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Effective adoptive transfer of haploidentical tumor-specific T cells in B16-melanoma bearing mice
CUI Nai-peng, XIE Shao-jian, HAN Jin-sheng, MA Zhen-feng, CHEN Bao-ping and CAI Jian-hui
Department of Surgery, Hebei Medical University, Shijiazhuang 050017, China (Cui NP, Han JS and CAI
Jian-hui)
Department of Gastrointestinal Surgery, Department of Oncology & Immunotherapy, Hebei General
Hospital, Shijiazhuang 050051, China (Cai JH)
Department of Surgical Oncology, Affiliated Hospital of Hebei University, Baoding 071000, China (Ma
ZF and Chen BP)
Department of Oncology & Immunotherapy, The 2nd Affiliated Hospital of Hebei Medical University,
Shijiazhuang 050000, China (Xie SJ)
Correspondence to: Dr. CAI Jian-hui, 348 Heping West Road, Shijiazhuang 050051, China (Tel:
86-311-85988977. Fax: 86-311-85988902. Email: [email protected])
This study was supported by grants from the Science & Technology Support Program of Hebei Province
(No. 09276418D-26, 10246139D), the Medical Applicable Technology Track Project of Hebei Province
(No. GL200938) and the Planning Project of Science and Technology bureau of Baoding (No. 10F08).
1
Keywords: B16 melanoma; adoptive transfer; adoptive immunotherapy; tumor microenvironment;
graft-versus-host disease
Background Adoptive transfer of allogeneic tumor-specific T cells often results in severe
graft-versus-host disease (GVHD). Here, we sought to maximize graft-versus-tumor and minimizing
GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice.
Methods C57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, or 7 Gy total body
irradiation (TBI), or 7 Gy TBI pus bone marrow transplantation. Tumor areas were measured every 3
days to assess the influences of irradiation treatment on tumor regression. B16-melanoma bearing mice
were irradiated with 7 Gy TBI; sera and spleens were harvested at days 1, 3, 5, 7, 9, 11 and 13 after
irradiation. White blood cell levels were measured and TGF-β1 and IL-10 levels in serum were detected
using ELISA kits. Real-time RT-PCR and flow cytometry were performed to test TGF-β1, IL-10 and
Foxp3 mRNA levels and the proportion of CD4+CD25+Foxp3+ T regulatory cells (Tregs) in spleens.
B16-melanoma bearing C57BL/6 mice were irradiated with 7 Gy TBI followed by syngeneic (Syn1/Syn2)
or haploidentical (Hap1/Hap2), dendritic cell-induced cytotoxic T lymphocytes (DC-CTLs) treatment,
tumor areas and system GVHD were observed every 3 days. Mice were killed 21 days after the DC-CTLs
adoptive transfer; histologic analyses of eye, skin, liver, lung and intestine were then performed.
Results Irradiation with 7 Gy TBI on the B16-melanoma-bearing mice did not influence tumor
regression compared with control group; however, it down-regulated the proportion of Tregs in spleens
and the TGF-β1 and IL-10 levels in sera and spleens, suggesting inhibition of autoimmunity and
intervention of tumor microenvironment. Adoptive transfer of haploidentical DC-CTLs significantly
inhibited B16-melanoma growth. GVHD assessment and histology analysis showed no significant
differences among the groups.
Conclusions Adoptive transfer of haploidentical tumor-specific T cells in irradiation-pretreated
B16-melanoma bearing mice preserved antitumor capacity without causing a GVHD response.
2
Adoptive immunotherapy is an appealing approach to cancer treatment, with the potential for more
precise targeting and reduced toxicity1-3. The main clinical concern in using adoptively transferred
self/tumor-specific T cells is the lower possibility of graft-versus-host disease (GVHD). However,
autologous tumor-specific T cells are often inconvenient to obtain, especially from patients with myeloma
or with advanced disease 4-6. Adoptive transfer of allogeneic tumor-specific T cells could provide an
alternative to bridging autologous T-cell therapy 7-9; however, its use could be hampered by GVHD. The
persistence of transferred cells might be crucial for a favorable clinical outcome 10-12. Allogeneic cells
with major MHC mismatches are rapidly rejected by the host immune system. As haploidentical donor
cells are more available than histocompatible allogeneic donor cells in clinical practice, we designed the
F1 (H-d/k)S(H-2b/d) mice donor in our study to observe antitumor effects and GVHD in a mouse model.
Adoptive lymphocytes have been genetically modified in many ways to improve activity and
circumvent tumor evasion, including transfer of transgenic T-cell receptors and chimeric antigen
receptors to redirect T cell and natural killer cell antigen specificity. Irradiation has been shown to reduce
the capacity of lymphocytes while preserving the cytotoxicity of these lymphocytes against tumor cells 13,
14
, and can also limit GVHD response. Data from the research of Boni7 showed that mice receiving a
preparative regimen of myeloablating (9 Gy) total body irradiation (TBI) experienced the significant
regression of large, vascularized tumors, whereas mice receiving preparative regimens of
nonmyeloablating (5 Gy) TBI experienced rapid rejection of tumor-specific allogeneic lymphocytes with
no impact on tumor growth.
In the present study, we pretreated B16-melanoma bearing C57BL/6 mice with nonmyeloablating (7
Gy) TBI without bone marrow transplantation (BMT), followed by adoptive transfer of haploidentical
tumor-specific T cells. The results showed that use of tumor-specific haploidentical T cells can result in
significant antitumor effects, without severe GVHD. Here, we describe a novel adoptive therapy that
could lead to a safe cancer treatment.
METHODS
Mice and tumor lines
Female C57BL/6 mice and male BABL/c mice, 8–10 weeks old, were purchased from the Hebei
Laboratory Animal Research Center (Shijiazhuang, Hebei, China). Female C57BL/6 mice were crossed
with male BABL/c mice to derive haploidentical F1 mice. F1 mice were sacrificed as donors at 6–8
weeks. B16-melanoma bearing C57BL/6 mice were used as recipients. All of the mice were bred and
housed at Hebei Laboratory Animal Research Center (Shijiazhuang, Hebei, China). Experiments were
conducted with the approval of the Animal Ethics Committee of Hebei Medical University.
The B16-melanoma cell line was purchased from the Shanghai Institute of Cell Biology. The B16 cells
were maintained in RPMI Media 1640 (Gibco, Grand Island, NY) supplemented with 10% (v/v) fetal calf
serum (FCS, HyClone Inc., Logan, UT), 100 IU/mL penicillin and 100 g/mL streptomycin at 37°C with
5%CO2 in a humidified atmosphere and harvested using 0.25% (v/v) trypsin-EDTA.
Total body irradiation (TBI) of tumor models
Mice were injected subcutaneously on their backs with 1×106 B16 melanoma cells. Treatment began after
10 days. We used a Co-60 source as radioactive source; the rate of delivery was 0.5 Gy per minute, and
the source–skin distance was 80 cm.
Flow cytometry
At various time points following TBI, spleens were removed, and single-cell suspensions were prepared
from spleens by passing the tissue through a wire mesh. The cells were stained for CD4+CD25+Foxp3+
using the Mouse Regulatory T cell Staining Kit (eBioscience), according to the manufacturer’s
instructions. All samples were analyzed on BD FACSCalibur Flow Cytometer (BD Biosciences), and the
3
data were analyzed using FlowJo software (version 6.4.7; Tree Star, Ashland, OR). The proportion of
CD4+CD25+Foxp3+/CD4+ T cells of splenocytes indicates the proportion of T regulatory cells (Tregs).
Peripheral white blood-cell (WBC) counting
The sample of peripheral blood was obtained from each mouse by cardiac puncture for WBC
determination. WBC count essay was done on a Coulter Counter (model FN).
Detection of TGF-β1 and IL-10 by ELISA
TGF-β1 and IL-10 concentrations in mouse serum samples were measured using specific enzyme-linked
immunosorbent assay (ELISA) kits for TGF-β1 and IL-10 (Bender MedSystems GmbH, Vienna, Austria)
according to the manufacturer’s protocol.
RNA Isolation and Quantitative RT-PCR
For analysis of TGF-β1, IL-10 and Foxp3 mRNA expression, total RNA was extracted from mouse spleen
tissues with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Complementary DNA was synthesized from the total RNA (0.5 μg) using the PrimeScript™ RT regent
Kit (Takara Biotechnology, Dalian, China) following the instructions provided by the manufacturer.
Subsequently, the cDNA was subject to real-time PCR using Power SYBR Green PCR Master Mix
(Takara Biotechnology, Dalian, China). Each real-time PCR reaction consisted of 2 μL diluted RT product,
10 μL SYBR Green PCR Master Mix and 250 nM forward and reverse primers (Table 1) in a total volume
of 20 μL. Reactions were carried out on a 7500 real-time PCR System (Applied Biosystems) for 40 cycles
(95 °C for 5 s, 60°C for 35 s) after an initial 30 s incubation at 95°C. The fold change in expression of
each gene was calculated using the ΔΔCt method, with the housekeeping gene β-actin mRNA as an
internal control.
Generation of bone marrow-derived DCs
Bone marrow-derived DCs were harvested from femur and tibia of C57BL/6 mice and haploidentical F1
mice, as described by Lutz15 with minor modifications. Briefly, 1×106 cells/mL erythrocyte-depleted
mouse bone marrow cells from flushed marrow cavities were cultured in complete medium (CM; RPMI
1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 mg/mL streptomycin, and 1 U/mL penicillin)
supplemented with 20 ng/mL rmGM-CSF and 20 ng/mL rmIL-4 (PeproTech, Rocky Hill, NJ, USA) in
six-well plates at 37°C in an atmosphere containing 5% CO2. On day 3, half of the medium was removed
and centrifuged for 5 min at 1,500 g. The collected cells were resuspended in the same volume of fresh
CM and replenished to their original plates. On day 5, 15 ng/mL rmTNF-α (PeproTech, Rocky Hill, NJ,
USA) was added to the system to stimulate the DCs to mature. After 24 h, mature DCs were enumerated
by FACS (FACScan, Becton Dickinson) analysis through staining with FITC anti-mouse CD11c, PE
anti-mouse CD80 and CD86, APC anti-mouse MHC-II molecules (eBioscience, Santiago, USA). The
corresponding labeled isotypes served as the controls. After that, the thawed frozen B16 tumor lysate was
added to the DC culture systems on day 6, at a ratio of five DC equivalents to one tumor cell, and
incubated at 37°C in an atmosphere containing 5% CO2. After 48 h of incubation, non-adherent cells that
included mature DCs were harvested by gentle pipetting. The DCs were then washed twice, enumerated
and resuspended in phosphate-buffered saline (PBS) at 5×106/mL.
Generation of tumor-specific cytotoxic T lymphocytes (CTL) in vitro
The single cell suspension was harvested from the spleens of C57BL/6 mice and haploidentical F1 mice,
and erythrocytes were lysed with ammonium chloride buffer (BD Biosciences, Heidelberg, Germany).
Splenocytes were washed in complete medium and passed through a nylon fiber column (Wako, Osaka,
Japan). Cells were cultured in complete medium (RPMI 1640 supplemented with 10% FCS, 2 mM
L-glutamine, 100 mg/mL streptomycin, and 1 U/mL penicillin), supplemented with 20 ng/mL rmIL-2
(PeproTech, Rocky Hill, NJ, USA) for 2 days. T cells were enumerated by FACS (FACScan, Becton
Dickinson) analysis through staining with FITC anti-mouse CD3 (eBioscience, Santiago, USA). The
4
mature DCs, loaded with B16 tumor lysate, were added to the T cell culture systems on day 3, at a ratio of
one DC equivalent to ten T cells, and incubated at 37°C in an atmosphere containing 5% CO2. The culture
systems were also supplemented with CM with 20 ng/mL rmIL-2. After 72 h of incubation, dendritic
cell-induced, tumor-specific cytotoxic T lymphocytes were harvested for further adoptive immunotherapy
for the mice; CTLs were washed twice, counted, and resuspended in PBS at 5×106/mL.
Tumor models and adoptive immunotherapy
C57BL/6 mice were each subcutaneously inoculated with 1×106 B16 cells in the back. Tumors were
measured with calipers every 3 days, and the tumor area was calculated as the products of the
perpendicular diameters. After 10 days, all of the mice receiving 7 Gy TBI without BMT on day 0.
Subsequently, syngeneic or haploidentical DC-CTLs were adoptively transferred by intravenous injection
(1 × 106 cells/mouse) into recipients on day 0. Some mice received 2 infusions, on day 0 and 7. Some of
the recipients were sacrificed on day 14; the other recipients were observed until day 21. All experiments
were performed in a blinded, randomized fashion and performed independently at least twice, with
similar results.
Histology analysis
Formalin-preserved eye, skin, liver, lung and intestine were collected 21 days after adoptive transfer,
fixed in 4% formalin, and embedded in paraffin. Five-μm thick sections were stained with hematoxylin
and eosin for histological examination. Images were obtained using a Nikon Eclipse E400 microscope
(Tokyo, Japan) equipped with Nuance Multispectral Imaging System VIS and related software (CRI,
Woburn, MA).
Assessment of GVHD
The appearance of mice was monitored daily. The degree of systemic GVHD was assessed by a scoring
system that sums changes in five clinical parameters: weight loss, poor posture (hunching), activity, fur
texture, and skin integrity (maximum index = 10)16.
Statistical analysis
All data are presented as means ± SEM, and analyzed as appropriate by Student’s t test or one-way
ANOVA. Statistical analysis was performed using SPSS (Chicago, IL) software. P < 0.05 was considered
statistically significant.
RESULTS
Irradiation treatment did not influence tumor regression, but could down-regulate Tregs with
immune inhibitory effect.
Reportedly, allogeneic adoptive immunotherapy caused serious GVHD response, resulting in
elevated mortality 17. Pre-treated recipient mice with chemotherapy or irradiation was reported to affect
the tumor microenvironment and result in immune inhibitory during tumor immunotherapy 17.To tested
whether irradiation had direct effects on tumor regression, C57BL/6 mice bearing B16-melanoma tumors
were irradiated with 0, 5, 7 Gy TBI, or 7 Gy TBI plus syngeneic bone marrow transplantation (BMT)
with 106 unsorted bone marrow cells the day after radiation (7Gy TBI+BMT)7. There was no statistical
difference in tumor growth between mice receiving 5 Gy TBI, 7 Gy TBI or 7 Gy TBI + BMT versus 0 Gy
TBI (Figure 1), which suggested that 7 Gy TBI did not influence tumor regression.
However, in recipient mice, pretreatment with 7 Gy irradiation significantly interfered in the tumor
microenvironment. As shown in Figure 2A, peripheral blood WBC levels in B16 bearing C57BL/6 mice
was significantly higher than those of normal mice. When B16 bearing mice were treated with 7 Gy TBI,
WBC levels decreased by 86–96% from day 1 to day 13 after TBI. Next, we measured the TGF-β1 and
IL-10 protein levels in serum, and mRNA levels in spleen tissues of B16 bearing mice using ELISA kit
and real-time PCR, each of which reduced significantly after irradiation treatment (Figure 2B, C).
5
Meanwhile, Foxp3 mRNA level in spleens of B16 bearing mice were measured by real-time PCR. Foxp3
mRNA levels decreased after 7 Gy TBI and reached their lowest level at day 9 after irradiation
(Figure 2D). Flow cytometry was used to test the proportion of CD4+CD25+Foxp3+ Tregs from spleen
cells of B16 bearing mice (Figure 2E). Compared with the non-irradiated group, Treg proportions
significantly dropped from day 1 to day 13 after TBI, reaching their lowest level at day 9, which was
parallel with Foxp3 mRNA levels. These results suggest that pre-treatment with 7 Gy TBI changes the
tumor microenvironment, causing an immune inhibitory effect in the B16 bearing C57BL/6 mice.
Adoptive transfer of haploidentical DC-CTLs inhibited B16-melanoma growth paralleled syngeneic
DC-CTLs treatment.
C57BL/6 mice bearing B16 tumors established for 10 days were irradiated with 7 Gy TBI. Mice
were injected with 106 Hoechst 33342 stained syngeneic DC-CTLs (Syn), or with 106 Hoechst 33342
stained haploidentical DC-CTLs (Hap) the day after irradiation. Hoechst 33342 stained syngeneic or
haploidentical DC-CTLs in C57BL/6 mice tumors or spleens could be observed 36 h after adoptive
transfer (Figure 3A). Fourteen days after adoptive transfer, nodosity alteration was observed in spleens of
recipient mice (Figure 3B). These results suggested that either syngeneic or haploidentical DC-CTLs
could persist in tumor or spleens in pre-irradiated B16 bearing mice.
Next, we wanted to investigate the B16-melanoma growth after syngeneic or haploidentical
DC-CTLs transfer. C57BL/6 mice bearing B16-melanoma were pre-irradiated with 7 Gy TBI. Mice were
injected with PBS on both day 0 and day 7 (PBS [i.e., control]); with 106 syngeneic DC-CTLs on day 0
and PBS on day 7 (Syn 1); with 106 haploidentical DC-CTL cells on day 0 and PBS on day 7 (Hap 1);
with 106 syngeneic DC-CTL cells on day 0 and day 7 (Syn 2); or with 106 haploidentical DC-CTL cells
on day 0 and day 7 (Hap 2). As shown in Figure 4, the tumor grew rapidly when mice were injected only
with PBS. Both syngeneic and haploidentical DC-CTLs treatments significantly inhibited tumor growth
from day 9 to day 21. When mice were subjected to DC-CTLs twice (Syn2, Hap2), we observed more
significant tumor regressions. However, there was no significant difference between syngeneic and
haploidentical DC-CTLs transfer groups. These results suggest that haploidentical DC-CTLs treatment
inhibits tumor growth, which paralleled syngeneic DC-CTLs treatment.
Assessments of GVHD after DC-CTLs treatment in B16-melanoma bearing mice.
Allogeneic cells can induce GVHD-like reactions 18, 19. We wanted to explore whether treatment with
F1 haploidentical DC-CTLs could induce GVHD-like reactions. We monitored the appearance of mice
daily and assessed the degree of systemic GVHD by a scoring system. As shown in Table 2, a slight
increase of GVHD scores was seen in Syn2 and Hap2 mice on days 15 and 21 compared with PBS group,
but there were no significant differences among the groups. On histopathological examination (Figure 5),
no obvious lesions were seen in eyes, skin, liver, lungs or intestines compared with PBS-treated mice.
These results indicate that the F1 haploidentical DC-CTLs treatment gave no significant GVHD response.
DISCUSSION
T lymphocytes mediate tissue destruction, and recognize antigens with a high degree of specificity,
and thus have great potential in treating malignancies7. As patients with myeloma or advanced-stage
disease may lack autologous tumor-specific T cells, adoptive transfer of allogeneic T cells could be an
alternative immunotherapy strategy. However, use of this strategy for melanoma is hampered by antigen
mismatch of allogeneic MHC following GVHD responses, and T cell trafficking to and persistence in
tumor tissues 7, 17.
Here, we sought to use haploidentical MHC, partially matched effector cells for adoptive cancer
immunotherapy. As described previously, in vivo, the microenvironment around tumors tends to change,
supporting tumor growth. Many methods, such as chemotherapy or irradiation, have been used to try to
6
affect the tumor microenvironment during immunotherapy. To maximize graft-versus-tumor effects and
minimize GVHD, we tried variant doses of radiation to inhibit autoimmunity and intervene in the tumor
microenvironment. CD4+CD25+Foxp3+ Tregs inhibit autoimmunity and protect against tissue injury 20.
Tregs are characterized by the expression of specific transcription factors, such as the Foxp3 21-23, the
proportion of which reflects changes in the tumor microenvironment. The present study showed that the
preparative regimen of nonmyeloablating (7 Gy) TBI without BMT diminished the abundance of
CD4+CD25+Foxp3+ Tregs and levels of Foxp3 mRNA in spleens in B16-bearing mice. The cytokines
TGF-β1 and IL-10 are associated with Treg production and are critical to immune homeostasis in vivo 24,
25
. We found that 7 Gy TBI irradiation downregulated expression of TGF-β1 and IL-10 in the serum and
spleen tissues, parallel with Treg levels, in B16 bearing mice.
The therapeutic strategy of allogeneic T cell adoptive transfer was often hampered by transferred
cells trafficking to and persistent in tumors, which might be crucial for a favorable clinical outcome 10,
11. Allogeneic cells with major MHC mismatches are rapidly rejected by the host immune system. In vivo
survival and persistence of haploidentical T cells were critically dependent on the immunosuppressive
treatment before the transfer. The data shown in the present study showed that the antitumor effects of
haploidentical tumor-specific T cells are similar to autologous tumor-specific T cells. The intervention of
tumor microenvironment by the preparative regimen of nonmyeloablating (7 Gy) TBI may be the possible
mechanism behind it. Furthermore, 7 Gy TBI irradiation can persistently inhibit the immune state of the
recipients for at least two weeks, to avoid the rapid rejection of tumor-specific haploidentical
lymphocytes with no impact on tumor growth. Moreover, the present model eliminated the GVHD
response, possibly because F1 lymphocytes are tolerant to all antigens in the C57BL/6 background and
irradiation treatment inhibits the autoimmunity of the recipient mice.
Taken together, our results indicate that this is a safe, and effective immunotherapeutic alternative to
allogeneic or autologous antitumor T cells in B16-melanoma mouse models. However, there are also
some adverse effects in allogeneic T cell adoptive transfer strategy26. Administration of high-dose of
allogeneic haploidentical donor lymphocytes was reported to cause severe adverse reactions27. The
existence of intrinsic cancer cell escape mechanisms may caused low response rates to immune-based
therapies28, 29. Combined therapy may provide better choices to improved antitumor activities30-32.
Although further refinement and validation are necessary, this approach may offer a new choice in cancer
immunotherapy.
Acknowledgments: The authors sincerely thank other investigators and coordinators who made
invaluable contributions to this study. We thank Xue Ping, Xu Fang, Liu Hui, and Shi Jian-hong for their
generous help in histology and statistical analysis.
7
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Tables
Table 1 Mouse primer sequences used for QRCR.
Gene
Sense
TGFβ
5′-ATGGTGGACCGCAACAAC-3′
IL-10
5′-ACCAAAGCCACAAAGCAG-3′
Foxp3
5′-TGCAGGGCAGCTAGGTACTTGTA-3′
β-actin
5′-TACCCAGGCATTGCTGACAGG-3′
Anti-sense
5′-AGCCACTCAGGCGTATCAG-3′
5′-GGAGTCGGTTAGCAGTATG-3′
5′-TCTCGGAGATCCCCTTTGTCT-3′
5′-ACTTGCGGTGCACGATGGA-3′
Table 2 Comparison of GVHD values after DC-CTLs treatment
Time
GVHD score value
(days)
PBS
Syn1
Hap1
Syn2
3
0.25±0.46 0.38±0.52 0.44±0.53 0.57±0.53
9
0.38±0.48 0.50±0.50 0.66±0.50 0.86±0.35
15
0.75±0.43 0.88±0.64 0.89±0.60 1.29±0.49
21
0.86±0.64 1.25±0.46 1.33±0.50 1.57±0.79
10
Hap2
0.55±0.53
0.89±0.73
1.25±0.46
1.50±0.76
F
P
0.532
1.268
1.552
1.575
0.713
0.300
0.209
0.203
Figure legends
Fig.1 Radiation treatment did not influence tumor regression.
C57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, 7 Gy TBI, or 7 Gy TBI pus BMT.
Tumor areas were measured every 3 days after irradiation. Data represent the means of measurements of
at least 6 mice per group (means ± SEM).
11
Fig.2 Effects of radiation on immune response and tumor microenvironment.
C57BL/6 mice bearing B16-melanoma tumors were irradiated with 7 Gy TBI. (A) Mouse Blood WBC
levels was shown at the indicated time points. (B) Quantitative detection of TGFβ1 and IL-10 levels in
mouse serum samples were measured using ELISA kit (Bender MedSystems GmbH, Vienna, Austria). (C)
In parallel experiments, total RNAs were isolated from mouse spleens, TGFβ and IL-10 mRNA levels
were measured by using real-time PCR. (D) Foxp3 mRNA levels in mouse spleens were measured by
using real-time PCR. (E) The percentage of CD4+CD25+ T cells in mice spleens was analyzed by flow
cytometry (left, upper). The percentage of CD25+Foxp3+ T cells in mice spleens was analyzed by flow
cytometry (left, down). The percentage of CD25+Foxp3+ T cells was analyzed by gating on the CD4
population. The percentage of Tregs in mice spleens (right). Data represent the means of measurements of
at least 6 mice per group (means ± SEM). *P < 0.05 compared with the Tumor group.
12
Fig.3 Syngeneic or haploidentical DC-CTLs persistence in vivo.
(A) Hoechst 33342 stained DC-CTLs was shown in tumor or spleen tissues 36 h after adoptive transfer.
(B) Nodosity alteration of mouse spleen 14 days after adoptive transfer.
13
Fig.4 Effects of syngeneic or haploidentical DC-CTLs treatment on B16 tumor growth in mice.
Tumor areas were measured every 3 days after DC-CTLs treatment. Data represent the means of
measurements of at least 8 mice per group (means ± SEM). P < 0.05 from day 9 of group
Syn1/Hap1/Syn2/Hap2 compared with group PBS.
14
Fig.5 GVHD pathological manifestations in the skin, liver, lung, intestine and eye after DC-CTLs
adoptive transfer. Magnification: 100×.
15