| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI 48101; and
Division of Hematology and Oncology, University of Michigan and Veterans Administration Ann Arbor Health Care Systems, Ann Arbor, MI 48105
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
indicative of a Th1-type immune response were observed upon ex
vivo stimulation of vaccine-primed lymph node cells. Adoptive transfer
of immune T cell-enriched lymphocytes was sufficient to protect naive
recipients from lethal tumor challenge. Furthermore, CD8+ T
cells were absolutely required for tumor protection. Although C6VL-DC
and control vaccines stimulated low levels of tumor-specific Ab
production in mice, Ab levels did not correlate with the protective
ability of the vaccine. Thus, tumor cell lysate-pulsed DC vaccines
appear to be an effective approach to generate potent T cell-mediated
immune responses against T cell malignancies without requiring
identification of tumor-specific Ags or patient-specific Id protein
expression. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The use of DC in the development of effective prophylactic and therapeutic vaccines for a variety of cancers has been the focus of intense investigation by many groups. DC have been targeted in this effort largely due to their unique ability to initiate potent primary and secondary immune responses (13) and to control the type of immune response that is induced (14). DC loaded with tumor-derived DNA, RNA, protein(s), or peptides have been used as an efficacious vaccine in multiple murine tumor models (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). DC have also been effective as cellular adjuvants for B cell Id protein vaccines. Loading DC with B cell Id:KLH stimulated levels of anti-tumor immunity superior to Id:KLH injected with a chemical adjuvant (27). In patients, loading DC with B cell Id protein with or without carrier protein (KLH) resulted in the generation of Id-specific cellular immune responses (9, 28, 29) in addition to the largely humoral immune response observed in patients receiving B cell Id vaccines without DC.
The ability to target multiple tumor Ags may increase the magnitude and
diversity of anti-tumor responses, thus preventing tumor from
escaping responses limited in repertoire (30). Loading DC
with proteins from tumor cell lysates may result in the presentation of
a broader array of tumor-specific Ags than what may be presented using
Id protein (31), including multiple epitopes for both
CD4+ and CD8+ T cells
(32). In addition, tumor cell lysates contain agents known
to induce maturation of DC (32). Mature DC have an
enhanced capacity to prime potent Ag-specific CTL activity and
stimulate IFN- release by CD4+ Th cells.
Recently, two groups have demonstrated that tumor lysate-specific
cytotoxic T lymphocytes can be generated ex vivo against autologous
primary multiple myeloma (33) and B cell chronic
lymphocytic leukemia (34) tumor samples using tumor
lysate-pulsed DC as stimulators. We demonstrate here that tumor
lysate-pulsed DC stimulate a potent anti-tumor response against
murine T cell lymphoma in vivo. Vaccination of mice with T cell
lymphoma-pulsed DC (C6VL-DC) significantly enhanced the survival of
tumor-challenged mice compared with the previously reported TCR Id:KLH
in QS-21 vaccines. Anti-tumor immunity stimulated by C6VL-DC was lysate
specific and required the presence of CD8+ T
cells. The vaccines stimulated a potent cellular response that was
characterized ex vivo by tumor-specific proliferation and IFN-
secretion by vaccine-primed lymphocytes. Lastly, C6VL lysate-pulsed DC
vaccines significantly enhanced the survival of tumor-bearing mice.
This study is the first to directly compare the efficacy of defined
protein epitope-based and tumor lysate-pulsed DC-based vaccine
approaches for the immunotherapy of lymphomas. Our results demonstrate
that tumor lysate-pulsed DC are efficacious in stimulating reactivity
against T cell lymphoma that is capable of clearing existing tumor as
well as preventing future tumor growth. Tumor lysate-pulsed DC vaccines
out-perform the idiotype-based vaccine strategy currently being pursued
for the immunotherapy of T cell lymphomas.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female C57BL/6 mice (H-2b) were purchased from The Jackson Laboratory (Bar Harbor, ME) at 6 wk of age. The animals were housed in the Ann Arbor Veteran’s Affairs animal medical unit under specific pathogen-free conditions according to the guidelines drafted in the Animal Component of Research Protocol that is implemented in Veterans Affairs research centers. The mice were between 7 and 9 wk of age when vaccine protocols were initiated.
Media and cytokines
AIM-V serum-free medium (Invitrogen, Carlsbad, CA) was used to
derive DC and to generate DC-based vaccines. DC used for in vitro
assays and C6VL for tumor challenges were grown in complete medium
(CM), consisting of RPMI 1640 supplemented with 10% FCS, 0.1 mM
nonessential amino acids, 1 µM sodium pyruvate, 2 mM
L-glutamine, 50 µg/ml gentamicin, 100 U/ml penicillin,
0.5 µg/ml Fungizone, and 50 µM 2-ME (35). CD hybridoma
serum-free medium (Invitrogen) was used to produce mAbs used in vivo to
avoid serum protein contamination of Abs. Murine rGM-CSF (sp. act.,
5.0 x 106 U/mg) and murine rIL-4
(6.1 x 108 U/mg) were purified from
overexpressing cell lines generated in our laboratory; murine rIFN-
(50 µg/ml; BD PharMingen, San Diego, CA) was used to prepare a
standard curve for ELISAs. Human rIL-2 (18.0 x
106 U/mg) was obtained from Chiron
(Emeryville, CA).
Cell lines
C6VL (H-2b) is a radiation-induced T cell
thymoma of the C57BL/Ka background (36). C6VL was used as
our tumor model in these studies. C6VL cells have a mature T cell
phenotype expressing TCR-
, Thy 1.2, CD3, CD4, and
H-2Db surface proteins (37). C6VL
does not express MHC class II or Fas (10). MBL-2
(H-2b) is a T cell lymphoma cell line of C57BL/6
origin (provided by I. L. Weissman, Stanford, CA) that was used as
a control T cell lymphoma lysate and a control tumor challenge. B16/F10
(American Type Culture Collection, Manassas, VA) is a melanoma derived
from C57BL/6 mice and was used as a non-lymphoid tumor lysate
control.
Antibodies
mAb 124-40 (mouse IgG1
) recognizes a determinant on the C6VL
TCR -chain V region (36). mAb 2.43 (Rat IgG2b)
recognizes mouse CD8.2. mAb SFR8-B6 (rat IgG2b) recognizes human
HLA-Bw6. The 2.43 hybridoma was purchased from American Type Culture
Collection. The SFR8-B6 hybridoma was provided by J. Parnes (Stanford,
CA). mAb 124-40 was purified from culture supernatant over a protein
A-Sepharose column. 2.43 and SFR8-B6 were purified from serum-free
hybridoma culture supernatants by ammonium sulfate precipitation,
followed by strong anion exchange chromatography. Ab concentrations
were determined by bicinchoninic acid assay (Pierce, Rockford, IL).
FITC-labeled anti-CD4 (GK1.5, isotype rat IgG2b,), anti-CD80
(16-10A1, hamster IgG), anti-CD86 (GL1, rat IgG2a,),
anti-CD11c (HL3, hamster IgG), anti-I-Ab
(AF6-120.1, mouse IgG2a,), anti-CD40 (3/23, rat IgG2a,),
anti-CD3
(145-2C11, hamster IgG), and anti-CD19 (1D3, rat
IgG2a,); PE-labeled anti-CD8b.2 (53-5.8, rat IgG1,); and
isotype controls were purchased from BD Biosciences (San Diego, CA).
PE-labeled anti-CD8b.2 (53-5.8) was used to determine
CD8+ T cell depletion in mice and is
non-cross-blocking with mAb 2.43. FITC-labeled goat anti-mouse
IgG+IgM (H+L) was purchased from Jackson ImmunoResearch Laboratories
(West Grove, PA). Purified and biotinylated forms of rat anti-mouse
IFN- and rat anti-mouse IL-4 mAbs for ELISA were purchased from
Caltag (Burlingame, CA). Streptavidin-HRP was purchased from Pierce
(Rockford, IL).
C6VL TCR Id vaccine preparation
The C6VL TCR Id protein vaccine was used for comparison with
prior experiments. The C6VL TCR Id protein was purified from a
recombinant overexpressing cell line. Briefly, DNA constructs of -
and -chains of the C6VL TCR were modified to contain coding
sequences for a thrombin cleavage site, coiled-coil (derived from a
leucine zipper protein), and six histidine residues. The DNA constructs
were cloned into the expression vector, pSRSD5 (10) and
transfected into BW5147 cells by electroporation. A cell line was
isolated that expressed 
3 mg/L C6VL TCR Id protein in the culture
supernatant. The TCR Id protein was purified from the culture
supernatant on a 124-40 mAb affinity column and then on a metal
affinity column (MAC). The protein was eluted from the MAC with
imidazole and then digested with thrombin. After cleaving the
coiled-coil/His tag from the TCR Id protein with thrombin, the digest
was run over a second MAC, and the TCR Id protein was collected in the
flow-through. The TCR Id vaccines were prepared by conjugating the
recombinant TCR Id protein to KLH (1:1 ratio, 35 µg TCR Id/vaccine)
and then mixing with QS-21 (10 µg/vaccine) for s.c. delivery.
Generation of bone marrow-derived DC
Bone marrow cells were flushed from the femurs and tibias of
C57BL/6 mice and depleted of erythrocytes using Sigma Red Cell Lysis
Buffer (8.3 g/L NH4Cl in 0.01 M Tris-HCl buffer,
pH 7.2). The resulting cells were
cultured at a density of 1.0 x 106 cells/ml
in either CM (Figs. 1
and 2B)
or AIM-V serum-free medium (all other experiments) containing 10 ng/ml
rGM-CSF and rIL-4. On day 3 of culture, fresh cytokines were added, and
the total culture volume was increased by 50% with the addition of
fresh medium. After 5 days of culture, nonadherent cells were harvested
by gentle pipetting and were suspended in CM or AIM-V to a density of
4.0 x 106 cells/ml. DC were enriched from
the nonadherent population by density centrifugation over medium
containing 14.5% (w/v) metrizamide (Sigma) (25). The
cells at the metrizamide solution/medium interface were washed three
times in the respective medium before use. The resulting DC population
was analyzed by flow cytometry (FACScan; BD Biosciences, San Jose, CA)
for the expression of cell surface markers characteristic of DC. Each
preparation was 80% positive for the coexpression of MHC II, CD11c,
CD40, CD80, and CD86 (data not shown). The cells did not express CD3
or CD19.
|
|
After purification, DCs were resuspended to 1.0 x 106 cells/ml in either CM or AIM-V medium containing lysate from either C6VL T cell lymphoma, MBL-2 (control) T cell lymphoma, B16/F10 melanoma, kidney tissue, or normal primary lymphocytes. The cell lysates were prepared by subjecting 20.0 x 106 cells/ml in CM or AIM-V to three cycles of rapid freezing in liquid nitrogen and thawing at 55°C. The lysates were spun at 5000 rpm to remove particulate cellular debris. The DCs were pulsed with the lysates from three tumor cells per DC for 18 h (25). DC derived in CM were pulsed with lysates of cells cultured in CM, while DC derived in AIM-V were pulsed with lysates of cells cultured in AIM-V. After pulsing, the DC were collected, washed several times in HBSS, and resuspended in HBSS to 5.0 x 106 cells/ml for delivery to mice.
Immunizations
Lysate-pulsed DC were injected s.c. in the right flank of the
mice biweekly. Mice vaccinated before tumor challenge or for in vitro
restimulation studies were immunized one to three times with 1.0
x 106 (Fig. 1
) or 5.0 x
105 (
Figs. 2–7) DC. Tumor-bearing mice were
given 7.5 x 105 lysate-pulsed DC every 10
days starting 1 day after receiving a lethal number of C6VL tumor cells
(Fig. 8).
|
|
|
|
|
|
C6VL and MBL-2 tumor cells used for tumor challenges were expanded in vitro and then frozen in aliquots in liquid nitrogen. With the exception of vaccinating tumor-bearing mice, mice were challenged with tumor 14 days following the last vaccination. Two days before tumor challenge, an aliquot of tumor cells was thawed and cultured in CM for 48 h. The cells were collected by centrifugation, washed, and counted using a hemocytometer. The viability of the tumor cells before tumor challenge was generally >98% by trypan blue exclusion. The cells were diluted in HBSS such that a lethal number of tumor cells could be delivered i.p. in 500 µl. The 100% lethal doses for C6VL and MBL-2 were 1000–5000 and 5000 cells, respectively. Groups of 10 mice were used for survival analysis. Survival of tumor-challenged mice was monitored for 60 days following tumor challenge. Survival curves were constructed according to the Kaplan-Meier method. Statistical significance between vaccine groups was determined using the log-rank test and was achieved when p < 0.05.
C6VL staining with anti-C6VL Abs
Flow cytometric analysis of C6VL cells stained with hyperimmune serum was used to detect anti-C6VL Abs. Serum samples were obtained before vaccination and 10 days following the third vaccination with 5.0 x 105 unpulsed or lysate-pulsed DC or 35 µg TCR:KLH with QS-21. Serum samples from 10 mice from each experimental group were pooled, diluted 1:10 in PBS to a volume of 50 µl, and incubated in polystyrene tubes with 5.0 x 105 C6VL tumor cells for 30 min on ice. As a positive control, mAb 124-40 was added to preimmune serum at a concentration of 1.0 µg/1.0 x 106 cells. The cells were washed with PBS plus 1% FBS and C6VL-bound IgG and IgM were detected with 1.0 µg/1.0 x 106 cells FITC-conjugated goat anti-mouse IgG+IgM (H+L) Ab. The cells were washed three times with PBS plus 1% FBS, fixed in 200 µl of 2% paraformaldehyde (w/v), and analyzed by flow cytometry using a FACScan (BD Biosciences).
In vitro proliferation assay
Mice were vaccinated s.c. twice with 5.0 x 105 C6VL lysate-pulsed DC, biweekly. Lymph nodes from untreated mice or vaccine-primed lymph nodes (VPLN) were harvested 10 days following the second vaccine, processed gently into single-cell suspensions, and passed through 70-µm pore size nylon mesh filters to remove cell clumps or debris. Triplicate samples of 1.5 x 105 cells in CM were seeded in 96-well, flat-bottom plates. The lymphocytes were stimulated with irradiated C6VL (2500 rad) or MBL-2 (7500 rad) at stimulator:responder (S:R) ratios of 1:2, 1:10, and 1:50. Con A stimulation was used as a positive proliferation control and was titrated from 0.25 to 0.01 µg/ml in triplicate wells. Lymphocytes from untreated mice were used as a negative control. The cells were incubated at 37°C in a humidified incubator for 4 days, with 1 µCi/well [3H]thymidine added for the last 16 h. Cells were harvested onto glass-fiber filters using a FilterMate cell harvester (Packard Bioscience, Meriden, CT) and counted on a scintillation counter (Packard Bioscience). The stimulation index (SI) for each sample was calculated as: [([3H]thymidine incorporation (cpm) with stimulation - cpm without stimulation)/cpm without stimulation] and is represented as the mean of triplicate samples ± SD. Statistics between groups at each S:R ratio were measured at the 95% confidence level using Student’s t test.
In vitro cytokine secretion assay
VPLN cells were collected as described above. VPLN (2.0 x
106) in 1 ml CM were plated in triplicate wells
of a 24-well plate with either 1.0 x
106/well irradiated C6VL or irradiated MBL-2,
0.25 µg/ml Con A, or no stimulation (medium only) and incubated at
37°C for 96 h. Wells containing only irradiated C6VL or MBL-2
were assayed to control for cytokine secretion by stimulator cells.
Sandwich ELISAs for IL-4 and IFN- were performed as described in
Caltag’s protocol for cytokine ELISA with modifications. Briefly, rat
anti-mouse IFN- or rat anti-mouse IL-4 capture Abs at 0.25
µg/ml in 50 mM carbonate (50 µl/well), pH 9.5, was used to coat
wells of a 96-well Nunc microtiter plate. After washing, 100 µl of
undiluted supernatant from each VPLN restimulation were added to the
plates and serially diluted 1:2 over eight wells with PBS with 1% BSA.
Wells with known amounts of rIFN- or rIL-4 were included on the
plates to generate standard curves. Biotinylated second-stage rat Abs
against either IL-4 or IFN- were used to detect bound cytokine.
Streptavidin-HRP was used to detect bound second-stage Ab. The
peroxidase reaction was performed in a solution containing 50 mM sodium
citrate (pH 4.0), 150 µg/ml ABTS, and 0.01%
H2O2.
OD405–450 was measured on a Vmax Microplate Reader
(Molecular Devices, Menlo Park, CA). Data are represented as the
average of triplicate samples ± SD. Statistics between groups
were measured using Student’s t test. Statistical
significance was reached at p < 0.05.
In vivo depletion of CD8+ T cells
CD8+ T cells were depleted in vaccinated mice using anti-CD8.2 mAb 2.43. Mice were injected i.p. with 0.5 ml HBSS containing 250 µg of the mAb on days 6, 5, and 4 before tumor challenge and with four weekly injections thereafter. Nondepleted mice received the isotype-matched control mAb SFR8-B6 on the same schedule. The level of CD8+ T cell depletion and the maintenance of depletion were assessed by flow cytometry using a non-cross-blocking anti-CD8 mAb, 53-5.8. The analysis was performed on PBL, splenocytes, and lymph node cells 1 day before tumor challenge and on splenocytes and lymph node cells 1 day before each of the four weekly injections following tumor challenge.
Adoptive transfer of VPLN cells
Groups of 15 donor mice were vaccinated twice with C6VL lysate-pulsed DC or the control vaccine, MBL-2 lysate-pulsed DC. The DC vaccination consisted of 2.5 x 105 DC injected s.c. in the right flank and 1.25 x 105 DC injected in each hind footpad. Nine days after the last immunization, donor mice were sacrificed, and VPLN (right inguinal and both popliteal) were harvested. The lymphocytes were processed gently into single-cell suspensions and passed through 70-µm pore size nylon mesh filters to remove cell clumps and debris. B cells were depleted using a nylon wool column. The B cell-depleted lymphocytes were washed twice with HBSS. Lymphocytes were analyzed pre- and post-nylon wool depletion to determine B and T cell composition, as determined by CD3+ and CD19+ populations. Five million T cell-enriched VPLN in 200 µl HBSS were injected i.v. into groups of 10 naive recipients that were exposed to a sublethal dose of whole-body gamma irradiation (400 rad) 1 day previously. Immediately after injection, 3.0 x 104 U of human rIL-2 in 200 µl HBSS were injected i.p. into each recipient and control mouse. IL-2 injections continued every 12 h for 4 days (eight total doses) (38). One day following the last IL-2 injection, mice were challenged with a lethal dose of C6VL tumor and followed for survival.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Prior experiments have shown that C6VL TCR Id protein vaccines are
able to stimulate a protective immune response. To determine whether
lysate-pulsed DC vaccines were also effective in stimulating
anti-tumor immunity against C6VL, groups of 10 mice were vaccinated
three times with either 1.0 x 106 C6VL
lysate-pulsed DC (C6VL-DC) or TCR Id:KLH+QS-21. Unpulsed DC and HBSS
vaccines were administered to groups of mice as controls. Fourteen days
following the third vaccine, mice received a lethal dose of C6VL and
were monitored for survival. C6VL-DC vaccines stimulated high levels of
anti-tumor immunity in vaccinated mice, resulting in 100% survival
of the mice (Fig. 1). The protection generated by C6VL-DC vaccines was
superior to the response generated by TCR Id protein vaccines
(p = 0.0042). TCR Id:KLH+QS-21 vaccination did,
however, still significantly enhance the survival of tumor-challenged
mice compared with HBSS controls (p < 0.0001)
as was previously reported (11). The survival of C6VL-DC
recipients was significantly increased compared with recipients of
unpulsed DC (p<0.0001), which did not enhance the
survival of mice compared with HBSS injections
(p = 0.214).
Protection from tumor challenge is lysate specific
To determine whether the anti-C6VL tumor protection conferred
by DC vaccines was lysate specific, groups of 10 mice were vaccinated
three times with 5.0 x 105 C6VL-DC or
control lysate-pulsed DC. Control lysates were prepared from an
irrelevant T cell lymphoma (MBL-2), an irrelevant tumor (B16/F10
melanoma), normal lymphocytes, or irrelevant tissue (kidney). Unpulsed
DC vaccines and HBSS injections were included as negative controls
(Fig. 2A). Mice were challenged with a lethal dose of C6VL
14 days following the third vaccine. C6VL-DC stimulated high levels of
protection compared with unpulsed DC (p =
0.0002) or HBSS injection (p = 0.0018). DC
pulsed with control tumor or tissue lysates did not stimulate a
protective immune response against C6VL, and survival was
indistinguishable among the groups (p >
0.6).
The lysate specificity of the tumor protection was further evaluated in
a cross-challenge experiment. Groups of 20 mice were vaccinated twice
with 5.0 x 105 C6VL-DC or MBL-2-DC. Two
weeks following the last vaccine, 10 of the mice from each vaccine
group were injected with a lethal dose of C6VL, while the other 10 were
injected with a lethal dose of MBL-2. C6VL-DC protected eight of 10
mice challenged with C6VL. When C6VL-DC vaccine recipients were
challenged with MBL-2, the survival of the mice was significantly
reduced compared with that of C6VL-challenged mice
(p = 0.014; Fig. 2B). Similarly,
MBL-2-DC-vaccinated mice were poorly protected against C6VL as opposed
to MBL-2 challenge (p<0.001; Fig. 2B).
The lack of C6VL protection by MBL-2-DC vaccines was not due to a lack
of an immune response stimulated by the vaccine, as MBL-2-DC
vaccination protected 100% of mice that were lethally challenged with
MBL-2 (Fig. 2B).
Two and three C6VL lysate-pulsed DC vaccines stimulate equal levels of protection
Three immunizations with 5.0 x 105
C6VL-DC resulted in nearly all the mice surviving a lethal inoculation
of tumor cells (Fig. 2A). We sought to determine whether we
could use fewer injections of C6VL-DC and still protect the immunized
mice (Fig. 3). Groups of 10 mice were
vaccinated with 5.0 x 105 C6VL-DC one, two,
or three times before tumor challenge with C6VL. Mice immunized with
three vaccines of 5.0 x 105 MBL-2-DC were
included as a control group. Two and three vaccines of 5.0 x
105 C6VL-DC stimulated equal levels of protection
against lethal tumor challenge (p = 0.54),
which was significantly different from the control vaccine
(p = 0.0002). C6VL-DC given once before tumor
challenge stimulated intermediate levels of survival (40%) that were
significantly lower than those observed in the two- or three-vaccine
groups (p < 0.05).
C6VL lysate-pulsed DC do not stimulate significant humoral responses against the tumor
As reported previously, TCR Id:KLH+QS-21 vaccines stimulate high
titers of anti-C6VL TCR Id-specific Abs (10, 11). To
assess whether C6VL-DC vaccines stimulated C6VL-specific Ab production,
hyperimmune serum from vaccinated mice was used to stain C6VL cells
(Fig. 4). Mice were vaccinated three
times with 5.0 x 105 C6VL-DC, MBL-2-DC, or
unpulsed DC (Fig. 4A). Mice were also vaccinated with TCR
Id:KLH+QS-21 as a positive control for C6VL-specific Ab production
(Fig. 4B). Preimmune sera were obtained before the first
vaccination, and hyperimmune sera from 10 mice/group were collected and
pooled 10 days following the third vaccine. Preimmune serum spiked with
anti-C6VL TCR V mAb 124-40 was used as a positive control for
C6VL staining. Three injections of TCR Id:KLH+QS-21 stimulated a high
titer of anti-C6VL Abs. Hyperimmune serum from mice vaccinated with
C6VL-DC had only a small amount of Abs that bound to C6VL compared with
preimmune serum staining (mean log fluorescence (MLF), 6.69 compared
with 3.48). The same modest increase in C6VL staining was observed with
the hyperimmune serum from mice vaccinated with unpulsed DC (MLF 11.11)
and MBL-2-DC (MLF 5.52). An ELISA using plate-bound protein from C6VL
lysate also showed low amounts of nonspecific Ab binding to C6VL
proteins (data not shown).
C6VL lysate-pulsed DC stimulate tumor-specific cellular responses
To characterize the cellular immune response generated by C6VL-DC
vaccines, VPLN were harvested from mice and a standard cell
proliferation assay was performed (Fig. 5A). VPLN from mice vaccinated
twice with 5.0 x 105 C6VL-DC were
stimulated in vitro with either irradiated C6VL or MBL-2 control T cell
lymphoma cells. Background proliferation values for each stimulation
group were determined for triplicate wells containing responder cells
only. Naive lymphocytes were stimulated with irradiated C6VL as a
negative proliferation control. Con A stimulation of VPLN cells was
included as a positive proliferation control. Stimulation of VPLN cells
with irradiated C6VL tumor cells resulted in high levels of
proliferation at each S:R ratio. The SI were 12.5 ± 1.7,
8.75 ± 0.61, and 5.53 ± 0.87 for ratios of 1:2, 1:10, and
1:50, respectively. Stimulation of vaccine-primed cells with MBL-2
resulted in moderate levels of proliferation (SI of 4.27 ± 0.26,
3.69 ± 0.73, and 2.16 ± 0.19 for ratios of 1:2, 1:10, and
1:50, respectively) that were not statistically different from the
negative control (p = 0.67, 0.08, and 0.07 for
ratios of 1:2, 1:10, and 1:50, respectively). Stimulation of VPLN cells
with irradiated C6VL resulted in significantly higher levels of
proliferation than were observed upon stimulation with MBL-2
(p = 0.0041, <0.0001, and 0.020 for ratios of
1:2, 1:10, and 1:50, respectively). No proliferation of irradiated
tumor cells was detected.
Cytokine ELISAs were used to measure cytokine secretion by VPLN
stimulated by C6VL. Triplicate samples of VPLN cells from mice
vaccinated twice with 5.0 x 105 C6VL-DC
were stimulated in vitro with irradiated C6VL or MBL-2 cells at a ratio
of two VPLN cells to one irradiated tumor cell. Con A stimulation was
included as a positive control for IFN- secretion. Unstimulated VPLN
cells served as a negative control. Irradiated tumor cells were
incubated without responder cells to control for stimulator cell
cytokine secretion. Stimulation of VPLN with irradiated C6VL resulted
in high levels of IFN- secretion (66.7 ± 5.4 ng/ml) compared
with stimulation with MBL-2 (21.4 ± 3.4 ng/ml; p
= 0.022). IL-4 was also specifically secreted by VPLN upon ex vivo
stimulation with irradiated C6VL (<1 ng/ml), although at levels
80-fold lower than IFN- (data not shown). Stimulation of VPLN
cells with irradiated MBL-2 cells resulted in IFN- secretion not
statistically different from that observed in the unstimulated VPLN
cultures (p = 0.11) and did not result in
secretion of IL-4 above the limit of detection of the ELISA (0.11
ng/ml; data not shown). Irradiated tumor cells incubated without
responder cells did not secrete measurable levels of IFN- or
IL-4.
T cells are essential for tumor protection
The importance of the T cell-mediated immune response in
protection against C6VL tumor in vivo was determined by two different
approaches. An adoptive transfer experiment was performed (Fig. 6) to determine whether tumor protection
against C6VL could be solely conferred by immune T lymphocytes. VPLN
were harvested from donor mice vaccinated twice with 5.0 x
105 C6VL-DC. This cell population contained 38%
CD19+ B cells and 49% CD3+
T cells as determined by flow cytometry (data not shown). After
depleting B cells with a nylon wool column, the cells were >95% T
cells (data not shown). Five million T cell-enriched VPLN were
transferred i.v. into sublethally irradiated naive recipient mice.
Negative control mice were injected with 5.0 x
106 of similarly treated vaccine-primed
lymphocytes from MBL-2-DC-vaccinated donor mice. Groups of 10 mice that
were injected with C6VL-DC or HBSS but were not transferred served as
positive and negative vaccine controls, respectively. All recipient and
control mice received 30,000 U of recombinant human IL-2 beginning
immediately after the transfer continuing every 12 h for eight
total injections, based on reports that IL-2 induces the proliferation
of the transferred cells and prolongs their survival in vivo
(38). Four days after transfer, lymphocytes were isolated
from one mouse from each group and used in a proliferation assay. The
proliferative responses to irradiated C6VL or MBL-2 stimulation
observed in transfer recipients were less than one-half the responses
in representative mice vaccinated directly with C6VL-DC or MBL-2-DC
(data not shown). Five days following transfer or 1 day after the IL-2
injections were completed, mice were challenged with a lethal dose of
C6VL tumor (Fig. 6). Recipient mice that received immune lymphocytes
from C6VL-DC were protected from C6VL challenge at a level near that in
the positive control mice (p = 0.22),
demonstrating that the protective immunity could be transferred with
5.0 x 106 vaccine-primed lymphocytes.
Recipient mice that received immune lymphocytes from MBL-2-DC were not
protected from C6VL challenge compared with recipients of
C6VL-DC-primed lymphocytes (p = 0.00043), as
their survival was equivalent to that of HBSS-injected control mice
(p = 0.71).
The role of CD8+ effector cells in mediating
protective anti-tumor immunity was assessed by depleting
CD8+ T cells in C6VL-DC-immunized mice with an
anti-CD8 mAb (Fig. 7). Mice were
immunized twice with 5.0 x 105
lysate-pulsed DC and then treated with either an anti-CD8 mAb or an
isotype-matched control mAb. CD8+ cells were
depleted (>98%) in peripheral blood, spleen, and lymph nodes, and the
depletion was maintained for 30 days after tumor challenge. The
isotype-matched irrelevant mAb had no effect on the
CD8+ T cell population of mice. Mice vaccinated
with C6VL-DC that were not depleted of CD8+ T
cells were highly protected from a C6VL challenge compared with mice
vaccinated with MBL-2-DC (p = 0.000003).
Depletion of CD8+ T cells completely abrogated
tumor protection in mice vaccinated with C6VL-DC
(p = 0.000003).
C6VL lysate-pulsed DC vaccines are therapeutic in tumor-bearing mice
To determine whether C6VL-DC vaccines were effective in treating
pre-existing C6VL tumors, groups of 10 unvaccinated mice were
challenged with a lethal dose of C6VL and immunized for the first time
1 day thereafter. The mice were vaccinated with 7.5 x
105 C6VL-DC, MBL-2-DC, or TCR Id:KLH + QS-21
every 10 days for three immunizations. C6VL-DC vaccines significantly
enhanced the survival of tumor-bearing mice compared + mice vaccinated
with MBL-2-DC (p = 0.05) or TCR Id:KLH + QS-21
(p = 0.03), with 40% of the tumor-challenged
mice surviving (Fig. 8). TCR Id protein
vaccine recipients were not protected from existing C6VL tumor.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). We hypothesized that a vaccine designed to stimulate potent
cell-mediated responses against a broader array of tumor Ags would be
more efficacious.
DC are widely known to be potent inducers of CD4+
and CD8+ T cell-mediated responses against tumor
cells (14). In addition, evidence suggests that
vaccination with a mixture of Ags may yield enhanced anti-tumor
immunity as well as decrease the possibility of tumor escaping the
immune response (30, 32). The generation of a vaccine for
T cell lymphoma consisting of multiple known epitopes is currently
limited due to the lack of well-characterized, tumor-specific Ags.
However, loading DC with tumor RNA, DNA, or cell lysates allows a
similar effect without necessitating the discovery of each Ag. In this
study we directly compare the abilities of TCR Id:KLH+QS-21 and tumor
lysate-pulsed DC vaccines to stimulate protective immunity against the
murine T cell lymphoma, C6VL. We report that vaccination of mice with
C6VL lysate-pulsed DC stimulates potent anti-tumor protection that
surpasses immunity stimulated by TCR Id:KLH+QS-21 vaccines (Fig. 1).
Our goal in using C6VL lysate-pulsed DC to stimulate anti-tumor immunity was to have a broader array of tumor-specific Ags at which to target an immune response. However, a point that cannot be overlooked is that tumor lysates contain self-Ags as well as tumor-specific Ags. The benefits of using whole tumor cell lysates as Ag sources for loading DC have been demonstrated in many cases without the development of autoimmune responses toward self-Ags (25, 39). Recently, Vierboom et al. (40) demonstrated that expression of some self Ags can serve as targets for CTL-mediated destruction without demonstrable damage to normal tissues. Notwithstanding, skin depigmentation (41), autoimmune diabetes, and myocarditis (42) have been induced in mice vaccinated with tissue-specific Ags. C6VL is a CD4+ T cell malignancy. Therefore, we were particularly interested in whether lymphocyte populations were depleted in our immunized mice. Based on our data, C6VL-DC vaccines did not promote the development of autoreactivity against self-CD4+ T cells or other immune cell populations in the spleen, lymph nodes, or peripheral blood (data not shown).
Immunizations with C6VL-DC vaccines stimulated lysate-specific survival
in vivo (Fig. 2). Injections of DC pulsed with melanoma lysate, normal
lymphocyte lysate, or kidney tissue lysate did not protect mice from
C6VL challenge, indicating that C6VL lysate contains antigenic epitopes
capable of stimulating C6VL protection that are not highly expressed in
nonmalignant tissues or by all malignancies. Vaccination of mice with
DC pulsed with lysates prepared from the control T cell lymphoma line,
MBL-2, did not protect mice against tumor challenge. Thus, the Ags
stimulating protective anti-tumor immunity against C6VL are not
shared by all T cell lymphoma tumors. Indeed, mice vaccinated with
MBL-2 lysate-pulsed DC were protected against subsequent MBL-2 tumor
challenge, but not against C6VL challenge (Fig. 3B),
demonstrating that tumor lysate-pulsed DC vaccines could be efficacious
in stimulating lysate-specific, anti-tumor responses in other T
cell lymphomas as well. Similar findings have been reported for the use
of primary multiple myeloma (33) and B cell chronic
lymphocytic leukemia (34) lysate-pulsed DC to stimulate
patient CTL activity against autologous tumors in vitro. In these
studies primary tumor lysate-pulsed DC stimulated CTL activity against
autologous malignant cells, but not against autologous nonmalignant
cells and rarely against allogeneic tumor cells. Collectively, these
data argue that lymphoma lysate-pulsed DC as therapeutics would require
the preparation of vaccines on a per-patient basis.
The finding that 100% of the mice vaccinated three times with 1.0
x 106 lysate-pulsed DC survived subsequent tumor
challenge was surprising (Fig. 1). Vaccinating mice with DC derived and
loaded with Ag under serum-free conditions ensured that protection from
tumor challenge was not due to an immune response generated against FBS
proteins on C6VL cells cultured in serum-containing media (Figs. 2A, 3, 4, and 6–8). Decreasing the number of C6VL-DC
delivered to mice to 5.0 x 105 for three
vaccines did not result in a significant decline in tumor protection
(Fig. 2A). Furthermore, two vaccines of 5.0 x
105 C6VL-DC stimulated levels of tumor protection
equal to three vaccines (Fig. 3), demonstrating that we could be even
less aggressive in our vaccination protocol. Based on these data, we
modified our vaccination protocol to two vaccines of 5.0 x
105 DC for the experiments depicted in Figs. 2B and 5–7. The delivery of one C6VL-DC vaccine before
tumor challenge generated levels of protection similar to those
observed when three TCR Id:KLH+QS-21 vaccines were delivered. Whether
this result is indicative of an enhanced ability of DC to prime immune
responses compared with TCR Id or of a broader immune response
generated against multiple or more potent Ags is unclear, and further
work is being performed to examine this question.
C6VL-DC vaccines did not stimulate strong humoral immune responses
against C6VL (Fig. 4A). Further, Ab production did not
correlate with the protective ability of the C6VL-DC vaccine. Instead,
our data demonstrate that a Th1-type cellular immune response is
required to mediate C6VL-DC-induced tumor protection. The cellular
response was characterized ex vivo by high levels of tumor-specific
proliferation (Fig. 5A) and IFN- release (Fig. 5B) by VPLN. In vivo, we demonstrated that adoptively
transferred immune T cells were sufficient to protect naive mice from
tumor challenge (Fig. 6), and that the effector mechanism stimulated by
C6VL-DC vaccines was dependent upon CD8+ T cells
(Fig. 7). Additional studies indicated that NK cells do not contribute
to the effector mechanism generated by C6VL-DC. NK cells were not
activated in the spleens or lymph nodes of C6VL-DC recipients, and the
survival of vaccinated mice was not influenced by the depletion of
NK1.1+ cells before C6VL challenge (data not
shown). The role of CD4+ T cells in the
anti-tumor immunity generated by C6VL-DC vaccines was not tested in
our experiments, as anti-CD4 Abs would also deplete C6VL cells used
for tumor challenge.
Immunotherapeutic approaches for T cell lymphoma are likely to be most
effective for the clearance of minimal residual disease in patients
following conventional therapies. To more closely model the clinical
situation of a patient with minimal residual disease, we determined the
efficacy of the C6VL-DC vaccine in mice already carrying a lethal
number of C6VL cells. Naive mice or recipients of control vaccines
succumb to lethal dose C6VL challenge in 30 days. Despite the
aggressive nature of C6VL growth, C6VL-DC vaccines were able to
stimulate a protective immune response quickly enough to significantly
enhance the survival of tumor-bearing mice (Fig. 8). These data
indicate that lysate-pulsed DC vaccines may have clinically relevant
therapeutic potential in patients with pre-existing disease. Future
experiments using this vaccine protocol will focus on the use of
cytokine adjuvants to enhance the anti-tumor activity of C6VL-DC
vaccines in the setting of pre-existing T cell lymphoma.
Collectively, C6VL-DC vaccines have proven to stimulate immune responses superior in their potential to prevent future tumor growth and clear existing C6VL T cell lymphoma compared with previously reported TCR Id protein and TCR Id adenoviral vaccines. The studies presented herein provide evidence that vaccination with T cell lymphoma lysate-pulsed DC may allow enhanced efficacy in the clinical treatment of T cell malignancies and result in increased disease-free survival rates of patients without necessitating the identification of tumor-specific Ags or patient-specific Id protein expression.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Erin Gatza, Graduate Program in Immunology, University of Michigan, Veterans Administration Ann Arbor Health Care Systems Research Service 11R, Ann Arbor, MI 48185. E-mail address: egatza{at}umich.edu
3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; CM, complete medium; DC, dendritic cells; C6VL-DC, C6VL lysate-pulsed DC; MAC, metal affinity column; MBL-2-DC, MBL-2 lysate-pulsed DC; MLF, mean log fluorescence; SI, stimulation index; S:R ratio, stimulator:responder ratio; TCR Id:KLH+QS-21, TCR Id conjugated to keyhole limpet hemocyanin and mixed with QS-21; VPLN, vaccine-primed lymph nodes.
Received for publication June 24, 2002. Accepted for publication September 3, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  F. Ito, Q. Li, A. B. Shreiner, R. Okuyama, M. N. Jure-Kunkel, S. Teitz-Tennenbaum, and A. E. Chang Anti-CD137 Monoclonal Antibody Administration Augments the Antitumor Efficacy of Dendritic Cell-Based Vaccines Cancer Res., November 15, 2004; 64(22): 8411 - 8419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. van Broekhoven, C. R. Parish, C. Demangel, W. J. Britton, and J. G. Altin Targeting Dendritic Cells with Antigen-Containing Liposomes: A Highly Effective Procedure for Induction of Antitumor Immunity and for Tumor Immunotherapy Cancer Res., June 15, 2004; 64(12): 4357 - 4365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Lambert, C. Y. Okada, and R. Levy TCR Vaccines against a Murine T Cell Lymphoma: A Primary Role for Antibodies of the IgG2c Class in Tumor Protection J. Immunol., January 15, 2004; 172(2): 929 - 936. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), or
MBL-2-DC and treated with an isotype-matched irrelevant mAb (SFR8-B6;
X). Two weeks following the last vaccination, mice were challenged with
5000 C6VL cells and were monitored for survival. CD8+ T
cells were depleted before tumor challenge, and depletion was
maintained for 30 days following tumor challenge.