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Dana-Farber Cancer Institute and
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4, and IL-10. These findings
indicate that both CD4+ and CD8+ T cells can be
primed in vivo by FC/MUC1 immunization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MUC1, a carcinoma-associated Ag, is a high molecular weight glycoprotein overexpressed in human breast, pancreatic, and other carcinomas (21). The MUC1-transgenic (MUC1.Tg) mouse model that express MUC1 is unresponsive to MUC1 Ag (22, 23). We demonstrated in our previous studies that antitumor immunity can be augmented using hybrid cells created by fusion of DC with MUC1-positive carcinoma cells (FC/MUC1). These fusion cells were effective in inducing immunity against MUC1 Ag and rejected established tumor metastases (24). The CTL induced by immunization with FC/MUC1 reversed unresponsiveness of T cells to MUC1 in MUC1.Tg and rejected MUC1-positive pulmonary metastases (25). Recently, vaccination of fusion DC with renal carcinoma cells has been reported to be effective in the treatment of patients with metastatic kidney cancer (26).
In this study, the kinetics of migration and homing of FC/MUC1 fusion cells in MUC1.Tg mice was evaluated. Our results demonstrate that s.c. injected fluorescence-labeled FC/MUC1 cells migrate to regional lymph nodes, reside in the T cell area, and function as APC. Moreover, immunization with FC/MUC1 cells was associated with reversal of T cell unresponsiveness to MUC1 and activation of MUC1-Ag-specific CD4+ and CD8+ T cells. Our data suggest that CD4+ T cells play a central role in modulation of effector function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The C57BL/6 mouse strain transgenic to human MUC1 was established as described previously (22). The MUC1.Tg mice express MUC1 at the apical surfaces of the epithelium lining the bronchi, mammary gland, pancreas (acinar cells), kidney (distal convoluted tubules and collecting ducts), gallbladder, salivary glands, stomach, and uterus at a level similar to that found in humans (22). PCR was performed to identify routinely MUC1.Tg-positive mice in the colony. The mice were maintained in microisolator cages under specific pathogen-free conditions. Age- and sex-matched mice were used for the experiments.
Cell culture and DC/tumor fusion
The murine MC38 carcinoma (C57BL/6) cell line stably expressing a MUC1 cDNA (MC38/MUC1) (27, 28) and MCF7 human breast carcinoma cells (MUC1 positive; American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. DC isolated from the bone marrow of wild-type C57BL/6 mice have been described previously (29). DC were cultured in 20 ng/ml recombinant murine GM-CSF (Sigma-Aldrich, St. Louis, MO) medium for 5 days. The purified DC were fused to MC38/MUC1 carcinoma cells in the presence of 50% polyethylene glycol (PEG; Sigma-Aldrich) (24). Briefly, DC and MC38/MUC1 cells were collected, washed twice in serum-free medium, and counted. DC were mixed with MC38/MUC1 cells at a 10:1 ratio. The fusion process was conducted with 50% PEG in prewarmed Dulbecco’s PBS without Ca2+ or Mg2+ at pH 7.4. After washing twice, the fused cells were plated in 24-well culture plates for 5 days. Then the cells were plated in six-well culture plates in complete RPMI 1640 medium supplemented with 20 ng/ml recombinant murine GM-CSF (Sigma-Aldrich). By day 5 of culture, the unfused tumor cells had become firmly attached to the tissue culture flask, whereas the fused cells could be dislodged by gentle pipetting. The latter were then collected and analyzed by flow cytometry for Ag expression.
Cell labeling and immunization
To study the migration of fusion cells, DC were labeled with 1 µM (5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR; excitation/emission spectra, 540/566 nm) and MC38/MUC1 with 1 µM 5-chloromethylfluorescein diacetate (CMFDA, 492/516 nm; Molecular Probes, Eugene, OR) by incubation for 30 min at room temperature, respectively. The labeled cells were washed extensively in PBS and fused in the presence of 50% PEG. The fusion cells (5 x 105) were injected s.c. in the posterior flank near the base of the tail of MUC1.Tg mice. To quantitate migration of the labeled cells, DLN from mice immunized with DC, MC38/MUC1, or FC/MUC1 cells were collected at varying time points. Frozen sections, 4-µm thick, were cut. Four slides per lymph node (LN) were enumerated by touch counting (30) under fluorescence microscopy. The average number of labeled cells for each LN was calculated with the total labeled cells divided by the counted area of LN (millimeter).
To visualize the interaction of fusion cells with T cells in the LN, FC/MUC1 cells were first sorted with FITC-conjugated anti-MUC1 (HMPV; BD PharMingen, San Diego, CA) and PE-conjugated anti-MHC class II (M5/114.15.2; BD PharMingen) mAb. The sorted FC/MUC1 were washed and then incubated with 1 µg/ml 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiIC18(5), excitation and emission spectra, 644 nm/663 nm; Molecular Probes) for 30 min at 37°C. The DiIC18(5)-labeled FC/MUC1 cells (5 x 105) were injected s.c. into MUC1.Tg mice. The inguinal LN were collected and sectioned at various time points. The frozen sections were stained with FITC-conjugated anti-CD4 (H129.19) and anti-CD8 mAb (53-6.7; BD PharMingen).
Flow cytometry and T cells sorting
Inguinal lymph node cells (LNC) after two immunizations were teased and suspended in medium. T-LNC were purified by passage through nylon wool and analyzed by staining with FITC-conjugated mAb, CD3 (145-2C11), CD4 (H129.19), and CD8 (53-6.7; BD PharMingen) for 30 min on ice. The cells were washed, fixed, and analyzed by FACScan (BD Biosciences, Bedford, MA) with CellQuest analysis software. For cell sorting, T-LNC were stained with FITC-conjugated anti-CD4 (H129.19) and PE-conjugated anti-CD8 (53-6.7) mAb and then sorted into CD4+ and CD8+ T cell subsets in separate tubes by MoFlo (Cytomation, Fort Collins, CO) with Summit version 3.0 analysis software.
Immunohistochemistry staining
Immediately after their removal, regional LN were frozen in liquid nitrogen with OCT freezing medium (Tissue-Tek, OT Embedding Medium; Sakura Finetek, Torrance, CA). Tissue sections (5 µm) were prepared in a cryostat and fixed in acetone for 10 min. Sections were incubated with mAb DF3 (anti-MUC1) for 30 min at room temperature and then subjected to indirect immunoperoxidase staining using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA).
51Cr cytotoxicity assays
MC38, MC38/MUC1, and MCF7 targets were labeled with 51Cr for 60 min at 37°C. The 51Cr-labeled cells (1 x 104) were added to 96-well V-bottom plates and incubated with various ratios of CD8+ LNC or splenocytes for 5 h at 37°C. The supernatants were collected and assayed in a gamma counter for 51Cr release. Spontaneous release of 51Cr was assessed by incubation of targets in the absence of effectors. Maximum or total 51Cr release was determined by incubation of targets in 0.1% Triton X-100. The percentage of specific 51Cr release was determined by the following equation: Percent specific release = [(experimental - spontaneous)/(maximum - spontaneous)] x 100.
RT-PCR detection
RNA from 1 x 106 T-LNC, sorted CD4,
or CD8 T cells was extracted by TRIzol reagent (Life Technologies,
Rockville, MD). Total RNA to cDNA was reverse transcribed using a
poly(dT) oligonucleotide and SuperScript (Life Technologies).
Semiquantitative PCR was performed by amplifying cDNA with the
following oligonucleotide primers (31, 32): murine IL-2
(5'-TCCACTTCAAGCTCTACAG-3' and 5'-GAGTCAAATCCAGAACATGCC-3');
IFN- (5'-CATTGAAAGCCTAGAAAGTCTG-3' and
5'-CTCATGGAATGCATCCTTTTTCG-3'); IL-4
(5'-GAGATCATCGGCATTTTGAAC-3' and
5'-GCTCTTTAGGCTTTCCAGGAAGTC-3'); IL-10 (5'-CTATGCTGCCTGCTCTTACTGA-3'
and 5'-TTCAGCAGACTCAATACACACT-3'); 
-actin
(5'-TGTGATGGTGGGAATGGGTCAG-3' and 5'- TTTGATGTCACGCACGATTTCC-3')
(Stratagene, La Jolla, CA). PCR-amplified products were analyzed on
a 2% agarose gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MC38/MUC1 carcinoma cells were fused with syngeneic DC. The fused
cells (FC/MUC1) were demonstrated to have dual expression of MUC1 and
MHC class II or costimulatory molecules by flow cytometry. FC/MUC1 and
MC38/MUC1, but not DC, expressed MUC1 (Fig. 1
A). FC/MUC1 also expressed
MHC class II (Fig. 1A) at a level comparable to that found
on DC. To directly visualize the migration of FC/MUC1 to the DLN, DC
were labeled with fluorescent cell tag CMTMR (orange) and MC38/MUC1
tumor cells with CMFDA (green). The labeled cells were then fused in
the presence of 50% PEG. The dual-labeled fusion cells (FC/MUC1), as
well as single-labeled DC and tumor cells, were injected into MUC1.Tg
mice in the flank near the base of the tail. Under the fluorescence
microscopy, DC with veiled morphology were visualized as orange (Fig. 1B, left panel) in the T cell zone of DLN. In the
same field adjacent to the DC, a FC/MUC1 cell was located by dual
labeling with orange and green (Figs. 1B, left
and right panels). The FC/MUC1 cells were observed in DLN as
early as 18 h after injection; their numbers peaked 24–48 h
postinjection and then gradually decreased after 96 h (Fig. 1C). These results indicate that FC/MUC1 fusion cells, like
DC, are able to migrate into DLN after s.c. injection. In contrast,
green-labeled MC38/MUC1 tumor cells were not detectable in the DLN
after s.c. injection (Fig. 1C). Collectively, these
observations suggest that FC/MUC1 migrate to DLN.
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, A–C). Red-labeled
FC/MUC1 cells were surrounded by green-stained CD4 (Fig. 2B)
and CD8 (Fig. 2C) T cells, forming clusters of cells. These
clusters were visualized as yellow. Sections of LN were stained with
anti-MUC1 Ab and examined for the presence of MUC1-positive fusion
cells. The MUC1-expressing cells were detected in the T cell area of LN
from mice immunized with FC/MUC1 (Fig. 2E), but not in those
of mice immunized with either DC (Fig. 2D) or MC38/MUC1
cells (data not shown). These findings demonstrate that the FC/MUC1
cells which migrate to DLN form clusters with T cells.
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Immune response is manifested by the increasing size of DLN and
the proliferation of LNC. In the following experiments, we studied the
effect of FC/MUC1 immunization on LNC. MUC1.Tg mice were immunized
twice with DC, FC/MUC1, or irradiated MC38/MUC1 cells by s.c. injection
in the flank near the base of the tail. Draining inguinal LN were
removed 7 days after each immunization. After the first immunization
with FC/MUC1, the size of DLN increased slightly compared with DLN from
mice immunized with PBS, DC, or irradiated MC38/MUC1 cells (data not
shown). After the second immunization, however, the DLN from
FC/MUC1-immunized mice were substantially larger (Fig. 3A). By contrast, there was
little increase in the size of DLN from mice immunized with PBS, DC, or
irradiated MC38/MUC1 cells (Fig. 3A). In a parallel study,
T-LNC were isolated and stained with anti-CD3 and CD4 or CD8 mAb to
quantitate the number of T cells. The number of T cells positive for
CD3 and CD4 or CD8 increased significantly on day 7 after the first
immunization with FC/MUC1. The number of positive T cells doubled after
the second injection of FC/MUC1 cells compared with the number of T
cells from mice immunized with PBS, DC, or MC38/MUC1 cells (Fig. 3B). These findings indicate that immunization with FC/MUC1
results in significant proliferation of T-LNC in vivo.
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To determine whether T cell proliferation was associated with T
cell activation, we studied the induction of Ag-specific CTL and
cytokine profiles of the activated T cells. MUC1.Tg mice were immunized
twice with FC/MUC1, irradiated MC38/MUC1 tumor cells, or PBS.
CD8+ T cells from DLN were selected by cell
sorting, cocultured with MC38, MC38/MUC1, or MCF7 targets, and examined
by the 51Cr release assay. Strong CTL activity
against MC38 (30%) or MC38/MUC1 (45%) (Fig. 4A and Table I) was demonstrated using
CD8+ T cells from mice immunized with FC/MUC1. In
contrast, there was no lysis of targets by CD8+ T
cells from mice immunized with irradiated MC38/MUC1 tumor cells or PBS
(Fig. 4A). To determine whether the induction of
MUC1-specific CTL is confined to DLN or found throughout the lymphoid
system, we examined the CTL activity of immunized splenocytes with
51Cr release assay. CD8+
splenocytes isolated from MUC1.Tg mice immunized with FC/MUC1, but not
those immunized with irradiated MC38/MUC1, exhibited strong CTL
activity against murine MUC1-positive targets (Fig. 4B and
Table I). The finding that there is CTL activity against MC38 tumor
cells supports the induction of polyclonal CTL by FC/MUC1 immunization
against known and unknown tumor Ags. Our results also indicate that the
CTL activity is MHC class I restricted as demonstrated by the lack of
lysis of MCF7 targets. These results are consistent with our previous
studies that s.c. immunization of FC/MUC1 induces immune responses and
MUC1-specific CTL in DLN and other secondary lymphoid tissues
(33).
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Development of an effective T cell response requires
interactions via cytokines among APC, CD4+, and
CD8+ T cells (20). To define the
cytokine profile of activated LNC, we used the RT-PCR to assess the
cytokine mRNA levels of LNC isolated 7 days after the second
immunization. Increased RNA transcripts of IL-2, IFN-, IL-4, and
IL-10 were detected in LNC from mice immunized with FC/MUC1 compared
with LNC from mice immunized with irradiated MC38/MUC1 or PBS (Fig. 5A). We fractionated the LNC
from mice immunized with FC/MUC1 into CD4+ and
CD8+ subsets by cell sorting. Active
transcription of IL-2, IFN-, IL-4, and IL-10 was demonstrated in
sorted CD4+ T cells. In contrast, only IFN-
was detected in the CD8+ population (Fig. 5B).
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and, to a
lesser extent, IL-10 were detected in CD4+ T
cells 7 days after the first immunization with FC/MUC1, whereas IL-4
was barely detectable (Fig. 5
C). The synthesis of cytokines
increased 7 days after the second immunization, especially for IL-4 and
IL-10 (Fig. 5C). However, CD8+ T cell
populations from FC/MUC1 immunization detected only IFN- RNA
synthesis at 7 days after the first immunization, and this synthesis
increased after the second immunization (Fig. 5C).
Collectively, these results indicate that immunization with FC/MUC1
cells results in active synthesis of cytokines by
CD4+ LNC. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It could be argued that fusion cells only act as a carrier of tumor Ags, such that the visualized fluorescence-labeled cells in the DLN are actual host DC or Langerhans cells containing degraded fusion cells and that the induction of CTL is the result of cross-presentation of tumor Ag expressed by fusion cells and mediated by host DC. This possibility could exist, provided that host DC, which are tolerant to MUC1 Ag expressed by MC38/MUC1 tumor cells, reverse tolerance to MUC1 Ag that is processed and expressed by the fusion cells. In either case, the fusion cells play an important role in the activation of T cells and induction of Ag-specific CTL. Alternatively, both direct Ag presentation by fusion cells and cross-presentation by host DC may participate in the T cell activation. The exact role of host DC, in our model, is currently under investigation.
The molecular events surrounding Ag processing and presentation by fusion cells need to be further investigated. As previously demonstrated, fusion cells express MHC class I and II and costimulatory molecules as well as tumor Ags. Under electron microscopy, fusion cells were observed to actively express MUC1 Ag and MHC class I and II molecules (our unpublished data). Considering the kinetics and magnitude of antitumor immunity induced by fusion cells in vitro and in vivo, it is tempting to speculate that fusion cells are able to activate T cells in multiple pathways. The finding that fusion cells express tumor Ag and MHC class I and II molecules leads us to believe that fusion cells are able to process tumor-derived peptides and MHC class I peptides derived from DC. They form MHC class I-peptide complexes, in the endoplasmic reticulum, which are transported to the surface and presented to CD8+ T cells through the MHC class I-restricted pathway. Similarly, fusion cells can synthesize MHC class II peptides derived from DC in the endoplasmic reticulum, which are transported to the cytoplasm where MHC class II-peptide complexes are assembled with tumor-derived peptides. These complexes are presented to CD4+ T cells, which are involved in CTL induction, through the MHC class II pathway. Furthermore, it is possible that cross-presentation mediated by host APC can participate in CTL induction as long as the latter are responsive to Ags which are processed and expressed by fusion cells.
Upon activation, T cells differentiate into the type I subset
expressing IFN- or type 2 expressing IL-4 through regulation by
cytokines, dose of Ag, and antigenic stimulation via TCR (35, 36). Traditionally, Th1 have been thought to be
associated with cell-mediated immunity and Th2 to be related to humoral
immunity. However, both types of T cells have been shown to participate
in the antitumor immune response (37, 38, 39, 40, 41, 42). The
heterogeneity of cytokine profiles has been extensively demonstrated
(43, 44, 45). The diversity of cytokine production patterns
reflects the polyclonal populations of activated T cells rather than
the cytokine profile of an individual cell (43, 44). These
results are consistent with our findings that immunization with fusion
cells induces polyclonal T cell responses (24). It is
possible that mixed subsets of T cells in our model can be induced by
epitopes presented by fusion cells with different affinity and
intensity to TCR (46). In contrast to the
CD4+ T cells, CD8+ T cells
only express IFN-. The failure to demonstrate heterogeneity of the
cytokine profile, as found for CD4+ T cells,
indicates differential regulation of the cytokine production of
CD4+ and CD8+ T cells.
Alternatively, the Th1 and Th2 differentiation is a stochastic process
(34). Nonetheless, the production of both Th1 and Th2
cytokines by CD4+ T cells in the present study
indicates the important role played by CD4+ T
cells in the regulation of effector function.
In summary, s.c. injected fusion cells migrate to DLN. The migration of FC/MUC1 cells and close interaction with T cells are associated with activation of CD4+ T cells and induction of Ag-specific CTL.
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Footnotes |
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2 S.K. and Y.T. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jianlin Gong, Beth Israel Deaconess Medical Center, KS 135, 330 Brookline Avenue, Boston, MA 02215. E-mail address: jgong{at}caregroup.harvard.edu
4 Abbreviations used in this paper: DC, dendritic cell; FC/MUC1, dendritic/MUC1-positive tumor fusion cell; MUC1.Tg, MUC1-transgenic; DLN, draining lymph node; LN, lymph node; PEG, polyethylene glycol; CMTMR, (5-(and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; CMFDA, 5-cholormethylfluorescien diacetate; DiIC18(5), 1,1'-diactadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate; LNC, LN cell.
Received for publication August 31, 2001. Accepted for publication December 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and DC
(n = 3, 
) were enumerated by touch counting at
various time points. We sampled four slides for each LN and calculated
the average number of labeled cells in a 1-mm area. Each dot represents
the average number of labeled cells in one LN. Similar results were
obtained in three individual experiments.
), CD4+ (
), and
CD8+ (
, striped bars) expression by flow cytometry. The
numbers 1 and 2 on the x-axis represent the first and
second immunizations. Similar results were obtained in three individual
experiments.
) target cells. CTL activity was determined by the 51Cr
release assay and the data are presented as mean ± SD from
triplicates in one experiment. Similar results were obtained in three
individual experiments and are presented in Table I