Published in Volume
123, Issue 6
(June 3, 2013)J Clin Invest.
Copyright #x000a9; 2013, American Society for Clinical
Adoptively transferred TRAIL+ T cells suppress GVHD and
augment antitumor activity
1Department of Immunology and Medicine, 2Department of
Cancer Biology, and 3Department of Radiology, Memorial Sloan-Kettering
Cancer Center, New York, New York, USA. 4Department of Clinical and
Experimental Medicine, University of Perugia, Perugia, Italy. 5Department
of Hematology and Tumor Immunology Charit#x000e9; CBF #x02013;
Universit#x000e4;tsmedizin Berlin, Berlin, Germany. 6Department of
Pathology, Brigham and Women#x02019;s Hospital, Boston, Massachusetts, USA.
7Department of Pathology, Immunology, and Laboratory Medicine,
University of Florida College of Medicine, Gainesville, Florida, USA.
8Department of Pediatric Hematology/Oncology, Medizinische Hochschule
Hannover, Hannover, Germany. 9Center for Cell Engineering and Molecular
Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Address correspondence to: Arnab Ghosh or Marcel R.M. van den Brink, Department
of Immunology and Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Ave.,
New York, New York 10065, USA. Phone: 646.888.2304; Fax: 646.422.0452; E-mail:
GhoshA1@mskcc.org (A. Ghosh), vandenBM@mskcc.org
(M.R.M. van den Brink).
First published May 15, 2013
Submitted: August 30,
2012; Accepted: March 14,
Current strategies to suppress graft-versus-host disease (GVHD) also compromise
graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to
separate GVHD and GVT responses merely spare GVT function without actually enhancing
it. We have previously shown that endogenously expressed TNF-related
apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against
certain malignancies in recipients of allogeneic hematopoietic stem cell
transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we
genetically engineered T cells to overexpress TRAIL and adoptively transferred
donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We
found that murine TRAIL+ T cells induced apoptosis of alloreactive T
cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine
TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT
response. Moreover, human TRAIL+ T cells mediated enhanced in vitro
cytotoxicity against both human leukemia cell lines and against freshly isolated
chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf,
donor-unrestricted antitumor cellular therapy, in vitro#x02013;generated
TRAIL+ precursor T cells from third-party donors also mediated enhanced
GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing
donor T cells could potentially enhance the curative potential of allo-HSCT by
increasing GVT response and suppressing GVHD.
While the safety of clinical allogeneic hematopoietic stem
cell transplantation (allo-HSCT) has improved significantly in recent years, its success
is limited by disease relapse and graft-versus-host-disease (GVHD) (1). Both allo-HSCT and a variety of immunotherapeutic
strategies have demonstrated that T lymphocytes can exert potent antitumor activity.
Most genetic engineering strategies have involved directing T cell specificity toward
tumor-associated antigens using chimeric antigen receptors (2, 3) or transgenic T cell
receptors (TCRs) (4). These strategies, while
promising, are limited by requirements for clearly defined tumor-associated antigens or
epitopes. They may have risks in the context of allo-HSCT, potentially by exacerbating
GVHD (5) or by producing the mispairing of TCRs,
leading to neoreactivity (6). In contrast,
currently used strategies to prevent GVHD almost uniformly impair T cell function, with
deleterious effects on graft-versus-tumor (GVT) response.
Among the major
cytolytic molecules, TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptotic
signals in target cells expressing TRAIL receptors, which in humans include death
receptor (DR) 4 and 5 molecules, and in mice include only DR5. Expression of DR5 is
higher in certain tumors (7, 8); furthermore, DR5 expression by tumor cells can be induced by
treatment with small molecules like proteasome inhibitors (9, 10), rendering them
susceptible to TRAIL-mediated killing. We have previously demonstrated that endogenous
TRAIL expression in alloreactive T cells is an important mediator of GVT effects (11). TRAIL is thus an attractive candidate for
genetic engineering of donor T cells to enhance their antitumor potential. Importantly,
in the setting of allo-HSCT, TRAIL does not appear to mediate GVHD lethality, although
we found that TRAIL can contribute to thymic GVHD (11, 12). Here, we present our studies
of the effects of genetically overexpressing TRAIL in allogeneic T cells transferred to
murine bone marrow transplantation (BMT) recipients. We found that these engineered T
cells indeed mediated enhanced GVT activity. However, to our surprise, these
TRAIL+ T cells also ameliorated GVHD through the suppression of
alloreactive T cells.
TRAIL+ T cells mediate strong GVT effects. To assess the effect of constitutive TRAIL expression on donor T cells, we
constructed the lentiviral vectors pLM-TRAIL-GFP to express murine TRAIL with a GFP
reporter and, as a control, pLM-GFP (Figure 1A).
T cells transduced with these vectors are termed TRAIL+ T cells and
GFP+ T cells, respectively. We determined high transduction
efficiencies measured by GFP with both vectors (Figure 1B) and also confirmed that murine T cells transduced with our
pLM-TRAIL-GFP vector had increased expression of TRAIL compared with cells transduced
with control vector (Figure 1C). Expression of
TRAIL or GFP did not affect the expression of other cytolytic molecules, such as
perforin, granzyme, or FasL (Supplemental Figure 1A; supplemental material available
online with this article; doi:
TRAIL+ T cells are strong antitumor agents. (A) Representation of pLM-TRAIL-GFP construct: pLM-GFP-2A-TRAIL.
(B) Prestimulated B6-derived T cells were transduced and
transduction was measured by the expression of GFP. (C) TRAIL
overexpression on transduced T cells was determined by flow cytometry.
(D) TRAIL+ T cells mediate stronger killing against
labeled LB27.4 targets in a 51Cr release cytolysis assay. Graphs
representing 3 independent experiments are shown. (E) Lethally
irradiated CBF1 recipients were reconstituted with 5 #x000d7; 106 cells
per recipient of WT B6 TCD BM and inoculated with 2.5 #x000d7; 105
cells per recipient (upper panel) or 1 #x000d7; 105 cells per recipient
of LB27.4 lymphoma cells (lower panel). Designated groups were treated with 0.5
#x000d7; 106 cells per recipient (upper panel) or 1 #x000d7;
106 cells per recipient (lower panel) of GFP+ or
TRAIL+ T cells. (F) Transduced allogeneic
GFP+ or TRAIL+ pre#x02013;T cells adoptively transferred
into a syngeneic BMT model. RENCA tumor cells were inoculated s.c. 2 weeks after
BMT. Tumor volume is expressed in centimeters cubed measured as 1/2 #x000d7;
length #x000d7; (width)2. Pooled data from 2 independent experiments
are depicted. *P lt; 0.05; **P lt; 0.01;
***P lt; 0.001; ****P lt; 0.0001. hPGK, human
phosphoglycerate kinase promoter; pA, #x0201c;self-cleaving#x0201d; 2A peptides;
WPRE, woodchuck hepatitis posttranscriptional regulatory element.
We next sought to determine the effect of
TRAIL overexpression in T cells on antitumor activity. In vitro, unsorted
TRAIL+ T cells mediated significantly stronger cytotoxicity against
LB27.4 lymphoma targets compared with GFP+ T cells (Figure 1D). We also evaluated TRAIL+ T cell
activity in vivo using the haploidentical B6#x02192;CBF1 model inoculated with 2 or 1
#x000d7; 105 cells per recipient of LB27.4 lymphoma cells. We found that
TRAIL+ T cells (at 0.5 or 1 #x000d7; 106 cells per
recipient) mediated strong antitumor activity, with 100% survival of mice inoculated
with lymphoma, while mice that received control GFP+ T cells succumbed to
lymphoma and GVHD (Figure 1E and Supplemental
Figure 1B). Interestingly, mice that received TRAIL+ T cells had
tumor-free survival and developed minimal signs of GVHD (Supplemental Figure 1C).
We next examined the effects of TRAIL overexpression in a clinically relevant
model in the absence GVHD by transferring ex vivo#x02013;generated allogeneic
precursor T cells (pre#x02013;T cells) in a syngeneic transplant. We previously
reported that adoptively transferred pre#x02013;T cells, generated ex vivo using the
OP9-DL1 coculture system, undergo maturation and selection in the recipient thymus
and mediate antitumor effects across MHC barriers without alloreactivity (13). We adoptively cotransferred GFP+
and TRAIL+ B6 pre#x02013;T cells into syngeneic BMT recipients
(BALB/c#x02192;BALB/c), where they comparably reconstituted the recipient thymus
(Supplemental Figure 2A) and spleen at days 14 and 28 (Supplemental Figure 2, B and
C). After subcutaneous challenge with a renal cell carcinoma cell line (RENCA),
syngeneic (BALB/c#x02192;BALB/c) BMT recipients of TRAIL+ B6 pre#x02013;T
cells had significantly reduced tumor burden, with delayed tumor growth compared with
bone marrow (BM) alone and GFP+ B6 pre-T cell#x02013;treated tumor
recipients (Figure 1F). We did not observe GVHD
in any of the pre#x02013;T cell experiments. We conclude that TRAIL overexpression
can enhance the antitumor activity of both mature and pre#x02013;T cells in BMT
recipients in allogeneic and syngeneic settings.
TRAIL+ T cells suppress GVHD. Having observed that TRAIL+ T cells produce a surprisingly mild degree of
GVHD, we further investigated this in experiments with transplanted mice in the
B6#x02192;CBF1 model in the absence of tumor. We again found that mice receiving
TRAIL+ T cells had significantly reduced GVHD mortality at T cell doses
of 0.5 #x000d7; 106 (Figure 2A) and 1
#x000d7; 106 (Figure 2B).
Corresponding with improved survival, recipients of TRAIL+ T cells also
showed significantly reduced GVHD-associated weight loss (Supplemental Figure 3, A
and B) and lower clinical GVHD scores (Figure 2,
D and E). We then evaluated TRAIL+ T cells in an additional MHC-disparate
BMT model (B10.BR#x02192;B6) and again found that TRAIL+ T cells similarly
protected BMT recipients from GVHD mortality and morbidity (Figure 2, C and F). Histologically, we found that
recipients of TRAIL+ T cells had significantly less GVHD in target organs,
including liver and small and large intestines (Figure 3, A and B). Skin GVHD, measured by apoptotic scores, was not
significantly increased in allo-BMT recipients of TRAIL+ T cells. Thymic
GVHD manifests as decreased thymic cellularity and reduced numbers of
CD4+CD8+ double-positive (DP) T cells (12). Thymic GVHD was also not significantly increased in
recipients of TRAIL+ T cells compared with GFP+ T cells as
shown by statistically similar cellularity and DP T cell numbers (Figure 3C). Overall, these findings in mice demonstrate
that overexpression of TRAIL by donor T cells diminishes GVHD morbidity, reduces
organ damage in the liver and intestines, and improves survival in clinically
relevant BMT models.
Adoptive transfer of TRAIL+ T cells does not cause lethal GVHD. (A, B, D, and E) Lethally
irradiated CBF1 recipients were reconstituted with 5 #x000d7; 106 cells
per recipient of WT B6 TCD BM. Designated groups were treated with (A
and D) 0.5 #x000d7; 106 cells per recipient or
(B and E) 1 #x000d7; 106 cells per
recipient of B6-derived GFP+ or TRAIL+ T cells. Survival was
monitored and clinical GVHD scores were recorded weekly in a blinded fashion.
(C and F) Lethally irradiated B6 recipients were
reconstituted with 5 #x000d7; 106 cells per recipient of WT B10.BR TCD
BM. Designated groups were treated with 1 #x000d7; 106 cells per
recipient of B10.BR GFP+ or TRAIL+ T cells. Survival was
monitored (A#x02013;C) and clinical GVHD scores were
recorded weekly (D#x02013;F) in a blinded fashion.
*P lt; 0.05; **P lt; 0.01;
***P lt; 0.001.
TRAIL+ T cells suppress GVHD. (A and B) Livers and small and large intestines from
B6#x02192;CBF1 mice treated with GFP+ or TRAIL+ T cells were
harvested on day 14 after BMT and skin was harvested on day 21 and scored for GVHD
pathology. Representative micrographs are shown (original magnification,
#x000d7;200 for liver, small and large intestines; and #x000d7;400 for skin). GVHD
scores pooled from 2 independent experiments are shown (n =
8#x02013;10 per group). (C) Thymi from B6#x02192;CBF1 mice treated
with GFP+ or TRAIL+ T cells were harvested on day 21 after
BMT. Total cellularity was obtained from counts of live thymocyte suspension and
numbers of DP T cells were derived from flow cytometric determination of
CD4+CD8+ T cell proportions. Data pooled from 2
independent experiments are shown (n = 8#x02013;10 per group).
(D and E) Lethally irradiated CBF1 recipients were
reconstituted with 5 #x000d7; 106 cells per recipient of B6 TCD BM.
Designated groups were treated with 0.5 #x000d7; 106 cells per
recipient of GFP+ or TRAIL+ T cells mixed with nontransduced
Luc+ T cells. Bioluminescence imaging of the transplanted mice was
performed weekly (D) and flux was measured (E). Animals
representative of 1 experiment (n = 7 per group) and flux pooled
from 3 independent experiments are shown. (F) Lethally irradiated
CBF1 recipients were reconstituted with 5 #x000d7; 106 cells per
recipient of B6 TCD BM. Designated groups were treated with 0.5 #x000d7;
106 cells per recipient of GFP+ or TRAIL+ T
cells mixed with nontransduced T cells (n = 10 per group).
Survival was monitored daily. *P lt; 0.05; **P
lt; 0.01; ****P lt; 0.0001. NS, not significant.
Recent studies have indicated that the endogenous
induction of TRAIL has suppressive effects on other T cells (14, 15). To investigate
whether TRAIL+ T cells can suppress alloreactive T cells, we adoptively
transferred luciferase-expressing (Luc+) nontransduced T cells at a 1:1
ratio with either GFP+ or TRAIL+ T cells (all B6 background)
into CBF1 allo-BMT recipients and measured the expansion of nontransduced
Luc+ T cells after transfer. We found that Luc+ T cells
underwent alloreactive expansion when cotransferred with GFP+ T cells,
which was significantly attenuated upon transfer with TRAIL+ T cells
(Figure 3, D and E). In addition, CBF1
recipients of B6 BM were adoptively transferred with (a) nontransduced T cells, (b)
nontransduced T cells and GFP+ T cells, (c) nontransduced T cells and
TRAIL+ T cells. Recipients of TRAIL+ T cells had
significantly improved survival compared with either those that received
nontransduced T cells cotransferred with GFP+ T cells or nontransduced T
cells alone (Figure 3F). These findings suggest
that TRAIL+ T cells suppress GVHD by limiting alloreactive T cell
We next examined whether TRAIL-mediated suppression of
alloreactive T cell expansion can inhibit antiviral responses. We assessed viral
burden after lymphocytic choriomeningitis virus (LCMV) infection in CBF1 recipients
of allografts treated with GFP+ or TRAIL+ T cells (Supplemental
Figure 3C). Recipients of GFP+ or TRAIL+ T cells had
statistically similar viral burden, which was significantly lower than the viral load
of allo-BMT recipients without adoptive transfer of T cells. These results indicate
that TRAIL+ T cells do not significantly impair antiviral responses.
TRAIL receptors are induced on host APCs following radiation. To explore the potential mechanisms by which TRAIL+ T cells suppress the
expansion of alloreactive T cells, we first examined the effects of TRAIL+
T cells on host APCs. In mice, TRAIL exerts cytolysis through its receptor, DR5,
which has been reported to be upregulated after irradiation (16). We indeed found increased expression of DR5 transcripts in
lymphoid organs, liver, and intestine within 4 hours after irradiation (Figure 4A) and significantly increased DR5 cell surface
expression by splenic APCs, including DCs, macrophages, and B cells within 1 to 4
days after irradiation (Figure 4B). To assess
whether negative regulation by TRAIL+ T cells interacting with
DR5+ host APCs contributed to the suppression of GVHD, we performed
experiments with B10.BR TRAIL+ T cells in B6 DR5 KO or B6 WT recipients
(Figure 4, C and D). DR5 KO recipients had
significantly increased GVHD morbidity and somewhat greater GVHD mortality, although
this did not reach statistical significance. Taken together, our results demonstrate
that DR5 expression is upregulated in host tissues following irradiation, and that
host DR5 expression plays a role in the suppressive effect of TRAIL+ T
cells on GVHD.
TRAIL+ T cells can eliminate residual host APCs. (A) Tissues from lethally irradiated CBF1 mice were harvested at
designated time points and qPCR was performed for DR5. (B) DR5 was
assessed by flow cytometry on splenocytes gated on CD11c+ DCs,
CD11b+ macrophages, and B220+ B cells. Representative
data from 2 independent experiments are shown (n = 3 per group).
*P lt; 0.05. (C and D) Lethally
irradiated WT or DR5 KO B6 recipients were reconstituted with 5 #x000d7;
106 cells per recipient of WT B10.BR TCD BM. Designated groups were
treated with 1 #x000d7; 106 cells per recipient of B10.BR
TRAIL+ T cells. Survival was monitored (C) and clinical
GVHD scores were recorded weekly (D) in a blinded fashion. Graph
representative of 2 independent experiments is shown. *P lt;
0.05; ***P lt; 0.001; ****P lt; 0.0001. mLN,
mesenteric lymph node.
Fratricidal TRAIL+ T cells suppress GVHD. We next investigated whether TRAIL+ T cells suppressed GVHD by directly
inhibiting other alloreactive donor T cells. Since naive human T cells express DR5 on
their surface following activation (17), we
assessed DR5 expression on alloactivated murine T cells. Four days after adoptive
transfer into lethally irradiated CBF1 recipients, the CD25hi
alloactivated donor T cells expressed significantly higher levels of cell surface DR5
than the CD25lo T cells (Figure 5A).
CD4+ T cells in particular expressed significantly higher levels of
DR5, with a similar trend in donor CD8+ T cells. Since activation of DR5
can induce cell death, we next studied whether TRAIL+ T cells could cause
fratricide of alloactivated T cells. We found that TRAIL+ T cells mediated
significantly enhanced cytotoxicity against activated T cells in vitro compared with
GFP+ T cell controls (Figure 5B).
To investigate the role of DR5 signaling in the suppression of alloreactive T cells
by TRAIL+ T in vivo, we adoptively transferred donor B6 WT
TRAIL+ T cells or DR5 KO TRAIL+ T cells into our
B6#x02192;CF1 haploidentical model. DR5 KO TRAIL+ T cells, which are
impervious to TRAIL-mediated fratricide, caused significantly greater GVHD mortality
and morbidity compared with B6 WT TRAIL+ T cells (Figure 5C). Similar GVHD mortality and morbidity were
observed in groups treated with DR5 KO TRAIL+ T and DR5 KO GFP+
T cells (Figure 5D). Together these findings
suggest that TRAIL+ T cells can suppress GVHD by fratricide of
alloactivated T cells.
Adoptive transfer of TRAIL+ T cells leads to suppression of
alloactivated T cells. (A) Purified T cells from B6 mice were injected into lethally
irradiated CBF1 hosts. Single-cell suspension of splenocytes was analyzed by flow
cytometry on day 4. DR5 MFI in CD25hi and CD25lo cells was
analyzed in total donor T cells, CD4+ T cells, and CD8+ T
cells. Representative data (n = 4 per group) from 2 independent
experiments are shown. (B) Activated B6 T cells were used as targets
of GFP+ and TRAIL+ T cells in a CTL assay. Graph
representative of 2 independent experiments is shown. (C and
D) Lethally irradiated CBF1 recipients were reconstituted with 5
#x000d7; 106 cells per recipient of WT B6 TCD BM and 1 #x000d7;
106 cells per recipient of B6 WT or DR5 KO B6 TRAIL+ T
cells. Survival was monitored (C) and clinical GVHD scores were
recorded weekly (D) in a blinded fashion. Graph representative of 2
independent experiments is shown. *P lt; 0.05;
**P lt; 0.01; ***P lt; 0.001.
TRAIL+ human T cells can eliminate tumors and alloreactive T cells. Given our promising findings in murine BMT models, we investigated the antitumor and
alloactivated T cell#x02013;killing potential of human T (huT) cells engineered to
express TRAIL. We designed a human (hu)TRAIL-expressing retroviral vector,
SFG-huTRAIL-IRES-GFP, and its control, SFG-CBRLuc-IRES-GFP (Figure 6A). We observed efficient transduction of
activated huT cells by the expression of GFP using flow cytometry (Figure 6B). We then evaluated the ability of
TRAIL+ huT cells to mediate cytotoxicity. TRAIL+ huT cells
mediated significantly stronger killing of the human erythroleukemia cell line K562
than did GFP+ huT cells (Figure 6C).
We then further tested the antitumor potential of TRAIL+ huT cells against
PBMCs obtained from 2 chronic lymphocytic leukemia (CLL) patients. Fifty-six percent
of PBMCs from patient CLL-1 and 60% of PBMCs from patient CLL-2 were
CD19+CD5+ cells (putative malignant cells) and had high
expression of huTRAIL receptors DR4 and DR5 (Supplemental Figure 4).
TRAIL+ huT cells mediated stronger cytolytic activity against PBMCs
obtained from the 2 CLL patients compared with the GFP+ huT cells,
demonstrating enhanced antitumor effects against primary tumors (Figure 6, D and E). As we had observed in our murine
models, TRAIL+ huT cells also mediated significantly enhanced cytotoxicity
against activated huT cells (Figure 6F).
Altogether, these data show that TRAIL overexpression in huT cells can enhance
cytolysis of both tumor cells and activated T cells, similar to our findings in
Genetically engineered TRAIL+ huT cells have increased GVT
potential and can suppress activated T cells. (A) Representation of SFG-huTRAIL-GFP construct:
SFG-huTRAIL-IRES-GFP. SFG-CBRLuc-IRES-GFP was used as a GFP control.
(B) Prestimulated huT cells were transduced and transduction was
measured by the expression of GFP. (C#x02013;E)
51Cr release assays comparing tumor cytolysis between
GFP+ and TRAIL+ huT cells against the (C)
K562 cell line and PBMCs derived from CLL patients (D) CLL-1 and
(E) CLL-2. (F) 51Cr release assay
comparing cytolysis mediated by GFP+ and TRAIL+ huT cells
against activated T cells. Representative graphs of at least 3 assays are shown.
*P lt; 0.05; **P lt; 0.01;
***P lt; 0.001; ****P lt; 0.0001. IRES,
internal ribosome entry site.
TRAIL is a potent inducer of apoptosis through either the DR4
or DR5 receptors (18). These receptors are often
expressed on cells upon malignant transformation. Systemic administration of recombinant
TRAIL has had limited success in clinical trials, possibly due to the rapid clearance of
recombinant TRAIL protein, as well as the presence of decoy receptors (19). The relative absence of toxicities observed in
trials of TRAIL therapies, however, argues that TRAIL remains a safe therapeutic option
for cancer immunotherapy.
We genetically engineered T cells with a TRAIL
overexpression vector and found that murine and human TRAIL+ T cells can
cause strong antitumor effects in vitro and in vivo. Antitumor activity mediated by
TRAIL+ T cells is antigen independent and directed against malignant cells
expressing the TRAIL receptors. While this strategy could easily be applied in syngeneic
settings for tumor immunotherapy, we focused on using TRAIL-overexpressing T cells
across MHC barriers to increase the feasibility and abundance of T cell availability. We
used donor-derived T cells in the context of allogeneic BMT and demonstrated strong
antitumor response across MHC barriers. In this setting, TRAIL+ T cells have
an added benefit of suppressing GVHD, leading to improved outcomes. In an additional
clinically applicable strategy, the adoptive transfer of allogeneic ex
vivo#x02013;generated pre#x02013;T cells expressing TRAIL mediated significant antitumor
activity in syngeneic BMT recipients. The development and persistence of GFP+
and TRAIL+ B6 pre-T#x02013;derived T cells indicate a relative tolerance
toward the adoptively transferred allogeneic pre#x02013;T cells, consistent with our
previous studies (13). TRAIL+
pre#x02013;T cells can therefore be used as an #x0201c;off-the-shelf#x0201d; cell
therapy in allogeneic and autologous settings.
TRAIL+ T cells were
also found to suppress GVHD. We found a marked improvement in GVHD survival with reduced
clinical and pathological scores with a variety of T cell doses in 2 different GVHD
models. Mechanistically, TRAIL+ T cells appear to inhibit the proliferation
of other alloreactive T cells. While a body of evidence suggests a role for the
TRAIL/DR5 pathway in immune regulation (15, 20), its physiological role in suppressing GVHD has
not been clear (11). We found that TRAIL receptor
DR5 expression is induced in APCs postirradiation and that GVHD suppression by
TRAIL+ T cells is less effective in DR5 KO recipients, suggesting that DR5
expression (possibly on host APCs) could play a role in GVHD suppression by
TRAIL+ T cells. In addition, we found that TRAIL-mediated suppression of
GVHD was critically dependent on the elimination of other alloactivated T cells
expressing DR5. The GVHD potential of DR5 KO TRAIL+ T cells was similar to
DR5 KO GFP+ T cells, further suggesting that TRAIL+ T cells
suppress GVHD by directly inducing apoptosis of alloreactive T cells.
Considering that TRAIL+ T cells suppress alloreactive expansion
independent of antigen specificity, it is possible that TRAIL+ T cells can
suppress T cells activated by pathogens. In fact, Brenden et al. showed that TRAIL can
be involved in the elimination of antiviral effector T cells (21). However, in our experiments with LCMV infection, we did not
find significant suppression of antiviral immunity by T cells. This could be due to
increased cytotoxicity of TRAIL-overexpressing T cells, as previously shown in an
influenza model (22).
We have previously
shown that the TRAIL/DR5 pathway is required for thymic GVHD mediated by alloreactive T
cells (12). In this study, we thus evaluated
whether TRAIL overexpression could result in aggravated thymic GVHD, but did not find
evidence for this. One possible explanation is that physiological expression of TRAIL by
alloactivated T cells develops with the coexpression of other cytolytic ligands, and
isolated overexpression of TRAIL may not be adequate to exacerbate thymic GVHD without
the contribution of multiple other cytolytic mediators downstream of the alloreactive
activation known to play roles in GVHD and GVT activity (23).
In summary, we demonstrate that overexpression of TRAIL by
murine or human T cells enhances tumor killing while simultaneously having the
unexpected benefit of reducing GVHD through fratricide of alloactivated T cells (and
possibly eliminating host APCs). Genetic engineering to induce constitutive TRAIL
expression on allogeneic T cells therefore represents a uniquely promising strategy to
enhance GVT without a requirement for antigen specificity, while actively reducing
Lentiviral vectors and mouse T cell transduction. Murine TRAIL (mTRAIL) cDNA was obtained from activated T cells and cloned into the
lentiviral construct pLM-GFP-2A (pLM-GFP) to create pLM-GFP-2A-TRAIL (pLM-TRAIL-GFP).
Lentiviral vectors were produced by tripartite transfection of 293T cells with
transfer genes (control pLM-GFP or pLM-TRAIL-GFP), pCMV#x00394;R8.92, and
pUCMD.G53 using TransIT-293 (Mirus Bio). Concentrated lentiviral vector
supernatants were used for transductions.
Mouse T cells were transduced as
described previously (24). Briefly, T cells
derived from splenocytes were stimulated with Concanavalin A (Sigma-Aldrich) in the
presence of IL-7 and IL-2 (Miltenyi Biotec). Concentrated lentiviral supernatants
containing vectors for control GFP and TRAIL GFP were used to transduce T cells to
obtain GFP+ and TRAIL+ T cells, respectively.
Generation of pre#x02013;T cells. GFP+ and TRAIL+ pre#x02013;T cells were generated ex vivo for
adoptive transfer (25).
Lin#x02013;Sca+Kithi (LSK) cells were obtained
from donor BM and prestimulated with SCF, TPO, IL-3, IL-6 (R#x00026;D Systems), and
Flt3L (Miltenyi Biotec). The prestimulated cells were then transduced with
concentrated lentiviral supernatants. Pre#x02013;T cells were generated from the
transduced LSK cells by coculturing with OP9-DL1 cells in the presence of IL-7 and
FLT3-L (Miltenyi Biotec).
Mice, syngeneic, and allo-BMT. C57BL6 (B6-H2b), BALB/c (H2d) CBF1 ([BALB/c #x000d7; B6]F1),
B10.BR (H2k), and B6-expressing congeneic marker Ly5.1
(B6-Ly5.1-H2b) were obtained from The Jackson Laboratory (11). DR5 KO (B6 strain) mice and their littermate
control mouse colonies were set up from cryopreserved spermatozoa (MMRRC-NIH) (16). Animals were housed in the Memorial
Sloan-Kettering Cancer Center#x02013;specific pathogen-free barrier facilities. For
syngeneic BMT, lethally irradiated mice (850 cGy; BALB/c) were reconstituted with
lineage-depleted BALB/c BM. For allo-BMT, we used a haploidentical B6#x02192;CBF1
model or complete MHC-mismatched B10.BR#x02192;B6 combinations. Lethally irradiated
(1,300 cGy for CBF1; 1,100 cGy for B6; and 850 cGy for BALB/c) mice were
reconstituted with 5 #x000d7; 106 cells per recipient of B6 WT T
cell#x02013;depleted donor BM (TCD BM) as described previously (11, 25, 26).
Retroviral vectors and huT cell transduction. In the TRAIL-expressing vector hTRAIL, cDNA (pORF-hTRAIL; InvivoGen) was placed under
the control of the retroviral long terminal repeat (LTR) promoter followed by the
IRES-GFP sequence. The final vector was labeled SFG-huTRAIL-IRES-GFP. The click
beetle red luciferase encoding the SFG-CBRLuc-IRES-GFP retroviral vector was used as
a GFP positive control vector (27). The PG13
viral producer cell line was transduced with the vectors mentioned above and selected
based on GFP expression.
T cells from consenting healthy volunteers were
isolated from buffy coat (New York Blood Center) using the density gradient
separation method. Forty-eight hours after stimulation with phytohemagglutinin (2
#x003bc;g/ml; Fisher Scientific), T cells were transduced using GALV-pseudotyped
retroviral particles obtained from transduced PG13 cells, as described previously
(27), in the presence of human recombinant
(hr)IL-2 (20 U/ml; R#x00026;D Systems). During T cell expansion, the medium was
supplemented with hrIL-15 at a concentration of 10 ng/ml (R#x00026;D Systems).
Cell lines and in vitro cytotoxicity assays. The cell lines K562, LB27.4, and RENCA have been described (11, 28). In vitro
cytotoxicity assays were performed by measuring chromium-51 (51Cr) release
from labeled target cells as described previously (11). In some experiments, activated murine T cells were generated by
incubating splenic T cells in wells coated with anti-CD3 and anti-CD28 in the
presence of IL-2 and IL-7 for 24 hours before the assay. For activated human T cells,
T cells derived from buffy coat were stimulated for 48 hours in the presence of
hrIL-2 and hrIL15.
Primary CLL cells. PBMCs from the CLL patients (CLL-1 and CLL-2) were isolated using density gradient
separation (Ficoll-Paque PLUS; GE Healthcare). No further manipulation was performed.
PBMCs were pelleted and resuspended in freezing medium composed of 90% FBS (Omega
Scientific) plus 10% DMSO (Sigma-Aldrich), then frozen and stored in liquid nitrogen.
The cells were thawed and then washed thrice prior to use.
GVHD and GVT. To model GVHD, GFP+ or TRAIL+ T cells were adoptively
transferred into allo-BMT recipients as described above. In some experiments
MACS-purified (Miltenyi Biotec) splenic T cells were admixed with the transduced T
cells at a 1:1 ratio and adoptively transferred. All BMT recipients were monitored
daily for survival and scored weekly on a 10-point scale in a blinded fashion for
signs of clinical GVHD (11, 29). Animals that scored greater than 5 were
immediately euthanized. For histopathological assessment of GVHD, liver, small
intestine, and large intestine were harvested 14 days after BMT, and skin was
harvested 21 days after BMT. The organs were formalin preserved, paraffin embedded,
sectioned, and stained with H#x00026;E. Blinded scoring was performed at
Massachusetts General Hospital (Boston, Massachusetts, USA), and University of
Florida (Gainesville, Florida, USA), as previously described (29).
To study GVT effects, LB27.4 (H2b/d)
lymphoma cells were i.v. inoculated separately at the time of allo-BMT and adoptive
transfer (11). Survival was monitored daily
and animals found dead were necropsied to determine the presence or absence of tumor.
RENCA (H2d) cells were s.c. inoculated in recipients 14 days after BMT.
Tumors were measured with calipers and the volumes are expressed in centimeters cubed
measured as 1/2 #x000d7; length #x000d7; (width)2.
Flow cytometry. Flow cytometric analyses of cells were performed with fluorochrome-labeled antibodies
(all antibodies were purchased from BD Biosciences and eBioscience) or TRAIL-R2-FcIg
fusion protein (Alexis Biochemicals) and used as described previously (11). The cells were acquired on an LSR II flow
cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
LCMV challenge. On day 14 after BMT, mice were challenged i.p. with 2 #x000d7; 105 LCMV
Armstrong PFUs. Splenocytes were harvested 8 days after challenge and weighed. PFU
assays were performed as previously described (30). The plaques were read the next day.
In vivo bioluminescence imaging. T cells from luciferase-expressing mice (a gift from Robert Negrin, Stanford
University, Palo Alto, California, USA) were admixed with TRAIL+ or
GFP+ T cells and visualized using in vivo bioluminescence imaging
systems (Caliper Life Sciences) (31). The
bioluminescent flux was analyzed using Living Image software, version 4.3 (Caliper
Quantitative PCR. Tissue was harvested, mRNA was extracted, and cDNA were prepared at the Memorial
Sloan-Kettering Cancer Center Genomics Core Facility. Quantitative real-time PCR
(qRT-PCR) was performed for DR5 expression using a Taqman assay according to the
manufacturer#x02019;s protocols (Applied Biosystems).
Statistics. Calculations were performed using Excel (Microsoft) and graphed using Prism V5.0
software (GraphPad Software). Survival curves were analyzed with a Mantel-Cox test,
and other comparisons were made using a Mann-Whitney U test or 2-way
ANOVA. Data represent the means #x000b1; SEM. P values less than
0.05 were considered statistically significant.
Study approval. All animal protocols were approved by the IACUC of Memorial Sloan-Kettering Cancer
Center. We used samples from 2 CLL patients and volunteers who provided signed,
informed consent to have their samples used for research purposes, in accordance with
the Declaration of Helsinki and approved by the IRB of Memorial Sloan-Kettering
This research was supported by grants from the NIH (R01-HL069929, R01-CA107096,
R01-AI080455, P01-CA33049, and R01-HL095075, all to M. van den Brink). The content is
solely the responsibility of the authors and does not necessarily represent the official
views of the NIH. Support was also received from the US Department of Defense (USAMRAA
Award W81XWH-09-1-0294, to M. van den Brink), the Radiation Effects Research Foundation
(RERF-NIAID, to M. van den Brink), the Ryan Gibson Foundation, The Lymphoma Foundation,
and Alex#x02019;s Lemonade Stand. A. Ghosh has been a recipient of a Dr. Mildred Scheel
fellowship of Deutsche Krebshilfe and the Judah Folkman Fellowship from the American
Association for Cancer Research (AACR). A. Ghosh is a fellow of the Lymphoma Research
Foundation. M.G. Sauer was supported by the Deutsche Forschungsgemeinschaft (SFB 738, TP
A3). E. Velardi was supported by a fellowship from the Italian Foundation for Cancer
Research (FIRC). M. Sadelain received support from the Major Family Foundation and the
Lewis Sander Fund.
We appreciate the help of Julie White, LCP, RARC, of
the Genomics Core Facility and the Flow Cytometry Core Facility of Memorial Sloan-Kettering
Cancer Center. We thank Robert Negrin of Stanford University for his gift of the luciferase mice.
We also appreciate the help of Kate Takvorian, Lauren Dimenna, Susan Puckett, Lao-Tzu
Allan-Blitz, Anna Maria Mertelsmann, and Kristina Carrigan for their contributions to this
Blazar BR, Murphy WJ, Abedi M. Advances in graft-versus-host disease biology and
therapy. Nat Rev Immunol. 2012;12(6):443–458.
Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid
leukemia. N Engl J Med. 2011;365(8):725–733.
Brentjens RJ, et al. Safety and persistence of adoptively transferred autologous
CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell
leukemias. Blood. 2011;118(18):4817–4828.
Doubrovina E, Carpenter T, Pankov D, Selvakumar A, Hasan A, O#x02019;Reilly RJ. Mapping of novel peptides of WT-1 and presenting HLA alleles that
induce epitope-specific HLA-restricted-T-cells with cytotoxic activity against
WT-1+ leukemias. Blood. 2012;120(8):1633–1646.
Bendle GM, et al. Lethal graft-versus-host disease in mouse models of T cell receptor
gene therapy. Nat Med. 2010;16(5):565–570.
van Loenen MM, et al. Mixed T cell receptor dimers harbor potentially harmful
neoreactivity. Proc Natl Acad Sci U S A. 2010;107(24):10972–10977.
Smyth MJ, Takeda K, Hayakawa Y, Peschon JJ, van den Brink MR, Yagita H. Nature#x02019;s TRAIL #x02014; on a path to cancer
immunotherapy. Immunity. 2003;18(1):1–6.
Frew AJ, et al. Combination therapy of established cancer using a histone deacetylase
inhibitor and a TRAIL receptor agonist. Proc Natl Acad Sci U S A. 2008;105(32):11317–11322.
Sayers TJ, et al. The proteasome inhibitor PS-341 sensitizes neoplastic cells to
TRAIL-mediated apoptosis by reducing levels of c-FLIP. Blood. 2003;102(1):303–310.
Sun K, et al. Inhibition of acute graft-versus-host disease with retention of
graft-versus-tumor effects by the proteasome inhibitor bortezomib. Proc Natl Acad Sci U S A. 2004;101(21):8120–8125.
Schmaltz C, et al. T cells require TRAIL for optimal graft-versus-tumor
activity. Nat Med. 2002;8(12):1433–1437.
Na IK, et al. The cytolytic molecules Fas ligand and TRAIL are required for murine
thymic graft-versus-host disease. J Clin Invest. 2010;120(1):343–356.
Zakrzewski JL, et al. Tumor immunotherapy across MHC barriers using allogeneic T-cell
precursors. Nat Biotechnol. 2008;26(4):453–461.
Janssen EM, et al. CD4+ T-cell help controls CD8+ T-cell memory via
TRAIL-mediated activation-induced cell death. Nature. 2005;434(7029):88–93.
Wolkers MC, et al. Nab2 regulates secondary CD8+ T-cell responses through
control of TRAIL expression. Blood. 2012;119(3):798–804.
Finnberg N, et al. DR5 knockout mice are compromised in radiation-induced
apoptosis. Mol Cell Biol. 2005;25(5):2000–2013.
Barblu L, Herbeuval JP. Three-dimensional microscopy characterization of death receptor 5
expression by over-activated human primary CD4+ T cells and apoptosis. PLoS One. 2012;7(3):e32874.
Sheridan JP, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy
receptors. Science. 1997;277(5327):818–821.
Martinez-Lostao L, Marzo I, Anel A, Naval J. Targeting the Apo2L/TRAIL system for the therapy of autoimmune
diseases and cancer. Biochem Pharmacol. 2012;83(11):1475–1483.
Iyori M, Zhang T, Pantel H, Gagne BA, Sentman CL. TRAIL/DR5 plays a critical role in NK cell-mediated negative
regulation of dendritic cell cross-priming of T cells. J Immunol. 2011;187(6):3087–3095.
Brincks EL, Katewa A, Kucaba TA, Griffith TS, Legge KL. CD8 T cells utilize TRAIL to control influenza virus
infection. J Immunol. 2008;181(7):4918–4925.
Hamada H, et al. Multiple redundant effector mechanisms of CD8+ T cells
protect against influenza infection. J Immunol. 2013;190(1):296–306.
Ramirez-Montagut T, et al. IFN-gamma and Fas ligand are required for graft-versus-tumor activity
against renal cell carcinoma in the absence of lethal graft-versus-host
disease. J Immunol. 2007;179(3):1669–1680.
Kessels HW, de Visser KE, Tirion FH, Coccoris M, Kruisbeek AM, Schumacher TN. The impact of self-tolerance on the polyclonal CD8+ T cell
repertoire. J Immunol. 2004;172(4):2324–2331.
Zakrzewski JL, et al. Adoptive transfer of T-cell precursors enhances T-cell reconstitution
after allogeneic hematopoietic stem cell transplantation. Nat Med. 2006;12(9):1039–1047.
Hanash AM, et al. Abrogation of donor T-cell IL-21 signaling leads to tissue-specific
modulation of immunity and separation of GVHD from GVL. Blood. 2011;118(2):446–455.
Dobrenkov K, et al. Monitoring the efficacy of adoptively transferred prostate
cancer-targeted human T lymphocytes with PET and bioluminescence
imaging. J Nucl Med. 2008;49(7):1162–1170.
Takeda K, et al. Involvement of tumor necrosis factor-related apoptosis-inducing ligand
in NK cell-mediated and IFN-gamma-dependent suppression of subcutaneous tumor
growth. Cell Immunol. 2001;214(2):194–200.
Waldman E, et al. Absence of beta7 integrin results in less graft-versus-host disease
because of decreased homing of alloreactive T cells to intestine. Blood. 2006;107(4):1703–1711.
Holland AM, et al. Extrathymic development of murine T cells after bone marrow
transplantation. J Clin Invest. 2012;122(12):4716–4726.
Edinger M, et al. CD4+CD25+ regulatory T cells preserve
graft-versus-tumor activity while inhibiting graft-versus-host disease after bone
marrow transplantation. Nat Med. 2003;9(9):1144–1150.