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Departments of
*
Immunology and
Cellular Biology, Second Military Medical University, Shanghai, People’s Republic of China, and
Department of Molecular Biotherapy Research, Japanese Foundation of Cancer Research, Toshima-ku, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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are also necessary.
When 3LL tumor-bearing mice were treated with 1 x 104
Mut1 peptide-pulsed Lptn-DC, their pulmonary metastases were
significantly reduced, whereas the same low dose of Mut1 peptide-pulsed
DC had no obvious therapeutic effects. Our data suggest that Lptn-DC
are more potent adjuvants for peptide delivery to induce protective and
therapeutic antitumor immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As adjuvants for Ag delivery, DC pick up Ags in the periphery and carry
them to T cell area in lymphoid organs to prime the immune responses
(31, 32, 33, 34, 35). The precise molecular mechanisms underlying DC in vivo
migration and their interaction with T cells are not well defined.
Chemokines capable of regulating the migration of immune cells may
contribute to the initiation of immune responses by DC. It is evident
that DC express some chemokine receptors, so chemokines participate in
the migration and recruitment of DC (36, 37, 38, 39). Moreover, DC can produce
several kinds of chemokines (40, 41, 42, 43) (e.g., macrophage-inflammatory
protein- (MIP-), monocyte chemotactic protein, RANTES,
MIP-1
, and DC-CK1) to actively attract T cells to initiate immune
responses. Lymphotactin (Lptn) is a recently defined C chemokine that
is specifically attractive to T cells (44, 45, 46). Cotransfection of Lptn
and IL-2 genes into tumor tissue could induce potent antitumor immunity
(47). We hypothesized that the improved preferential chemotaxis of DC
on T cells by genetically modifying DC with T cell-attracting chemokine
might be capable of facilitating the in vivo stimulation of T cells by
DC and consequently favoring DC Ag presentation and T cell activation.
Recently, different gene transfer approaches have been explored to genetically modify DC for vaccination, and it has been found that DC genetically modified with tumor Ags or immunoregulatory cytokines are potentially advantageous in inducing antitumor immunity (12, 13, 24, 25, 26, 27, 30). Adenovirus (Ad) vector capable of mediating gene transfer with high efficiency and acting as adjuvants to boost CTL response were demonstrated to be the potentially promising viral vector for DC genetic modification (12, 25, 26, 27, 28, 30). One of our previous studies showed that granulocyte-macrophage CSF (GM-CSF) gene-modified DC pulsed with tumor Ag could induce antitumor immunity more potently (12).
The primary aim of this study is to improve the efficacy of DC-based vaccines for cancer treatment by genetically modifying DC with a T cell-attracting chemokine. So in this study, replication-defective Ad vector harboring mouse Lptn was constructed and used to genetically modify mouse bone marrow DC. In the tumor model of 3LL Lewis lung carcinoma, normal mice were vaccinated with the Lptn gene-modified DC (Lptn-DC) pulsed with 3LL cell-specific Mut1 peptide (FEQNTAQP) (48), and the protective and therapeutic effects were investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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293 (CRL1573; American Type Culture Collection (ATCC, Manassas, VA)) is a human embryonic kidney cell line transformed with Ad5 E1A and E1B genes and supporting propagation of E1-deleted recombinant Ads. 3LL is a murine Lewis lung carcinoma cell line derived from C57BL/6 mice (H-2Kb). The above cell lines were cultured in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The B16.F10 melanoma cell line was cultured in complete RPMI 1640. Female C57BL/6 and BALB/c mice, 5–6 wk old, were purchased from Joint Ventures Sipper BK Experimental Animal Co. (Shanghai, People’s Republic of China).
Peptides
Peptides with a purity of >95% were synthesized by an automatic solid-phase peptide synthesizer (Applied Biosystems, Foster City, CA) by Cybersyn Co. (Lenni, PA) and purified by reversed-phase HPLC. The Mut1 peptide FEQNTAQP consists of the 52–59 amino acid positions of the mutated connexin 37 protein expressed in the 3LL cell line (48). The sequence of OVA257–264 peptide is SIINFEKL (49). Peptides were dissolved in serum-free Iscove’s modified Dulbecco’s medium/50 mM 2-ME and stored at -20°C.
Recombinant Ads
Recombinant Ad vector harboring LacZ reporter gene has been
described previously (50). Lptn cDNA was obtained from the activated
splenic T cells by RT-PCR. Briefly, mouse splenic T lymphocytes were
enriched by passing through a nylon wool column and stimulated with PMA
and A23187 at the final concentrations of 0.9 and 200 ng/ml,
respectively, for 4 h. Lptn cDNA was cloned from the activated T
cells by RT-PCR using mouse Lptn-specific primer ends modified to
include EcoRI and BamHI sites at 5' and 3'
termini, respectively (44), and confirmed by automatic sequencer
(ABI377). The recombinant Ads harboring mouse Lptn or LacZ gene were
generated by the method of cosmid/terminal peptide complex
homologous recombination previously described elsewhere (51). Lptn cDNA
was placed under the control of CMV promoter in the modified pCI
expression vector (Promega, Madison, WI). Subsequently, the expression
cassette was inserted into the ClaI cloning site of cosmid
vector pAdex1cw (kindly provided by I. Saito, Tokyo University, Tokyo,
Japan), which bears an Ad5 genome spanning 0–99.3 map units with
deletions of E1A, E1B, and E3. The resultant recombinant cosmid
was cotransfected into 293 cells with EcoT22I-digested Ad5
DNA-terminal peptide complex by calcium phosphate precipitation, and
the recombinant Ads were generated by homologous recombination (51).
The incorporation of the expression cassette was confirmed by digestion
with appropriate restriction enzymes. Subsequently, the recombinant
viruses were propagated in 293 cells and purified on CsCl density
gradient, the titers of which were determined by plaque assay on the
293 cells. The Ad solutions were stored at -80°C. The DNA structure
of Lptn recombinant Ad is shown in Fig. 1
.
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The procedure used in this study was previously described by Porgador and Gilboa (15), with some minor modifications. Briefly, bone marrow cells from C57BL/6 mice were depleted of red blood cells with ammonium chloride and depleted of lymphocytes, granulocytes, and Ia+ cells using a mixture of mAbs and rabbit complement. The mAbs were 2.43 (anti-CD8), GK1.5 (anti-CD4), RA3–3A1/6.1 (anti-B220/CD45R), B21-2 (anti-Ia), and 2.4G2 (anti-FcRII; tumor immunology bank 210, 207, 146, 229, and HB197; ATCC). Cells were plated in six-well culture plates (106 cells/ml, 3 ml/well) in RPMI 1640 supplemented with 10% heat-inactivated FCS, 50 µM 2-ME, 10 mM HEPES (pH 7.4), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 3.3 ng/ml recombinant murine GM-CSF (Sigma, St. Louis, MO). At day 3 of culture, floating cells were gently removed and fresh medium was added. At day 7 of culture, nonadherent and loosely adherent cells were collected and replated in a six-well culture plate (106 cells/ml, 3 ml/well). At 9 or 10 days of culture, nonadherent cells (DC) were harvested for identification and genetic modification.
Gene modification and peptide pulsing of DC
DC (1
2 x 106) were washed twice with HBSS
and resuspended in 100 200 µl serum-free RPMI 1640 with LacZ or
Lptn recombinant Ad with a multiplicity of infection of 100. After
1 h of incubation at 37°C (with gentle agitation every 20 min),
the cells were washed with HBSS and resuspended in 1 ml of RPMI 1640
supplemented with 10% FCS (1 x 106/ml). Twenty-four
hours after gene modification, LacZ gene-modified DC (LacZ-DC) were
collected for X-Gal staining to evaluate gene transfer
efficiency, and the culture supernatants from DC or gene-modified DC
were harvested for chemotaxis assay of Lptn. In addition, RT-PCR was
also performed on gene-modified DC to analyze Lptn expression. Before
PCR amplification, the reverse transcripts were digested with
RNase-free DNase to degrade the potentially contaminated DNA templates.
The upstream primer of mouse ß-actin was
5'-TGGAATCCTGTGGCATCCATGAAAC3-', and the downstream primer was
5'-TAAAACGCAGCTCAGTAACAGTCCG-3', with an expected size of 359 bp. The
specific upstream primer for mouse Lptn was
5'-TGGGGACTGAAGTCCTAGAAG-3', and the downstream primer was 5'-TTACC
CAGTCAGGGTTACTGCTGTG-3', with the product size of 300 bp. For peptide
pulsing, 12 x 106 DC cultured overnight after gene
transfection were washed with Iscove’s modified Dulbecco’s medium/50
mM 2-ME and resuspended in 0.8 ml of the same medium containing 100
µg of peptide. After 3 h incubation at 37°C (with gentle
shaking every 30 min), the peptide-pulsed DC were irradiated (5000
rad), washed twice with HBSS, and resuspended in HBSS for injection.
In vitro microchemotaxis assay
The microchemotaxis assay was conducted using a modified 48-well Boyden chamber migration assay (52). Duplicate wells of the lower half of the microchemotaxis chamber (NeuroProbe, Cabin John, MD) were filled with the appropriate dilutions of DC supernatants or standard human Lptn (PharMingen, San Diego, CA), and the upper chambers of the assembly were filled with 40 µl of the appropriate cell suspension (2 x 106 cells/ml). CD4+ and CD8+ T cells were negatively selected from splenocytes of C57BL/6 mice using the mixtures (RA3–3A1/6.1 (anti-B220/CD45R), B21-2 (anti-Ia), PK136 (anti-NK), 2.4G2 (anti-FcRII), and 2.43 (anti-CD8) or GK1.5 (anti-CD4)) and complement. Data were obtained by counting five nonoverlapping high power microscope fields from each well. Cells were considered to be chemoattracted if the chemotactic index (number of cells migrating in experimental well/number of cells migrating in medium only) was >2.
Immunization and tumor challenge
Peptide-pulsed DC (1 x 104 or 1 x 105) in 0.2 ml HBSS were injected s.c. into both thighs of normal C57BL/6 mice. One week after vaccination with peptide-pulsed DC with or without genetic modification, the mice were injected s.c. in the flank of the abdomen with a lethal dose of 5 x 105 3LL Lewis lung carcinoma cells. To evaluate the specificity of the antitumor immunity induced by Mut1 peptide-pulsed DC or Lptn-DC, the immunized mice were also challenged with B16 tumor cells. The tumor size was monitored at regular intervals and calculated as the product of the maximal perpendicular diameters. Mice were killed when the challenged tumors reached 3 cm in diameter or severe ulceration developed. All experiments were performed three times using individual treatment groups of six mice. Data are representative of three experiments performed.
Immunotherapy of preestablished 3LL carcinoma model
The spontaneous metastasis model of 3LL lung carcinoma was
established by inoculating 3LL tumor cells (2 x
105/mouse) into the footpad (53). Eighteen days later,
tumor-bearing legs were amputated when the tumor size in the footpad
reached 78 mm in diameter. Postsurgical mortality was <2%. Two
days after amputation, mice were vaccinated twice s.c. with 1 x
105 or 1 x 104 peptide-pulsed DC at
weekly intervals. Mice were killed when HBSS-treated mice died 30–35
days postamputation. Metastatic loads were recorded with lung weights.
Cytotoxicity assay
One week after immunization, the immunized mice were killed, and
their splenic lymphocytes (2 x 106 cells/ml) were
restimulated in vitro with 50 U/ml IFN--pretreated and irradiated
(5000 rad) 3LL cells (2 x 105 cells/ml) in six-well
culture plates (4 ml/well). The culture medium consisted of RPMI 1640
and NCTC109 (1/1, v/v) supplemented with 10% FCS, 50 mM 2-ME, 2 mM
glutamine, 10 mM HEPES (pH 7.4), 100 U/ml penicillin, and 100 µg/ml
streptomycin. After 5 days of restimulation, the viable lymphocytes
were collected and cultured with 2 x 10451Cr-labeled 3LL or B16 target cells in a
round-bottom 96-well microtiter plate (Nunclon, Naperville, IL) in
triplicate at different E:T ratios. After incubation at 37°C for
4 h, 100 µl of supernatants were harvested, and their
radioactivity was measured by a gamma counter (model 1275, Wallac,
Turku, Finland). The percentages of specific 51Cr release
were calculated according to the following formula: % 51Cr
release = 100 x [(cpm experiment - cpm spontaneous
release)/(cpm maximum - cpm spontaneous release)], where the
spontaneous release was obtained from target cells cultured with medium
alone, and the maximum release was obtained from target cells cultured
with 0.1% NP40 instead of effector cells. The spontaneous release
was <15%.
In vivo depletion of immune cell subsets and immunoregulatory molecules
Mice were immunized once with 1 x 105 Mut1
peptide-pulsed Lptn-DC and challenged with 5 x 105
3LL tumor cells 11 days after immunization. Four days before DC
immunization or tumor challenge, the mice started to receive a total of
five i.p. injections of the ascites (0.1 ml/mouse/injection) from
hybridoma-bearing mice at the intervals of 3 days. The mAbs used were
GK1.5 (anti-CD4), 2.43 (anti-CD8), PK136 (anti-NK; ATCC
HB191), and R4-6A2 (anti-IFN-; ATCC HB170) mAbs. Normal rat IgG
(Sigma) was given as mock control. Depletion of T cell subsets and NK
cells was monitored by flow cytometry, which showed >90% specific
depletion in splenocytes. To block the CD28/CTLA4 pathway of T
costimulation, peptide-pulsed DC (1 x 106 cells/ml)
were pretreated with 50 µg of CTLA-Ig in the volume of 1 ml at 4°C
for 45 min before being coinjected into mice. CTLA-Ig is a chimeric
fusion protein comprising the extracellular domain of CTLA4 Ag and the
Fc fragment of human IgG. CTLA-Ig fusion cDNA was inserted into the
pCAAGS expression vector (kindly provided by I. Saito, Tokyo
University, Tokyo, Japan) under the control of CMV promoter and was
subsequently transduced into the P3U1 myeoloma cell line. The CTLA-Ig
was purified from its culture supernatant with a protein A affinity
column (Pharmacia).
Statistical analyses
Data are presented as mean value ± SD. Student’s t test was used for comparison of two groups. p values of <0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow DC from C57BL/6 mice were expanded in vitro for 9–10
days, followed by characterization using composite criteria of typical
morphology, cell surface markers, and MLR. DC comprised >90% of the
nonadherent cells present 9–10 days after bone marrow culture. Surface
phenotype analysis by flow cytometry showed that bone marrow DC
expressed high levels of MHC-I, MHC-II, CD40, and CD80 and
moderate levels of DC-specific Ag DEC205 (data not shown). MLR revealed
that bone marrow DC were potent stimulators in allogenic MLR (data not
shown). The genetic modification of DC was mediated by Ad vector. The
DNA structure of Lptn Ad vector (Ad.Lptn) is demonstrated in Fig. 1
.
The recombinant Ad vector of LacZ was previously described (50). Using
LacZ as a reporter gene, we evaluated the DC gene transfer efficiency
mediated by Ad vector. Twenty-four hours after infection of day 9 DC
with LacZ recombinant Ads at a multiplicity of infection of 100,
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining
showed that gene transfer efficiency was >95% (data not shown), which
confirms that Ad vector can efficiently mediate gene transfer into DC.
Chemotaxis assay revealed that the culture supernatants from DC or
LacZ-DC had no significant chemotactic activity on CD4+ or
CD8+ T cells, but the 24-h culture supernatants from DC
transfected with Ad.Lptn (Fig. 1) could attract both CD4+ T
cells and CD8+ T cells markedly (Fig. 2). This indicates that Lptn Ad vector
can mediate the expression of bioactive Lptn in DC. Lptn expression in
Lptn-DC was further confirmed by RT-PCR analysis. Before PCR, the
reverse transcripts were digested with RNase-free DNase I to degrade
the potentially contaminated DNA templates; RNA samples without reverse
transcription were directly subjected to PCR as control. As shown in
Fig. 2, neither DC nor LacZ-DC expressed any detectable Lptn by RT-PCR,
whereas Lptn mRNA expression was detected 4 h after gene
modification of day 9 DC with Ad.Lptn. In addition, neither LacZ nor
Lptn gene transfer into DC by their replication-defective Ad
vectors changed their phenotype obviously.
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One of our primary aims was to determine whether vaccination of
Lptn-DC pulsed with tumor peptide could induce peptide-specific CTL
response more effectively. Accordingly, Lptn-DC were pulsed with
peptide (Mut1 or OVA) and injected s.c. into mice at a dose of 1
x 104 or 1 x 105 cells/mouse. The CTL
were determined after 5 days of in vitro restimulation with 3LL
tumor cells. Mut1 is an H-2Kb-restricted Ag peptide of 3LL
carcinoma cells. H-2Kb OVA peptide was used as an
irrelevant peptide control in this study. Although immunization with a
single dose of 1 x 105 Mut1 peptide-pulsed DC or Mut1
peptide-pulsed LacZ-DC induced CTL response specifically against 3LL
cells, these cells were less potent CTL inducers than were Mut1
peptide-pulsed Lptn-DC (Fig. 3).
Immunization with a lower dose of Mut1 peptide-pulsed DC or Mut1
peptide-pulsed LacZ-DC (1 x 104) induced poor CTL
response, but the same low-dose Mut1 peptide-pulsed Lptn-DC did induce
higher CTL activity specifically against 3LL tumor cells, which was
comparable with that induced by vaccination with 10-fold peptide-pulsed
DC. On the other hand, immunization of Mut1 peptide-pulsed DC or Mut1
peptide-pulsed Lptn-DC induced poor CTL activity against B16 cells
(data not shown), and immunization with OVA peptide-pulsed DC or OVA
peptide-pulsed Lptn-DC induced no significant CTL response against 3LL
cells. This implies that Lptn-DC are more potent to deliver peptide to
induce specific CTL in vivo.
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Then, we tested whether vaccination of mice with Lptn-DC pulsed
with tumor peptide was capable of inducing protective immunity against
tumor challenge more potently. Seven days after a single vaccination of
DC, the vaccinated mice were challenged s.c. with 5 x
105 3LL or B16 tumor cells. Consistent with the CTL
response, vaccination with 1 x 105 Mut1
peptide-pulsed DC could provide protection specifically against 3LL
tumor challenge, but less effectively than the vaccination with Lptn-DC
counterpart. Vaccination with a low dose (1 x 104) of
Mut1 peptide-pulsed DC could not protect the immunized mice from 3LL
tumor challenge, and tumors grew in all mice. But all the mice
immunized with 1 x 104 Mut1 peptide-pulsed Lptn-DC
were free of tumor 20 days after 3LL tumor challenge (Fig. 4), and 83.3% of mice were free of tumor
90 days after tumor challenge in three different experiments.
Immunization of Mut1 peptide-pulsed DC or Mut1 peptide-pulsed Lptn-DC
induced poor protection against B16 cells (data not shown), and
immunization with OVA peptide-pulsed DC or OVA peptide-pulsed Lptn-DC
failed to elicit any protective immunity against 3LL tumor challenge.
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To investigate the potential roles of T cell subpopulations and NK
cells in the induction of protective immunity by Lptn-DC pulsed with
MHC-I-restricted peptide, mice were depleted of CD4+ or
CD8+ T cells or NK cells during immunization or during
challenge. As shown in Fig. 5, depletion
of CD8+ T cells during immunization or during challenge
abrogated the protective immunity induced by peptide-pulsed Lptn-DC.
Mice depleted of CD4+ T cells during immunization failed to
reject tumor challenge. However, the mice could reject tumor challenge
when CD4+ T cells were depleted during challenge. These
results are consistent with the report by Porgador and Gilboa (15), in
which they found that CD8+ T cells were the predominant
effector cells in the MHC-I-restricted peptide-induced antitumor
immunity and that CD4+ T cells were required for the
induction of CD8+-dependent T cell immunity but were
unnecessary in the effector phase. Although Lptn was recently reported
to be capable of attracting NK cells besides T cells (25), mice
depleted of NK cells during immunization or during challenge were
protected from tumor challenge, suggesting that interaction with T
cells and subsequent activation of T cells are responsible for the
antitumor immunity induced by peptide-pulsed Lptn-DC. To evaluate the
potential role of T cell costimulation in peptide-pulsed
Lptn-DC-induced immune response, the CD28/CTLA4 costimulation pathway
was functionally blocked by the chimeric fusion protein CTLA-Ig. The
results showed that blockade of the CD28/CTLA4 pathway during
immunization abrogated the protective immunity completely. IFN- is
regarded as an important Th1-associated cytokine and plays an important
role in the induction of Th1-mediated immunity. Consistent with this
notion, blockage of IFN- during immunization or during challenge
abrogated protective immunity induced by peptide-pulsed Lptn-DC.
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To be more stringent and clinically relevant, the therapeutic
effects of vaccination with Lptn-DC pulsed with tumor-associated
peptide were further evaluated in the treatment of mice with the
preestablished 3LL metastasis model. The 3LL tumor cell line is poorly
immunogenic and highly metastatic. The spontaneous metastasis model of
3LL was established by injection of 2 x 105 3LL tumor
cells into the footpad. The average lung weight of a normal mouse was
190210 mg. Thirty to thirty-five days after amputation, the mean
lung weight of the HBSS-treated control group was about 600 mg. As
shown in Fig. 6, vaccination with 1
x 105 Mut1 peptide-pulsed DC or Mut1 peptide-pulsed
LacZ-DC could slightly reduce lung metastases (with the mean lung
weights of 436 and 402 mg, respectively), but less effectively than the
Lptn-DC counterpart (256 mg in mean lung weight). The therapeutic
effects of Mut1 peptide-pulsed DC or Mut1 peptide-pulsed LacZ-DC
disappeared when the DC dose was reduced to 1 x 104,
but the same dose of Mut1 peptide-pulsed Lptn-DC could still inhibit
pulmonary metastases markedly, yielding a mean lung weight of 312 mg.
The survival time of the mice treated with Mut1 peptide-pulsed Lptn-DC
was also greatly extended, much more effectively than with the mock
control (data not shown), whereas vaccination with Mut1 peptide-pulsed
DC or Mut1 peptide-pulsed Lptn-DC could not inhibit B16 pulmonary
metastasis (data not shown), and OVA peptide-pulsed DC or OVA
peptide-pulsed DC Lptn-DC did not exhibit any therapeutic effects on
3LL pulmonary metastases. These findings suggest that peptide-pulsed
Lptn-DC can induce specific therapeutic antitumor immunity more
potently.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is evident that chemokine can regulate the migration of T cells as
well as DC; thus, chemokine may play potentially regulatory roles in
the priming of T cell immunity by DC. Recent investigations
demonstrated that DC could express chemokine receptors (e.g., CCR1,
CCR2, CCR5, CCR6, CXCR1, CXCR2, and CXCR4) and respond to
chemokines to migrate directionally (36, 37, 38, 39). In a more active manner,
DC can attract T cells by secreting several chemokines (e.g., MIP-,
monocyte chemotactic protein, RANTES, and MIP-1) (40, 41, 42). More
interestingly, a CC chemokine named DC-CK1 was cloned recently
from human DC, which can preferentially attract human naive T cells
(43). Although the murine analogue of DC-CK1 has not been defined, this
suggests that chemoattraction between DC and T cells would favor DC Ag
presentation to prime T cell immunity. In our study, Lptn-DC were found
to be capable of attracting T cells more efficiently without any
obvious changes of cell phenotypes. The culture supernatants from
normal DC or LacZ-DC have no significant chemotactic activity on T
cells, which implies that the autocrine of chemokine by DC may
be very low and under the threshold of chemotaxis assay. Nevertheless,
the supernatants from Lptn-DC can attract CD4+ and
CD8+ T cells markedly, which indicates that Lptn gene
modification of DC can improve their preferential chemotaxis on T cells
and consequently may optimize the microenviroment of Ag presentation
and favor DC Ag presenting to T cells. Although Lptn is also capable of
attracting NK cells (46), in vivo depletion of NK cells during
immunization did not abrogate the protective immunity induced by
peptide-pulsed Lptn-DC.
Consistent with previous reports about peptide-pulsed DC (15), both
CD4+ and CD8+ T cells are necessary for the
induction of MHC-I-restricted peptide immunity by Lptn-DC, and only
CD8+ T cells are required in the effector phase. In
addition, T cell costimulation and IFN- are also found to be
required for the induction of T cell immunity by peptide-pulsed
Lptn-DC. The above data supported our hypothesis that Lptn autocrine by
DC in the local microenviroment enables them to preferentially attract
T cells more efficiently, favors DC Ag presentation, and, hence,
improves the efficacy of DC-based vaccines. Whether Lptn autocrine by
Lptn-DC affects their in vivo migration and tissue localization is
under further investigation.
Genetic modification of DC is receiving much attention in DC-based vaccines. Different viral vectors, including retroviral vector (20, 21, 22, 23, 24), Ad vector (25, 26, 27, 28), and vaccinia vector (26), have been evaluated to genetically modify DC. By coculture with retrovirus-producing packaging cells or repeated rounds of infection, mouse and human DC could be transduced with reporter genes (e.g., ß-galactosidase, CD2, and LNGFR) and human tumor-associated Ag genes (e.g., mucin and MART-1), but the gene transfer efficiency is relatively low (20–75%) compared with that mediated by Ad vector. In addition, the former protocol is not compatible with the current guideline about the clinical use of retroviral vector. Ad vector could mediate gene transfer into DC with high efficiency (>95%). In animal models, a single vaccination with 1–3 x 105 DC genetically modified with Ad vector harboring cDNA for model Ags (e.g., ß-galactosidase and OVA) have been documented to be capable of inducing protective and therapeutic antitumor immunity (25, 26). In our previous studies (12), mouse DC were transduced with GM-CSF Ad vector, and it was found that GM-CSF gene-modified DC acquired more potent costimulatory activity and could induce protective and therapeutic antitumor immunity against B16 melanoma after they were pulsed with tumor Ag. Although Ad vector is highly immunogenic, it is reported that repeated injections of Ad-infected DC induced only low titers of neutralizing Abs, and that the presence of neutralizing Abs specific for Ad did not affect the usefulness of infected DC to boost CTL response by repeated applications (26). Thus, Ad vector seems to be a promising tool to be used to genetically modify DC for vaccination. Besides viral vectors, nonviral methods were used to transduce DC, including lipofectin, calcium phosphate precipitation, and electroporation, but none of them yielded efficient gene transfer compared with Ad vector (27).
Our finding that a low dose of Lptn-DC (1 x 104) pulsed with tumor Ag peptide can exhibit a marked therapeutic effect on a preestablished tumor reduces the conventional dose of DC at least 10 times. Moreover, immunization with low-dose DC facilitates a reduction of the risk of autoimmune diseases related with DC-based vaccines. To our knowledge, this is the first demonstration that the improved chemotaxis of DC on T cells via genetic modification of DC with chemokine can increase the efficacy of DC-based vaccines. This study also provides an applicable approach to utilizing chemokines in the immunologic intervention.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Xuetao Cao, Department of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China. E-mail address:
3 X.C. and W.Z. contributed equally to this work.
4 Abbreviations used in this paper: DC, dendritic cell; Ad, adenovirus; GM-CSF, granulocyte-macrophage colony-stimulating factor; Lptn, lymphotactin; Lptn-DC, Lptn gene-modified DC; LacZ-DC, LacZ gene-modified DC; CTLA, cytotoxic T lymphocyte-associated protein; MIP-, macrophage-inflammatory protein-.
Received for publication May 21, 1998. Accepted for publication August 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Z. Guo, M. Zhang, H. Tang, and X. Cao Fas signal links innate and adaptive immunity by promoting dendritic-cell secretion of CC and CXC chemokines Blood, September 15, 2005; 106(6): 2033 - 2041. [Abstract] [Full Text] [PDF]
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 Y. Wu, T. Wan, X. Zhou, B. Wang, F. Yang, N. Li, G. Chen, S. Dai, S. Liu, M. Zhang, et al. Hsp70-Like Protein 1 Fusion Protein Enhances Induction of Carcinoembryonic Antigen-Specific CD8+ CTL Response by Dendritic Cell Vaccine Cancer Res., June 1, 2005; 65(11): 4947 - 4954. [Abstract] [Full Text] [PDF]
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B. Wang, H. Chen, X. Jiang, M. Zhang, T. Wan, N. Li, X. Zhou, Y. Wu, F. Yang, Y. Yu, et al. Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein Blood, July 1, 2004; 104(1): 200 - 206. [Abstract] [Full Text] [PDF]
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T. Wan, X. Zhou, G. Chen, H. An, T. Chen, W. Zhang, S. Liu, Y. Jiang, F. Yang, Y. Wu, et al. Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant Blood, March 1, 2004; 103(5): 1747 - 1754. [Abstract] [Full Text] [PDF]
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H. Matsuyoshi, S. Senju, S. Hirata, Y. Yoshitake, Y. Uemura, and Y. Nishimura Enhanced Priming of Antigen-Specific CTLs In Vivo by Embryonic Stem Cell-Derived Dendritic Cells Expressing Chemokine Along with Antigenic Protein: Application to Antitumor Vaccination J. Immunol., January 15, 2004; 172(2): 776 - 786. [Abstract] [Full Text] [PDF]
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Z. Guo, M. Zhang, H. An, W. Chen, S. Liu, J. Guo, Y. Yu, and X. Cao Fas ligation induces IL-1{beta}-dependent maturation and IL-1{beta}-independent survival of dendritic cells: different roles of ERK and NF-{kappa}B signaling pathways Blood, December 15, 2003; 102(13): 4441 - 4447. [Abstract] [Full Text] [PDF]
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L. Stievano, V. Tosello, N. Marcato, A. Rosato, A. Sebelin, L. Chieco-Bianchi, and A. Amadori CD8+{alpha}{beta}+ T Cells That Lack Surface CD5 Antigen Expression Are a Major Lymphotactin (XCL1) Source in Peripheral Blood Lymphocytes J. Immunol., November 1, 2003; 171(9): 4528 - 4538. [Abstract] [Full Text] [PDF]
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C. Palena, J. Schlom, and K.-Y. Tsang Differential Gene Expression Profiles in a Human T-cell Line Stimulated with a Tumor-associated Self-peptide versus an Enhancer Agonist Peptide Clin. Cancer Res., May 1, 2003; 9(5): 1616 - 1627. [Abstract] [Full Text] [PDF]
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 P. Middel, P. Thelen, S. Blaschke, F. Polzien, K. Reich, V. Blaschke, A. Wrede, K. M. Hummel, B. Gunawan, and H.-J. Radzun Expression of the T-Cell Chemoattractant Chemokine Lymphotactin in Crohn's Disease Am. J. Pathol., November 1, 2001; 159(5): 1751 - 1761. [Abstract] [Full Text] [PDF]
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P. Ghia, P. Transidico, J. P. Veiga, C. Schaniel, F. Sallusto, K. Matsushima, S. E. Sallan, A. G. Rolink, A. Mantovani, L. M. Nadler, et al. Chemoattractants MDC and TARC are secreted by malignant B-cell precursors following CD40 ligation and support the migration of leukemia-specific T cells Blood, August 1, 2001; 98(3): 533 - 540. [Abstract] [Full Text] [PDF]
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S. Liu, Y. Yu, M. Zhang, W. Wang, and X. Cao The Involvement of TNF-{{alpha}}-Related Apoptosis-Inducing Ligand in the Enhanced Cytotoxicity of IFN-{{beta}}-Stimulated Human Dendritic Cells to Tumor Cells J. Immunol., May 1, 2001; 166(9): 5407 - 5415. [Abstract] [Full Text] [PDF]
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C. J. Kirk, D. Hartigan-O’Connor, B. J. Nickoloff, J. S. Chamberlain, M. Giedlin, L. Aukerman, and J. J. Mulé T Cell-dependent Antitumor Immunity Mediated by Secondary Lymphoid Tissue Chemokine: Augmentation of Dendritic Cell-based Immunotherapy Cancer Res., March 1, 2001; 61(5): 2062 - 2070. [Abstract] [Full Text]
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X. Cao, W. Zhang, T. Wan, L. He, T. Chen, Z. Yuan, S. Ma, Y. Yu, and G. Chen Molecular Cloning and Characterization of a Novel CXC Chemokine Macrophage Inflammatory Protein-2{gamma} Chemoattractant for Human Neutrophils and Dendritic Cells J. Immunol., September 1, 2000; 165(5): 2588 - 2595. [Abstract] [Full Text] [PDF]
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C. Cerdan, E. Serfling, and D. Olive The C-class chemokine, lymphotactin, impairs the induction of Th1-type lymphokines in human CD4+ T cells Blood, July 15, 2000; 96(2): 420 - 428. [Abstract] [Full Text] [PDF]
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 C. Klein, H. Bueler, and R. C. Mulligan Comparative Analysis of Genetically Modified Dendritic Cells and Tumor Cells as Therapeutic Cancer Vaccines J. Exp. Med., May 15, 2000; 191(10): 1699 - 1708. [Abstract] [Full Text] [PDF]
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J. T. Tan, J. K. Whitmire, K. Murali-Krishna, R. Ahmed, J. D. Altman, R. S. Mittler, A. Sette, T. C. Pearson, and C. P. Larsen 4-1BB Costimulation Is Required for Protective Anti-Viral Immunity After Peptide Vaccination J. Immunol., March 1, 2000; 164(5): 2320 - 2325. [Abstract] [Full Text] [PDF]
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