cells is a
good model to mimic human lymphomas.
www.aacrjournals.org
Although NK cells are able to recognize tumors using a variety of
activating receptors, their in vivo activities are dampened by many
different inhibitory receptors (29). Our preliminary data showed
that chNKG2D-bearing T cells lack the expression of inhibitory
receptors such as Ly49A or Ly49C/I.1 In addition, chNKG2D T cells
predominantly produce Th1 cytokines, not Th2 cytokines, whereas
NK cells can express Th2 cytokines such as interleukin (IL)-10 and
transforming growth factor-h, which may inhibit their antitumor
activity (30, 31). Therefore, chNKG2D-bearing T cells are expected
to achieve more significant antitumor effects than NK cells.
ChNKG2D T cells show cytotoxicity against RMA/Rae1 cells
in vitro. This cytotoxic activity uses both perforin and FasL
pathways. Expression of either one of these two molecules
conferred killing of RMA/Rae1 cells. Similarly, neither perforin
nor FasL alone was required for chNKG2D T cell–mediated in vivo
antitumor activity. It is interesting that even though deficiencies
in these cytotoxic pathways caused a 5-fold drop in cytotoxicity
in vitro, they had no effect on the efficacy of chNKG2D T cells
in vivo. A study by Winter et al. showed similar results in which
effector T cell–mediated tumor regression following adoptive
transfer was independent of perforin or FasL (32). The reasons
for these results may be that both perforin- and FasL-mediated
pathways could be used for cytotoxicity. Using chNKG2D T cells
generated from Pfp/FasL/ mice, we showed that direct killing
was involved in the chNKG2D T cell–mediated antitumor effects
in vivo. Unlike the critical role of IFN-g generated by chNKG2D T
cells in controlling tumor burden, chNKG2D T cells that could not
kill were still able to reduce tumor burden by >70%. Thus,
cytotoxicity by chNKG2D T cells is important, but chNKG2D T cells
promote tumor elimination even in the absence of their cytotoxic
function.
Although chNKG2D T cells produce multiple cytokines and chemokines, including IL-3, CCL3, and CCL5 in vitro upon engagement with RMA/Rae1 cells (9), IFN-g production was essential to
11033
1
Unpublished data.
Cancer Res 2007; 67: (22). November 15, 2007
Cancer Research
mediate antitumor effects by chNK2D T cells in vivo. It is wellknown that IFN-g coordinates a diverse array of cellular programs
(14, 33). In some models, IFN-g has been shown to be critical in
inhibiting tumor growth by inducing apoptosis or inhibiting
angiogenesis (34, 35). Corthay et al. showed that IFN-g was critical
for CD4+ T cell–mediated macrophage activation and tumor
rejection (36). Cytokines (such as IL-12, IL-18, and IL-21) and
costimulatory molecules (CD40 and LIGHT) have also been shown
to mediate antitumor effects in an IFN-g–dependent manner
(34, 37–39). In this systemic lymphoma model, we speculate that
the IFN-g production by chNKG2D T cells upon recognition of
NKG2D ligand–positive tumor cells may activate host immune cells
(macrophages, dendritic, NK, and/or T cells) and enhance the
development of Th1 responses leading to systemic antitumor
effects in vivo. Although not as critical as IFN-g, GM-CSF was
necessary for maximum therapeutic benefit because GM-CSF/
chNKG2D T cells did not promote tumor clearance as well T cells
that produced GM-CSF. GVAX, a GM-CSF gene-transduced tumor
vaccine, has been shown to be immunostimulatory and to have
antitumor activity in cancer patients in several clinical trials (40).
GM-CSF may recruit and activate dendritic cells and macrophages,
thereby improving tumor antigen presentation. In this model,
direct killing of RMA/Rae-1 tumor cells by chNKG2D-bearing
T cells in situ may help release tumor antigens in combination
with concurrent production of GM-CSF, which may provide a good
milieu for the recruitment of dendritic cells and antigen presentation.
This study, and our previous results, showed that adoptive
therapy of RMA/Rae1 tumor-bearing mice with chNKG2D T cells
could elicit hosts to generate memory responses against NKG2D
ligand–negative RMA cells and these responses required host
adaptive immunity (9, 20). Generation and maintenance of memory
responses are important for reducing recurrent tumor disease
because (a) memory T cells respond to recall antigens at a much
faster pace and greater magnitude than naı̈ve T cells; and (b)
memory T cells survive much longer than their naı̈ve counterparts
(41, 42). Tumor cells are also known to mutate (15); therefore,
induction of polyclonal tumor–specific T cells will minimize the
chances of tumor cells to ‘‘escape.’’ Generation of memory T cells
can be negatively affected by an immunosuppressive environment
in tumor-bearing hosts (43–45). Regulatory T cells and immunosuppressive cytokines such as transforming growth factor-h and
IL-10 have been shown to impair memory generation as well as
effector functions (43–45). In contrast, proinflammatory cytokines
and Toll-like receptor agonists are able to rescue antitumor
memory responses in immunosuppressive environments (45, 46).
Therefore, it is possible that chNKG2D T cell–tumor cell interaction
may create a proinflammatory environment, thus helping overcome immunosuppressive conditions and promoting the generation of memory T cells.
One of the hurdles to adoptive T cell transfer for cancer
immunotherapy is that most infused cells failed to persist to
mediate an effective response (47). An easy means to enhance the
activity and survival of transferred cells is administration of
cytokines, such as IL-2, IL-7 and IL-15, etc (48–50). These cytokines
share the common g chain receptor and regulate lymphocyte
growth and survival. The administration of low, nontoxic doses of
IL-2 and IL-7 can improve the survival of transferred tumorreactive T cells in vivo and lead to the establishment of persistent T
cell memory (48, 49). Although we observed significant therapeutic
Figure 5. ChNKG2D T cells use both
perforin and FasL to kill Rae1-positive RMA
cells in vitro . Effector T cells derived from
B6 (5, n), Pfp/ (4, E), FasL/ (o, .) or
TRAIL/ ( w , y) mice that were modified
with either wtNKG2D (wt, open symbols )
or chNKG2D (ch, filled symbols ) were
cocultured with RMA (A and C ) or RMA/
Rae-1 (B and D ) cells, respectively, at ratios
from 1:1 to 25:1 in 5-h 51Cr release assays.
Points, means of triplicates from two to six
independent experiments; bars, SE. C and
D, T cells derived from mice deficient in both
perforin and FasL (Pfp//FasL/) were
also used as effector cells. Points, means of
two sets of triplicates in two independent
experiments; bars, SD.
Cancer Res 2007; 67: (22). November 15, 2007
11034
www.aacrjournals.org
Mechanisms of ChNKG2D Receptors against Lymphoma
Figure 6. The effector mechanisms of
chNKG2D T cell–mediated therapeutic
effects in vivo. A, tumor-bearing mice that
were inoculated i.v. with 2.5 106 RMA/
RG cells on day 0 were treated with a
single dose of chNKG2D T cells (7.5 106)
derived from either wild-type B6, Pfp/,
FasL/, TRAIL/, or Pfp/FasL/ mice
at day 2. B, tumor-bearing mice that were
inoculated i.v. with 2.5 106 RMA/RG cells
on day 0 were treated with a single dose
of 7.5 106 chNKG2D T cells derived
from either wild-type B6, IFN-g/, or
GM-CSF/ mice on day 2. wtNKG2D T
cells from B6 mice were used as negative
controls. The percentages of GFP+ cells in
spleens were determined by flow cytometry
on day 13. Points, data pooled from two
independent experiments. Significance
was determined by ANOVA. **, P < 0.01,
compared with B6 wtNKG2D T cell
treatment; x, P < 0.01, compared with B6
chNKG2D T cell treatment.
effects with chNKG2D T cell–infusion without coadministration of
cytokines, we expect that cytokine administration in combination
with chNKG2D-bearing T cell transfer may give better results in
tumor inhibition.
Antigen-specific T cells represent heterogeneous populations of
differentiated cells that include effector, effector-memory, and
central-memory subsets that differ in functional properties and the
differential expression of homing receptors and costimulatory
molecules (51). Recent data showed that administration of naı̈ve
and early effector T cells, in combination with active immunization and IL-2, resulted in the eradication of large, established
tumors (52). However, more-differentiated effector T cells were less
effective for in vivo tumor treatment. In our study, genetically
modified T cells were transferred into mice within 10 days after
initial activation to avoid potential reduced functional activity.
Phenotypically, these chNKG2D T cells are early effectors (i.e.,
CD62L+/, IL-7RaLow, CD27+, CCR7+, and KLRG1).2
Although the direct killing of tumor cells by chNKG2D T cells via
the combination of perforin and FasL played a role in controlling
RMA/Rae1 growth, neither perforin nor FasL alone were required
by chNKG2D T cells to achieve in vivo therapeutic efficacy. In
2
Unpublished data.
References
1. Rossig C, Brenner MK. Genetic modification of T
lymphocytes for adoptive immunotherapy. Mol Ther
2004;10:5–18.
2. Ho WY, Blattman JN, Dossett ML, Yee C, Greenberg
PD. Adoptive immunotherapy: engineering T cell
responses as biologic weapons for tumor mass destruction. Cancer Cell 2003;3:431–7.
www.aacrjournals.org
contrast, cytokines, especially IFN-g were critical for chNKG2Dmediated in vivo antitumor effects. IFN-g/ chNKG2D T cells
could not control tumor growth even though they could kill RMA/
RG tumor cells very well. Therefore, elimination of RMA/Rae1 cells
may be primarily done by host immune cells with the help of
inflammatory cytokines produced by chNKG2D T cells. In
summary, chNKG2D T cells could significantly inhibit systemic
lymphoma growth and lead to the tumor-free survival of mice. The
antitumor effects were dependent on IFN-g, GM-CSF, and direct
killing (through a combination of perforin and FasL), but not
TRAIL expressed by chNKG2D receptor–bearing T cells.
Acknowledgments
Received 6/18/2007; revised 7/31/2007; accepted 9/7/2007.
Grant support: Hitchcock Foundation at Dartmouth Medical School, the
Department of Microbiology and Immunology; NIH (AI 07363), and a Leukemia and
Lymphoma Society Special Fellowship (T. Zhang). The contents are solely the
responsibility of the authors and do not necessarily represent the official views of the
NIH and the LLS.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Jeff Whitsett (University of Cincinnati, Cincinnati, OH) and Jacques
Peschon (Amgen, Seattle, WA) for providing GM-CSF/ and TRAIL/ mice,
respectively; Alice Givan and Gary Ward for assistance with flow cytometry; and the
staff of the Animal Resources Center for assistance with animal care.
3. Rosenberg SA. Progress in human tumour immunology and immunotherapy. Nature 2001;411:380–4.
4. Ribas A, Butterfield LH, Glaspy JA, Economou JS.
Cancer immunotherapy using gene-modified dendritic
cells. Curr Gene Ther 2002;2:57–78.
5. Sadelain M, Riviere I, Brentjens R. Targeting tumours
with genetically enhanced T lymphocytes. Nat Rev
Cancer 2003;3:35–45.
6. Kessels HW, Wolkers MC, van den Boom MD, van der
11035
Valk MA, Schumacher TN. Immunotherapy through
TCR gene transfer. Nat Immunol 2001;2:957–61.
7. Zhang T, He X, Tsang TC, Harris DT. Transgenic TCR
expression: comparison of single chain with full-length
receptor constructs for T-cell function. Cancer Gene
Ther 2004;11:487–96.
8. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer
regression in patients after transfer of genetically
engineered lymphocytes. Science 2006;314:126–9.
Cancer Res 2007; 67: (22). November 15, 2007
Cancer Research
9. Zhang T, Lemoi BA, Sentman CL. Chimeric NKreceptor-bearing T cells mediate antitumor immunotherapy. Blood 2005;106:1544–51.
10. Zhang T, Barber A, Sentman CL. Generation of
antitumor responses by genetic modification of primary
human T cells with a chimeric NKG2D receptor. Cancer
Research 2006;66:5927–33.
11. Smyth MJ, Swann J, Cretney E, Zerafa N, Yokoyama
M, Hayakawa Y. NKG2D function protects the host from
tumor initiation. J Exp Med 2005;202:583–8.
12. Trapani JA, Smyth MJ. Functional significance of the
perforin/granzyme cell death pathway. Nat Rev Immunol 2002;2:735–47.
13. Davidson WF, Giese T, Fredrickson TN. Spontaneous
development of plasmacytoid tumors in mice with
defective Fas-Fas ligand interactions. J Exp Med 1998;
187:1825–38.
14. Schroder K, Hertzog PJ, Ravasi T, Hume, DA.
Interferon-g: an overview of signals, mechanisms and
functions. J Leukoc Biol 2004;75:163–89.
15. Dunn GP, Koebel CM, Schreiber RD. Interferons,
immunity and cancer immunoediting. Nat Rev Immunol
2006;6:836–48.
16. Schmaltz C, Alpdogan O, Muriglan SJ, et al. Donor T
cell-derived TNF is required for graft-versus-host disease
and graft-versus-tumor activity after bone marrow
transplantation. Blood 2003;101:2440–5.
17. Nagoshi M, Goedegebuure PS, Burger UL, Sadanaga
N, Chang MP, Eberlein TJ. Successful adoptive cellular
immunotherapy is dependent on induction of a host
immune response triggered by cytokine (IFN-g and
granulocyte/macrophage colony-stimulating factor)
producing donor tumor-infiltrating lymphocytes.
J Immunol 1998;160:334–44.
18. Helmich BK, Dutton, RW. The role of adoptively
transferred CD8 T cells and host cells in the control of
the growth of the EG7 thymoma: factors that determine
the relative effectiveness and homing properties of Tc1
and Tc2 effectors. J Immunol 2001;166:6500–8.
19. Hollenbaugh JA, Dutton RW. IFN-g regulates donor
CD8 T cell expansion, migration, and leads to apoptosis
of cells of a solid tumor. J Immunol 2006;177:3004–11.
20. Sentman CL, Barber MA, Barber A, Zhang T. NK cell
receptors as tools for cancer immunotherapy. Adv
Cancer Res 2006;95:249–92.
21. Eriksson M, Meadows SK, Basu S, Mselle TF, Wira
CR, Sentman CL. TLRs mediate IFN-g production by
human uterine NK cells in endometrium. J Immunol
2006;176:6219–24.
22. Roby KF, Taylor CC, Sweetwood JP, et al. Development of a syngeneic mouse model for events related to
ovarian cancer. Carcinogenesis 2000;21:585–91.
23. Chakraborty M, Abrams SI, Camphausen K, et al.
Irradiation of tumor cells up-regulates Fas and enhances
CTL lytic activity and CTL adoptive immunotherapy.
J Immunol 2003;170:6338–47.
24. Kataoka T, Shinohara N, Takayama H, et al.
Concanamycin A, a powerful tool for characterization
and estimation of contribution of perforin- and Fasbased lytic pathways in cell-mediated cytotoxicity.
J Immunol 1996;156:3678–86.
25. Gilboa E. The makings of a tumor rejection antigen.
Immunity 1999;11:263–70.
26. Raulet DH. Roles of the NKG2D immunoreceptor and
its ligands. Nat Rev Immunol 2003;3:781–90.
27. Salih HR, Antropius H, Gieseke F, et al. Functional
expression and release of ligands for the activating
immunoreceptor NKG2D in leukemia. Blood 2003;102:
1389–96.
28. Sconocchia G, Lau M, Provenzano M, et al. The
antileukemia effect of HLA-matched NK and NK-T cells
in chronic myelogenous leukemia involves NKG2Dtarget-cell interactions. Blood 2005;106:3666–72.
29. Koh CY, Blazar BR, George T, et al. Augmentation of
antitumor effects by NK cell inhibitory receptor
blockade in vitro and in vivo . Blood 2001;97:3132–7.
30. Mehrotra PT, Donnelly RP, Wong S, et al. Production
of IL-10 by human natural killer cells stimulated with
IL-2 and/or IL-12. J Immunol 1998;160:2637–44.
31. Gray JD, Hirokawa M, Ohtsuka K, Horwitz DA.
Generation of an inhibitory circuit involving CD8+ T
cells, IL-2, and NK cell-derived TGF-h: contrasting
effects of anti-CD2 and anti-CD3. J Immunol 1998;160:
2248–54.
32. Winter H, Hu HM, Urba WJ, Fox BA. Tumor
regression after adoptive transfer of effector T cells is
independent of perforin or Fas ligand (APO-1L/CD95L).
J Immunol 1999;163:4462–72.
33. Blankenstein T, Qin Z. The role of IFN-g in tumor
transplantation immunity and inhibition of chemical
carcinogenesis. Curr Opin Immunol 2003;15:148–54.
34. Wigginton JM, Gruys E, Geiselhart L, et al. IFN-g and
Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy. J Clin Invest
2001;108:51–62.
35. Beatty G, Paterson Y. IFN-g-dependent inhibition of
tumor angiogenesis by tumor-infiltrating CD4+ T cells
requires tumor responsiveness to IFN-g. J Immunol
2001;166:2276–82.
36. Corthay A, Skovseth DK, Lundin KU, et al. Primary
antitumor immune response mediated by CD4+ T cells.
Immunity 2005;22:371–83.
37. Fan Z, Yu P, Wang Y, et al. NK-cell activation by
LIGHT triggers tumor-specific CD8+ T-cell immunity to
reject established tumors. Blood 2006;107:1342–51.
38. Oshikawa K, Shi F, Rakhmilevich AL, Sondel PM,
Mahvi DM, Yang NS. Synergistic inhibition of tumor
growth in a murine mammary adenocarcinoma model
Cancer Res 2007; 67: (22). November 15, 2007
11036
by combinational gene therapy using IL-12, pro-IL-18,
and IL-1h converting enzyme cDNA. Proc Natl Acad Sci
U S A 1999;96:13351–6.
39. Di Carlo E, Comes A, Orengo AM, et al. IL-21 induces
tumor rejection by specific CTL and IFN-g-dependent
CXC chemokines in syngeneic mice. J Immunol 2004;
172:1540–7.
40. Eager R, Nemunaitis J. GM-CSF gene-transduced
tumor vaccines. Mol Ther 2005;12:18–27.
41. Sprent J, Surh CD. T cell memory. Annu Rev Immunol
2002;20:551–79.
42. Wang LX, Kjaergaard J, Cohen PA, Shu S, Plautz
GE. Memory T cells originate from adoptively transferred effectors and reconstituting host cells after
sequential lymphodepletion and adoptive immunotherapy. J Immunol 2004;172:3462–8.
43. Kursar M, Bonhagen K, Fensterle J, et al. Regulatory
CD4+CD25+ T cells restrict memory CD8+ T cell
responses. J Exp Med 2002;196:1585–92.
44. Ahmadzadeh M, Rosenberg SA. TGF-h1 attenuates
the acquisition and expression of effector function by
tumor antigen-specific human memory CD8 T cells.
J Immunol 2005;174:5215–23.
45. Wang RF. Immune suppression by tumor-specific
CD4+ regulatory T-cells in cancer. Semin Cancer Biol
2006;16:73–9.
46. Kilinc MO, Aulakh KS, Nair RE, et al. Reversing tumor
immune suppression with intratumoral IL-12: activation
of tumor-associated T effector/memory cells, induction
of T suppressor apoptosis, and infiltration of CD8+ T
effectors. J Immunol 2006;177:6962–73.
47. Leen AM, Rooney CM, Foster AE. Improving T cell
therapy for cancer. Annu Rev Immunol 2007;25:243–65.
48. Yee C, Thompson JA, Byrd D, et al. Adoptive T cell
therapy using antigen-specific CD8+ T cell clones for
the treatment of patients with metastatic melanoma:
in vivo persistence, migration, and antitumor effect of
transferred T cells. Proc Natl Acad Sci U S A 2002;99:
16168–73.
49. Melchionda F, Fry TJ, Milliron MJ, McKirdy MA,
Tagaya Y, Mackall CL. Adjuvant IL-7 or IL-15 overcomes
immunodominance and improves survival of the CD8+
memory cell pool. J Clin Invest 2005;115:1177–87.
50. Teague RM, Sather BD, Sacks JA, et al. Interleukin-15
rescues tolerant CD8+ T cells for use in adoptive
immunotherapy of established tumors. Nat Med 2006;12:
335–41.
51. Lanzavecchia A, Sallusto F. Progressive differentiation and selection of the fittest in the immune response.
Nat Rev Immunol 2002;2:982–7.
52. Gattinoni L, Klebanoff CA, Palmer DC, et al.
Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively
transferred CD8+ T cells. J Clin Invest 2005;115:1616–26.
www.aacrjournals.org