|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 91-99
GENE THERAPY
Dendritic cells modified to express CD40 ligand elicit therapeutic
immunity against preexisting murine tumors
Toshiaki Kikuchi,
Malcolm A. S. Moore, and
Ronald
G. Crystal
From the Division of Pulmonary and Critical Care Medicine, Weill
Medical College of Cornell University New York Presbyterian Hospital;
James Ewing Laboratory of Developmental Hematopoiesis, Memorial
Sloan-Kettering Cancer Center; and Institute of Genetic Medicine,Weill
Medical College of Cornell University, New York, NY.
 |
Abstract |
CD40 ligand (CD40L) is essential for the initiation of
antigen-specific T-cell responses. This study is based on the
hypothesis that dendritic cells (DCs) genetically modified ex vivo to
express CD40L will enhance in vivo presentation of tumor antigen to the cellular immune system with consequent induction of antitumor immunity
to suppress tumor growth. To examine this concept, subcutaneous murine
tumors were injected with bone marrow-derived DCs that had been
modified in vitro with an adenovirus (Ad) vector expressing murine
CD40L (AdmCD40L). In B16 (H-2b, melanoma) and CT26
(H-2d, colon cancer) murine models, intratumoral injection
of 2 × 106 AdmCD40L-modified DCs (CD40L-DCs) to
established (day 8) subcutaneous tumors resulted in sustained tumor
regression and survival advantage. This antitumor effect was sustained
when the number of CD40L-DCs were reduced 10-fold to
2 × 105. Analysis of spleens from CD40L-DC-treated
animals demonstrated that CD40L-DCs injected into the subcutaneous CT26
flank tumors migrated to the spleen, resulting in activation of
immune-relevant processes. Consistent with this concept, intratumoral
administration of CD40L-DCs elicited tumor-specific cytotoxic
T-lymphocyte responses, and the transfer of spleen cells from
CD40L-DC-treated mice efficiently protected naive mice against a
subsequent tumor challenge. In a distant 2-tumor model of metastatic
disease, an untreated B16 tumor in the right flank regressed in
parallel with a left B16 tumor treated with direct injection of
CD40L-DCs. These results support the concept that genetic modification
of DCs with a recombinant CD40L adenovirus vector may be a useful
strategy for directly activating DCs for cancer immunotherapy.
(Blood. 2000;96:91-99)
© 2000 by The American Society of Hematology.
| |
Introduction |
Although many tumors have antigens recognizable by the
immune system, the ability of tumors to escape a functional immune system suggests that the immune mechanism is often insufficient to
effectively overtake the potential of many tumors to grow. In an
attempt to bolster antitumor immunity, this study focuses on a strategy
to enhance the ability of the adaptive immune system to recognize the
tumor cells as foreign by activating dendritic cells (DCs) by the
genetic strategy of modifying them to express CD40 ligand (CD40L), a
ligand normally found on the surface of activated CD4+ T
cells that play a central role in activating DCs as antigen-presenting cells (APCs).1-3
CD40L is a 33-kd type II membrane protein that is preferentially
expressed on activated CD4+ T cells.2,4-6 The
receptor for CD40L, CD40 (40 kd), is expressed on APCs, including DCs,
B cells, activated macrophages, and follicular DCs.2 DCs
are professional APCs specialized in sampling antigen throughout the
body, migrating to lymphoid organs, and presenting antigen to naive T
cells.1 Stimuli from CD4+ T-helper cells via
the CD40/CD40L interaction is essential in bringing the DCs to a state
in which they can autonomously trigger antigen-specific T-cell
responses.7-9 In this context, the CD40L on
antigen-stimulated CD4+ T-helper cells activates DCs, with
up-regulation of T-cell costimulatory molecules such as B7 and
intercellular adhesion molecule (ICAM)-1, and consequent direct
stimulation of CD8+ T-killer cells.10,11 As
part of the activation of DCs, the CD40L-CD40 interaction induces
the production of cytokines that favor the development of a
T-helper 1 (Th1) response.10-12
On the basis of the hypothesis that augmentation of APC activation will
augment antigen-specific immune responses against a tumor, and with the
knowledge that CD40L (normally expressed on activated CD4+
T cells) can trigger activation of APCs via the CD40 receptor on DCs,
we and others have used gene transfer of the coding sequence of CD40L
to tumor cells, leukemia cells, or fibroblasts, or intradermally as a
strategy to activate APCs.13-20 In this study, with the
knowledge that the triggering of CD40 with anti-CD40 antibodies on DCs
will substitute for the requirement of activated CD4+ T
cells in activating DCs,7-9 we have extended the concept of using gene transfer to activate the DCs directly by hypothesizing that,
by genetically modifying DCs to express CD40L, the DC would be able to
activate itself and thus enhance the function of DCs within a tumor to
augment antitumor immune responses. With this background, this study
evaluates the concept of suppressing the growth of preexisting tumors
by direct administration of syngenic bone marrow-derived DCs that have
been genetically modified with the CD40L coding sequence in vitro. To
accomplish this, we have used an E1 adenovirus (Ad)
gene vector (AdmCD40L) to transfer the murine CD40L complementary DNA
(cDNA) to DCs, and then injected the modified DCs into preexisting
tumors in syngenic hosts. The data demonstrate that direct
administration of AdmCD40L-modified DCs will elicit specific antitumor
immunity and inhibit the growth of preexisting tumors mediated by
tumor-specific systemic immune processes.
| |
Materials and methods |
Mice
Female C57Bl/6 (H-2b), Balb/c (H-2d) mice,
and CD4 knockout (CD4/) mice that had been
backcrossed to the C57Bl/6 background for 8 generations, 6 to 8 weeks
old, were purchased from the Jackson Laboratories (Bar Harbor, ME), and
housed under specific pathogen-free conditions.
Cell culture
CT26 is an undifferentiated colon adenocarcinoma cell line
(H-2d) originally derived by intrarectal injections of
N-nitroso-N-methylurethane in a female Balb/c mouse
(provided by N. P. Restifo, National Cancer Institute, Bethesda,
MD).21 The SVBalb fibroblast cell line is also syngenic to
Balb/c mice (provided by L. Gooding, Emory University, Atlanta,
GA).22 C3 is a cell line originally derived by transfecting
C57Bl/6 mouse embryonal fibroblasts (H-2b) with a plasmid
containing the entire genome of the human papilloma virus type 16 (provided by C. J. M. Melief, University Hospital Leiden, The
Netherlands).23 The CL7 fibroblast cell line
(H-2d), the B16 murine melanoma cell line
(H-2b), and Lewis lung carcinoma (LLC) cell line
(H-2b) were obtained from the American Type Culture
Collection (Manassas, VA). The CT26 and C3 cell lines were maintained
in complete RPMI-1640 media (10% fetal bovine serum, 2 mmol/L
L-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin). DCs
were generated from mouse bone marrow precursors in complete RPMI-1640
media with recombinant murine granulocyte-macrophage colony-stimulating
factor (GM-CSF) (100 U/mL; Sigma, St Louis, MO) and recombinant murine interleukin-4 (IL-4; 2 ng/mL; R & D Systems, Minneapolis, MN), as
described previously.24 By flow cytometric analysis, more than 70% of DCs generated in this way displayed the characteristic DC
surface markers CD11c and MHC class II, with less than 7%
contaminating T cells (not shown). All other cell lines were maintained
in complete DMEM media.
Adenovirus vectors
The replication-deficient adenovirus vectors used in this study are
based on human Ad5 genome with E1 and E3 deletions. The AdmCD40L vector
and the AdNull control vector express the murine CD40L cDNA and no
transgene, respectively, under the control of the cytomegalovirus (CMV)
early/immediate promoter/enhancer.18,25 Propagation,
purification, and titration of the adenovirus vectors were as
previously described.26,27
Flow cytometry
To quantify expression of surface molecules on DCs, DCs from Balb/c
mice were transduced with AdmCD40L or AdNull at a multiplicity of
infection (moi) 40 for 4 hours, and cultured at
2 × 106 or 2 × 105 cells/mL.
After incubation for 72 hours, 4 × 105 DCs were
stained for 30 minutes at 4°C with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAb) to CD86 (B7-2, GL1), CD80
(B7-1, 16-10A1), CD54 (ICAM-1, 3E2), CD48 (BCM-1, HM48-1), CD40
(HM40-3), or CD25 (IL-2 receptor  chain, 7D4), and phycoerythrin (PE)-conjugated anti-mCD40L mAb MR1 (Pharmingen, San Diego, CA). Isotype-matched antibodies served as FITC- or PE-conjugated controls (Pharmingen). Stained cells were analyzed in an Elite flow cytometer (Coulter, Hialeah, FL). For coexpression analysis, an FL1 (fluorescence 1, 530 nm for FITC)/FL2 (fluorescence 2, 585 nm for PE) (FL-1/FL-2) contour plot was used to calculate the percentage of positive cells.
Cytokines
To demonstrate the function of the AdmCD40L to confer to DCs the
ability to express functional mCD40L, DCs from Balb/c mice were
transduced with AdmCD40L, AdNull, or phosphate-buffered saline (PBS)
alone (ie, no transduction) at a moi 40 for 4 hours, and plated on
24-well plates at 5 × 106 cells/mL. Where
indicated, transduced DCs were incubated with anti-mCD40L mAb MR1 (10 µg/mL; Pharmingen) or the same amount of the control hamster IgG
(Pharmingen), or were placed at several concentrations,
5 × 106, 1 × 106,
5 × 105, and 1 × 105 cells/mL.
After incubation for 72 hours, 37°C, the supernatant (400 µL) was
harvested and centrifuged to remove debris. The levels of murine IL-12
or macrophage inflammatory protein 1 (MIP-1) released into the
culture medium were assessed by enzyme-linked immunosorbent assay
(ELISA), using the mouse IL-12 p40 or MIP-1 immunoassay (R & D
Systems), respectively.
Tumor therapy model
Tumor cells (5 × 105 B16 or
2 × 105 CT26) were injected subcutaneously in the
right flank of mice. On day 8, tumors of the mice were inoculated with
100 µL of DCs or CL7 cells, infected or mock-infected with the
AdmCD40L or AdNull vector (moi 40, 24 hours). The size of each tumor
was assessed 3 times weekly and recorded as the average tumor area
(mm2) ± standard error by measuring the
largest perpendicular diameters. When animals became moribund or the
tumors reached 15 mm in diameter, the mice were killed and this was
recorded as the date of death for survival studies. For some studies,
where indicated, mice were challenged in both flanks with tumor cells:
5 × 105 B16 in the right flank, and
5 × 105 B16 or LLC in the left flank.
Tumor-specific cytotoxic T lymphocytes
To assess the ability of AdmCD40L-modified DCs to induce
tumor-specific cytotoxic T lymphocytes (CTL), 10 days after the
treatment (intratumoral injection of AdmCD40L-modified DCs to 8-day
established tumors), splenocytes were isolated from 2 mice, pooled, and
restimulated for 5 days at 3 × 106 or
4 × 106 cells/mL with 106 cells/mL CT26
or B16 cells treated with 100 µg/mL mitomycin C (Sigma). After
restimulation, viable cells were collected and tested in a
51Cr-release assay for their ability to lyse CT26 or B16
cells. Where indicated, antibodies to H-2d or
H-2b (Pharmingen) were included at a final concentration of
10 µg/mL. The percentage of specific 51Cr release
was calculated as 100 × ([experimental release  spontaneous release]/[maximal release spontaneous
release]).
Adoptive transfer of splenocytes
Ten days after the treatment of day 8 tumor-bearing mice, the
spleens were removed from 11 to 15 mice, pooled, and a single cell
suspension prepared by mechanical dissociation. Splenocytes (5 × 107 cells per mouse) were administered into
recipient animals via tail vein. After 7 days, recipient animals were
challenged by subcutaneous injection in the right flank with
2 × 105 CT26 cells or 5 × 105
B16 cells (day 0). Survival was then recorded as described above.
Reverse transcriptase-polymerase chain reaction
To demonstrate expression of CD40L mediated by the AdmCD40L vector,
total cellular RNA was extracted from infected cells in vitro or
spleens by the guanidinium thiocyanate-phenol chloroform extraction
method.28 Synthesis for cDNA from total cellular RNA (1 µg) was performed using the Advantage RT-for-PCR kit (Clontech, Palo
Alto, CA). One tenth of the cDNA was amplified with primers specific
for either vector-derived mCD40L or the control
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts: for
mCD40L, 5'-TGCCAAGAGTGACGTGTCCA-3' and
5'-TCAGAGTTTGAGTAAGCCAAAAGA-3'; for the control GAPDH,
5'-ATGGTGAAGGTCGGTGTGAACGGA-3' and
5'-TTACTCCTTGGAGGCCATGTAGGC-3'. Amplification conditions
were 94°C, 2 minutes, and then 30 cycles of 94°C, 1 minute;
55°C, 1 minute; and 68°C, 1 minute, followed by 68°C, 7 minutes. For detection of vector-derived mCD40L mRNA in spleens, 2% of
the amplified product was used as a template for nested polymerase
chain reaction (PCR). Nested PCR was conducted for 25 cycles with the
same thermal cycling profile as above using inner primers specific for
vector-derived mCD40L transcripts:
5'-CCTAATACGACTCACTATAC-3' and
5'-CTCTGTGGATCACTTGGCTT-3'. Each PCR was carried out in 50 µL, and 5 µL of PCR products were resolved on a 1% agarose gel.
Immunohistochemistry
To examine the ability of AdmCD40L-modified DCs to elicit
immune-related process in vivo, spleens or tumors from treated animals were assessed by immunohistochemistry for cells expressing
interleukin-2 receptor chain (IL-2R), an activation marker of T
lymphocytes, or CD4 and CD8, respectively. After frozen sections (6 µm in thickness) were fixed in cold acetone, the samples were treated
with 10 µg/mL antimouse CD25 mAb (IL-2R; Pharmingen), 2.5 µm/mL
antimouse CD8a mAb (Pharmingen), or antimouse CD4 mAb (Pharmingen), 30 minutes, and then stained with 10 µg/mL goat antirat IgG Oregon Green
antibody (Molecular Probes, Eugene, OR) for 30 minutes.
Sections were assessed by counting the number of positive cells in 10 random high power fields (hpf; magnification ×600).
Statistical analysis
All data are reported as mean ± standard error. Statistical
comparison was made using Fisher's exact method, and a value of P < .05 was accepted as indicating significance. Survival
evaluation was carried out using Kaplan-Meier analysis.
| |
Results |
Cell surface phenotype of AdmCD40L-modified dendritic cells
AdmCD40L infection of DCs enhanced expression of CD40L and other
DC-lymphocyte costimulatory molecules (Figure
1). AdmCD40L-modified DCs cultured at the
standard density (2 × 106 cells/mL) had a 3- to
6-fold increase in the number of CD80 (B7-1) and CD54 (ICAM-1)
expressing DCs compared with AdNull-infected DCs. When DCs were
cultured at a lower density (2 × 105 cells/mL)
after AdmCD40L modification, DCs expressed CD80+ and
CD54+, but at a slightly decreased level compared with DCs
cultured at a higher density, suggesting that at least some of the DC
activation was via a bystander mechanism. In this regard, despite the
similar percentage of CD40L-expressing DCs cultured at a higher or
lower density, the lower-density culture was associated with a 3- to 4-fold decrease in the percentage of DCs expressing
CD80+CD40L (11.3% vs 2.9%) or
CD54+CD40L (19.2% vs 6.5%). DCs
cultured at a lower density expressed no less
CD80+CD40L+ or
CD54+CD40L+ than DCs cultured at a higher
density. Other surface molecules CD86 (B7-2) and CD48 (the mouse
homologue of LFA-3) expression was also augmented in the higher density
cultures by AdmCD40L modification (CD86+: AdNull 2.8% vs
AdmCD40L 27.8%; CD48+: AdNull 17.6% vs AdmCD40L 27.1%;
not shown), but CD40L-transduced DCs displayed lower frequency
expression of CD40, the receptor for CD40L (AdNull 72% vs AdmCD40L
45%; not shown). CD25 (IL-2R), an activation marker of lymphocytes
and macrophages was expressed on only a small percentage of
AdmCD40L-modified DCs. A similar proportion of cultures infected with
the control vector AdNull expressed CD25. The percentages of cells
expressing CD25 were less than 10% and were independent of
AdmCD40L-mediated CD40L expression, indicating that there were
minimal numbers of macrophages and lymphocytes contaminating the DC
cultures.
View larger version (70K):
[in this window]
[in a new window]
| Fig 1.
Accessory molecules expressed by AdmCD40L-modified DCs.
Bone marrow-derived DCs were transduced with AdmCD40L or AdNull at moi
40 for 4 hours, and cultured at 2 × 106 or
2 × 105 cells/mL for 72 hours. The modified DCs
were then analyzed for surface coexpression of CD40L with CD80 (B7-1),
CD54 (ICAM-1), or CD25 (IL-2 receptor -chain) by 2-color flow
cytometry. The percentage of cells in each quadrant is listed.
|
|
Cytokine production of AdmCD40L-modified dendritic cells
ELISA analyses confirmed that Ad vector-mediated transfer of the
CD40L cDNA to DCs induced the DCs to secrete cytokines (not shown).
Because stimulation of CD40 on DCs is known to induce IL-12 and
MIP-1 expression,10-12 the AdmCD40L-transduced DCs
cultures were assessed for IL-12 and MIP-1 in the supernatant.
Infection of DCs by AdmCD40L stimulated the production of IL-12
(P < .0001), whereas AdNull-infection did not
(P > .9). For AdmCD40L-transduced DCs, the amount of IL-12
secretion per cell was not affected by decreasing the cell
concentration of DCs culture after CD40L transduction, even to
105 cells/mL (not shown). AdmCD40L, but not AdNull control
infection of DCs, also induced MIP-1 secretion
(P < .0001). Both IL-12 and MIP-1 secretion were
inhibited by addition of anti-mCD40L mAb MR1 compared with control IgG
(IL-12, P < .0001; MIP-1, P < .01),
suggesting that the AdmCD40L vector directed CD40L expression on the DC
surface was responsible for stimulating the DCs to secrete IL-12 and
MIP-1.
Antitumor effects of mCD40L-modified dendritic cells
Treatment of CT26-bearing Balb/c mice (H-2d) with
2 × 106 CD40L-DCs induced significant inhibition of
tumor growth (P < .05 to all other groups) at time points
15 to 20 days, and resulted in the long-term survival in most mice
(Kaplan-Meier analysis, P < .05 to all other groups; Figure
2A,B). Administration of
2 × 106 AdNull- or mock-infected DCs also had some
beneficial effect compared with no treatment (tumor size days 15 to 22, P < .05; survival, P < .05). In the B16 tumor
model in C57Bl/6 mice (H-2b), tumor growth was suppressed
significantly by 2 × 106 CD40L-DCs treatment
compared with that of all other control groups (days 12 to 23, P < .005), resulting in survival advantage
(P < .005; Figure 2C,D). To a lesser extent, the B16 tumor
size treated with 2 × 106 AdNull- or mock-infected
DCs was also suppressed significantly compared with the
tumors without any treatment (tumor area days 14 to 21, P < .005; survival, P < .05).
View larger version (30K):
[in this window]
[in a new window]
| Fig 2.
Suppression of growth of preexisting tumors and survival
of mice with preexisting tumors by intratumoral administration of
AdmCD40L-modified DCs.
(A) Tumor growth, CT26 tumors, Balb/c mice. Tumor cells
(2 × 105) were implanted subcutaneously in the
midflank. On day 8, tumor-bearing mice were treated by intratumoral
injection of 2 × 106 bone marrow DCs modified by in
vitro infection with AdmCD40L ( ), AdNull ( ), or PBS alone ( )
(vector moi 40, 24 hours). (B) Survival, CT26 tumors, Balb/c mice.
Shown is the survival of animals in panel A. (C) Tumor growth, B16
tumors, C57Bl/6 mice. The study was similar to that in panel A, but
5 × 105 tumor cells were used. (D) Survival, B16
tumors, C57Bl/6 mice. Similar to B, using mice in panel C. For panels A
and C, the size of each tumor was assessed 3 times per week, and is
reported as the average tumor area (mm2) ± standard
error of n = 5 per group. Asterisks in panels A and C indicate
significant differences at 95% confidence limits between AdmCD40L and
all other surviving groups; on days 22 and 25 in panel A,
AdmCD40L-infected DCs had no significant effect compared with
mock-infected DCs. For panels B and D, survival was recorded as the
percentage of animals in each group (Kaplan-Meier analysis, for all
panels, P < .05 AdmCD40L compared with all other groups).
In all panels, controls included tumor-bearing mice without any
treatment (`).
|
|
Marked tumor suppression was also observed with administration to the
tumors of one tenth the numbers (2 × 105) of
AdmCD40L-infected DCs. In this context, 2 × 105 AdmCD40L-DCssuppressed the growth of established tumors
(both CT26 and B16) when injected intratumorally, but AdNull- or
mock-infected DCs did not (Figure 3).
Balb/c mice bearing CT26 8-day established tumors were treated by
direct injection with 2 × 105 AdmCD40L-transduced
DCs. This therapy significantly inhibited tumor growth days 13 to 22 (P < .0001) and survival at 12 weeks in 60%
(P < .005), in contrast with AdNull- or mock-infected DCs as well as no treatment (Figure 3A,B). Intratumoral injection with
AdNull- or mock-infected DCs had no therapeutic effect on CT26-bearing
mice days 11 to 22 (P > .7). Similar results were achieved in the B16 established tumors. The growth of B16 tumors injected with AdmCD40L-modified DCs was suppressed significantly over
days 13 to 24 (P < .0005) with enhanced survival
(P < .005), whereas tumors injected with AdNull- or
mock-infected DCs grew in a similar fashion to naive tumors from days
10 to 22 (P > .2) and did not have enhanced survival
(P > .2; Figure 3C,D). This antitumor effect of
2 × 105 AdmCD40L-modified DCs against B16 tumors
was also observed in CD4 knockout mice over days 10 to 18 (P < .005 compared with tumor-bearing wild-type mice
without any treatment; not shown).
View larger version (27K):
[in this window]
[in a new window]
| Fig 3.
Treatment of established tumors with intratumoral
administration of reduced numbers of AdmCD40L-modified DCs.
The study was carried out in a fashion parallel to that in Figure 2,
but with administration of 10% the number of AdmCD40L-modified DCs.
(A) Growth of CT26 tumors in Balb/c mice. Balb/c mice were injected in
the flank with 2 × 105 CT26 tumor cells as
described in Figure 2A (day 0). On day 8, the tumor-bearing mice
received intratumor injections of 2 × 105 DCs that
had been transduced in vitro with AdmCD40L (), AdNull (), or PBS
alone () at moi 40 for 24 hours. (B) Survival, CT26 tumors, Balb/c
mice. Shown is the survival of animals in panel A. (C) Growth of B16
tumors in C57Bl/6 mice. The study was identical to that described in
panel A, except for the different tumor type and its number; ie,
5 × 105 B16 cells were used as described in Figure
2C. (D). Survival, B16 tumors, C57Bl/6 mice. Similar to B, using mice
in panel C. For panels A and C, the size of each tumor was assessed 3 times per week, and is reported as the average tumor area
(mm2) ± standard error of n = 5 mice per group.
Asterisks indicate significant differences at 95% confidence limits
between AdmCD40L-transduced DCs and all other surviving groups. For
panels B and D, survival was recorded as the percentage of animals in
each group (Kaplan-Meier analysis, for both panels, P < .05
AdmCD40L compared with all other groups). In all panels, controls
included tumor-bearing mice without any treatment ().
|
|
Antitumor cytotoxic T lymphocyte induced by intratumoral injection
with mCD40L-transduced dendritic cells
Direct injection of AdmCD40L-modified DCs to CT26 or B16 tumors
elicited tumor-specific CTL activity (Figure
4). Balb/c mice bearing CT26 tumors were
intratumorally inoculated with AdmCD40L- or AdNull-modified DCs, or
were not inoculated. Effector cells were generated from splenocytes
obtained 10 days after the inoculation by culture with mitomycin
C-treated CT26 cells. Cells from only mCD40L-DC-treated mice exhibited
specific lysis of CT26 target cells (Figure 4A). This cytotoxicity was
inhibited by addition of the relevant MHC class I antibody
(H-2d), but not by addition of isotype-matched irrelevant
class I antibody (H-2b) (H-2d: 85%-96%
inhibition; H-2b: 11%-24% inhibition; not shown). No
apparent lysis was observed against irrelevant but syngenic fibroblast
SVBalb cells (Figure 4B). C57Bl/6 mice treated with mCD40L-transduced
DCs exhibited a strong B16-specific splenic CTL response (Figures
4C,D). Controls for this analysis included lymphocytes obtained from
tumor-bearing mice injected with AdNull-transduced DCs or without any
treatment. Splenocytes were restimulated with B16 cells as described
above, and the resulting effector cells were evaluated for cytolytic activity against B16 and C3 cells. Injection with AdNull-transduced DCs
induced minimal CTL with reactivity against B16 cells, but AdmCD40L
transduction markedly enhanced this specific cytolytic activity. No
specific lysis with effector cells in the B16 model was observed
against C3 cells.
View larger version (29K):
[in this window]
[in a new window]
| Fig 4.
Induction of tumor-specific cytotoxic T cells after
intratumoral administration of AdmCD40L-modified DCs.
(A,B) CT26 tumors. DCs purified from bone marrow were transduced in
vitro with AdmCD40L () or AdNull () at moi of 40 for 24 hours,
and 2 × 105-modified DCs were administered
intratumorally to day 8 established CT26 tumors in Balb/c mice.
Controls included tumor-bearing mice without any treatment (). Ten
days after treatment, splenocytes were isolated and restimulated in
vitro for 5 days with mitomycin C-treated CT26 cells and assayed for
cytolytic function against 51Cr-labeled CT26 targets (panel
A) or SVBalb targets (panel B). (C,D) B16 tumors. The study was
identical to that described in panel A, except for the different tumor
type. Ten days after administration of transduced DCs, the splenocytes
were isolated, restimulated with mitomycin C-treated B16 cells, and
assayed as in A,B. Shown are data for 51Cr-labeled B16
targets (panel C) and C3 targets (panel D). All panels, results are
presented as the mean ± standard error (n = 3/data point).
|
|
The relevance of the in vitro specific cytolysis to in vivo function
was demonstrated by showing that adoptive transfer of splenocytes
protected against a subsequent challenge with the identical tumor cells
(Figure 5). In this context, adoptive
transfer of 5 × 107 splenocytes isolated 10 days
after intratumoral administration of AdmCD40L- or AdNull-transduced DCs
mediated 80% or 20% protection against a CT26 challenge over a
12-week period, respectively (AdmCD40L-DCs compared with naive control,
P < .005; AdmCD40L-DCs compared with AdNull-DCs,
P < .05; Figure 5A). Splenocytes from B16 tumor-bearing mice treated with AdmCD40L-transduced DCs provided 20% protection (compared with naive, P < .005; Figure 5B). In contrast,
transfer of lymphocytes isolated from mice injected with
AdNull-modified DCs mediated only a minor enhancement in survival
compared with no infusion of splenocytes (P < .05;
AdmCD40L-DCs vs AdNull-DCs, P < .0005).
View larger version (13K):
[in this window]
[in a new window]
| Fig 5.
Ability of splenocytes from tumor-bearing mice treated
with AdmCD40L-modified DCs to transfer tumor-specific immunity.
(A) Splenocytes from mice with CT26 tumors treated with
AdmCD40L-infected DCs. The 8-day established subcutaneous CT26 tumors
in Balb/c mice were inoculated with 2 × 105 DCs
modified with AdmCD40L () or AdNull () at moi of 40 for 24 hours.
Ten days after administration, splenocytes were transferred
intravenously to naive recipients (5 × 107 cells
per mouse). Controls included mice without any infusion of splenocytes
(). Recipient mice were challenged 7 days later with subcutaneous
administration of 2 × 105 CT26 cells (day 0). (B)
Splenocytes from C57Bl/6 mice with B16 tumors. The study was similar to
that in panel A, but B16 established tumors in C57Bl/6 mice were
inoculated with modified DCs and recipient mice were challenged with
5 × 105 B16 cells. In both panels, survival was
recorded as the percentage of surviving animals (Kaplan-Meir analysis,
P < .05 AdmCD40L compared with all other groups).
|
|
Antitumor CTL activity was also associated with a systemic therapeutic
effect in a distant 2-tumor model of metastatic disease (Figure
6). Mice bearing bilateral B16 flank tumors
were treated by intratumoral injection to the left tumor with
CD40L-transduced DCs. This therapy actively suppressed growth of the
untreated right tumor as well as the treated tumor, with 20% of the
mice alive at the end of the experiment on day 84, with no detectable tumors in both flanks. In contrast, tumors grew progressively and were
eventually lethal in control mice treated with AdNull-infected DCs or
without any therapy. As a further control, administration of
CD40L-transduced DCs to a left Lewis lung carcinoma tumor had no
beneficial effect on inhibiting B16 tumor growth in the right flank,
confirming the tumor specificity of the antitumor effect.
View larger version (14K):
[in this window]
[in a new window]
| Fig 6.
Treatment of contralateral tumors with intratumoral
administration of AdmCD40L-modified DCs.
(A) Tumor growth. C57Bl/6 mice were injected in both flanks with
5 × 105 B16 tumor cells (day 0). On day 8, the
bilateral tumor-bearing mice intratumorally received
2 × 105 DCs that had been modified in vitro with
AdmCD40L (), or AdNull ( ) in the tumors in the left flank.
Controls included bilateral tumor-bearing mice without any treatment
(). A parallel group of control mice were injected in the left Lewis
lung carcinoma tumors with AdmCD40L-transduced DCs (treatment for a
different tumor type than the right B16 tumors; ). The size of right
tumors is reported as at the average tumor area (mm2) ± standard error of n = 5 per group. Asterisks indicate significant
differences at 95% confidence limits between AdmCD40L-transduced DCs
in syngenic tumors and all other surviving groups. (B) Survival. The
survival of animals in panel A was recorded as the percentage of
animals in each group (Kaplan-Meir analysis, P < .05
AdmCD40L-transduced DCs for syngenic tumors compared with all other
groups).
|
|
Immunohistochemical analysis demonstrated a marked intratumoral
CD8+ T-cell infiltration (58 CD8+ T cells/10
hpf) and to a lesser extent of CD4+ T cells (12 CD4+ T cells/10 hpf) in CT26 tumors treated with
2 × 105 AdmCD40L-modified DCs 3 days after
treatment, but a minimal infiltration in those treated with
AdNull-modified DCs (14 CD8+/10 hpf, 10 CD4+/10 hpf) or without any treatment (17 CD8+/10 hpf, 14 CD4+/10 hpf; not shown).
Spleen analyses
In the spleens from the mice treated with CD40L-DCs,
AdmCD40L-derived mCD40L messenger RNA (mRNA) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and the
splenocytes expressed the activation marker CD25 by immunocytochemical analysis (Figure 7). Expression of CD40L
mRNA driven by AdmCD40L-modified DCs was first confirmed in vitro by
RT-PCR analysis (Figure 7A). As expected, an 877-base pair (bp)
fragment corresponding to the exogenous mCD40L cDNA was amplified from
total cellular RNA from both AdmCD40L-transduced DCs and control CL7
cells at a similar intensity. The intactness of the RNA was confirmed
by amplification of GAPDH cDNA. Importantly, nested RT-PCR analysis
demonstrated the expression of exogenous mCD40L transcripts in the
spleens from CD40L-DC treated mice, but no expression in the spleens
from mice that had been intratumorally injected with AdNull-infected DCs or AdmCD40L-modified CL7 cells (Figure 7B). These data suggested that the AdmCD40L-modified DCs injected into the flank tumors migrated
to the spleens in vivo. With the use of immunocytochemical analyses, an
assessment of spleens from mice injected with AdmCD40L-modified DCs
demonstrated cells expressing CD25 (IL-2 receptor chain; 39 positive cells per 10 hpf; Figure 7C). In contrast, control spleens
from mice injected with AdNull-modified DCs or AdmCD40L-modified CL7
had minimal CD25 expression (9 or 6 positive cells per 10 hpf,
respectively).
View larger version (54K):
[in this window]
[in a new window]
| Fig 7.
Demonstration of the ability of intratumoral
administration of AdmCD40L-transduced DCs to traffic to the spleen and
induce T-cell activation.
The expression of CD40L mRNA by the AdmCD40L-modified DCs (panel A) was
used to tract the genetically modified DCs, and immunocytochemistry for
the IL-2 receptor chain used to identify activated T cells in the
local milieu. (A) AdmCD40L-mediated CD40L expression assessed by RT-PCR
in DCs or control CL7 cells transduced with AdmCD40L. The RT-generated
cDNA from DCs transduced with AdmCD40L or AdNull, or CL7 cells
transduced with AdmCD40L was amplified with primers for exogenous
mCD40L or control GAPDH mRNA. PCR products were resolved on a 1%
agarose gel and stained with ethidium bromide. (B) Detection of DCs
expressing AdmCD40L-mediated CD40L by nested RT-PCR in spleens from
mice treated with intratumoral administration of AdmCD40L-transduced
DCs. Balb/c mice were injected subcutaneously in the right midflank
with 2 × 105 CT26 cells (day 0). On day 8, tumor-bearing mice were treated by intratumoral injection of
2 × 106 AdmCD40L- or AdNull-transduced DCs or
AdmCD40L-transduced CL7 cells. On day 11, the RT-generated cDNA from
the spleens was amplified with outer and inner primer pairs for
exogenous mCD40L by nested PCR. PCR products were resolved on a 1%
agarose gel and stained with ethidium bromide. PCR for the control
GAPDH mRNA is shown in the lower part of the panel. (C)
Immunohistochemical evaluation of spleens in panel B for CD25 (IL-2
receptor chain). On day 11, spleens were dissected, and frozen
spleen sections were stained using 10 µg/mL rat antimouse CD25 mAb,
followed by the visualization with 10 µg/mL antirat IgG Oregon Green
antibody. Specific immunoreactivity showed the presence of
CD25-expressing cells in spleens from mice treated with
AdmCD40L-modified DCs, whereas only a low level of tissue
autofluorescence was evident in control spleens (AdNull/DCs and
AdmCD40L/CL7).
|
|
No antitumor effect of AdmCD40L-modified fibroblasts
When injected intratumorally, CD40L-transduced DCs induced
therapeutic tumor immunity, but CD40L-transduced fibroblasts did not
(Figure 8). Growth of subcutaneous CT26
tumors was profoundly affected by 2 × 106
AdmCD40L-modified DCs (Figure 2A), whereas tumors treated with 2 × 106 of CL7 fibroblasts that had been infected
with AdmCD40L at the identical moi grew, as did the control group
without any treatment (P > .3; Figure 8A). This antitumor
effect in vivo correlated to tumor-specific CTL activity demonstrated
by splenocytes from mice treated with the same regimen (Figure 8B).
Animals bearing untreated CT26 tumors generated a 15% lysis of
51Cr-labeled CT26 target cells at an effector/target
(E/T) ratio of 60/1. This lytic activity was enhanced to
34% after CD40L-transduced DC treatment, but not after intratumoral
injection of CD40L-transduced CL7 cells. Taken together with the
adoptive transfer and spleen mRNA and immunohistochemical data (Figures
5, 7, 8), these data suggest that optimal therapeutic immunity of
CD40L-transduced DCs depends, at least in part, not on regional
stimulation by CD40L expression in tumors, but on migration of
activated DCs to the lymphoid tissues to stimulate tumor
antigen-specific T cells.
View larger version (14K):
[in this window]
[in a new window]
| Fig 8.
Specificity of suppression of tumor growth after
treatment with intratumoral administration of AdmCD40L-transduced DCs
compared with AdmCD40L-modified fibroblasts.
(A) Tumor growth. Groups of Balb/c mice were challenged subcutaneously
with 2 × 105 CT26 cells (day 0). On day 7, bone
marrow DCs () or syngenic fibroblast CL7 cells () were transduced
in vitro with AdmCD40L at moi of 40 for 24 hours. Mice bearing day 8 established tumors were given 2 × 106 modified
cells intratumorally. Controls included tumor-bearing mice without any
treatment (). The size of each tumor was assessed 3 times per week,
and is reported as the average tumor area (mm2) ± standard error (n = 5 per group). Asterisks indicate significant
differences at 95% confidence limits between AdmCD40L-transduced DCs
and all other groups. (B) Tumor-specific cytotoxic T lymphocyte
activity. Ten days after treatment described for panel A, the
splenocytes were isolated, restimulated, and assayed as in Figure 4A,B.
Shown are data for 51Cr-labeled CT26 targets. Results are
presented as the mean ± standard error (n = 3/data point). In all
groups, lysis of control 51Cr-labeled SVBalb targets was
within 8% (not shown).
|
|
| |
Discussion |
This study is based on the hypothesis that DCs that have been
genetically modified with a recombinant adenovirus to express CD40L
will be self-activated, and when placed in the milieu of a tumor, will
initiate a tumor-specific cellular immune response that will suppress
growth of the tumor and increase survival of the host. The data support
this hypothesis. Intratumoral injection of AdmCD40L-modified syngenic
DCs to 2 different types of murine tumors elicited therapeutic
antitumor immunity that suppressed the growth of established tumors and
enhanced survival. Administration of AdmCD40L-infected DCs elicited
tumor-specific cytolytic T-lymphocyte responses that protected naive
syngenic mice against a subsequent tumor challenge and impaired the
growth of distant established tumors.
AdmCD40L-modified dendritic cells elicit antitumor
immune responses
The observations in this study are consistent with the concept that
DCs transduced to express CD40L efficiently initiates protective
antitumor immunity. DCs are professional antigen-presenting cells that
sample antigens in all body tissues and migrate to lymphoid organs
where they present antigens to T cells.1 Several recent
reports suggest that DCs cannot stimulate cytotoxic T cells directly
unless they are first stimulated via CD40 on their
surface.7-9 This is usually accomplished by CD40L expressed
on CD4+ helper T cells.5,29 The
T-helper-mediated CD40 triggering up-regulates adhesion and
costimulatory molecule in the DCs, bringing the DCs to a state where
they can autonomously stimulate a T-killer response.2,7-11
This study extends this concept by demonstrating that genetic
modification of DCs to express CD40L accomplishes the goal of
activating DCs to induce functionally relevant cell-mediated adoptive
immune responses such as suppression of tumor in an antigen-specific fashion. After adenovirus gene transfer of the CD40L cDNA to DCs to
express CD40L, the transduced DCs most likely self-trigger CD40 and
activate themselves to present tumor antigen to naive CD8+
CTL. When administered to tumors, these genetically modified DCs likely
capture tumor antigens and present them to naive CD8+ CTL,
probably after migrating to lymphoid organs. Consistent with this
hypothesis, CD40L-transduced DCs administration to tumors resulted in
the up-regulation of IL-2R in spleen cells. Further evidence for
this concept comes from the observation that exogenous CD40L mRNA
expression originating from injected AdmCD40L-modified DCs was detected
in the spleen of treated mice, suggesting the modified, activated DCs
had migrated from the tumor to the spleen.
The triggering of CD40 on DCs leads to increased production of several
inflammatory cytokines and chemokines, including interleukin-12 (IL-12) and macrophage inflammatory protein-1
(MIP-1).10-12 IL-12, a cytokine that promotes
the development of Th1 CD4+ T cells and the maturation of
CTLs, likely plays a supportive role for generation of T-killer
responses for tumor immunity.30 In contrast, MIP-1, a
chemokine known to preferentially induce the migration of
CD8+ T cells, helps DCs to encounter and stimulate rare
tumor antigen-specific CD8+ CTLs.31
The expression of CD40L is normally restricted almost exclusively to
activated CD4+ helper T cells, and exquisitely regulated in
concert with other receptor-ligand pairs within a specialized
microenvironment.32,33 In this regard, studies with murine
models have shown that inappropriate presence of the CD40L protein may
have adverse consequences. Wiley et al34 showed that
soluble CD40L protein administration to the murine airway induced
pulmonary inflammation. Brown et al35 demonstrated that
constitutive CD40L expression in bone marrow or thymic cells by a
retroviral vector produced monoclonal T-lymphoblastic lymphomas of
thymic origin. In this context, the use of genetic modification of DCs
will have to be closely monitored for adverse effects. However, the use
of Ad vectors to transfer to CD40L cDNA to DCs may have an advantage,
in that expression by Ad in vivo is generally transient and locally
restricted to the genetically modified cells without induction of
systemic adverse effects.36-39
Because tumor-specific immunity relies on professional APC activated
through CD40-CD40L interactions, genetic modification of tumor cells,
lymphoma cells, or bystander fibroblasts to express CD40L has been
evaluated in an attempt to stimulate APCs to induce tumor-specific
cellular immunity. Kato et al17 and we18 have used adenovirus vectors to transfer CD40L cDNA to human lymphoma cells
and murine tumor cells, respectively, and demonstrated that this
genetic modification induced generation of specific CTLs. Grossmann et
al15 and Couderc et al13 transduced murine
tumor cells ex vivo with a retroviral construct containing the CD40L cDNA, and induced a systemic immune response capable of impeding tumor
growth in vivo. Dilloo et al14 showed that triggering CD40
on murine lymphoblastic cells with retrovirus-mediated expression of
CD40L on bystander fibroblasts enhanced the antigen-presenting potential of those lymphoma cells. Finally, Nakajima et
al20 transfected the CD40L-expressing plasmid into murine
mastocytoma cells, and investigated its potentiation in enhancing host
APC functions. On the basis of this concept, Mendoza et
al19 and Gurunathan et al16 have shown that
intradermal or subcutaneous coinjection of a plasmid expressing CD40L
with a plasmid expressing  -galactosidase DNA enhances cellular
immune response to -galactosidase as a model of a tumor antigen. As
an alternative strategy, culture of DCs with recombinant soluble CD40L
protein, fibroblasts, or hybridoma cells transfected with CD40L cDNA,
or anti-CD40 antibody, induced APC functions of DCs.7-12,40
This is associated with up-regulation of accessory molecules such as
ICAM-1, B7-1, and B7-2, on CD40-triggered DCs, and high levels of
production of cytokines such as IL-12, MIP-1, IL-8, and
TNF-.10-12 Ridge et al,8 Bennett et
al,7 and Schoenberger et al9 clearly illustrated that anti-CD40 antibody (acting as a surrogate of CD40L
triggered CD40 on DCs) brings the DCs to a state in which they can
autonomously present antigen to CD8+ killer T cells and
induce antigen-specific CTL responses. Finally, Labeur et
al40 has recently shown that DCs incubated with soluble CD40L protein became mature as evident by morphology, up-regulation of
adhesion and costimulatory molecules, and high levels of IL-12 secretion, to promote antitumor immunity in vivo.
Dendritic cell-based cancer immunotherapy
With the use of methods for isolation and in vitro culture of murine
and human DCs, a variety of strategies for cancer immunotherapy have
been developed using DCs as vaccines.1,41-46 After tumor antigens are incubated with DCs ex vivo, these antigen-loaded DCs are
then reinfused as vaccines. Several approaches for delivering tumor
antigens to DCs have been evaluated, including whole tumor lysates,
defined tumor antigenic peptides, tumor antigen-encoding DNA or RNA,
recombinant viral vectors (adenovirus, poxvirus, vaccinia virus, and
retrovirus), encoding tumor antigens, tumor-derived mRNA, coculture
with tumor cells, or fusion with whole tumor cells.24,47-73 Immunization using these antigen-pulsed DCs elicit host protective and
therapeutic antitumor immunity associated with the induction of tumor
reactive CD8+ T cells.46
In contrast to the strategy of loading DCs with tumor antigens ex vivo,
the strategy in this study of using DCs genetically modified to express
CD40L has several theoretical advantages of potential clinical
interest. First, the mCD40L-modified DCs can directly interact with
tumor antigens in vivo without the possible alteration or concern for
alteration or loss of tumor antigen-associated RNA or peptides induced
in vitro manipulation. Second, in vivo interaction between the
mCD40L-modified DCs and tumor cells expressing the entire repertoire of
tumor antigens should allow the host defense system to be stimulated
against multiple tumor antigens. The involvement of oligoclonal
effector subpopulations specific for diverse antigenic tumor epitopes
may be advantageous for an optimal antitumor response. Third,
intratumoral administration of the mCD40L-modified DCs does not depend
on the prior identification of appropriate tumor antigens and is not
limited to individuals expressing a particular corresponding MHC
allele. In this context, although it is possible to use some defined
tumor antigens in DCs adoptive transfer therapy, potential MHC-binding
tumor-specific peptides remain unknown for most human
tumors.46,74 This advantage is not unique to CD40L-modified
DCs in that some tumor cell-based DC-pulsing approaches, such as
unfractionated peptides eluted from tumor cells, tumor-cell-derived
polyadenylated RNA, and coculture or fusion with tumor cells, do not
also require prior knowledge of the relevant tumor antigenic
peptides.47,52,56,58,64,72,73 Finally, CD40L expressed
exogenously on DCs may protect modified DCs from both CD95-mediated and
spontaneous apoptosis through self-CD40 triggering, as does recombinant
soluble CD40L,75 resulting in long-term stimulation of host
antitumor immunity. There, however, is no evidence that constitutive
expression of CD40L on transduced DCs has similar effects on their
survival as an appropriate extrinsic triggering of CD40. Definitive
proof awaits further studies addressing this issue.
| |
Acknowledgments |
We thank Dr Tsuneyoshi Hamada, Division of Hematology-Oncology, for
technical support; and N. Mohamed for help in preparing this manuscript.
| |
Footnotes |
Submitted August 20, 1999; accepted February 13, 2000.
Supported in part by NIH R01 CA75192; the Will Rogers Memorial
Fund, Los Angeles, CA, and GenVec, Inc, Rockville, MD. M.A.S.M. was
supported, in part, by NCI-P30-CA-08748 and the Gar Reichman Fund of
the Cancer Research Institute, New York, NY.
Reprints: Institute of Genetic Medicine, 520 E 70th St,
ST 505, New York, NY 10021; e-mail:
geneticmedicine{at}mail.med.cornell.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Banchereau J, Steinman RM.
Dendritic cells and the control of immunity.
Nature.
1998;392:245-252[Medline]
[Order article via Infotrieve].
2.
Grewal IS, Flavell RA.
CD40 and CD154 in cell-mediated immunity.
Annu Rev Immunol.
1998;16:111-135[Medline]
[Order article via Infotrieve].
3.
Schuler G, Steinman RM.
Dendritic cells as adjuvants for immune-mediated resistance to tumors.
J Exp Med.
1997;186:1183-1187[Free Full Text].
4.
Armitage RJ, Fanslow WC, Strockbine L, et al.
Molecular and biological characterization of a murine ligand for CD40.
Nature.
1992;357:80-82[Medline]
[Order article via Infotrieve].
5.
Roy M, Waldschmidt T, Aruffo A, Ledbetter JA, Noelle RJ.
The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells.
J Immunol.
1993;151:2497-2510[Abstract].
|
Top
Abstract
Introduction