Disease without Exacerbating Graft-versus-Host Cell

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Disease without Exacerbating Graft-versus-Host Cell
This information is current as
of August 11, 2017.
A TLR5 Agonist Enhances CD8+ T
Cell-Mediated Graft-versus-Tumor Effect
without Exacerbating Graft-versus-Host
Disease
Xilai Ding, Guanglin Bian, Nicholas D. Leigh, Jingxin Qiu,
Philip L. McCarthy, Hong Liu, Semra Aygun-Sunar,
Lyudmila G. Burdelya, Andrei V. Gudkov and Xuefang Cao
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Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 189:4719-4727; Prepublished online 8
October 2012;
doi: 10.4049/jimmunol.1201206
http://www.jimmunol.org/content/189/10/4719
The Journal of Immunology
A TLR5 Agonist Enhances CD8+ T Cell-Mediated Graftversus-Tumor Effect without Exacerbating Graft-versus-Host
Disease
Xilai Ding,*,1 Guanglin Bian,*,1 Nicholas D. Leigh,* Jingxin Qiu,† Philip L. McCarthy,‡
Hong Liu,‡ Semra Aygun-Sunar,x Lyudmila G. Burdelya,x Andrei V. Gudkov,x and
Xuefang Cao*
A
llogeneic bone marrow transplantation (allo-BMT) is
an established treatment for leukemia, lymphoma, and
other hematologic and nonhematologic malignancies (1).
Donor-derived immune cells were found to be able to identify and
attack tumor cells in the host, producing a graft-versus-tumor
(GVT) effect (2). Initially, GVT effect was regarded as a beneficial by-product of graft-versus-host disease (GVHD) (3). It has
since been recognized as a unique immune response that is critical
for the therapy of hematologic and other malignancies (4, 5).
Multiple cell types such as NK cells, CD4+, and CD8+ T cells
have been implicated in the GVT mechanisms (6–8). Among
them, CD8+ T cells were shown to be a major contributor for GVT
activity because of the strong alloantigen specificity (9, 10).
*Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263;
†
Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263;
‡
Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263; and
x
Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY
14263
1
X.D. and G.B. contributed equally to this work.
Received for publication April 25, 2012. Accepted for publication September 6,
2012.
This work was supported by a Young Investigator Development Award (to X.C.) from
the Roswell Park Alliance Foundation and Institutional Research Grant IRG-02197-09 (to X.C.) from the American Cancer Society.
Address correspondence and reprint requests to Dr. Xuefang Cao, Department of
Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY
14263. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; allo-BMT, allogeneic bone marrow transplantation; BM, bone marrow; DC, dendritic cell; GVHD,
graft-versus-host disease; GVT, graft-versus-tumor; TCD-BM, T cell-depleted bone
marrow.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201206
Therefore, it remains critical to modulate CD8+ T cell response in
allo-BMT to enhance GVT activity whereas limiting GVHD.
TLRs can recognize pathogen-associated molecular patterns of
bacterial or viral origin. TLR-mediated recognition of “danger
signals” allows the immune system to rapidly respond to pathogens via innate immune cells and subsequently may activate
adaptive immune responses (11). So far, 13 members of the TLR
family have been identified in mice (12). Because of their
immune-modulating function, various TLR ligands have been
investigated as adjuvants to induce favorable immune responses
(13–15). Flagellin, the structural component of bacterial flagellum, is a natural ligand for TLR5 (16). TLR5 binding by flagellin
was shown to convert tolerogenic dendritic cells (DCs) into activating APCs that preferentially induce Th1 responses (17). Flagellin was also shown to promote Th2 and humoral immune
response through direct stimulation of TLR5-expressing CD11c+
cells (18–20). However, there have been conflicting reports regarding the ability of flagellin to promote CD8+ T cell response
(21–25). It was shown that flagellin was not able to stimulate
CD8+ T cells when administered separately from Ag (24). In
contrast, flagellin was able to promote CD8+ T cell response when
fused with OVA protein, SIINFEKL peptide, or viral Ags (21–25).
In addition, flagellin has been reported to modulate antitumor
immune responses (26). However, studies with tumor models have
yielded conflicting results with regard to whether flagellin actually
promotes or suppresses tumor growth (26–28).
Recently, a pharmacologically optimized form of flagellin,
CBLB502, was generated and shown to be as efficacious as flagellin in inducing NF-kB activation. CBLB502 demonstrated
powerful radioprotection to lethally irradiated hosts in both mouse
and monkey models (29). Because of its lower toxicity and lower
immunogenicity, CBLB502 emerges as a more attractive TLR5
agonist and is currently undergoing phase I clinical trial. Mean-
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Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and nonhematologic malignancies. Donorderived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the
effectiveness of the transplantation therapy. CBLB502 is a novel agonist for TLR5 derived from Salmonella flagellin. On the basis of
TLR5-mediated immunomodulatory function, we examined the effect of CBLB502 on GVT activity. Using two tumor models that
do not express TLR5, and thereby do not directly respond to CBLB502, we found that CBLB502 treatment significantly enhanced
allogeneic CD8+ T cell-mediated GVT activity, which was evidenced by decreased tumor burden and improved host survival.
Importantly, histopathologic analyses showed that CBLB502 treatment did not exacerbate the moderate graft-versus-host disease
condition caused by the allogeneic CD8+ T cells. Moreover, mechanistic analyses showed that CBLB502 stimulates CD8+ T cell
proliferation and enhances their tumor killing activity mainly indirectly through a mechanism that involves the IL-12 signaling
pathway and the CD11c+ and CD11b+ populations in the bone marrow cells. This study demonstrates a new beneficial effect of
CBLB502, and suggests that TLR5-mediated immune modulation may be a promising approach to improve GVT immunity
without exacerbating graft-versus-host disease. The Journal of Immunology, 2012, 189: 4719–4727.
4720
while, preclinical studies with mouse models have shown that
CBLB502 can inhibit acute renal ischemic failure and can protect
mice from dermatitis caused by local cancer radiotherapy (30, 31).
These reports support a notion that CBLB502 may be an effective
agent in modulating beneficial immune responses. Therefore, the
goal of this study is to test the hypothesis that CBLB502 can
improve CD8+ T cell response in the context of allo-BMT and
whether it affects the balance between GVT immunity and
GVHD.
Materials and Methods
Mice and cell lines
Reagents and Abs
CBLB502 was derived from flagellin as described previously (29). D-luciferin was used as the luciferase substrate for bioluminescence imaging to
determine the tumor burden in vivo or in vitro. Kits or microbeads used for
the isolation of CD8+ T cells (Pan T cell kit), T cell-depleted bone marrow
(TCD-BM) cell (CD90.2 beads), CD11b+ cell (CD11b+ beads), and
CD11c+ cells (CD11c+ beads) were purchased from Miltenyi Biotec. Flow
cytometry Abs, including anti-CD8 (53.67), anti-CD3 (145-2C11), antiCD4 (RM4-4), anti-CD4 (RM4-5), H-2Kb (AF6-88.5.5.3), anti-CD11b
(M1/70), and anti-CD11c (N418), were purchased from eBioscience.
RT-PCR
For analysis of TLR5 mRNA expression, total RNA from A20, P815, and
SCCVII were extracted using TRIzol reagent according to manufacturer’s
instructions (Invitrogen, Carlsbad, CA). To eliminate any eventual contamination with genomic DNA, we treated isolated RNAs (1 mg) with DNaseI
(Invitrogen, Carlsbad, CA). cDNAs were synthesized using SuperscriptTM
II Reverse Transcriptase and oligo(dT)12-18 primer (Invitrogen), according to manufacturer’s protocol. A total of 40 ng cDNA was amplified by
PCR using primer specific for TLR5 (GenBank accession number
NM_016928.2) gene (forward, 59-AGTCCCCCAGCTCCAGTTTC-39; reverse, 59-GGAGCCCCCTAGCAGTGAGT-39). The RT-PCR conditions were
95˚C for 2 min, followed by 30 cycles of 95˚C for 30 s, 55˚C for 20 s, and
72˚C for 1 min, and 1 cycle of 72˚C for 10 min (for final extension). Primer
specific for GAPDH (forward, 59-ACCACAGTCCATG CCATCAC-39;
reverse, 59-TCCACCACCATGTTGCTGTA-39) was used as a control. The
RT-PCR conditions for GAPDH were 95˚C for 2 min, followed by 20
cycles of 95˚C for 30 s, 50˚C for 20 s, and 72˚C for 1 min, and 1 cycle of
72˚C for 10 min (for final extension).
Preparation of CD8+ T cells, TCD-BM cells, and CD11c+ and
CD11b+ bone marrow cells
CD8+ T cells (purity 85–90%) were purified with autoMACS from the
spleens by using Pan T isolation kit II combined with biotin-conjugated
anti-CD4 according to the protocols (Miltenyi). TCD-BM cells were
prepared by using anti-CD90.2 microbeads. For CD11c+ bone marrow
(BM) cells, positive selection was applied to total BM cells with CD11c
microbeads (Miltenyi) (purity ∼80%). For CD11b+ BM cells, positive
selection was applied to total BM cells with CD11b microbeads (Miltenyi)
(purity . 99%).
GVT model
BALB/c (H-2d) host mice were irradiated with 9 Gy from [137Cs] sources
at Roswell Park Cancer Institute. DBA/2 (H-2d) host mice were irradiated
with 14 Gy by two separate doses with 24-h interval. Two days (for BALB/c
mice) or 1 d (for DBA/2 mice) after the last irradiation, all host mice were
inoculated with 1.0 3 106 A20 tumor cells or 1.0 3 104 P815 tumor cells
i.v. followed by another i.v. injection of 2.0 3 106 TCD-BM cells only or
combined with 0.3 3 106 CD8+ T cells isolated from C57BL/6 (H-2Kb)
mice. CBLB502 was given s.c. by 1 mg/dose per mouse at days 1, 3, 5, 7,
and 9 after allo-BMT. Bioluminescence imaging was performed to monitor
tumor burden as described previously (32–34). Tumor burden was
expressed as photon flux (photons/s).
MLR
A standard one-way MLR was performed in 1 ml media on a 48-well plate
by mixing total splenocytes (1.0 3 106) derived from C57BL/6 mice
(responders) with irradiated (100 Gy) BALB/c or DBA/2 splenocytes (1.0 3
106) as stimulators. For a modified one-way MLR, 0.2 3 106 CD8+ T cells,
purified from C57BL/6 mice, were cultured with 1.0 3 106 irradiated
BALB/c or DBA/2 splenocytes (stimulators) in the presence or absence of
0.5 3 106 C57BL/6-derived TCD-BM cells. For in vitro tumor burden
assay, tumor cells were added after 4 d of MLR and then cocultured for
another 12 h before bioluminescence imaging.
CFSE-based proliferation assay
Purified CD8+ T cells (1.0 3 107/ml) were stained with CFSE at a final concentration 0.3 mg/ml for 5 min at 37˚C. A total of 0.2 3 106 CFSE-labeled
CD8+ T cells were plated as MLR responders in 1 ml media on 48-well
plate with or without 0.5 3 106 TCD-BM cells in the presence of irradiated
BALB/c splenocytes treated with either CBLB502 or PBS. After culturing
for 3 or 5 d, cells were harvested and stained with mouse H-2Kb and CD8
Abs. CFSE dilution on the 7-aminoactinomycin D (7-AAD)2H-2Kb+CD8+
cells was analyzed with FCS express software (De Novo Software).
Histopathologic analysis of GVHD target organs
Mice were euthanized on day 31 after allo-BMT. Large and small intestines
were removed, formalin fixed, sectioned, and stained with H&E. Intestine
tissues were examined for the presence of crypt epithelial cell apoptosis,
crypt loss, surface colonocyte vacuolization, surface colonocyte attenuation, lamina propria inflammatory cell infiltrate, mucosal ulceration, luminal sloughing of cellular debris, leukocyte infiltration of the lamina
propria, and villous blunting. A previously established semiquantitative
scoring system was used to assess abnormalities associated with GVHD
(35). The scoring system designated 0 as normal, 0.5 as focal and rare, 1.0
as focal and mild, 2.0 as diffuse and mild, 3.0 as diffuse and moderate, and
4.0 as diffuse and severe.
Results
CBLB502 enhances CD8+ T cell-mediated GVT effect
This study is designed to determine the effect of CBLB502 on GVT
activity that is mediated by donor-derived immune cells. Previously, CBLB502 demonstrated potent radioprotection effect to
lethally irradiated animals if given before or immediately after
irradiation exposure (29). CBLB502 did not show detectable radioprotection effect if administered 2 d after lethal irradiation. To
exclude any radioprotection effect that may interfere with the
GVT activity, we established a 2-d delayed allo-BMT model. The
BALB/c (H-2d) host mice were lethally irradiated (9 Gy)
2 d before being transplanted with TCD-BM cells derived from
the C57BL/6 (H-2b) donor mice. In addition, to exclude any direct
response to CBLB502 by tumor cells, we have used two tumor
models that do not express TLR5 and therefore do not directly
respond to CBLB502 (Fig. 1A). Specifically for the A20 lymphoma model, the BALB/c (H-2d) host mice were lethally irradiated 2 d before an i.v. inoculation with 1.0 3 106 A20 cells,
followed by an i.v. injection of 2.0 3 106 TCD-BM cells only or
combined with 0.3 3 106 CD8+ T cells purified from C57BL/6
donors. These mice were then treated with CBLB502 (1.0 mg in
0.1 ml PBS per mouse) or PBS via s.c. route for five times at 1, 3,
5, 7, and 9 d after allo-BMT. To determine the effect of CBLB502
on GVT activity, we performed bioluminescence imaging to
measure tumor burden. Compared with PBS control, CBLB502
treatment showed no significant effect on tumor burden in the
hosts receiving TCD-BM only (Fig. 1B), suggesting that CBLB502
did not affect tumor growth in the absence of donor T cells.
Transplant of CD8+ T cells in addition to TCD-BM significantly
reduced tumor burden (p , 0.0001), indicating that donor CD8+
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C57BL/6 (H-2b) mice were purchased from The Jackson Laboratory.
BALB/c (H-2d) and DBA/2 (H-2d) mice were purchased from National
Cancer Institute. A20 lymphoma, derived from BALB/c strain, and P815
mastocytoma cells, derived from DBA/2 strain, were transduced to express
luciferase as described previously (32). SCCVII, derived from C3H/HEN
(H-2k) mice, is a squamous cell carcinoma tumor line that is known to
express TLR5 (31). All mice were maintained in specific pathogen-free
housing, and all experiments were conducted in accordance with the animal care guidelines for Roswell Park Cancer Institute, using protocols
approved by the animal studies committee.
CBLB502 ENHANCES GRAFT-VERSUS-TUMOR ACTIVITY
The Journal of Immunology
4721
T cells are responsible for tumor eradiation. Notably, CBLB502
treatment significantly enhanced the ability of donor CD8+ T cells
to control tumor growth (p = 0.009). To further examine the impact of transplant and CBLB502 treatment on the tumor-bearing
mice, we monitored survival (Fig. 1C). As expected, all of the
hosts receiving TCD-BM only died of tumor outgrowth, and
CBLB502 treatment had no significant effect on their survival.
Transplant of CD8+ T cells significantly prolonged host survival
(p , 0.0001) but failed to rescue the hosts from death caused by
tumor growth. CBLB502 treatment combined with CD8+ T cell
transplant rescued 60% (4/7) of the tumor-bearing hosts
(p = 0.0219), which were found tumor free at day 60 and gained
.10% of weight compared with their initial body weight. To rule
out a tumor model-specific or host strain-specific effect, we inoculated DBA/2 (H-2d) mice with the syngeneic P815 mastocytoma cells and performed similar transplantation with C57BL/6
mice as donors. DBA/2 mice were irradiated with a total of 14 Gy
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FIGURE 1. CBLB502 treatment enhances CD8+ T
cell-mediated GVT effect, reduces tumor burden, and
improves host survival. (A) TLR5 mRNA expression
was not detected in A20 and P815 tumor cells. SCCVII
tumor cell line, expressing TLR5, was used as a positive control. GAPDH mRNA expression is used as an
internal loading control. (B) Two days after lethal irradiation, BALB/c mice were i.v. inoculated with 1.0 3
106 luciferase-expressing A20 tumor cells and then
transplanted with 2.0 3 106 TCD-BM cells alone or
combined with 0.3 3 106 CD8+ T cells harvested from
C57BL/6 donor mice. These mice were then treated with
CBLB502 (1.0 mg in 0.1 ml PBS/mouse) or PBS via s.c.
route for five times at 1, 3, 5, 7, and 9 d after allo-BMT.
Tumor burden was measured by bioluminescence imaging. Representative data from one of three independent experiments are shown as mean 6 SD, with three to
four mice in each group. The p value was acquired by
two-way ANOVA analysis. (C) Kaplan–Meier survival
curves of the BALB/c hosts are summarized from two
independent experiments as described in (B). (D) One
day after receiving total 14 Gy irradiation split into two
separate doses with 24-h interval, DBA/2 mice were
inoculated with 1.0 3 104 luciferase-expressing P815
tumor cells and then transplanted with 2.0 3 106
TCD-BM cells alone or combined with 0.3 3 106
CD8+ T cells harvested from C57BL/6 donor mice.
CBLB502 treatment and tumor burden measurement
were performed as described earlier. Representative data
from one of two independent experiments are shown as
mean 6 SD, with three to four mice in each group. The
p value was acquired by two-way ANOVA analysis.
split into two separate doses with 24-h interval. One day after the
last irradiation, 1.0 3 104 P815 cells were inoculated, followed by
2.0 3 106 TCD-BM only or combined with 0.3 3 106 CD8+
T cells. Similar to what was observed in the A20 model,
CBLB502 treatment had no effect on P815 tumor burden in the
hosts receiving TCD-BM only (Fig. 1D), suggesting that CBLB502
did not affect P815 tumor growth in the absence of donor T cells.
Transplant of CD8+ T cells reduced P815 tumor burden
(p = 0.0187), and CBLB502 treatment enhanced the ability of
donor CD8+ T cells to control tumor growth (p = 0.0543). Together,
these results indicate that CBLB502 treatment enhances CD8+
T cell-mediated GVT activity in two independent tumor models.
CBLB502 enhances allogeneic CD8+ T cell-mediated tumor
killing in vitro and in vivo
To recapitulate the in vivo GVT effect and to test what cellular
compartments are responding to CBLB502, we moved onto a
4722
CBLB502 ENHANCES GRAFT-VERSUS-TUMOR ACTIVITY
CBLB502 augments allogeneic CD8+ T cell proliferation
in vitro and in vivo
FIGURE 2. CBLB502 treatment enhances CD8+ T cell-mediated tumorkilling activity in vitro and in vivo. (A and B) A modified one-way MLR
was set up in 1 ml culture in triplicate on 48-well plate. Purified CD8+
T cells (0.2 3 106; responders), derived from C57BL/6 mice, were cultured with irradiated BALB/c or DBA/2 splenocytes (1.0 3 106; stimulators) in the presence or absence of C57BL/6-derived TCD-BM cells
(0.5 3 106) and treated by CBLB502 (100 ng) or PBS. Four days later,
A20 (0.2 3 106) (A) or P815 (0.1 3 106) (B) target tumor cells were added
and incubated for another 12 h. Bioluminescence imaging was performed
to measure tumor burden in each well. Triplicate wells cultured with irradiated stimulators alone without adding TCD-BM or CD8+ T cells were
used as the control. All tumor burden data were normalized relative to the
average tumor burden in the control wells. Data were representative of three
Flagellin was previously reported to enhance CD8+ T cell response
only when it is fused with a peptide or protein Ag (22, 24). To
investigate whether CBLB502 can promote CD8+ T cell response
in this allo-BMT model, we used a CFSE dilution assay to measure CD8+ T cell proliferation in the modified MLR system. In
independent experiments. (C) Allo-BMT and CBLB502 treatment were
performed as described in Fig. 1B. At day 10 after allo-BMT, spleen cells
were harvested from the host. A total of 1 3 106 spleen cells from each
host were coincubated with 2.5 3 103 luciferase-expressing A20 tumor
cells in 96-well plates with triplicate samples for 20 h. Tumor burden was
then measured by bioluminescence imaging. All tumor burden data were
normalized relative to the average tumor burden in the control wells cultured with 2.5 3 103 A20 tumor cells alone. Data were representative of
two independent experiments. The p value was acquired by Student t test.
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modified MLR system (36). We used 1.0 3 106 irradiated BALB/c
(H-2d) splenocytes to stimulate 0.2 3 106 CD8+ responder T cells
in the absence or presence of 0.5 3 106 TCD-BM cells purified
from C57BL/6 mice (H-2b). After 4 d of culture, 0.2 3 106 A20
tumor cells were added as target and incubated for another 12 h.
Tumor burden was then measured by bioluminescence imaging. In
the absence of TCD-BM cells, CD8+ T cells did not show significant killing activity (Fig. 2A). CBLB502 treatment appeared to
slightly increase the killing activity of CD8+ T cells (p = 0.0135).
However, this system might not be stringent enough to conclude
whether CBLB502 directly stimulate CD8+ T cells, because the
purity of the isolated CD8+ T cells is typically 85–90% and
contamination by other cell types (Supplemental Fig. 1) could
mediate an indirect activation. Nevertheless, when TCD-BM cells
were added in the MLR culture, the killing activity of CD8+
T cells was significantly increased (p = 0.0046), probably because
BM-derived APCs augmented CD8+ T cell activation in this
system. Moreover, CBLB502 treatment in the presence of TCDBM further enhanced the killing activity of CD8+ T cells
(p = 0.0101). Finally, it is important to emphasize that the presence of TCD-BM cells caused the highest stimulation effect of
CBLB502 on the killing activity CD8+ T cells (p , 0.0001),
suggesting that BM-derived APCs may be the principal responder
to CBLB502. We also performed equivalent experiments with the
P815 tumor model and observed similar results (Fig. 2B). Together, these results demonstrate that, in this modified MLR system, CBLB502 treatment significantly enhances the tumor-killing
activity of allogeneic CD8+ T cells, and that some BM-derived
cells, probably APCs, can respond to CBLB502 and mediate this
stimulation effect.
In addition, to examine whether CBLB502 enhances the in vivo
cytotoxic activity of donor CD8+ T cells, we transplanted C57BL/
6-derived TCD-BM and CD8+ T cells into BALB/c hosts. These
mice were then treated with CBLB502 or PBS five times as described earlier. At day 10 after allo-BMT, we harvested the spleen
cells from the hosts. These in vivo activated cells were then
coincubated in 96-well plates with luciferase-expressing A20 tumor cells for 20 h. Tumor burden was then measured by bioluminescence imaging. As shown in Fig. 2C, spleen cells harvested
from BM-only recipients showed no tumor-killing activity because their tumor burden was the same as the control wells seeded
with only tumor cells. In contrast, spleen cells harvested from the
hosts receiving both BM and CD8+ T cells showed significant
tumor-killing activity. CBLB502 treatment clearly enhanced the
tumor-killing activity of these CD8+ T cells (Fig. 2C).
The Journal of Immunology
brief, purified CD8+ T cells were labeled with CFSE and added
into the MLR culture as outlined earlier. After 3 or 5 d, the cultured cells were harvested and stained with Abs for cell-surface
markers. As a marker for active proliferation, CFSE dilution on
the 7-AAD2H-2Kb+CD8+ T cells was analyzed by flow cytometry. Only slight CD8+ T cell proliferation was observed at day 3,
whereas significant proliferation was observed at day 5 in the
MLR (Fig. 3A). CBLB502 enhanced CD8+ T cell proliferation,
and so did the presence of TCD-BM cells. Notably, CBLB502
treatment in the presence of TCD-BM cells promoted CD8+ T cell
proliferation to the maximum level, correlated with the highest
killing activity of CD8+ T cells under this condition. These data
clearly indicate that CBLB502 can augment CD8+ T cell proliferation in vitro through BM-derived cells.
In addition, to examine whether CBLB502 can augment CD8+
T cell proliferation in vivo, we transplanted C57BL/6-derived
TCD-BM and CD8+ T cells into BALB/c hosts inoculated with
A20 tumor cells. These mice were then treated with CBLB502 or
PBS for five times as described earlier. At day 9 after allo-BMT,
we harvested the spleen cells from the hosts and used flow cytometry
to analyze these cells. Although we transplanted only 0.3 3 106
CD8+ T cells into each host, we harvested an average of nearly
3 3 106 H-2Kb+CD8+ donor T cells from each host spleen, indicating that the CD8+ donor T cells underwent significant expansion
(Fig. 3B). Notably, CBLB502 treatment increased not only the
frequency of CD8+ donor T cells, but also the total cell number in
the spleen. Overall, CBLB502 treatment resulted in an expansion of
donor CD8+ T cells that was .2-fold that in the PBS-treated hosts.
CBLB502 increases CD8+ T cell response through CD11c+
and CD11b+ BM cells
Previous studies using RT-PCR and a polyclonal Ab suggested that
TLR5 may be expressed on many cell types, including DCs, macrophages, and epithelial cells (37, 38). However, because of the
lack of a reliable mAb, the cellular expression pattern of TLR5
remains poorly defined. To explore which cell type in the BM
FIGURE 4. CBLB502 enhances CD8+ T cell-mediated tumor-killing
activity through CD11c+ and CD11b+ BM cells. Purified CD8+ T cell
responders (0.2 3 106) derived from C57BL/6 mice were cultured alone in
a one-way MLR or with syngeneic CD11c+ (0.1 3 106) (A) or CD11b+
(0.25 3 106) (B) cells purified from BM in the presence of irradiated
BALB/c splenocytes as stimulators (1.0 3 106). The MLR cultures were
treated with CBLB502 (100 ng) or PBS in the beginning. After 4 d, A20
tumor cells were added and incubated for 12 h, followed by tumor burden
assay by bioluminescence imaging. Triplicate wells cultured with irradiated BALB/c splenocytes alone without adding TCD-BM or CD8+ T cells
were used as the control. All tumor burden data were normalized relative
to the average tumor burden in the control wells. Data were representative
of three independent experiments. The p value was acquired by Student t
test.
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FIGURE 3. CBLB502 enhances allogeneic CD8+ T cell proliferation
in vitro and in vivo. (A) Purified CD8+ T cell responders (0.2 3 106),
derived from C57BL/6 mice, were first labeled with CFSE (0.3 mg/ml) and
then cultured with irradiated BALB/c splenocytes (1.03 106) as stimulators in the presence or absence of C57BL/6-derived TCD-BM cells
(0.5 3 106). The MLR cultures were treated with CBLB502 (100 ng) or
PBS in the beginning. At day 3 or 5, cells were harvested and stained for
CD8 and H-2Kb, and dead cell exclusion marker 7-AAD. On the left are
representative histograms of CFSE dilution on the gated 7-AAD2H-2Kb+
CD8+ T cells. On the right are summary data of CFSE dilution (mean 6
SD) derived from three independent experiments with triplicate samples.
(B) Allo-BMT and CBLB502 treatment were performed as described in
Fig. 1B. At day 9 after allo-BMT, spleen cells were harvested from the
hosts and analyzed with flow cytometry. On the left are representative dot
plots of the H-2Kb+CD8+ donor T cells in the host spleen. On the right are
the total numbers of CD8+ donor T cells (mean 6 SD) in each host spleen,
with four mice assessed in each group. Total numbers of CD8+ donor
T cells were acquired by the equation: (total cell count) 3 (the percentage
of H-2Kb+CD8+ cells). The p value was acquired by Student t test.
4723
4724
CBLB502 ENHANCES GRAFT-VERSUS-TUMOR ACTIVITY
responds to CBLB502 and stimulates CD8+ T cell response in our
model, we first tested whether DC could mediate the CBLB502
effect. CD11c+ cells were purified from fresh BM by positive
selection (purity ∼80%). We used 0.1 3 106 CD11c+ cells to
replace the 0.5 3 106 TCD-BM cells in the modified MLR system.
Although CD11c+ cells alone did not show any direct tumorkilling activity (Fig. 4A), they exhibited a T cell activation effect similar to that of TCD-BM cells. In response to CBLB502
treatment, CD11c+ cells were able to significantly enhance the
killing activity of CD8+ T cells. Because CD11b+ population is the
most frequent myeloid population (39), we tested whether
CD11b+ cells could also mediate the effect seen with CBLB502
treatment. We purified CD11b+ cells from BM (purity . 99%) and
added them into the modified MLR culture. As shown in Fig. 4B,
CD11b+ cells alone did not show any killing activity but displayed
a T cell activation effect similar to that of CD11c+ cells. In response to CBLB502 treatment, CD11b+ cells were also capable of
enhancing the killing activity of CD8+ T cells. Together, these
results indicate that both CD11c+ and CD11b+ cells in the BM can
respond to CBLB502 and stimulate CD8+ T cell function in this
allo-BMT model.
cocultured for another 12 h, followed by bioluminescence imaging
to measure tumor burden. Compared with the stimulator-only
control in which no responder splenocytes were added, both WT
and IL-12Rb22/2 responders exhibited significant tumor-killing
activity. CBLB502 treatment of the MLR significantly enhanced
the tumor-killing activity of WT responders, whereas IL-12Rb22/2
responders showed a significantly decreased response to CBLB502
treatment (p , 0.01). To mimic the in vivo allo-BMT model, we
used purified TCD-BM cells and CD8+ T cells as responders to
perform a modified MLR as described earlier, which yielded very
similar results (Fig. 5B). Taken together, these results demonstrate
that the IL-12 signaling pathway is involved in CBLB502mediated stimulation of allogeneic T cell function.
TLR5 binding initiates a signal transduction cascade that leads to
the activation of NF-kB, which subsequently promotes the expression of a variety of inflammatory cytokines (40–42). Among
them, IL-12 is known to be produced by DCs and macrophages,
and to play a critical role for T cell activation (43). To examine the
role of the IL-12 signaling pathway in CBLB502-mediated activation of T cell function, we used two MLR systems. First, in
a standard one-way MLR, we used total splenocytes harvested
from C57BL/6 WT and IL-12Rb22/2 mice as responders stimulated by irradiated BALB/c or DBA/2 splenocytes (Fig. 5A). After
4 d of MLR, A20 or P815 tumor cells were added as targets and
FIGURE 5. CBLB502 enhances CD8+ T cell-mediated tumor-killing activity through the IL-12 signaling
pathway. (A) A standard one-way MLR was performed
by mixing total splenocytes (1.0 3 106) derived from
C57BL/6 WT or IL-12Rb22/2 mice (responders) with
irradiated BALB/c or DBA/2 splenocytes (1.0 3 106)
as stimulators. (B) A modified one-way MLR was
performed by using purified CD8+ T cells (0.2 3 106)
and TCD-BM cells (0.5 3 106) derived from C57BL/6
WT or IL-12Rb22/2 mice as responders and irradiated
BALB/c or DBA/2 splenocytes (1.0 3 106) as stimulators. The MLR cultures were treated with CBLB502
(100 ng) or PBS in the beginning. After 4 d, A20 or
P815 tumor cells were added and incubated for 12 h,
followed by tumor burden assay by bioluminescence
imaging. Triplicate wells cultured with irradiated stimulators alone without adding TCD-BM or CD8+ T cells
were used as the control. All tumor burden data were
normalized relative to the average tumor burden in the
control wells. Data were representative of three independent experiments. The p value was acquired by
Student t test.
We have shown earlier that CBLB502 treatment enhances GVT
effect in the allo-BMT model. To explore any potential effect on
GVHD in the tumor-bearing mice, we collected tissue samples at
31 d after transplantation, a critical period when some of the host
mice started to die of tumor growth. We performed histopathologic
analyses of the skin, liver, and small and large intestines. No evidence of GVHD was observed in the skin. The liver samples did
not yield conclusive results because of various amounts of tumor
cell infiltrates making it difficult to distinguish GVHD damage
from tumor-induced damage. Nevertheless, we observed reliable
evidence of GVHD in both large and small intestines that were free
from tumor infiltration. We used an established scoring system to
evaluate GVHD severity (35). As shown in Fig. 6A, transplant
of CD8+ T cells induced mild to moderate condition of GVHD.
However, CBLB502 treatment did not cause any significant histopathologic difference in the tumor-bearing mice. Body weight
was also monitored, and CBLB502 administration did not cause
significant weight loss. Together, these results indicate that the
posttransplant administration of CBLB502 did not exacerbate
GVHD in the tumor-bearing mice.
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CBLB502 promotes allogeneic CD8+ T cell response through
IL-12 signaling pathway
CBLB502 does not exacerbate GVHD in the tumor-bearing mice
The Journal of Immunology
4725
CBLB502 does not affect donor cell engraftment or CD8+
T cell differentiation
In allo-BMT model, donor cell engraftment is a critical aspect that
can impact GVT effect and host survival. To determine whether
CBLB502 treatment influences donor cell engraftment, we used
flow cytometry to measure the frequency of H-2Kb+ donor cells in
host spleens at day 31 after allo-BMT. As shown in Supplemental
Fig. 2A and 2B, .95% of host spleen cells were found to be
H-2Kb+ donor derived, and CBLB502 treatment did not cause
any significant change. These results indicate that donor cells
engrafted successfully, and that our CBLB502 treatment scheme
did not affect donor cell engraftment. To further check whether
CBLB502 treatment could cause any change in donor T cell
phenotype, we determined the frequencies of naive (CD442
CD62L+), central memory (CD44+CD62L+), and effector memory
(CD44+CD62L2) cells in the host spleens at days 10 and 31 after
allo-BMT. As shown in Supplemental Fig. 2C and 2D, CBLB502
treatment did not cause any significant change for these phenotypes. These data indicate that this scheme of CBLB502 treatment
does not change the phenotypic differentiation of donor T cells.
Discussion
Using an allo-BMT model, we show that a novel TLR5 agonist
CBLB502 significantly enhances CD8+ T cell-mediated GVT immunity. CBLB502 improves the proliferation and function of al-
logeneic CD8+ T cells mainly indirectly through a mechanism that
involves donor BM-derived CD11c+ and CD11b+ cells and the IL12 signaling pathway. As a result, CBLB502 treatment leads to
reduced tumor burden, prolonged survival for the tumor-bearing
mice, and tumor-free survival for about half of hosts prechallenged
with tumor cells. Importantly, CBLB502 treatment induces the
improved GVT effect without causing more severe GVHD.
A great challenge in allo-BMT is to identify novel approaches
that can separate the beneficial GVT effect from the undesirable
GVHD damage. Therefore, it is a significant finding that CBLB502
treatment enhances GVT effect but does not cause more severe
GVHD. However, our results are not entirely consistent with a
recent report, which used tumor-free hosts to show that peritransplant administration of flagellin reduced GVHD in an alloBMT model whereas enhancing donor-derived antiviral immunity (44). The discrepancy may be because of several factors,
besides the difference between flagellin and CBLB502. We used
BALB/c hosts, whereas the previous report used C57BL/6 hosts.
For the GVT study, we transplanted 0.3 3 106 CD8+ T cells that
the hosts could tolerate without developing lethal GVHD, whereas
the previous report transplanted 5 3 106 total splenocytes to study
lethal GVHD. In addition, the different administration scheme
may be another factor. In the previous report, flagellin was given
at 3 h before irradiation and 24 h after transplantation. In our
study, CBLB502 treatment initiated 3 d after irradiation and
1 d after transplantation, based on a rationale that its potent ra-
Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017
FIGURE 6. CBLB502 treatment does not affect
GVHD in the tumor-bearing mice. Thirty-one days
after tumor inoculation and allo-BMT, large and
small intestine tissues were prepared and stained
with H&E. A previously established semiquantitative scoring system was used to assess abnormalities
associated with GVHD. The scoring system designated 0 as normal, 0.5 as focal and rare, 1.0 as focal
and mild, 2.0 as diffuse and mild, 3.0 as diffuse and
moderate, and 4.0 as diffuse and severe. (A) Microscopic analysis (with 3400 magnification) of
large intestines of tumor-bearing mice treated with
CBLB502 or PBS control. Blue arrows indicate
apoptotic cells. Green arrows indicate crypt destruction. (B) GVHD scores summarized from two
independent experiments are shown, with 9–11 mice
assessed in each group. The p value was acquired by
Student t test.
4726
Acknowledgments
We thank the Roswell Park Cancer Institute flow cytometry facility and molecular imaging facility, and Scott Abrams, Mary Lynn Hensen, Jeremy
Waight, Kelvin Lee, Maegan Capitano, and Elizabeth Repasky for helpful
advice and technical assistance.
Disclosures
L.G.B. and A.V.G. are paid consultants of Cleveland BioLabs, Inc. A.V.G. is
a shareholder of Cleveland BioLabs, Inc. The other authors have no financial conflicts of interest.
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As shown in Supplemental Fig. 2, our treatment scheme successfully excluded any undesirable effect on donor cell engraftment,
which is a prerequisite for donor-derived GVT effect. Furthermore, this scheme induces transient proliferation and activation of
donor T cells without changing their long-term phenotypic differentiation, which may explain why CBLB502 enhances GVT
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Flagellin is a highly effective adjuvant for the CD4+ T cell and
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In summary, this study shows that CBLB502 can significantly
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model, this study demonstrates a new beneficial effect of CBLB502
and also provides evidence to support a rational scheme of
CBLB502 administration that may be a promising approach to improve the effectiveness of allo-BMT without exacerbating GVHD.
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