Download in MUC1-Transgenic Mice Cells CD8 T Cells by Dendritic/Tumor

The Kinetics of In Vivo Priming of CD4 and
CD8 T Cells by Dendritic/Tumor Fusion Cells
in MUC1-Transgenic Mice
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of June 17, 2017.
Shigeo Koido, Yasuhiro Tanaka, Dongshu Chen, Donald
Kufe and Jianlin Gong
J Immunol 2002; 168:2111-2117; ;
doi: 10.4049/jimmunol.168.5.2111
http://www.jimmunol.org/content/168/5/2111
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References
The Kinetics of In Vivo Priming of CD4 and CD8 T Cells by
Dendritic/Tumor Fusion Cells in MUC1-Transgenic Mice1
Shigeo Koido,2* Yasuhiro Tanaka,2* Dongshu Chen,* Donald Kufe,* and Jianlin Gong3*†
Previous work has demonstrated that dendritic/tumor fusion cells induce potent antitumor immune responses in vivo and in vitro.
However, little is known about the migration and homing of fusion cells after s.c. injection or the kinetics of CD4ⴙ and CD8ⴙ T
cell activation. In the present study, fluorescence-labeled dendritic/MUC1-positive tumor fusion cells (FC/MUC1) were injected s.c.
into MUC1-transgenic mice. The FC/MUC1 migrated to draining lymph nodes and were closely associated with T cells in a pattern
comparable with that of unfused dendritic cells. Immunization of MUC1-transgenic mice with FC/MUC1 resulted in proliferation
of T cells and induced MUC1-specific CD8ⴙ CTL. Moreover, CD4ⴙ T cells activated by FC/MUC1 were multifunctional effectors
that produced IL-2, IFN-␥, IL-4, and IL-10. These findings indicate that both CD4ⴙ and CD8ⴙ T cells can be primed in vivo by
FC/MUC1 immunization. The Journal of Immunology, 2002, 168: 2111–2117.
*Dana-Farber Cancer Institute and †Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA 02115
Received for publication August 31, 2001. Accepted for publication December
20, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Cancer Institute Grant R01 CA87057-01, U.S.
Department of Defense Breast Cancer Research programs Grant 990344, and Susan
G. Komen Breast Cancer Foundation Grant 9825.
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: [email protected]
4
Abbreviations used in this paper: DC, dendritic cell; FC/MUC1, dendritic/MUC1positive 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.
Copyright © 2002 by The American Association of Immunologists
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.
Materials and Methods
MUC1.Tg mice
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 ␮g/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,
0022-1767/02/$02.00
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T
here are two different pathways for Ag presentation (1).
Endogenously synthesized proteins, such as that in viral
infections, are processed and presented through the MHC
class I-restricted pathway to CTL (2). In contrast, exogenous proteins from the extracellular environment are processed and displayed in association with class II molecules and recognized by
CD4⫹ T cells (3). Certain exogenous Ags can also be presented to
CTL through cross-presentation (4 –12). Uptake of Ags by APCs is
required for cross-presentation and thereby activation of CD8⫹
CTL. Such cross-presentation of Ags captured by APC has been
shown to induce the priming of tumor-specific CTL (12, 13). Other
studies have demonstrated that cross-presentation of self-Ags induces the peripheral deletion of autoreactive CD8⫹ T cells (14,
15). These results indicate that cross-presentation of Ags can result
in both priming and tolerance of CTL (7). In either direct- or crosspresentation of Ag, dendritic cells (DC),4 a potent APC, play a
central role (3, 7, 16 –19). DC or Langerhans cells in the epidermis
are thought to capture exogenous Ags, migrate into draining lymph
nodes (DLN), and reside in the T cell parafollicular and paracortical zones. The initial priming of T cells takes place in the DLN
(9, 20).
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
2112
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
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 (1452C11), 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 PEconjugated 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 ␮m) 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).
51
Cr cytotoxicity assays
MC38, MC38/MUC1, and MCF7 targets were labeled with 51Cr for 60 min
at 37°C. The 51Cr-labeled cells (1 ⫻ 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)] ⫻ 100.
RT-PCR detection
RNA from 1 ⫻ 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⬘-GAGTCAAATCCAGAA
CATGCC-3⬘); IFN-␥ (5⬘-CATTGAAAGCCTAGAAAGTCTG-3⬘ and 5⬘CTCATGGAATGCATCCTTTTTCG-3⬘); IL-4 (5⬘-GAGATCATCGGC
ATTTTGAAC-3⬘ and 5⬘-GCTCTTTAGGCTTTCCAGGAAGTC-3⬘); IL-10
(5⬘-CTATGCTGCCTGCTCTTACTGA-3⬘ and 5⬘-TTCAGCAGACTCAAT
ACACACT-3⬘); ␤-actin (5⬘-TGTGATGGTGGGAATGGGTCAG-3⬘ and 5⬘TTTGATGTCACGCACGATTTCC-3⬘) (Stratagene, La Jolla, CA). PCRamplified products were analyzed on a 2% agarose gel.
Results
Kinetics of FC/MUC1 migration to DLN and close interaction
with T cells
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. 1A). 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.
Priming of naive T cells requires physical contact between APC
and T cells. To determine whether the fusion cells interact with T
cells after migration, FC/MUC1 were stained with mAb against
MHC class II and MUC1 Ag and subjected to cell sorting. The
purified FC/MUC1 were labeled with red-DiIC18(5) fluorescence
and injected into MUC1.Tg mice. The DLN were collected, sectioned, counterstained for FTIC-conjugated anti-CD4 or CD8 mAb
(green), and examined by fluorescence microscopy. Numerous red
DiIC18(5)-labeled FC/MUC1 were visible at 48 h postinjection in
the parafollicular and paracortical zones of the LN (Fig. 2, 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.
Proliferation of T-LNC in MUC1.Tg mice immunized with
FC/MUC1
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.
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To study the migration of fusion cells, DC were labeled with 1 ␮M (5-(and
6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR;
excitation/emission spectra, 540/566 nm) and MC38/MUC1 with 1 ␮M
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 ⫻ 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-␮m 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 ␮g/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 ⫻ 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).
T CELL PRIMING BY DC/TUMOR FUSION IMMUNIZATION
The Journal of Immunology
2113
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.
Induction of MUC1-specific CTL
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 MUC1specific 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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FIGURE 1. FC/MUC1 migration to regional LN. A, Phenotypes of DC, MC38/
MUC1, and FC/MUC1 were determined by bidimensional flow cytometry for dual
expression of MUC1 and MHC class II. B, DC
and MC38/MUC1 were labeled with 1 ␮M
CMTMR (orange) and CMFDA (green), respectively. The CMTMR-labeled DC and
CMFDA-labeled MC38/MUC1 cells were
fused in the presence of 50% PEG. Fused cells
(5 ⫻ 105) were injected s.c. into MUC1.Tg
mice in the posterior flank near the base of the
tail. DLN were collected at 24 h, frozen, and
sectioned. The slides were observed under fluorescence microscopy (magnification, ⫻40).
C, The labeled cells in DLN of MUC1.Tg mice
immunized with FC/MUC1 (n ⫽ 4, F), MC38/
MUC1 (n ⫽ 3, ‚), and DC (n ⫽ 3, E) 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.
2114
T CELL PRIMING BY DC/TUMOR FUSION IMMUNIZATION
Cytokine production by CD4 and CD8 effectors
Development of an effective T cell response requires interactions
via cytokines among APC, CD4⫹, and CD8⫹ T cells (20). To
FIGURE 3. Induction of proliferative immune response in MUC1.Tg mice immunized with FC/MUC1.
MUC1.Tg mice were immunized twice in the posterior
flank near the base of the tail with 5 ⫻ 105 DC, irradiated MC38/MUC1, and FC/MUC1 cells on days 0
and 7. PBS injection was used as a control. A, Inguinal
LNC were harvested on day 14 after immunization. B,
T-LNC were isolated on days 7 and 14 and analyzed
for CD3⫹ (f), CD4⫹ (p), and CD8⫹ (z, 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.
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,
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FIGURE 2. Interaction of FC/MUC1 with T cells in the regional LN. A, LN section was stained with H&E. Magnification, ⫻20. B and C, The FC/MUC1
cells were selected by immunofluorescence cell sorting with FITC-MUC1 and PE-MHC class II Ab staining and then labeled with 1 ␮g/ml DiIC18(5)
fluorochrome. The DiIC18(5)-labeled FC/MUC1 cells (5 ⫻ 105) were injected s.c. into mice at the posterior flank near the base of the tail. The inguinal
LN were collected 48 h postinjection, frozen, and sectioned. The cryosections were stained with FITC-conjugated CD4 (B) and CD8 (C) mAb and viewed
under fluorescence microscopy (magnification, ⫻20). D and E, Sections of DLN from mice treated with DC (D) or FC/MUC1 (E) were stained with mAb
DF3/MUC1 (anti-MUC1) at 48 h postinjection. MUC1-positive cells were observed in the T cell area of DLN from mice immunized with FC/MUC1 cells
(magnification, ⫻20). B, B cell area. Similar results were obtained in repeated experiments.
The Journal of Immunology
2115
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).
To assess the kinetics of FC/MUC1 immunization on T cell
activation, LNC were isolated from mice immunized with FC/
MUC1, MC38/MUC1, or PBS 7 days after the first and second
vaccinations. The isolated LNC were selected into CD4⫹ and
CD8⫹ subsets by immunofluorescence cell sorting. IL-2 and
IFN-␥ 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. 5C). 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
In our previous work, immunization with DC/tumor fusion cells
reversed the unresponsiveness of T cells to tumor Ag and induced
specific antitumor immunity. In this study, we have analyzed the
kinetic effects of fusion cells in priming naive T cells in vivo. The
fusion cells, like DC, migrated into regional LN. FC/MUC1 were
visible in the DLN as early as 18 h after s.c. injection as demonstrated by fluorescence labeling. Importantly, the fusion cells localized to the T cell area and formed clusters with CD4⫹ and
CD8⫹ T cells in the LN. In concert with these findings, the FC/
MUC1 induced Ag-specific CD4⫹ and CD8⫹ T cells. The fusion
cells, like DC, express MHC class I and II and costimulatory molecules and, unlike DC, tumor Ags (24), and thus are well equipped
Table I. Percentage of CTL activity from FC/MUC1-immunized MUC1.Tg mice
CD8⫹ T-LNCa
a
b
CD8⫹ T Splenocytesa
Expt.
E:T ratio
MC38/MUC1
MC38
MCF7
E:T ratio
MC38/MUC1
MC38
MCF7
1
30:1
10:1
3:1
1:1
45.4
38.77
30.51
17.07
30.06
25.62
20.9
19.3
12.77
10.13
9.42
5.66
50:1
17:1
6:1
2:1
68.4
56.3
31.71
28.97
50.11
45.2
24.89
21.3
10.77
9.53
7.66
7.1
2
60:1
20:1
6:1
2:1
31.6
16.38
13.14
8.09
26.70
16.83
12.66
6.84
NDb
ND
ND
ND
60:1
20:1
6:1
2:1
31.19
23.23
10.75
5.54
16.90
11.95
2.68
5.83
ND
ND
ND
ND
3
60:1
20:1
6:1
2:1
52.12
23.37
6.95
8.47
27.6
14.75
4.32
0.0
2.9
0.0
0.0
0.0
60:1
20:1
6:1
2:1
46.34
29.45
9.27
5.76
36.39
22.23
0.0
2.08
2.66
0.0
0.0
0.0
CD8⫹ T-LNC and CD8⫹ T splenocytes from MUC1. Tg mice immunized with FC/MUC1.
ND, Not done.
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FIGURE 4. Induction of MUC1-specific CTL by
FC/MUC1 immunization. MUC1.Tg mice (n ⫽
6/group) were immunized twice in the posterior flank
near the base of the tail with PBS, 5 ⫻ 105 irradiated
MC38/MUC1, or 5 ⫻ 105 FC/MUC1. A, LN from all
six mice were collected on day 7 after the second immunization. The CD8⫹ T-LNC were isolated and purified with fluorescent cell sorting. B, CD8⫹ splenocytes
were isolated on day 7 after the second immunization.
The CD8⫹ LNC and splenocytes were incubated at the
indicated E:T ratio with MC38 (E), MC38/MUC1 (F),
and MCF7 (䡺) 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.
2116
T CELL PRIMING BY DC/TUMOR FUSION IMMUNIZATION
FIGURE 5. Cytokine synthesis in activated CD4⫹
and CD8⫹ T cells by FC/MUC1 immunization.
MUC1.Tg mice were immunized twice s. c. with PBS,
5 ⫻ 105 irradiated MC38/MUC1, or 5 ⫻ 105 FC/
MUC1. A, T-LNC from MUC1.Tg mice immunized
with PBS, irradiated MC38/MUC1, or FC/MUC1
were isolated and purified through nylon wool. B, TLNC were sorted into CD4⫹ or CD8⫹ T subsets with
FITC-conjugated CD4 and PE-conjugated CD8 mAb.
C, T-LNC were isolated on day 7 after the first and
second immunization and sorted into CD4⫹ and
CD8⫹ subsets. IL-2, IFN-␥, IL-4, and IL-10 cytokine
mRNA synthesis were determined by RT-PCR
analysis.
cytokines, dose of Ag, and antigenic stimulation via TCR (35, 36).
Traditionally, Th1 have been thought to be associated with cellmediated 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– 42). The heterogeneity of cytokine profiles has been extensively demonstrated (43– 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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