A HLA-A2-restricted CTL epitope induces anti-tumor effects against human lung cancer in mouse xenograft model
Yuh-Pyng Sher1,3*, Su-I Lin6,7*, I-Hua Chen6, Hsin-Yu Liu6, Chen-Yuan Lin4, I-Ping Chiang5,
Steve Roffler8, Hsin-Wei Chen 2,6, Shih-Jen Liu2,6, 7
1Graduate Institute of Clinical Medical Science, 2Graduate Institute of Immunology, China
Medical University, Taichung, Taiwan
3Center for Molecular Medicine, 4Division of Hematology and Oncology, 5Department of
Pathology, China Medical University Hospital, Taichung, Taiwan
6National Institute of Infectious Diseases and Vaccinology, National Health Research
Institutes, Zhunan, Miaoli, Taiwan,
7Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 8 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
* These authors contributed equally to this work
Correspondence to: Shih-Jen Liu
National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, No. 35 Keyan Road, Zhunan Town, Miaoli County, 350, Taiwan
E-mail: levent@nhri.org.tw Fax: 886-3-758-3009 Tel: 886-3-724-6166 ext. 37709 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Abstract
Cancer immunotherapy is attractive for antigen-specific T cell-mediated anti-tumor therapy, especially in induction of cytotoxic T lymphocytes. In this report, we evaluated human CTL epitope-induced anti-tumor effects in human lung cancer xenograft models. The tumor associated antigen L6 (TAL6) is highly expressed in human lung cancer cell lines and tumor specimens as compared to normal lung tissues. TAL6 derived peptides strongly inhibited tumor growth, cancer metastasis and prolonged survival time in HLA-A2 transgenic mice immunized with a formulation of T-helper (Th) peptide, synthetic CpG ODN, and adjuvant Montanide ISA-51 (ISA-51). Adoptive transfer of peptide-induced CTL cells from HLA-A2 transgenic mice into human tumor xenograft SCID mice significantly inhibited tumor growth. Furthermore, combination of CTL-peptide immunotherapy and gemcitabine additively improved the therapeutic effects. This pre-clinical evaluation model provides a useful platform to develop efficient immunotherapeutic drugs to treat lung cancer and demonstrates a promising strategy with benefit of antitumor immune responses worthy of further development in clinical trials.
Key words: peptide, cytotoxic T lymphocytes, TAL6, lung cancer 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
Introduction
Lung cancer is one of the leading causes of malignancy-related death because of its
frequency and its highly metastatic potential. Currently, immunotherapy for lung cancer is
considered as a promising treatment capable of inducing systemic tumor-specific immune
responses without provoking serious side effects [1, 2]. The critical factor for
immunotherapy is to choose potential cancer specific antigens as targets without affecting
normal tissues.
The tumor-associated antigen L6 (TAL6), is a tumor-specific antigen which is a distant
member of the transmembrane-4 superfamily (TM4SF) and is often overexpressed in human
lung, breast, and colon cancer tissues but not in normal tissues [3-5]. Antibody-based
immunotherapy against membrane TAL6 protein was used to treat breast cancer in clinical
studies [6-12], but the therapeutic effects were limited. Several strategies are used to
improve immunotherapy for cancer treatment such as induction of cytotoxic T-cells (CTL)
responses to lung cancer antigens [13-15]. Utilization of synthetic peptides for CTL
epitopes–based vaccines are safe and easy to manufacture for clinical use. Recently, the
multi-peptides vaccine (IDM-2101) was designed to induce CTLs against five
tumor-associated antigens (TAAs) frequently overexpressed in NSCLC (i.e. carconoembryonic
antigen (CEA), p53, Her2, and melanoma antigens (MAGE)), and it provided clinical
efficacy in metastatic NSCLC [16]. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Although immunotherapy or cancer vaccines to induce CTLs for NSCLC treatment has
revealed promising effects [1, 2], lack of proven clinical benefits continues to block
development of immunotherapy. Developing a convenient and predictive model to evaluate
the CTL activity before clinical trials is critical and essential to increase the successful rate
of immunotherapy. In this study, we generated a HLA-A2 transgenic mouse model and
human tumor xenograft in SCID mice model to investigate the pre-clinical therapeutic effect
of immunotherapy to human tumors via adoptive transfer of HLA-A2 restricted CD8+ CTLs
recognizing human tumor antigens. Because TAL6 is a tumor specific antigen and correlates
with cancer metastasis, the TAL6-derived CTL peptide was investigated to elicit tumor
specific CTL responses and additive therapeutic effects with gemcitabine in pre-clinical
trials. 61 62 63 64 65 66 67 68 69 70 71
Materials and Methods Detection of TAL6 expression
TAL6 protein on cells was detected by flow cytometry using a mouse monoclonal antibody
against TAL6 [17]. A rabbit anti-human TM4SF1 antibody (Sigma) was used to detect
TAL6 in lung cancer tissues by immunohistochemical (IHC) staining with horseradish
peroxidase-conjugated avidin biotin complex (ABC) from the Vectastain Elite ABC Kit
(Vector Laboratories, Burlingame, CA) and AEC chromogen (Vector Laboratories). The
sections were counterstained with hematoxylin and mounted. An Asian lung cancer tissue
array was purchased from US Biomax (LC1006, Rockville, MD). All stainings were
evaluated by experienced histologists.
Animals and Cell lines
Human lung adenocarcinoma cell lines CL1-0 (low invasiveness) and CL1-5 (high
invasiveness) were established from the same lung cancer origin [18]. Primary cultured cell
lines, PE53 and PE8, were established from the pleural effusion of two individual
adenocarcinoma lung cancer patients with written informed consent from each patient. Other
lung cancer cell lines were obtained from the Bioresource Collection and Research Center
(Hsin-Chu, Taiwan). The EL4-TAL6-A2 cells stably express TAL6 and HLA-A2 in EL4
cells. The EL4-TAL6 cells are EL4 cells that stably express TAL6. These cells were cultured 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
transgenic mice were kindly provided by professor Show-Li Chen (National Taiwan
University, Taiwan). All animal experiments were performed in specified pathogen-free
(SPF) conditions under protocols approved by the Animal Committee of the National Health
Research Institutes (NHRI).
Peptide immunization of HLA-A2 transgenic mice
Peptide A2-5 (LLMLLPAFV) from the TAL6 protein was identified as an HLA-A2 specific
CTL epitope in our previous study [17]. An irrelevant peptide (LYLTQDLFL, from the
spike protein of SARS CoV) was included for immunization. Peptides with purity >90 %
were synthesized by the peptide synthesis core facility of the National Health Research
Institutes (NHRI) in Taiwan. All peptides were dissolved in 100% DMSO at 10mg/mL as
stock solutions, stored at -80°C, and analyzed by HPLC and mass spectrometry to verify
their purity. Peptide immunization was performed as previously described [17]. In brief, 1
mg CTL peptide and 1 mg Th epitope peptide (PADRE: AKFVAAWTLKAAA) in 0.5 ml
PBS were mixed with 0.5 ml of Montanide ISA 51 (ISA, from SEPPIC company) and then
the mixtures were injected s.c. in HLA-A2 Tg mice twice at a 7-day interval. The
splenocytes were collected 7 days after the second injection for further ELISPOT assay or
CD107a+ CD8+ cells detection.
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
The ELISPOT assay was performed as previously described [19]. In brief, 5x105 spleen cells
with 10 μg/ml of the indicated peptides were added to a 96-well PVDF-membrane plate coated with anti-IFN-γ antibody. Spots were developed using a 3-amine-9-ethyl carbazole (AEC, Sigma) solution. The reaction was stopped after 4-6 minutes by running the plate under tap water. The spots were then counted using an ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH).
Tumor model and treatment
HLA-A2 Tg mice were injected s.c. twice at a 2-week interval with A2-5 formulated in
ISA-51 with 50 μg of Th and 10 μg of CpG. CpG was purchased from GeneDirex. It consisted of
a sequence of 5’-TCCATGACGTTCCTGACGTT-3’ with a phosphorothioate backbone.
The CD8+ T cells were harvested by mouse CD8+ T cell Dynabeads (invitrogen). The
EL4-TAL6 or EL4-EL4-TAL6-HLA-A2 cells (2x105) were inoculated s.c. at the opposite site of
peptide injection seven days after the second immunization. Tumor size was measured 3
times per week by using the formula: tumor volume = length x width x width /2. In a
metastatic mouse model of melanoma, HLA-A2 Tg mice were immunized with
ISA/A2-5/Th/CpG by the above procedure and then B16 or B16-TAL6-A2 (5×105) cells were
intravenously injected at 7 days post final immunization. Lung tissues were collected and
fixed in 8% formalin for detecting the tumor nodules after 20 days of tumor inoculation. In
human tumor xenograft model, human lung cancer H2981 cells (1×108) were subcutaneously
112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131
model. Purified CD8+ T cells (1x107) from peptide-immunized HLA2 Tg mice were
intravenously delivered into SCID mice on day 7 post tumor inoculation. In combination
therapy, 22 or 30 days after H2981 inoculation, gemcitabine was administered i.p. as a single
dose (3mg/mouse) in the cancer xenotransplanted mice. At 5 days after gemcitabine
administration, isolated CD8+ T cells (1x107) were adoptively transferred into mice.
CD107a cytotoxicity assay
The CD107a cytotoxicity assay has been described in previous reports [17]. HLA-A2 Tg
mice were injected s.c. twice with the indicated peptide (50μg/ml) emulsified in ISA in the
presence of Th peptide (50μg/ml) and CpGODN (10μg/mouse). On day 7 after the second
immunization, splenocytes were harvested and then suspended at 2x107 cell/ml in medium
that contained 10μg/ml of the indicated peptides (50μg/ml) or cells (2x106 cell/ml) and
PE-conjugated anti-CD107a monoclonal antibody (1:100) in 96-well round-bottom plates. After
2 hours of incubation at 37°C, brefeldin A (10μg/ml) and monensin (0.66μg/ml) were added
for another 2-6 hours. The plates were washed with PBS containing 0.1% FBS, and rat
anti-mouse Fc antibody (1:100) was added for 5 minutes, followed by addition of the
FITC-conjugated rat anti-mouse CD8 antibody (1:100) for 30 minutes. The cytotoxic CD107a+
CD8+ cells were analyzed on a flowcytometer (FACS Calibur, BD Bioscience).
133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
51Cr release cytotoxicity assays
To analyze T cell cytotoxicity, HLA-A2 transgenic mice were subcutaneously immunized
twice with peptide (50μg/mouse), CpGODN (10μg/mouse) and ISA. 7 days after the final
immunization, spleens and lymph nodes were isolated and stimulated with peptide A2-5
(10μg/ml) and IL-2 (10U/ml) for 5 days as effector cells. Effector cells were incubated with
mouse anti-HLA-A2, rat anti-CD8 and mouse or rat IgG isotype control antibodies at 37°C
for 1 hour. H2981 cells (5x106) were labeled with chromium (100μCi) for 2 hour as target
cells. The target cells (5x103/well) were incubated at E: T cell ratios of 100:1 in a final
volume of 100 l/well at 37°C for 5 hours. Supernatants (100μl) were harvested, and 51Cr
release was measured. Spontaneous release was measured in wells containing target cells
alone. Maximum 51Cr release in target cells was measured by adding 2% Triton X-100.
Specific lysis (%) was calculated as: 100x (test 51Cr release – spontaneous 51Cr)/ (maximum
51Cr release – spontaneous 51Cr).
Tumor-infiltrating cells analysis
Cell surface marker staining and flow cytometry were performed as previously described
[20]. Briefly, cell suspensions from tumor-bearing mice were mechanically disrupted into
fragments at 24 hr after CD8+ T cells adoptively transferred. Cells were stained with
PE-conjugated anti-CD11b and PE-Cy7-PE-conjugated anti-Gr-1 antibodies to quantify MDSCs, 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
FITC-conjugated CD8 to quantify CD8 T cells, and a PE-conjugated HLA-A2/A2-5 tetramer
to detect A2-5-specific T cells. The cell populations were determined via flow cytometry
(FACSCalibur, BD Bioscience, San Jose, CA, USA). All data were acquired using a
FACSCalibur device and were plotted using FCS express version 3.0 software (research
edition, De Novo software TM).
Statistical analysis
The statistical significance of differences between mean values of the experimental
groups was determined using one way analysis of variance (ANOVA). The differences were
considered statistically significant if the P value was <0.05. 172 173 174 175 176 177 178 179 180 181
Results
High TAL6 protein expression in lung cancer cell lines and clinical lung tumor tissues To determine whether TAL6 protein was overexpressed in lung cancer cells as a tumor
specific antigen for treatment, lung cancer cell lines and primary lung tumor tissues from
NSCLC patients were stained with an anti-TAL6 monoclonal antibody. By using flow
cytometry, high levels of TAL6 protein was detected on most of the lung cancer cell lines
except NCI-H157 (Fig. 1A). By comparing two cell lines from the same original cell
population, we found higher TAL6 expression on CL1-5 cells with high metastatic ability as
compared to parental CL1-0 cells with low metastatic ability (Fig. 1A), which is consistent
with previous reports that TAL6 expression on tumor cells is associated with cancer
metastatic ability [21, 22]. In addition, TAL6 was detected in primary cultured lung cancer
cells from lung cancer patients’ pleural effusion (Fig. 1B) and high TAL6 expression was
maintained in human tumor xenografts in SCID mice (Fig. 1C). To assess the clinical
relevance of TAL6 protein expression in lung cancer patients, we performed IHC staining of
TAL6 in a lung cancer tissue array, which contains 45 tumor and 55 adjacent normal tissues
from Asian patients (Fig. 1D). For comparison, we set a score over 0 as positive and found
60% positive staining of TAL6 in lung cancer tissues and 12.7% in normal lung tissues (Fig.
1E). Notably, most normal lung tissues were in negative and the maximal score of positive
staining in normal lung tissues was 1, which was only detected in paired tumors with 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200
positive staining (Fig. 1F). TAL6 expression was high in lung cancer tissue but remained
low in matched adjacent normal lung tissue (P < 0.0001; Fig. 1F). Furthermore, to assess the
diagnostic accuracy, we performed a receiver operating characteristic (ROC) curve analysis
which is used in medicine to determine a cutoff value for the TAL6 IHC result of the tissue
array [23]. Area under the curve (AUC) can range from 0.5 (random chance, or no predictive
ability) to 1 (perfect discrimination/accuracy). On ROC curve analysis, the area under the
curve (AUC) was 0.75 and its sensitivity and specificity was 70.6% and 70.6% (Fig. 1G),
indicating TAL6 expression is indeed abundant in lung cancer tissues.
Immunization of a TAL6-derived CTL epitope can suppress tumor growth in HLA-A2 transgenic mice
In our previous study, we identified a HLA-A2 specific CTL epitope of TAL6, called
peptide A2-5, that could induce HLA-A2-restricted immunity and TAL6 specific
cytotoxicity of CTLs by the immunization of A2-5 formulated in incomplete Freund’s
adjuvant (IFA) with a universal Th epitope Pan-DR peptide against TAL6-expressing breast
tumors [17]. To improve the immune-stimulatory activity of the TAL6-derived CTL epitope,
we formulated peptides with adjuvant ISA (see material and method) to boost host
immunity. Splenocytes from Th and A2-5 immunized A2 Tg mice were restimulated with
EL4-TAL6-A2 (EL4 cells that expressed TAL6 and HLA-A2) or EL4-TAL6 (EL4 cells that 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219
expressed TAL6 alone) cells in vitro. We found a higher frequency of CD107a+CD8+ cells
(cytolytic T cells) when cultured with EL4-TAL6-A2 cells (2.05 ± 0.40) as compared to
cultures with EL4-TAL6 cells (0.5 ± 0.44). That indicated cytolytic T cells from HLA-A2
specific CTL peptides immunized mice can be activated to specifically recognize cancer cell
expressed TAL6 and HLA-A2, but not to cells without HLA-A2 expression, supporting the
function of HLA-A2-restricted immunity (Fig. 2A). Because immunization of the A2-5
epitope could induce ex vivo CTL activity in the presence of HLA-A2, we further
investigated the function of A2-5 immunization for in vivo tumor development by
inoculating EL4-TAL6-A2 or EL4-TAL6 cancer cells in HLA-A2 transgenic mice. After
peptide immunization, the growth of EL4-TAL6-A2 tumors was significantly suppressed
compared with EL4-TAL6 tumors (Fig 2B). These results indicate that A2-5 peptide
immunization can induce HLA-A2-restricted CTL responses and provide therapeutic activity
in TAL6 and HLA-A2 co-expressing cancer cells.
Improved immunization of TAL6-derived CTL epitope can suppress tumor metastases and prolong survival in HLA-A2 transgenic mice
To further improve the immunization of peptide A2-5, TLR9 ligand CpG adjuvant was
included with the peptide and Montanide ISA-51 in HLA-A2 transgenic mice. Splenocytes
were harvested after the final immunization and T cell activation was analyzed using the 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238
IFN- secreting ELISPOT assay. Formulation of ISA/A2-5/Th/CpG (combination of
Montanide ISA-51, A2-5 peptide, Th peptide, and TLR9 ligand CpG) induced substantial
IFN- secretion as compared to A2-5/Th/CpG (161.1±18.38 v.s. 9±3.81) (Fig. 3A).
Comparison of the efficacy of each adjuvant showed that ISA/A2-5/Th/CpG induced the
strongest IFN- secretion as compared to ISA/A2-5/Th and A2-5/Th/CpG. In addition, ISA
provided greater adjuvant efficacy than CpG (Fig. S1). The ISA/A2-5/Th/CpG formulation
induced more CD107a+CD8+ cells, which were activated specifically with peptide A2-5 ex
vivo (Fig. 3B). Consistently, ISA/A2-5/Th/CpG induced more activated cytotoxic CD8+ T
cells after stimulation with EL4-TAL6-A2 cells (Fig. 3C). To determine whether the CTL
response elicited by the peptide A2-5 can inhibit cancer metastases in HLA-A2 Tg mice,
EL4-TAL6-A2 cells (2x104) were injected intravenously to develop a tumor metastatic
animal model. No metastatic tumors in lungs were observed in ISA/A2-5/Th/CpG
immunized mice at 20 days after tumor inoculation, whereas lung tumor nodules were
detected in the other groups (Fig. 3D). Moreover, the survival was significantly prolonged in
ISA/A2-5/Th/CpG immunized mice and moderately enhanced in A2-5/Th/CpG immunized
mice, compared to control mice (Fig. 3E). To further detect the effect of A2-5 peptide
specific TAL6-derived immunity in suppressing metastasis, melanoma B16 or
B16-TAL6-A2 cells (B16 cells that expressed TAL6 and HLA-B16-TAL6-A2) were intravenously injected in naïve
and ISA/A2-5/Th/CpG immunized HLA-A2 transgenic mice (Fig. 3F). Gross examination of 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
whole lung specimens demonstrated that tumor metastasis to lungs were dramatically
suppressed in ISA/A2-5/Th/CpG immunized mice bearing B16-TAL6-A2 tumors compared
to B16 cells group. Such protection in mice was reversed in naïve mice, indicating the A2-5
immunization can induce specific TAL6-derived immunity to reduce metastasis. Thus, these
results suggest that ISA/A2-5/Th/CpG could induce strong HLA-A2 specific CTL responses
against cancer metastases in HLA-A2 Tg mice.
The TAL6-derived CTL peptide immunization can suppress tumor growth in a human tumor xenograft animal model with adaptive T cell transfer from HLA-A2 transgenic mice
To determine whether the induction of HLA-A2 specific CTL responses by peptide A2-5
could inhibit the growth of human lung cancer cells, a human tumor xenograft animal model
was established for assessing the therapeutic effect of the TAL6 CTL peptides elicited
immune response. After identifying HLA types in human lung cancer cell lines, H2981 cells
with HLA A11 and A2 were used to establish a lung cancer animal model for further
treatment. After immunization of 5/Th/CpG/ISA twice in HLA-A2 transgenic mice,
A2-5-induced CTLs destroyed lung cancer H2981 cells (28.5 ± 1.78 ％ ) and this specific
cytotoxicity was inhibited with either anti-HLA-A2 (9.0 ± 0.74 ％) or anti-CD8 (10.0 ± 0.86
％) monoclonal antibodies in vitro (Fig. 4A). To evaluate the therapeutic efficacy of peptide-258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276
induced CTL responses in human tumor xenograft animal models, purified CD8+ T cells
from peptide immunized HLA-A2 Tg mice were then intravenously transferred into SCID
mice bearing H2981 tumors on day 7. The tumor progress in mice receiving adoptively
transferred CD8+ T cells from A2-5/Th/CpG/ISA immunized mice was reduced significantly
as compared to the control group (Fig. 4B), indicating anti-tumor activity of peptide A2-5
immunized CD8+ CTL cells against human lung cancer cells in vivo.
Combined therapy with gemcitabine and peptide A2-5 derived CTLs suppresses tumor growth and prolongs animal survival
Myeloid derived suppressor cells (MDSCs) represent one of the critical barriers to effective
immune responses for detecting and eliminating cancer cells. Gemcitabine (GEM), a
pyrimidine antimetabolite in clinical use for cancer treatment, has been reported to reduce
the frequency of MDSCs [24]. We found that the percentage of MDSCs increased in SCID
mice bearing H2981 tumors, but decreased with GEM treatment, indicating a role for GEM
modulating MDSCs (Fig. 5A). To determine whether combination treatment of GEM and
TAL6-derived CTL peptides immunization can enhance anti-cancer activity, CTLs derived
from peptide-immunized HLA-A2 transgenic mice were adoptively transferred into SCID
mice bearing H2981 tumors with or without GEM treatment. Although treatment with GEM
or adoptive transfer of peptide A2-5 vaccinated CD8+ T cells provided significant anti-tumor
277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
effects, combination of these two treatments reduced the tumor size more dramatically and
more effectively prolonged mice survival (Fig. 5B and C). Comparison of the median
survival time showed that the GEM+A2-5 CD8+T cells group had the longest survival of 97
days, better than A2-5 CD8+ (81 days), GEM+control CD8+ (63 days), GEM (65 days),
control CD8+ (60 days), and PBS (55 days) groups.
To investigate further whether MDSC elimination and peptide A2-5 derived CTLs are
critical for the additive effect of combined antitumor therapy, the tumor-infiltrated cell
population was analyzed. The percentage of MDSC was significantly decreased in GEM and
GEM combined with the peptide A2-5 vaccinated CD8+ T cells groups (Fig. 6A). Large
numbers of tumor-infiltrated CD8+T cells were detected in mice receiving control CD8+T
cells, A2-5 vaccinated CD8+ T cells, and GEM combined with a peptide A2-5 vaccinated
CD8+ T cells groups (Fig. 6B). However, the percentage of A2-5-HLA-A2 tetramer-binding
cells, which indicate the specific A2-5 derived CTLs, was greatly increased in the mice
treated with both GEM and peptide A2-5 vaccinated CD8+ T cells as compared to the A2-5
CD8+ group (Fig. 6C), even though the two groups had similar CD8+ T cells percentages
(Fig. 6B). It is likely that reduction of MDSC by gemcitabine preserved the specific A2-5
derived CD8+ T cells in tumors. These results suggest improved therapeutic application with
GEM and adoptive transfer of peptide A2-5 vaccinated CD8+ T cells. Furthermore, to
evaluate if the vaccine could suppress established tumors, we treated tumors after 30 days of 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314
cancer cell inoculation. GEM or adoptive transfer of peptide A2-5 vaccinated CD8+ T cells
suppressed tumor growth, and combination treatment showed additive anti-tumor activity
(Fig. 6D).
Discussion
Although TAL6 was considered as a target for antibody-based immunotherapy against
breast and lung cancer during the past decade, the therapeutic effects of TAL6
antibody-based immunotherapy are limited in humans. Alternatively, T cell-antibody-based immunotherapy
may be a feasible approach for cancer treatment. Recently, we have identified a
HLA-A2-restricted CTL epitope of TAL6 that can stimulate CTL activation to kill TAL6-expressing
MCF-7 breast cancer cells in vitro [17]. In this study, we further extended this T cell-based
immunotherapy for lung cancer treatment. TAL6 protein was previously detected with
antibody in most NSCLC tissues [4], but the expression frequency of TAL6 protein in lung
cancer is less reported in Asian patients. In this report, we found that TAL6 was highly
expressed in over 80% of lung cancer tissues in Asian patients, suggesting thatTAL6 could
be a tumor antigen. The CTL epitope A2-5 peptide induced anti-tumor effects against lung
cancer in HLA-A2 transgenic mice, and adoptive transfer of CD8+ T cells from peptide
A2-5 immunized mice into SCID mice inhibited tumor growth of human lung cancer xenografts.
More importantly, additive improvement of the therapeutic effects with T cell-based 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333
immunotherapy and gemcitabine chemotherapy was observed in a human cancer xenograft
model. These results demonstrate that peptide A2-5 immunization to elicit CTL responses is
feasible for lung cancer treatment.
Immunotherapy to induce cytotoxic T lymphocyte (CTL) activity is important to
inhibit human tumor growth and is a promised strategy. The deficiency of good evaluation
systems for pre-clinical therapeutic studies is a major challenge for T cell-based
immunotherapy assessment due to the human HLA restriction of therapeutic CTL peptides.
Here, we developed a convenient model to evaluate CTL activity before clinical trials in
HLA-A2 transgenic mice immunized with different formulations. ISA-51 adjuvant has been
used in human clinical trials and is safer than incomplete adjuvant (IFA). We found that
formulation with ISA-51, peptide A2-5, a Th epitope and TLR 9 agonist (CpG)
(ISA/A2-5/Th/CpG) provided stronger anti-tumor effects than formulation with ISA/A2-5/Th. The
results demonstrated that emulsion type adjuvant ISA may prolong the CpG release and
enhance CTL responses. The findings are similar to our previous reports that emulsion type
adjuvant PELC and PELA73 could enhance CTL responses and anti-tumor effects in the
presence of CpG ODN [25, 26]. Thus, combination of emulsion type adjuvant and a TLR9
agonist is a potent formulation for peptide-based therapeutic vaccines for cancer therapy. We proved that adoptive transfer of A2-5-induced CTL from HLA-A2 Tg mice could
inhibit H2981 cells growth in SCID mice, supporting the notion that peptide A2-5 could be 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352
effective in humans. However, there is still room to improve the anti-tumor effects of peptide
A2-5 immunization. The tumor-infiltrating immunosuppressive cells impair CTL functions
in the tumor microenvironment and are major barriers for cancer therapy. To overcome the
immunosuppressive microenvironment, several approaches are aimed at depleting
immunosuppressive cells including antibody deletion [27], chemotherapeutic drugs [28, 29],
or TLR ligand modulation [20, 30, 31]. We selected gemcitabine for combination therapy
because gemcitabine has been reported to inhibit Tregs and MDSCs in the tumor
microenvironment for enhancement of anti-tumor immunity [32-34]. We confirmed that the
number of MDSCs was reduced in human tumor-bearing SCID mice treated with
gemcitabine. Gemcitabine combined with CD8 T cells adoptively transferred from A2-5
immunized HLA-A2 Tg mice significantly suppressed the growth of human tumors in SCID
mice. These observations implied that combination of chemo-drugs and CD8+ T cell-based
immunotherapy might benefit cancer patients.
We provide direct evidence that A2-5-induced CD8+ T cells are able to kill human lung
cancer that expressed high-levels of TLA6 in vitro and in vivo. In addition, we showed that
peptide formulated with ISA/Th/CpG elicited strong anti-tumor immunity. To achieve the
maximum anti-tumor ability, selection of suitable adjuvants is important. In the future, the
Th epitope could be conjugated with the CTL epitope to simplify the peptide-synthetic
process. Furthermore, multiple CTL epitopes from different functional tumor-associated 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371
antigens could be added to boost the CTL activity.
Recently, TAL6 was found to be critical for endothelial cell function and tumor
angiogenesis [35] and could be a vascular therapeutic target in cancer therapy [36]. Another
TAL6 family protein, TM4SF5, is overexpressed in hepatocellular carcinoma and colon
cancer [37, 38]. The TM4SF5-specific monoclonal antibody could inhibit colon cancer
growth in a mouse model [39]. These studies demonstrated that TAL6 family proteins may
be good targets for antibody-based cancer immunotherapy. Therefore, our current study
provides a promising strategy to facilitate successful cancer therapy for cancer that expresses
TAL6 family proteins.
Acknowledgments
We thank Dr. Show-Li Chen (National Taiwan University, Taiwan) for supplying HLA-A2
Tg mice. This work was supported by a National Health Research Institutes intramural grant
(IV-103-PP20) and National Research Program for Biopharmaceuticals grants from the
Ministry of Science and Technology awarded to S.J. Liu (NSC 99-2323-B-400 -009 and
MOST 103-2325-B-400 -015). References 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
antigens: towards a new therapy for minimal residual disease. Expert Opinion on Biological
Therapy. 2008; 8(7):951-968.
2. Hirschowitz EA and Yannelli JR. Immunotherapy for Lung Cancer. Proc Am Thorac
Soc. 2009; 6(2):224-232.
3. Marken JS, Schieven GL, Hellstrom I, Hellstrom KE and Aruffo A. Cloning and
expression of the tumor-associated antigen L6. Proc Natl Acad Sci U S A. 1992;
89(8):3503-3507.
4. Hellstrom I, Horn D, Linsley P, Brown JP, Brankovan V and Hellstrom KE.
Monoclonal mouse antibodies raised against human lung carcinoma. Cancer research. 1986;
46(8):3917-3923.
5. Hellstrom I, Beaumier PL and Hellstrom KE. Antitumor effects of L6, an IgG2a
antibody that reacts with most human carcinomas. Proc Natl Acad Sci U S A. 1986;
83(18):7059-7063.
6. DeNardo SJ, DeNardo GL, Miers LA, Natarajan A, Foreman AR, Gruettner C,
Adamson GN and Ivkov R. Development of tumor targeting bioprobes ((111)In-chimeric L6
monoclonal antibody nanoparticles) for alternating magnetic field cancer therapy. Clinical
cancer research : an official journal of the American Association for Cancer Research. 2005;
11(19 Pt 2):7087s-7092s.
7. DeNardo SJ, DeNardo GL, Natarajan A, Miers LA, Foreman AR, Gruettner C, 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410
nanoparticle AMF--induced thermoablative therapy for human breast cancer in mice. J Nucl
Med. 2007; 48(3):437-444.
8. Denardo SJ, O'Grady LF, Richman CM, Goldstein DS, O'Donnell RT, Denardo DA,
Kroger LA, Lamborn KR, Hellstrom KE, Hellstrom I and Denardo GL.
Radioimmunotherapy for advanced breast cancer using I-131-ChL6 antibody. Anticancer
Res. 1997; 17(3B):1745-1751.
9. DeNardo SJ, Warhoe KA, O'Grady LF, Hellstrom I, Hellstrom KE, Mills SL, Macey
DJ, Goodnight JE and DeNardo GL. Radioimmunotherapy for breast cancer: treatment of a
patient with I-131 L6 chimeric monoclonal antibody. Int J Biol Markers. 1991;
6(4):221-230.
10. Goodman GE, Hellstrom I, Brodzinsky L, Nicaise C, Kulander B, Hummel D and
Hellstrom KE. Phase I trial of murine monoclonal antibody L6 in breast, colon, ovarian, and
lung cancer. J Clin Oncol. 1990; 8(6):1083-1092.
11. Goodman GE, Hellstrom I, Yelton DE, Murray JL, O'Hara S, Meaker E, Zeigler L,
Palazollo P, Nicaise C, Usakewicz J and et al. Phase I trial of chimeric (human-mouse)
monoclonal antibody L6 in patients with non-small-cell lung, colon, and breast cancer.
Cancer immunology, immunotherapy : CII. 1993; 36(4):267-273.
12. Ziegler LD, Palazzolo P, Cunningham J, Janus M, Itoh K, Hayakawa K, Hellstrom I,
Hellstrom KE, Nicaise C, Dennin R and et al. Phase I trial of murine monoclonal antibody 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430
the breast, colorectum, and lung. J Clin Oncol. 1992; 10(9):1470-1478.
13. Harao M, Hirata S, Irie A, Senju S, Nakatsura T, Komori H, Ikuta Y, Yokomine K,
Imai K, Inoue M, Harada K, Mori T, Tsunoda T, Nakatsuru S, Daigo Y, Nomori H, et al.
HLA-A2-restricted CTL epitopes of a novel lung cancer-associated cancer testis antigen, cell
division cycle associated 1, can induce tumor-reactive CTL. International Journal of Cancer.
2008; 123(11):2616-2625.
14. Karanikas V, Colau D, Baurain J-F, Chiari R, Thonnard J, Gutierrez-Roelens I, Goffinet
C, Schaftingen EV, Weynants P, Boon T and Coulie PG. High Frequency of Cytolytic T
Lymphocytes Directed against a Tumor-specific Mutated Antigen Detectable with HLA
Tetramers in the Blood of a Lung Carcinoma Patient with Long Survival. Cancer research.
2001; 61(9):3718-3724.
15. Nishizaka S, Gomi S, Harada K, Oizumi K, Itoh K and Shichijo S. A New
Tumor-Rejection Antigen Recognized by Cytotoxic T Lymphocytes Infiltrating into a Lung
Adenocarcinoma. Cancer research. 2000; 60(17):4830-4837.
16. Ishioka G, Disis M, Morse M, Cunningham CC, Figlin R, Groshen S, Lenz H-J,
Chesnut R and Fikes J. Multi-Epitope CTL Responses Induced by a Peptide Vaccine
(EP-2101) in Colon and Non-Small Cell Lung Cancer Patients. Journal of Immunotherapy. 2004;
27(6):S23-S24.
17. Tu SH, Huang HI, Lin SI, Liu HY, Sher YP, Chiang SK, Chong P, Roffler S, Tseng 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450
antigen L6 can inhibit tumor growth in vivo. J Immunother. 2012; 35(3):235-244.
18. Chu YW, Yang PC, Yang SC, Shyu YC, Hendrix MJ, Wu R and Wu CW. Selection of
invasive and metastatic subpopulations from a human lung adenocarcinoma cell line.
American journal of respiratory cell and molecular biology. 1997; 17(3):353-360.
19. Chen HW, Leng CH, Liu HY, Cheng WF, Chang YW, Wu PY, Lien SP, Huang TY,
Chiang SK, Lin MH, Tao MH, Chong P and Liu SJ. Identification of HLA-A11-restricted
CTL epitopes derived from HPV type 18 using DNA immunization. Cancer Biol Ther. 2009;
8(21).
20. Chang LS, Leng CH, Yeh YC, Wu CC, Chen HW, Huang HM and Liu SJ. Toll-like
receptor 9 agonist enhances anti-tumor immunity and inhibits tumor-associated
immunosuppressive cells numbers in a mouse cervical cancer model following recombinant
lipoprotein therapy. Molecular cancer. 2014; 13:60.
21. Chang Y-W, Chen S-C, Cheng E-C, Ko Y-P, Lin Y-C, Kao Y-R, Tsay Y-G, Yang P-C,
Wu C-W and Roffler SR. CD13 (aminopeptidase N) can associate with tumor-associated
antigen L6 and enhance the motility of human lung cancer cells. International Journal of
Cancer. 2005; 116(2):243-252.
22. Kao Y-R, Shih J-Y, Wen W-C, Ko Y-P, Chen B-M, Chan Y-L, Chu Y-W, Yang P-C,
Wu C-W and Roffler SR. Tumor-associated Antigen L6 and the Invasion of Human Lung
Cancer Cells. Clinical Cancer Research. 2003; 9(7):2807-2816. 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470
and predictive biomarker research. J Clin Pathol. 2009; 62(1):1-5.
24. Suzuki E, Kapoor V, Jassar AS, Kaiser LR and Albelda SM. Gemcitabine selectively
eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and
enhances antitumor immune activity. Clinical cancer research : an official journal of the
American Association for Cancer Research. 2005; 11(18):6713-6721.
25. Song YC, Cheng HY, Leng CH, Chiang SK, Lin CW, Chong P, Huang MH and Liu SJ.
A novel emulsion-type adjuvant containing CpG oligodeoxynucleotides enhances CD8+
T-cell-mediated anti-tumor immunity. Journal of controlled release : official journal of the
Controlled Release Society. 2014; 173:158-165.
26. Chen WL, Liu SJ, Leng CH, Chen HW, Chong P and Huang MH. Disintegration and
cancer immunotherapy efficacy of a squalane-in-water delivery system emulsified by
bioresorbable poly(ethylene glycol)-block-polylactide. Biomaterials. 2014; 35(5):1686-1695. 27. Menetrier-Caux C, Curiel T, Faget J, Manuel M, Caux C and Zou W. Targeting
regulatory T cells. Targeted oncology. 2012; 7(1):15-28.
28. Zheng Y, Dou Y, Duan L, Cong C, Gao A, Lai Q and Sun Y. Using chemo-drugs or
irradiation to break immune tolerance and facilitate immunotherapy in solid cancer. Cellular
immunology. 2015; 294(1):54-59.
29. Apetoh L, Vegran F, Ladoire S and Ghiringhelli F. Restoration of antitumor immunity
through selective inhibition of myeloid derived suppressor cells by anticancer therapies. 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490
30. James BR, Anderson KG, Brincks EL, Kucaba TA, Norian LA, Masopust D and
Griffith TS. CpG-mediated modulation of MDSC contributes to the efficacy of Ad5-TRAIL
therapy against renal cell carcinoma. Cancer immunology, immunotherapy : CII. 2014;
63(11):1213-1227.
31. Shirota Y, Shirota H and Klinman DM. Intratumoral injection of CpG oligonucleotides
induces the differentiation and reduces the immunosuppressive activity of myeloid-derived
suppressor cells. J Immunol. 2012; 188(4):1592-1599.
32. Sawant A, Schafer CC, Jin TH, Zmijewski J, Tse HM, Roth J, Sun Z, Siegal GP,
Thannickal VJ, Grant SC, Ponnazhagan S and Deshane JS. Enhancement of antitumor
immunity in lung cancer by targeting myeloid-derived suppressor cell pathways. Cancer
research. 2013; 73(22):6609-6620.
33. Shevchenko I, Karakhanova S, Soltek S, Link J, Bayry J, Werner J, Umansky V and
Bazhin AV. Low-dose gemcitabine depletes regulatory T cells and improves survival in the
orthotopic Panc02 model of pancreatic cancer. International journal of cancer Journal
international du cancer. 2013; 133(1):98-107.
34. Ghansah T, Vohra N, Kinney K, Weber A, Kodumudi K, Springett G, Sarnaik AA and
Pilon-Thomas S. Dendritic cell immunotherapy combined with gemcitabine chemotherapy
enhances survival in a murine model of pancreatic carcinoma. Cancer immunology,
immunotherapy : CII. 2013; 62(6):1083-1091. 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510
L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis. Cancer
research. 2009; 69(8):3272-3277.
36. Lin CI, Merley A, Sciuto TE, Li D, Dvorak AM, Melero-Martin JM, Dvorak HF and
Jaminet SC. TM4SF1: a new vascular therapeutic target in cancer. Angiogenesis. 2014;
17(4):897-907.
37. Lee SA, Kim TY, Kwak TK, Kim H, Kim S, Lee HJ, Kim SH, Park KH, Kim HJ, Cho
M and Lee JW. Transmembrane 4 L six family member 5 (TM4SF5) enhances migration
and invasion of hepatocytes for effective metastasis. Journal of cellular biochemistry. 2010;
111(1):59-66.
38. Lee SA, Lee SY, Cho IH, Oh MA, Kang ES, Kim YB, Seo WD, Choi S, Nam JO,
Tamamori-Adachi M, Kitajima S, Ye SK, Kim S, Hwang YJ, Kim IS, Park KH, et al.
Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal
transition in human hepatocarcinoma. The Journal of clinical investigation. 2008;
118(4):1354-1366.
39. Kim YE, Kwon S, Wu G, Kim D, Park BK, Park JA, Choi KC, Kim DS, Kwon HJ and
Lee Y. Therapeutic effect of a TM4SF5-specific monoclonal antibody against colon cancer
in a mouse model. Oncotarget. 2014; 5(18):8402-8415. 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530
Figure Legends
Figure 1. TAL6 protein is over-expressed in lung cancer cells and clinical lung tumor tissues.
(A) TAL6 protein levels were measured on the cell surface by flow cytometry using mouse
anti-TAL6 antibody (green line) or isotype control antibody (shaded), and then probed with
goat anti-mouse FITC-conjugated antibody in 9 lung cancer cell lines. (B) TAL6 expression
in primary cultured lung cancer cells from pleural effusion was detected by flow cytometry
as in panel A. PE53 and PE8 were established from two individual lung cancer patients. (C)
TAL6 expression was stained in xenograft tumors from SCID mice by subcutaneously
injecting primary cultured lung cancer cells. (D) The IHC staining from stage III lung cancer
patients and cancer adjacent normal pneumonic lung tissue. An enlarged image is shown
below the original IHC stains. (E) IHC analysis of TAL6 in lung cancer tissue array scored
by staining intensity from 0 to 3+ (0, negative; 1, weak; 2, moderate; 3, strong) by
histologists. Positive is taken as a score of greater than 0; negative is indicated by score = 0.
(F) Analysis of IHC scores in tumor and adjacent normal tissue. (G) ROC curve analysis for
prediction of TAL6 expression in lung tumor tissues from IHC results of lung cancer tissue
array. The AUC is 0.75 and the sensitivity and specificity is 70.6% and 70.6%.
Figure 2. Anti-tumor effect of HLA-A2-restricted TAL6 peptide immunization in HLA-A2
transgenic mice. (A) HLA-A2 transgenic mice were immunized s.c. twice with peptide A2-5 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549
splenocytes were harvested from the mice and co-cultured with TAL6-A2 or
EL4-TAL6 cells for 2 hr. The percentage of CD107a+CD8+ cells was detected by flow cytometry.
Error bars, SD from three independent experiments; *, P < 0.05; **, P < 0.01. (B) At 7 days
after final immunization of PBS or peptide A2-5 in HLA-A2 transgenic mice,
EL4-TAL6-A2 or EL4-TAL6 cancer cells (2×105 cells per mouse) were injected subcutaneously and the
tumor growth was monitored. Each group contains 5 mice. Error bars, SEM; **, P < 0.01.
Figure 3. Improved immunization in HLA-A2 transgenic mice prolongs animal survival time
and prevents lung metastases. HLA-A2 transgenic mice were subcutaneously immunized
twice with peptide A2-5 (50μg/mouse), Th epitope peptide, CpGODN (10μg/mouse) and
ISA and then the anti-tumor effects were monitored. (A) Splenocytes were harvested and
incubated with peptide A2-5 or irrelevant peptide (10g/ml). IFN-γ secreting cells were
detected by IFN-γ ELISPOT assay. Error bars, SD. ***P< 0.001. (B) Splenocytes harvested
from peptide immunized mice were stimulated with 10μg/ml of peptidesfor 6 hr in the
presence of PE-conjugated anti-CD107a. After stimulation, FITC-conjugated anti-CD8
antibody was used to detect CD8+ T cell. The percentage of CD107a+CD8+ cells in individual
immunized groups was determined by flow cytometry. (C) Irradiated EL4-TAL6-A2 or
EL4-TAL6 cells (2×104) were used to stimulate splenocytes for 2 hr. The percentage of
CD107a+CD8+ cells was determined by flow cytometry as panel B described. (D) After
551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569
peptides immunization, EL4-TAL6-A2 cells (2×105 cells) were inoculated through the i.v.
routes. Lung tissues from each group were collected for tumor nodule detection 20 days after
tumor implantation. (E) Mice survival time was monitored and analyzed. *, P < 0.05; **, P <
0.01. (F) Melanoma B16 or B16-TAL6-A2 cells (B16 cells with expression of TAL6 and
HLA-A2) were intravenously injected in naïve and ISA/A2-5/Th/CpG immunized HLA-A2
transgenic mice. Whole lungs were examined.
Figure 4. Peptide A2-5 immunization induces HLA-A2 specific cytotoxic T
lymphocyteresponses in HLA-A2 transgenic mice. (A) Effector cells (splenocytes) from
immunized HLA-A2 transgenic mice were stimulated with peptide A2-5 (10μg/ml) and IL-2
(10U/ml) and subjected to a standard 51Cr-release assay with H2981 cells as targets.
Antibodies against HLA-A2, CD8 and mIgG isotype control were incubated with effector
cells at 37°C for 1 hour. CTL assays were done with 3 mice per group and observed with
effector/target (E/T) ratios of 100. *** P< 0.001. (B) The treatment schedule is shown in the
top. H2981 cells (1×108) were subcutaneously inoculated into SCID mice. Then, the purified
CD8+ T cells (1x107) from peptide A2-5 immunized HLA2 Tg mice were adoptively
transferred through the i.v. route into individual SCID mouse bearing H2981 tumors on day
7. Tumor size was monitored until day 57. Each group contains 6 mice. Error bars, SEM; **,
P < 0.01. 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588
Figure 5. Combination of gemcitabine and peptide A2-5 induced CD8+ T cells provides
anti-tumor activity and prolongs animal survival time in a human lung anti-tumor xenograft model.
(A) Lung cancer H2981 cells (1×108) were subcutaneously injected into SCID mice.
Gemcitabine was i.p. injected (3mg/mouse) into mice when tumors reach ~ 100 mm3. At day
5 after gemcitabine injection, splenocytes from tumor xenografted SCID mice were isolated
and the frequency of myeloid-derived suppressor cells (MDSCs) was determined by using
PE-conjugated anti-GR-1 antibody and FITC-conjugated anti-CD11b antibody. Error bars,
SD. *P< 0.05. (B) Tumor size was measured at 2-3 day intervals in each group of mice.
Gemcitabine was i.p. injected (3mg/mouse) on day 25 post tumor inoculation. Peptide A2-5
induced CD8+ T cells (1x107) from HLA-A2 Tg mice were adoptively transferred into human
tumor xenograft mice on day 30. (C) Survival rate of mice determined with different
treatments as shown in (B). Median survival time and log-rank test in SPSS analysis were
analyzed.
Figure 6. Reduced MDSC and increased peptide A2-5 induced CD8+ T cells were detected in
xenograft tumors. After treatment as described in Figure 5, H2981 tumors were collected on
day 26 from SCID mice bearing human lung tumor xenografts. All tumors were subjected to
enzymatic digestion to obtain single-cell suspensions for cell population analysis. (A) The 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607
percentage of MDSC was detected with FITC-conjugated anti-GR-1 antibody and
PE-conjugated anti-CD11b antibody. (B) Tumor infiltrated CD8+T cells were stained with
FITC-conjugated anti-CD8. (C) The percentage of A2-5-HLA-A2 tetramer-binding cells was
detected by flow cytometry. Error bars show mean and SD. **P< 0.01. (D) H2981 cancer
cells (1×108)were subcutaneously injected in SCID mice. After 30 days, gemcitabine was
i.p. injected (3mg/mouse). Isolated CD8+ T cells (1x107) were adoptively transferred into
SCID mice bearing xenograft tumors at day 35.
Figure S1. Immunization of ISA/A2-5/Th/CpG elicited high IFN- secretion cells in
HLA-A2 transgenic mice. HLA-HLA-A2 transgenic mice were subcutaneously immunized twice with
A2-5 (50μg/mouse) and CpGODN (10μg/mouse)/ISA. Splenocytes were harvested and
incubated with various peptides (10g/ml). Error bars, SD. *P< 0.05. **P< 0.01. 608 609 610 611 612 613 614 615 616 617 618 619
