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*
Center for Surgery Research and
Departments of Hematology/Medical Oncology and Immunology, The Cleveland Clinic Foundation, Cleveland, OH 44195; and
Department of Immunology, Second Military Medical University, Shanghai Peoples Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the past several years, dendritic cells (DCs)3 have been identified as the most effective APC. DCs possess an exceptional capability to capture Ags, process and present antigenic peptide fragments, migrate to lymphoid organs, and induce primary immune responses of both CD8+ and CD4+ T cells (15). The ability of DCs to act as potent APC for the induction of T cell responses is attributed to the high expression of MHC Ags and adhesion and/or costimulatory molecules as well as their ability to produce cytokines essential for the activation and proliferation of the T cells. Lack of tumor Ag presentation by DCs in vivo could be one of the reasons why the host immune system fails to eradicate most immunogenic tumors (16). Several strategies of using tumor Ag-charged DCs as vaccines for cancer immunotherapy have been developed. Immunization using DCs pulsed with purified tumor-associated peptides or proteins has been shown to be a powerful method of priming tumor-reactive T cells and inducing host protective and therapeutic antitumor immunity in mice (17, 18, 19, 20) and humans (21, 22, 23). However, this approach is currently limited for clinical application, as few human tumor rejection Ags have been identified. The high polymorphism of the human HLA system has also made it difficult to identify tumor-associated peptides as a vaccine for cancer therapy. In the best studied human melanoma where a class of tumor-associated proteins have been identified (4, 24), it is unclear which of the identified tumor-associated Ags is the best choice to induce effective tumor rejection in vivo or how effective they are (25). Immunization with multiple tumor Ags may be superior to the use of a single dominant epitope to induce antitumor immunity more effectively.
In this study we examined whether fusion of DCs with syngeneic poorly immunogenic tumors could generate hybrid cellular vaccines to effectively elicit host T cell-mediated antitumor immunity. It is hypothesized that the fusion of tumor cells with DCs would generate a hybrid vaccine with potent Ag-processing and -presenting capabilities and expression of multiple tumor Ags, thereby inducing more effective antitumor immunity. A recent study by Gong et al. demonstrated that fusion of a relatively immunogenic mouse tumor, MC38 carcinoma, with syngeneic DCs as a vaccine induced T cell protective immunity against tumor challenge and immune rejection of established tumor (26). In this study, B16 (B16.F10) melanoma and RMA-S lymphoma cell lines were selected for the fusion experiments. B16 tumor is poorly immunogenic and lacks the expression of MHC and costimulatory molecules. Immunization with irradiated B16 tumor cells fails to induce systemic immunity or elicit functional tumor-reactive T cells. RMA-S is a Rauscher MuLV (R-MuLV)-induced T cell lymphoma of B6 mouse origin that is genetically defective of TAP and thus does not process endogenous Ags (27). Our results demonstrate that fusion of DCs with syngeneic tumor generated hybrid cells that express both DC-derived accessory molecules and tumor-derived Ags. The tumor/DC fusion hybrids were capable of processing and presenting tumor-associated Ags to elicit tumor-reactive CTLs. Vaccination of B6 mice with B16/DC fusion preparations induced partial protective immunity against tumor challenge. In addition, immunization with B16/DC or RMA-S/DC vaccine could prime lymph node (LN) T cells for adoptive immunotherapy. The transfer of such vaccine-primed, ex vivo expanded LN T cells into tumor-bearing mice specifically reduced the number of established B16 pulmonary metastases and effectively eradicated disseminated FBL-3 tumor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old C57BL/6 (denoted B6, H-2b) mice and BALB/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were maintained in specific pathogen-free conditions and were used at the age of 8–10 wk.
Tumor cell lines
B16.F10, a spontaneous melanoma cell line of B6 mouse origin, was a gift from Dr. Steven A. Rosenberg’s laboratory (National Cancer Institute, Bethesda, MD). A B16 tumor line expressing the murine costimulatory molecule B7.1 (B16.B7.1) was provided by Dr. Suzanne Ostrand-Rosenberg (University of Maryland, Baltimore, MD). RMA-S is a genetically TAP-defective subline of RMA, a R-MuLV-induced T cell lymphoma of B6 mouse origin, and thus does not process endogenous Ags (27). FBL-3 is a F-MuLV-induced leukemia of B6 origin that expresses tumor-associated Ags cross-reactive with syngeneic leukemia and lymphoma induced by F-, M-, and R-MuLV tumors (28). EL-4 is a dimethylbenzanthrene-induced T cell lymphoma of B6 origin. MCA 207 is a fibrosarcoma of B6 mouse origin induced by 3-methylcholanthrene. LSTRA is a Moloney MuLV-induced lymphoma of BALB/c mouse origin. All tumor cell lines were maintained in culture in complete medium (CM) consisting of RPMI 1640 supplemented with 10% heat-inactivated FCS, 0.1 mM nonessential amino acid, 1 µM sodium pyruvate, 2 mM fresh L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 50 µg/ml gentamicin, 0.5 µg/ml fungizone (all from Life Technologies, Grand Island, NY), and 5 x 10-5 M 2-ME (Sigma, St. Louis, MO).
Preparation of DCs
The procedure used for generation of DCs from bone marrow (BM) culture was described previously with some modification (29). In brief, the BM cells prepared from femurs and tibias of normal B6 mice were depleted of RBC with ammonium chloride. The BM cells were treated with a mixture of mAbs (PharMingen, San Diego, CA) against IAb, B-220/CD45R, Lyt-2, and L3T4 plus low-tox-M rabbit complement (Accurate Chemical and Science Corp., Westbury, NY). The lymphocyte-depleted BM cells were then incubated in CM supplemented with recombinant mouse GM-CSF (10 ng/ml; PharMingen) and IL-4 (10 ng/ml; Sigma) in 24-well plates at 5 x 105 cells/well. On day 5, one-half of the media in each well was replaced with fresh CM containing mouse GM-CSF (10 ng/ml) and IL-4 (10 ng/ml). On day 6, the nonadherent DCs were harvested by gentle pipetting and were used in the studies. The average cell yield of DCs derived from the BM culture was 25 x 106 cells/mouse, and >90% of the cells expressed the N418 Ag.
Fusion protocol
DCs derived from BM culture were fused with tumor cells at a 3:1 (DC:tumor) ratio using polyethylene glycol (PEG; m.w. 1450)/DMSO solution (Sigma). In brief, tumor cells were cultured in CM supplemented with 20% FCS and 1x OPI solution (oxaloacetate, pyruvate, and insulin; Sigma) for 4–6 h before fusion. Tumor cells and DCs were then mixed and washed with serum-free medium. After removing the medium, 1 ml of PEG was added to the cell pellet while resuspending the cells by stirring for 2 min. An additional 10 ml of serum-free medium was added to the cell suspension over the next 3 min with continued stirring. The cells were centrifuged at 400 x g for 5 min. The cells were resuspended with 20% FCS CM and cultured for 24 h before staining or being used as targets or vaccines. Fusion preparations of DCs with B16 or RMA-S were termed B16/DC and RMA-S/DC, respectively.
Phenotype staining
B16, RMA-S, DCs, and their fusion hybrids were analyzed by staining with FITC- or PE-conjugated mAbs (PharMingen) against MHC Ags (Db, Kb, IAb), adhesion and costimulatory molecules (B7.1, ICAM-1), and lymphocyte Ags (Thy-1.2, SmIg) at 4°C for 45 min. DCs were identified by labeling with mAb against CD11c (N418) (30). B16, B16/DC or B16/B16 fused cells were stained with mAb against AKV Env gp85 protein (M562, provided by Dr. Masaru Taniguchi, Ciba University, Tokyo, Japan) as a B16 tumor-specific marker (31). RMA-S and RMA-S/DC fused cells were stained with Thy-1.2 or mAb against the R-MuLV-encoded Gag p12 protein (584, provided by Dr. Bruce Chesebro, National Institute of Allergy and Infectious Diseases, Hamilton, MO) as RMA-S tumor-derived markers (32). The method for labeling cells with TRITC (rhodamine) was similar as previously described (33). Briefly, cells were resuspended in RPMI 1640 at 1 x 106 cells/ml and incubated with TRITC (0.5 µg/ml) in 37°C for 45 min. The labeled cells were washed three times and used for fusion studies. The phenotypes of fresh and cultured LN T cells were determined by FACS analysis following staining with FITC- or PE-conjugated mAbs against Thy-1.2, Lyt-2, and L3T4 (PharMingen). All cells were washed twice with HBSS and fixed with 0.2% paraformaldehyde. Fluorescence intensity and positive cell percentage were measured on a FACScan flow microfluorometer (Becton Dickinson, Sunnyvale, CA).
T cell lines
FBL-3-specific CD8+ CTLs were generated from B6 mice immune to FBL-3 as described previously (28). B16 tumor-reactive CTLs were generated from spleens of B6 mice immunized with B16/DC fusion preparation. Briefly, B6 mice were s.c. immunized twice in a 2-wk interval on the flanks with 2 x 106 (106/side) irradiated (15,000 rad) B16 or with B16/DC suspended in 0.1 ml of HBSS. One week after final immunization, spleens from each group of mice were harvested. Splenocytes (4 x 106) from normal B6 mice or mice vaccinated with irradiated B16 or B16/DC were cultured with 105 irradiated (15,000 rad) B16.B7.1 tumor cells in wells of 24-well plates in CM. On days 2 and 4, one-half of the medium was changed with CM containing 5 U/ml human rIL-2 (Chiron, Emeryville, CA). After 5–7 days, lymphocytes were harvested and used as effectors in a cytotoxicity assay. FMR tumor-reactive CTLs were generated from spleens of B6 mice immunized with irradiated RMA-S/DC fusion preparation or RMA-S tumor cells in a similar fashion.
Proliferative assay
For MLR, graded doses (102-104/well) of irradiated (3000 rad) DCs or B6 splenocytes were placed in 96-well plates as stimulators and coincubated with BALB/c splenocytes (105/well) as responders. The plates were incubated in a humidified atmosphere under 5% CO2 tension at 37°C for 96 h and were pulsed for 16 h with 1 µCi of [3H]thymidine/well before harvesting. All determinations were conducted in triplicate, and thymidine incorporation was determined. Stimulation indexes were calculated by dividing the counts per minute (mean) obtained from each group by the counts per minute (mean) from irradiated BALB/c spleen cells alone.
The methods for assessing the ability of DCs to process and present FBL-3 tumor proteins to FBL-3 specific CD4+ T cells were similar as described previously (34). Briefly, graded doses (103-105/well) of irradiated DCs or B6 spleen cells were incubated with varying concentrations (1:80,000 to 1:640,000 dilution) of purified F-MuLV proteins in wells of 96-well plates as stimulators. Resting FBL-3 env gp70-specific CD4+ Th clone (C8) was used as the responder (2 x 104/well). The plates were incubated at 37°C for 96 h and were pulsed for 16 h with 1 µCi of [3H]thymidine/well before harvesting. All determinations were conducted in triplicate, and thymidine incorporation was determined. Stimulation indexes were calculated by dividing the sample cpm by the mean cpm of C8 clone incubated with irradiated DCs or B6 spleen cells without tumor Ags.
Cytotoxicity assay
Target cells were incubated at 37°C with 250 µCi of 51Cr (New England Nuclear, Boston, MA) in 1 ml of 20% FCS RPMI 1640 medium for 45 min. Labeled targets were washed three times with HBSS and resuspended in 20% FCS RPMI at 105 cells/ml. 51Cr-labeled target cells (104 cells in 100 µl) were placed into each well of 96-well plates, and 100 µl/well of each dilution of T cells as effectors was added. Plates were incubated at 37°C for 4 h. The supernatant from each well was harvested, and the amount of 51Cr released was counted in a gamma counter. The percentage of specific lysis was calculated. All determinations of cytotoxicity were conducted in triplicate, with a minimum of three E:T cell ratios.
In vitro activation of LN T cells
B6 mice were s.c. immunized twice in a 2-wk interval on the flanks with 2 x 106 (106/side) irradiated (15,000 rad) tumor, tumor/DC fusion preparation, or tumor mixed with DCs (1/1) suspended in 0.1 ml of HBSS. One week after the final immunization, inguinal LNs from each group of mice were harvested. LN cells from each group of mice were activated and expended in culture using anti-CD3 plus IL-2 as described previously (35). In brief, LN cells (3–4 x 106 cells/well) were activated on 24-well plates coated with anti-CD3 mAb (145-2C11) and incubated at 37°C for 2 days. The activated cells were suspended at 1–2 x 105 cells/ml in CM containing IL-2 (4 U/ml) and incubated in gas-permeable culture bags (Baxter Healthcare, Deerfield, IL) for an additional 3 days. The derived LN T cells were harvested and used as effector cells for adoptive immunotherapy.
Tumor challenge
B6 mice were s.c. immunized twice in a 2-wk interval with 106 irradiated (15,000 rad) B16, B16 mixed with DCs (1/1; unfractionated cells from overnight culture), or B16/DC fusion preparation (adherent cells from overnight culture). Ten days following the final immunization, each group of mice was injected s.c. with varying doses (104, 105, or 106 cells/mouse) of viable B16. Tumor growth and survival time of each group of mice were recorded. The size of the tumor in each mouse was measured in two perpendicular dimensions with a vernier caliper twice weekly after tumor challenge. Tumor incidence was considered positive when the average diameters of the tumor exceeded 3 mm.
Adoptive immunotherapy models
For therapy of B16 pulmonary metastases, B6 mice were injected i.v. with 105 live B16 tumor cells in 1 ml of PBS to initiate pulmonary metastases. Three days after tumor inoculation, mice were randomly divided into several groups to receive treatments by i.v. injection of 5 x 107 cultured LN T cells suspended in 1 ml of PBS. On day 21 after tumor inoculation, mice from each group were killed, and lungs were insufflated with Fekete’s solution. Lung metastases were counted. In some experiments, tumor-bearing mice were i.p. administered IL-2 (15,000 U, twice/day for 5 days) following the adoptive transfer of cultured LN T cells. For therapy of FBL-3 tumor, B6 mice were inoculated i.p. with 5 x 106 viable FBL-3 tumor cells on day 0. By day 5, the tumor was disseminated, and mice were treated with cyclosphosphamide (CY) at a dose of 180 mg/kg followed in 6 h by i.p. injection of cultured LN T cells (5 x 107 cells/mouse) suspended in 0.5 ml of PBS. The tumor growth and the survival time of each group of mice were monitored and recorded on a regular basis.
Statistical analysis
The significant differences in the numbers of pulmonary metastases and the survival time between groups were determined by the Mann-Whitney rank sum test. Two-sided p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs from B6 mice were prepared and enriched from BM culture
supplemented with recombinant mouse GM-CSF and IL-4. The purity and
phenotype of the BM-derived DCs were identified by labeling with a
panel of Abs against mouse DCs or lymphoid cell surface markers. The
results (Fig. 1
) showed that DCs derived
from BM culture were positive for Db, Kb,
IAb, B7.1, ICAM-1, and CD11c, but were negative for
Thy-1.2, SmIg, B16 tumor marker (MuLV AKV Env gp 85), and RMA-S tumor
marker (R-MuLV Gag p12), as expected. The purity of DCs
(N418+ cells) was >90%. The results shown in Fig. 1
confirmed that B16 tumor expressed very low amounts of Db
and Kb, and was negative for IAb, B7.1, ICAM-1,
CD11c, Thy-1.2, and SmIg but expressed high levels of Env gp85 on the
cell surface. RMA-S cells were positive for Thy-1.2 and Gag p12, but
expressed very low amounts of Db, Kb, and
ICAM-I and were negative for IAb, B7.1, SmIg, and CD11c.
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Fusion of DCs with syngeneic tumor B16 or RMA-S cells by PEG was
conducted at a DC:tumor ratio of 3:1. Fusion efficiency was determined
by staining the fusion preparations with mAbs reactive to cell surface
markers derived from DCs and parental tumor. The B16/DC fusions were
stained with M562 mAb and/or mAbs against IAb,
Db, B7.1, and CD11c. A fusion efficiency of 15–25% was
obtained reproducibly. The results (Fig. 2A) showed that fusion of B16
tumor with DCs produced a hybrid cell population that expressed cell
surface marker from B16 (Env gp85) and was positive for
IAb, Db, B7.1, and CD11c. No such positive cell
population was detected in preparations of B16 fused with B16 by PEG or
B16 mixed with DCs (1:1). The fusion between B16 and DCs was also
confirmed in experiments using TRITC (rhodamine)-labeled B16 cells
fused with DCs and then staining the overnight-cultured fusion
preparations with FITC-conjugated mAbs against IAb, or
CD11c (Fig. 2B). In similar experiments, RMA-S/DC fusion
preparations were stained with anti-Thy-1.2 or R-MuLV gag gp12
(584) mAb and/or with mAbs against IAb, Db,
Kb, or B7.1. A fusion efficiency of 15–25% was obtained
reproducibly. The RMA-S/DC-fused cells expressed Thy-1.2 and Gag gp12
and were positive for IAb, Db, B7.1, and CD11c
(data not shown).
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To assess the ability of tumor/DC fusion hybrids to process and
present intracellular proteins derived from tumor cells, RMA-S/DC-fused
cells were used. RMA-S is genetically defective of TAP and thus does
not process endogenous Ags. Our previous study has identified the
immunodominant CTL epitope of FMR-MuLV-induced tumors in B6 mice as a
Db-restricted nonamer peptide (p85–93) located in the
leader sequence of the gPr80gag protein encoded by the
FMR-MuLV gag gene. CD8+ CTLs specific against
FMR-MuLV-induced tumors in B6 mice can effective lyse FBL-3, MBL-2, and
RMA, but fail to recognize and lyse RMA-S (28). To determine whether
RMA-S/DC-fused cells can process and present tumor-associated proteins,
the RMA-S/DC fusion preparation (containing 15–25% fused cells) was
labeled with 51Cr and used as a target in the CTL assays.
The results (Fig. 3A) showed
that CD8+ CTLs specific for FBL-3 tumor lysed FBL-3 and
RMA-S/DC, but did not lyse RMA-S, or RMA-S mixed with DCs as targets.
To determine whether vaccination with RMA-S/DC can elicit
tumor-reactive T cells, B6 mice were immunized with irradiated RMA-S or
RMA-S/DC fusion preparation. Spleens from each group of mice were
harvested and stimulated in vitro with irradiated FBL-3 tumor. T cells
derived from the mixed tumor-lymphocyte cultures were tested for
cytotoxicity against FBL-3, RMA-S, EL-4, or LSTRA tumors. The results
showed that cultured lymphocytes from mice vaccinated with RMA-S had no
detectable cytotoxicity against FBL-3, RMA-S, EL-4, or LSTRA (Fig. 3B). By contrast, lymphocytes from mice vaccinated with
RMA-S/DC elicited CTLs that specifically lysed FBL-3, but not RMA-S,
EL-4, or LSTRA (Fig. 3C). The results demonstrated the
ability of RMA-S/DC fusion hybrids to process and present intracellular
Gag proteins derived from parental tumor to T cells.
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A). By contrast, cultured
lymphocytes from mice vaccinated with B16/DC specifically lysed B16,
but not EL-4, MCA 207, or LSTRA (Fig. 4B). A higher level of
cytolytic activity against B16.B7.1 was observed.
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To determine whether B16/DC-fused cells could induce host
protective immunity against B16 tumor, groups of naive B6 mice were
s.c. immunized twice with irradiated B16, B16/DC, B16 mixed with DCs,
or HBSS as control. Ten days after the final immunization, mice were
s.c. injected with varying doses of B16 tumor and monitored for tumor
growth and survival time. The results (Fig. 5) showed that mice immunized with B16 or
HBSS were all tumor positive in 2 wk and died within 45 days after
challenge with B16 tumor (104 cells/mouse). Mice immunized
with B16/DC demonstrated a significant delay in tumor development
(22.3 ± 5.2 days) and reduced tumor incidence, with 40% of the
mice (4 of 10) free of tumor. In mice vaccinated with B16 mixed with
DCs, 90% of the mice (9 of 10) developed tumor (17.7 ± 5.1 days)
and died within 45 days, with one mouse free of tumor. There was a
statistically significant difference between mice vaccinated with
irradiated B16 and B16/DC in tumor development
(p < 0.001) and survival time
(p < 0.05). However, when challenged with
higher doses (105 or 106 cells/mouse) of B16,
mice vaccinated with B16/DC all developed tumor and died within 36 days
similar to mice of other vaccination groups (data not shown). These
results demonstrated that vaccination with B16/DC fusion preparations
induced partial protective immunity against low dose B16 tumor
challenge.
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In a variety of animal tumor models, studies have shown that LNs
draining tumor or the vaccination site are an excellent source for
obtaining in vivo primed T cells for adoptive immunotherapy (35, 36).
LNs draining the vaccination site contain a much higher frequency of
Ag-primed T cells than spleen or peripheral blood (37). B6 mice were
s.c. immunized on the flanks with irradiated B16, B16/DC, or B16 mixed
with DCs. Draining inguinal LNs from each group of mice were harvested.
In a normal control B6 mouse, the inguinal LNs contain approximately
2.2 x 106 cells/LN. Vaccination with irradiated B16
or B16 mixed with DCs, or B16/DC-induced hypertrophy of the inguinal
LNs with total cell number increased 3.3-, 6.1-, and 7.5-fold,
respectively (Table I). The average LN
cell yield was much higher in mice vaccinated with B16 mixed with DCs
or B16/DC groups. Similar results were obtained from mice vaccinated
with irradiated FBL-3, RMA-S, RMA-S mixed with DCs, or RMAS/DC fusions.
The total fresh cell yield of inguinal LNs was increased 3.7-, 4.4-,
12.4-, and 12.8-fold, respectively (Table I). In vitro stimulation of
the LN T cells derived from each vaccination group with anti-CD3
mAb plus low dose IL-2 expanded the cell number 2- to 3-fold in 5 days.
The LN T cells were greatly enriched in the 5-day short term culture
from initially 35–40% Thy-1.2+ cells to a purity of
99.8% Thy-1.2+ T cells (65% CD8+, 30%
CD4+).
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A)
showed that without therapy all mice developed >250 lung metastases on
day 21. Adoptive transfer of LN T cells derived from normal B6 mice or
mice vaccinated with irradiated B16 or with B16 mixed with DCs had no
therapeutic effect on established pulmonary metastases
(p > 0.05). All tumor-bearing mice in these
treatment groups developed >200 lung metastases on day 21. By
contrast, a significant reduction of pulmonary metastases was achieved
in mice treated with LN T cells derived from mice vaccinated with
B16/DC. The mean number of lung metastatic nodules per mouse (33.1
± 12.2) was significantly less than that in the other treatment groups
(p < 0.01). In a separate experiment, B6 mice
bearing 3-day pulmonary metastases were treated by adoptive transfer of
cultured draining LN T cells plus i.p. administration of IL-2. The
results (Fig. 6B) showed that mice that received no therapy
or that were treated with IL-2 alone developed >250 lung metastases on
day 21. Adoptive transfer of cultured LN T cells from mice vaccinated
with B16 mixed with DCs plus in vivo administration of IL-2 had no
significant therapeutic effect on established pulmonary metastases
(p > 0.05). However, a dramatic reduction of
pulmonary metastases was achieved in mice treated with LN T cells
derived from mice vaccinated with B16/DC. The mean number of lung
metastatic nodules per mouse (4.8 ± 3.5) was significantly less
than that in the other treatment groups (p <
0.01). The specificity of therapeutic efficacy of the cultured draining
LN T cells derived from mice vaccinated with B16/DC was validated by
adoptive transfer of cultured LN T from mice vaccinated with irradiated
B16, B16/DC, or B16 mixed with DCs into B6 mice with disseminated FBL-3
leukemia. No significant therapeutic effect was observed in mice
treated with CY plus adoptive transfer of cultured LN T cells from mice
vaccinated with irradiated B16, B16/DC, or B16 mixed with DCs compared
with that in mice treated with CY alone (p >
0.05; data not shown).
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A). By contrast, treatment
with CY plus adoptive transfer of cultured LN T cells from mice
vaccinated with irradiated RMA-S/DC was effective and cured 80–100%
of FBL-3 tumor-bearing mice (Fig. 7, B and C). No
significant therapeutic effects were observed in mice treated with CY
plus cultured LN T cells primed with irradiated RMA-S mixed with DCs
(Fig. 7B) or cultured LN T cells primed with B16/DC (Fig. 7C) compared with those in mice treated with CY alone
(p > 0.05). These results demonstrated that
vaccination of B6 mice with B16/DC or RMA-S/DC elicited T cells with
therapeutic effectiveness against established B16 and FBL-3 tumor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the past several years the methods for isolation and in vitro
culture of DCs from mouse and human have been well developed. DC
precursors obtained from blood, bone marrow, or lymphoid organs can
mature and grow in the presence of GM-CSF, IL-4, and/or TNF-
to
large numbers for prolonged periods of time, with maintenance of Ag
processing and presenting functions (29, 39, 40, 41, 42, 43, 44, 45). Clinically, DCs can
be generated from blood or BM of cancer patients, and tumor cells can
be obtained from biopsy samples or surgically removed tumor mass. Thus,
immunization with tumor/DC fused cells as vaccines is potentially
applicable for human cancer treatment. Compared with other currently
used strategies for developing DC-based vaccines such as loading DCs
with tumor antigenic proteins or peptides, fusion of DCs with tumor
cells have several advantages. First, the hybrid cells have the
potential for allowing a nature processing antigenic tumor protein to
be present in MHC-peptide complexes on the cell surface. Second,
vaccination with tumor/DC hybrids may allow relatively stable,
persistent Ag production and presentation in vivo in contrast to
peptide-loaded DCs. Third, the fusion of tumor cell with DCs expressing
the entire repertoire of tumor Ags should allow immunization of host
with multiple tumor Ags without knowing the identity of the tumor Ags.
An optimal antitumor response may require the involvement of polyclonal
effector subpopulations directed to a broad range of tumor epitopes
rather than a response restricted to a single tumor determinant.
Immunization with multiple tumor Ags should be more effective than that
with a single epitope. Vaccination with DCs pulsed with unfractionated
peptides eluted from tumor cells (46) or tumor-derived mRNA (47) may
also offer the advantage of providing multiple Ags to the immune
system. However, immunization with tumor/DC fusion preparations
represents a much simpler and straightforward strategy to elicit T
cell-mediated antitumor immunity.
Host T cell-mediated antitumor immunity is known to involve both CD4+ and CD8+ subsets in MHC class I- and class II-restricted responses, respectively (48). Our results showed that fusion of DCs with poorly immunogenic tumors could generate hybrid cells that expressed tumor-derived Ags and were positive for MHC class I, MHC class II, as well as costimulatory molecules essential for the activation of T cells. Fused RMA-S/DC cells were sensitive to lysis by CTL, thus confirming the capability of the tumor/DC fused cells to process and present tumor-associated proteins to T cells. Studies by Öhlén et al. (49) reported that fusion of RMA-S with an allogeneic fibroblast line (A9, KkDk/ß2 ma) generated RMA-S/A9 fusion hybrids that were capable of processing and presenting influenza virus nucleoprotein to Db-restricted influenza-specific CTLs after infection with recombinant influenza virus. Our results showed that FMR-MuLV gag-specific CTLs recognized and lysed RMA-S/DC fused cells, but not RMA-S or RMA-S mixed with DCs. The capability of the tumor/DC fused cells to process and present tumor-associated proteins to T cells was also supported by the finding that vaccination of B6 mice with RMA-S/DC or B16/DC fusion preparations elicited detectable specific CTL activities against FBL-3 or B16 tumor cells, respectively. The CTL generated lysed B16.B7.1 more effectively than B16 cells. It is possible that B7 expression enhanced the sensitivity of target cells to lysis. There is also a possibility that tumor/DC fusion hybrids could express a broader range of antigenic epitopes and allow the activation of polyclonal effector subpopulations of T cells. Recently, such a mechanism was demonstrated using genetic modification of tumor cells with the B7 gene to elicit CTLs against otherwise silent subdominant tumor Ags (50). Resent studies have identified at least two antigenic peptides derived from mouse tyrosinase-related protein-2 (51) and mouse gp100 (N. P. Restifo, unpublished observation) as CTL epitopes of B16 tumor cells. Although not defined, B16 cells may carry MHC class II-restricted T cell epitopes that are capable of inducing CD4+ T cell-mediated antitumor immunity. In a recent study our results demonstrated that vaccination of B6 mice with B16/DC fused cells elicited both tumor-reactive CD4+ and CD8+ T lymphocytes and that both T cell subsets were essential for the therapeutic antitumor effect.4
Efficient separation of the tumor/DC hybrids from parental tumor cells
in the fusion preparations remains a technical challenge. Without
selection, the unfused parental tumor cells almost invariably overgrow
the fusion hybrid in several days. A potential approach is drug
selection of fused cells. In Gong’s report, the fused cells were
selected in hypoxanthine-aminopterin-thymidine (HAT)-containing medium,
as the parental MC38/MUC1 and MC38 tumor cells were HAT sensitive but
the fused cells were not (26). However, this method may not be
applicable to most tumor cell lines because it would require
establishment of an autologous HAT-sensitive tumor line from individual
patients for fusion. In our study immunization with preparations
containing about 20% fused cells proved effective in eliciting
tumor-reactive T cells and host protective antitumor immunity.
Apparently, the contaminating unfused cells do not impede the
immunogenicity of the tumor/DC fused cells, so that selection may not
be a clinically relevant problem. The host protective antitumor
immunity induced by vaccination with tumor/DC fusion could be improved
by repeated administration and alternative immunization strategies to
elicit more effective responses. Indeed, vaccination with purified
B16/DC fused cells separated by using an anti-Ia mAb-mediated
MiniMACS separation column (Miltenyi Biotec, GmbH; Germany) as
vaccine has been shown to induce much stronger host protective immunity
against high dose B16 tumor challenge (106 cells/mouse)
with a greater percentage of tumor-free mice (see Footnote 4). In this
study we used BM DCs from 6-day culture in GM-CSF- plus
IL-4-supplemented medium. It is known that such relatively immature DCs
have more active Ag processing capability but are less effective in T
cell activation than the mature DCs. Whether fusion of tumor cells with
mature DCs (5- to 7-day GM-CSF plus IL-4 culture followed by treatment
with TNF-) could generate more potent tumor/DC vaccine is yet to be
determined.
One of the major reasons to use tumor/DC fused cells for vaccination is that the tumor/DC fusion hybrids can process and present endogenous tumor-associated proteins to elicit MHC class I-restricted tumor-reactive CTLs. The most recent study by Ashley et al. demonstrated that vaccination with DCs pulsed with unfractionated tumor extracts could induce specific CTLs and potent antitumor immunity (52). The relative potency of Ag-pulsed DCs compared with tumor/DC fusion cells in eliciting anti-tumor immunity remains unclear.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Wei Chen, The Cleveland Clinic Foundation, Research Building/FF50, 9500 Euclid Ave., Cleveland, OH 44195. E-mail address:
3 Abbreviations used in this paper: DCs, dendritic cells; F-, M-, and R-MuLV, Friend, Moloney, and Rauscher murine leukemia virus; LN, lymph node; CM, complete medium; BM, bone marrow; GM-CSF, granulocyte-macrophage CSF; PEG, polyethylene glycol; PE, phycoerythrin; CY, cyclosphosphamide; HAT, hypoxanthine-aminopterin-thymidine.
4 X. Cao, W. Zhang, J. Wang, M. Zhang, X. Huang, H. Hamada, and W. Chen. Therapy of established tumor with a hybrid cellular vaccine generated by using GM-CSF genetically modified dendritic cells. Submitted for publication.
Received for publication February 13, 1998. Accepted for publication July 8, 1998.
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