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Immunology |
University of Pittsburgh Cancer Institute [T. K. H., N. M., G. D., H. K., T. L. W.] and Department of Pathology [T. L. W.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-IFN enzyme-linked immunospot and
cytotoxicity assays. Unmodified tumor cells were found to be the best
stimulators of antitumor activity of the established T-cell line, and
ATCs alone were minimally stimulatory. However, DCs that ingested ATCs
were able to present tumor antigens to CTLs, and DCimm and
DCmat were almost equally stimulatory. When DCs plus
various tumor-derived preparations were used as antigen-presenting
cells with autologous HLA-A2+ T cells obtained from normal
donors, DCs that had ingested ATCs were more effective in generating
CD8+ CTLs than tumor cells alone or DCs pulsed with tumor
lysates. The results indicate that human DCs fed with ATCs and then
matured effectively generated T cell-mediated antitumor responses
in vitro. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, it has been demonstrated that human DCs can acquire viral antigens from apoptotic cells and stimulate antigen-specific MHC class I-restricted CD8+ T cells to mediate antiviral CTL responses (7 , 8) . In this and other in vitro models (9, 10, 11) , apoptotic death was a critical trigger for the antigen processing pathway, and apoptotic cells were a preferred source of antigen, because antigens derived from necrotic cells were not presented on the MHC class I molecules. In addition, the use of ATCs fed to DCs led to effective priming of tumor-specific CTLs in several recent in vivo animal studies (11 , 12) .
In the present study, the ability of human DCs coincubated with ATCs or tumor cell lysates to stimulate tumor-specific T cells was evaluated in an in vitro TAA presentation model. We compared monocyte-derived DCimm and DCmat for their capacity to phagocytose apoptotic SCCHN and cross-present TAAs to tumor-specific CTLs. In a separate in vitro model of TAA cross-priming, we observed that DCs fed with apoptotic SCCHN were capable of cross-priming naïve T cells obtained from HLA-A2-matched healthy donors for tumor-specific responses.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human DCs were generated according to a modified method by Sallusto and Lanzavecchia (14) . Briefly, peripheral blood or a leukapheresis product was obtained from HLA-A2+ normal donors, and PBMCs were isolated by sedimentation over Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ). The PBMCs were incubated for 1 h at 37°C in AIM-V medium, and nonadherent cells were removed by gentle washing with warm medium. The remaining (adherent) cells were incubated in AIM-V medium + 1000 units/ml granulocyte macrophage colony-stimulating factor (Immunex, Seattle, WA) and IL-4 (Schering Plough, Kennilworth, NJ). The cultures were supplemented with additional cytokines on day 4 of culture. DCs were harvested at day 6 using cold Hanks’ solution (Life Technologies, Inc.).
The PCI-13-specific CTL bulk cell line was established from peripheral
blood lymphocytes of a patient with SCCHN, as described previously
(15)
. The CTLs were thawed and maintained in the presence
of IL-2 and IL-4 with repeated sensitization on tumor cell monolayers.
The CD8+ T cell line was derived from the
original bulk T cell line by negative selection, using anti-CD4
Ab-coated magnetic beads. The CD8+ cells
recognized a shared antigen on SCCHN: they lysed autologous SCCHN
targets as well as HLA-A2+ allogeneic (but not
HLA-A2-) SCCHN targets, including PCI-13
(16)
. This lysis was blocked with anti-CD3, anti-CD8,
anti-TCR 
/ß, and anticlass I MHC Abs (w6/32) as well as anti
HLA-A2 Abs. The CTL line or clones derived from it did not lyse K562 or
Daudi targets, normal tissue cells, or HLA-A2+
phytohemagglutinin-stimulated T cells. For the experiments
described here, the CD8+ T cell line was cultured
in AIM-V containing 10% FCS and 300 IU IL-2/IL-4. It was stimulated
twice with -irradiated PCI-13 cells (10,000 rad) and incubated for 7
days before being used in 51Cr-release assays or
ELISPOT assays.
Cytokines and Antibodies.
The following cytokines were used for cell cultures: IL-1ß (National
Cancer Institute, Biological Resources Branch, Frederick, MD), IL-2
(Chiron-Cetus, Emeryville, CA), IL-4 (Schering Plough), IL-6 (Sandoz,
Basle, Switzerland), PGE2 (Sigma Chemical Co.,
St. Louis, MO), IFN- (Genentech, San Francisco, CA), granulocyte
macrophage colony-stimulating factor (Immunex), and TNF- (Knoll
Pharmaceuticals, Whippany, NJ).
The antibodies used for staining of cells or blocking of responses were either unlabeled or labeled with PE or FITC and included: anti-MHC class I mAbs (HB95; w6/32), as well as anti-HLA-A2 mAbs (BB7.2) obtained from Dr. Albert DeLeo (University of Pittsburg Cancer Institute); anti-MHC class II, anti-CD14, anti-CD25, and anti-CD80 (Becton Dickinson, San Jose, CA); anti-CD40 and anti-CD86 (Ancell, Bayport, MN), anti-CD83 mAbs (Immunotech, Marseille, France) and respective IgG isotype controls (either from Becton Dickinson or PharMingen, San Diego, CA).
DCs, lymphocytes, or tumor cells (2 x 105/200 µl) were incubated with mAbs on ice for 30 min and washed twice in PBS containing 0.1% (w/v) BSA and 0.1% (w/v) NaN3. After staining, the cells were fixed with 1% (w/v) paraformaldehyde in PBS for 30 min at room temperature prior to flow cytometry. Flow cytometry analysis was performed as described previously (17) , using a FACScan (Becton Dickinson) equipped with a single 488-nm argon ion laser. At least 10,000 events were acquired for each sample.
Apoptosis Induction and Detection.
PCI-13 cells cultured in AIM-V were irradiated with 1500
µW/cm2 UVB (UVB bulb BLE-GT 302; Spectronics
Corp., Westbury, NY) for 2 min or 15 min. To minimize the UVB absorbing
effect of phenol red in AIM-V, the medium level was reduced to a
minimum during irradiation. Apoptosis was detected by
DiOC6 staining and in the TUNEL assay.
Additionally, apoptotic bodies were stained by propidium iodide (10
µg/ml; Sigma Chemical Co.) for 15 min at room temperature after cell
membrane permeabilization and examined by confocal microscopy, as
described below.
For DiOC6 staining, aliquots (50 nM) of the lipophilic cationic fluorochrome DiOC6 (Molecular Probes, Eugene, OR) were added to 5 x 105 cells/ml of culture medium and incubated for 15 min at room temperature. In apoptotic cells, mitochondria show a decrease in green fluorescence intensity, which is quantitated by flow cytometry (18) . For the TUNEL assay, tumor cells were fixed with 2% (w/v) paraformaldehyde in PBS and permeabilized with 0.1% (w/v) sodium citrate in PBS containing 0.1% (w/v) Triton X-100 for 7 min on ice. After washing, cells were incubated with FITC-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase enzyme solution for 1 h at 37°C, using reagents purchased from Boehringer Mannheim (Indianapolis, IN). After incubation, the cells were washed, and 10,000 events were acquired and analyzed by flow cytometry. Negative controls included cells incubated without the enzyme in the labeling buffer, and positive controls included the same cells treated with DNase (Sigma Chemical Co.).
Tumor Lysates.
Lysates were produced by exposing tumor cells to four rapid
freeze-and-thaw cycles until the cell membrane integrity was lost. Cell
debris was removed by centrifugation (30 min at 15,000 x g), and the protein content was measured by a
Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Aliquots of
the lysate (1 mg/ml) were used to pulse DCs.
Tumor Uptake by DCs.
To study their uptake by DCs, tumor cells were stained green
with 2 µg/ml DiOC16 (Molecular Probes) for 30
min at 37°C in PBS and washed three times in medium before induction
of apoptosis. After a 12–24-h incubation in medium to allow for the
tumor cells to undergo apoptosis, they were cocultured with DCs at
various DC:tumor cell ratios. The cells were harvested 2–18 h later,
and DCs were stained with PE-labeled anti-CD80 Ab. Two-color flow
cytometry was performed to determine the percentage of cells that
phagocytosed apoptotic SCCHN, based on the number of double-positive
cells (green/red). The same experiments were also
performed at 4°C to show that the uptake of tumor cells by DCs was
inhibited at low temperatures.
To prepare cells for confocal microscopy, sterilized glass coverslips were placed on the bottom of a 6-well plate. DiOC16-stained PCI-13 cells were added to these wells and exposed to UVB light as described above. DCs were added 12–24 h after induction of apoptosis. After overnight coculture, the glass coverslips were removed and washed with PBS. The DCs attached to glass were stained with anti-CD80 Ab in combination with a secondary Cy3-conjugated rabbit antimouse Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). After fixation with 1% (w/v) paraformaldehyde, coverslips were mounted on a slide and analyzed by confocal laser scanning microscopy at x600 original magnification (Leica TCS NT confocal LSM; Leica Lasertechnik, Heidelberg, Germany). Images were edited using the Adobe Photoshop software program (Adobe Systems, Mountain View, CA).
Maturation of DCs.
Maturation of DCs was induced by the addition of proinflammatory
cytokines (10 ng/ml IL-1ß, 1000 units/ml IL-6, 10 ng/ml TNF-, and
1 µg/ml PGE2), as described previously
(19)
. Changes in expression of MHC class I and II
molecules as well as CD14, CD25, CD40, CD80, and CD86 on DCs were
monitored by flow cytometry, and the level of expression is shown as
mean fluorescence intensity for DCimm and
DCmat.
Processing and Cross-Presentation of Tumor-derived Epitopes.
DCimm and DCmat that had
phagocytosed ATCs or DCs pulsed with tumor lysates, as described above,
were harvested, washed, and counted. To determine the ability of these
DCs to process and cross-present tumor-derived epitopes to the
PCI-13-specific CD8+ CTL line and, thus, be
recognized by the CTLs, the DCs were used as stimulators in 24-h
ELISPOT assays for IFN- production or as targets in 4 h
51Cr-release cytotoxicity assays.
IFN- ELISPOT Assay.
The ELISPOT assay was performed as described elsewhere
(20)
. Briefly, wells of 96-well plates with nitrocellulose
membrane inserts (Millipore, Bedford, MA) were coated with 50 µl of
primary Ab solution [10 µg/ml in 1x PBS (pH 7.4), clone MAB1-D1K;
Mabtech, Nacka, Sweden] and incubated for 24 h at 4°C. Then,
the plates were washed four times with PBS, and a 100-µl aliquot of
AIM-V supplemented with 10% (w/v) human serum was added for 1–3 h to
block nonspecific binding. Next, 1 x 104 to 2 x 104 responder T cells with an equal number of
stimulator cells (PCI-13 or DC) were added in a final volume of 200
µl of AIM-V medium. The assay was performed in quadruplicate wells
for each experimental condition. The plates were then incubated in a
humidified atmosphere of 5% CO2 in air at 37°C
for 24 h. After the incubation period, cells were removed by
washing the plates six times with 0.05% (w/v) Tween 20 in PBS (Fisher
Scientific, Pittsburgh, PA). A 50-µl aliquot of biotinylated
secondary anti-IFN- Ab (2 µg/ml, clone Mab7-B6–1; Mabtech) was
added to each well. The plates were again incubated in a humidified
atmosphere of 5% CO2 in air at 37°C for 2 h. The washing steps were repeated, and after a 1-h incubation at room
temperature with the avidin-peroxidase complex reagent (Vectastain
Elite Standard ABC-Kit; Vector Laboratories, Burlingame, CA), the
plates were washed again three times with PBS/0.05% Tween and then
three times with PBS alone. Aliquots (100 µl) of the
aminoethylcarbazole staining solution (Sigma Chemical Co.) were added
to each well to develop the spots. The reaction was stopped after 4–6
min under running tap water. The spots were counted by
computer-assisted image analysis (Zeiss ELISPOT 4.14.3.; Zeiss,
Jena, Germany). If the mean number of spots against DCs plus tumor
preparation (experimental values) was significantly different from the
mean number of spots against nonpulsed DCs (background values), as
determined by a two-tailed Wilcoxon rank sum test, the background
values were subtracted from the experimental values.
For Ab-blocking experiments, PCI-13 cells were preincubated with w6/32 Ab, anti-HLA-A2 (BB7.2) Ab or anti-HLA-DR Ab (clone L 243; kindly provided by Dr. Albert DeLeo), or purified mouse IgG1 (clone S1-68.1; PharMingen) for 30 min. As a control for the assay reproducibility, PBMCs obtained from the same normal donor and cryopreserved in a series of vials were used each time the assay was performed. These control cells were thawed, washed, and used at the concentration of 2 x 104/ml in AIM-V in the ELISPOT assay. The control PBMCs were stimulated with phorbol 12-myristate 13-acetate (1 ng/ml) and ionomycin (1 µM), both from Sigma Chemical Co. The coefficient of variation for this assay was determined as 15% based on 30 independent determinations.
Cytotoxicity Assay.
The 4-h 51Cr-release assay was performed at four
E:T ratios, as described previously (21)
. Briefly, targets
(PCI-13, K562 or DC +/- different tumor preparations) were labeled
with 51Cr for 45 min at 37°C, washed, and added
to wells of 96-well plates (1 x 104 cells/well). Effector T cells were then added
to give various E:T ratios. The assays were performed in triplicate.
The percentage of specific lysis was calculated according to the
formula:
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Enrichment of CD8+ Cells.
Cultured PBMCs or the bulk CTL line were enriched for
CD8+ cells by positive immunoselection, using
magnetic beads (MiniMacs; Miltenyi Biotec, Auburn, CA) according to the
manufacturer’s recommendations. The purity of selected
CD8+ cell fractions was checked by flow
cytometry.
Cross-Priming of T Cells.
PBMCs were obtained as leukapheresis products from normal
HLA-A2+ donors, and monocytes were separated by
adherence to plastic. The adherent cells were used for DC generation,
whereas the recovered lymphocytes were stimulated with autologous DCs,
which have ingested ATCs at the ratio of 10:1. The lymphocytes were
cultured in AIM-V medium + 10% human serum supplemented with
25 ng/ml IL-7 for the first 72 h and then in AIM-V supplemented
additionally with 20 IU/ml IL-2 for the remaining time in culture. The
lymphocytes were restimulated after the first week and weekly
thereafter for up to four total stimulations. Responses of T cells to
PCI-13, PCI-13 + w6/32 Ab, PCI-13 + HLA-A2.1,
PCI-13 + anti-HLA-DR, and PCI-13 + IgG or the
controls HR (HLA-A2+ gastric carcinoma), Fem-X
(HLA-A2+ melanoma), and
HLA-A2+ normal human fibroblasts were tested in
24-h ELISPOT and cytotoxicity assays.
Statistical Analysis.
A two-tailed Wilcoxon rank sum test was performed to analyze ELISPOT
data. Unpaired two-tailed Student’s t test was used for
statistical analysis of flow cytometry data.
Differences were considered significant when P was <0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or by increased TUNEL reactivity, which measures DNA
fragmentation (Fig. 1B)
. To allow for apoptosis to
progress, UVB-treated tumor cells were incubated for 4–48 h before
flow cytometry analysis. Optimal effects were observed following a
24–48-h incubation of UVB-treated PCI-13 cells (Fig. 1)
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We next coincubated apoptotic PCI-13 cells with
DCimm or DCmat for various
periods of time to determine the optimal conditions for internalization
of ATCs. We observed that DCimm ingested ATCs
already after 2 h of coincubation (data not shown). Overnight
coculture of viable PCI-13 cells with DCs resulted in a significant
increase in the double-stained cell population (27%), as shown in Fig. 2
. This proportion of double-stained DCs was further increased to 69%
when DCs were cocultured with ATCs. After a 24-h coincubation, no
"free" ATCs could be detected (Fig. 2F)
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Cross-Presentation by DCs of TAAs to PCI-13-specific
CD8+ T Cells.
To determine whether human DCs ingesting ATCs are able to process and
present tumor-derived epitopes to T cells, we used an in
vitro antigen-presentation model, consisting of a tumor-specific
CTL line and monocyte-derived DCs. The semi-allogeneic,
HLA-A2-restriced and PCI-13-specific CTL line was generated as
described previously by us (15
, 16)
, and cryopreserved T
cells were thawed, maintained in culture, and restimulated with PCI-13
tumor cells at weekly intervals to expand the cells, as needed. The
characteristics of this CTL line were extensively evaluated and
described before (16
, 22)
. Before their use as responders
in cross-presentation experiments with DCs, the expanded CTLs were
tested in 4-h 51Cr-release assays against PCI-13
as well as K562 targets. As shown in Fig. 4
, the CTLs were HLA-A2 restricted, and they efficiently killed
HLA-A2+ PCI-13 but not K562 targets. Not shown
are results demonstrating that the CTLs did not lyse
HLA-A2+ T cell blasts,
HLA-A2+ tumor targets that were not SCCHN or
HLA-A2- SCCHN targets.
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, the highest number of IFN- spots was always observed with viable
PCI-13 cells (P < 0.05), indicating that
tumor cells were the best stimulators. This finding was confirmed in
the cytotoxicity assays performed in parallel with ELISPOT assays (data
not shown). This was not surprising because the CTL line was generated
by coincubation with PCI-13 and had been repeatedly stimulated by
irradiated PCI-13 cells. ATCs alone were poor stimulators of CTLs,
possibly because these ATCs expressed significantly lower levels of MHC
class I molecules than PCI-13 tumor cells (mean fluorescence intensity
of 77 for viable PCI-13 cells versus 15 for ATCs and 10 for
IgG control). DCs ingesting ATCs or pulsed with tumor lysates were
recognized better than ATCs alone, and DCmat + ATCs tended to be somewhat more stimulatory than
DCimm, although the differences between
experiments using different DC preparations were not statistically
significant (Table 2)
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-irradiated
PCI-13 cells, ATCs alone, DCs + ATCs, DCs + nonapoptotic PCI-13, DCs pulsed with tumor lysates, or DCs alone as a
control. Various priming conditions +/- DCs were compared for the best
generation of tumor-reactive T cells, as detectable in ELISPOT assays.
The data in Table 3
indicate that in three of four cultures the percentage of
CD3+/CD8+ T cells was
increased after priming with DCs + ATCs compared with the
other priming conditions. Also, in two of four cultures primed with
irradiated tumor cells alone, increased proportions of natural
killer cells were observed. Overall, however, priming with
DCs + ATCs favored an enrichment in T cells.
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are shown in Fig. 5
. The ELISPOT results indicate that T cells derived by priming with
DCs + ATCs were significantly more responsive
(P < 0.05) to the tumor (PCI-13) than were T
cells derived by priming of PBMCs with -irradiated (viable) PCI-13
cells (Fig. 5A)
, ATCs alone (data not shown), or DCs alone
(Fig. 5, A and B)
. More importantly, the response
was blocked by anti-MHC class I and HLA-A2.1 mAbs but not isotype
control immunoglobulin. These T cells did not respond to irrelevant
HLA-A2+ HR (gastric carcinoma) or Fem-X
(melanoma) tumor cells, or HLA-A2+ normal
fibroblasts used as control (Fig. 5, A and C)
. In
only one of four cross-priming experiments -irradiated PCI-13 cells
were as stimulatory as DCs + ATCs.
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Most important, blocking experiments with anti-HLA class I and
anti-HLA-A2 mAbs confirmed that recognition of PCI-13 by these T cells
was MHC class I and HLA-A2 restricted, respectively (Fig. 5C)
. The results obtained with T2 cells, expressing
unoccupied HLA-A2 molecules, which were pulsed with fresh tumor lysate
and used as stimulators in ELISPOT assays, confirmed the results
obtained with PCI-13 or DC stimulators (data not shown). In a limited
number of experiments, we had sufficient numbers of T cells available
to perform cytotoxicity assays in addition to ELISPOT assays. Our data
consistently showed that DCs + ATCs generated MHC class
Irestricted CTLs, whereas the other priming regimens did not (data
not shown). Overall, these results clearly indicated that the
cross-priming protocol, using DCs + ATCs was optimal for the
generation of tumor-specific CTLs ex vivo. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Current evidence suggests that significant differences exist in the efficiency of TAA processing and presentation by human DCs, depending on the source or form of tumor-derived materials, the maturation stage of DCs, or responsiveness of T-cell populations available for stimulation with DCs (8, 9, 10, 11, 12 , 33 , 34) . These differences are likely to be important for the outcome of immunizations, and, thus, there exists a need to optimize the design of tumor vaccines, using different tumor preparations (tumor cell lysates, ATCs, whole tumor cells, tumor cell fractions) and ex vivo-generated DCs. Clearly, a selection of the optimal method for antigen delivery to be available for future DC-based vaccine clinical trials is important for their success.
In the present study, we have evaluated human monocyte-derived DCs for
their ability to: (a) take up ATCs; (b) present
tumor-derived epitopes to already sensitized and committed
tumor-specific T cells in the cross-presentation ex vivo
model; and (c) prime T cells from normal donors to develop
into antitumor effector cells in the cross-priming ex vivo
model. To this end, two in vitro models had been developed.
In the first model (cross-presentation) it was possible to evaluate
tumor-specific T-cell responses to human DCs presenting TAAs derived
from processed ATCs or tumor lysate. The CTLs used as responders in
this model recognized a shared antigen on HLA-A2+
SCCHN, as described previously (16)
, and again confirmed
in the current experiments. Human DCs were
HLA-A2+ semi-allogenic monocyte-derived APCs,
which were either immature or were matured by ex vivo
culture in the presence of proinflammatory cytokines. ELISPOT assays
for IFN- production or cytotoxicity assays were used to monitor
responses of CTLs to tumor-derived epitopes presented by DCs, tumor
cells alone, ATCs alone, or DCs + tumor lysates. This
in vitro cross-presentation model was used to quantitate the
magnitude of CTL responses, and it allowed for a comparison of
variously pretreated DCs for their capability to present a
tumor-derived epitope(s) to CTLs known to be able to recognize and
efficiently kill tumor cells.
Initially, while developing the model, it was necessary to show
that SCCHN cells, subjected to an apoptotic signal (UVB light), were
taken up by human monocyte-derived DCs. We showed a high level of
uptake of ATCs by these DCs, as determined by flow cytometry as well as
confocal microscopy. In fact, nearly all ATCs were taken up by DCs
during a 24-h period of coincubation. We also observed that maturation
of DCs was not visibly affected by the uptake of ATCs at the ATC:DC
ratio of 1:2. On the other hand, previous reports indicated that in
mice DC maturation was induced at a much higher (5:1) ATC:DC ratio,
whereas lower ratios, similar to those used in our experiments, failed
to mature DCs (11)
. To achieve maturation of human DCs,
we, therefore, resorted to the use of a mixture of proinflammatory
cytokines (19)
. When the ability of
DCmat to uptake ATCs and present TAAs to T cells
was compared with those of DCimm, it seemed that
the uptake of ATCs was found to be reduced after 8–12 h of
coincubation, but the T-cell stimulatory activity of
DCmat was improved as compared with that of
DCimm. Conceivably, up-regulation of
costimulatory molecules and MHC class I molecules or increased
stability of peptide-MHC-class I complexes on
DCmat could be responsible for this effect. In
addition, PGE2 in combination with TNF- could
synergistically induce high levels of IL-12 production in human
monocyte-derived DCs, stimulate T-cell proliferation (35)
,
and increase IFN- production by responder CD8+
T cells without inducing type 2 cytokines, as reported previously
(19)
.
In our cross-presentation model, DCmat ingesting ATCs were recognized by the PCI-13-specific T cells somewhat better than DCimm and ATCs, but untreated PCI-13 cells were always eliciting the best T-cell responses. This observation is as expected because the CTL line used was generated by IVS with PCI-13 cells and was repeatedly stimulated with PCI-13 cells at weekly intervals. Similar results were reported by Bellone et al. (9) in a murine system. It was interesting to note that ATCs alone were not stimulatory, possibly due to a dramatic decrease in the expression of MHC class I molecules in ATCs relative to PCI-13 cells, as observed by flow cytometry. Several recent reports indicated that ATCs have reduced immunogenicity compared with live cells (12 , 36 , 37) . Many different mechanisms have been proposed to account for this low immunogenicity (12 , 36 , 37) , but our findings of low levels of MHC class I expression in ATCs is a novel observation that seems to fit well with the requirement for DCs, which express both MHC and costimulatory molecules, to process and present these ATCs to generate an effective immune response. It is reasonable to assume that ATCs become immunogenic, when they are cross-presented to T cells by professional APCs equipped with an efficient antigen-processing and presenting machinery and expressing costimulatory molecules. Processing of phagocytosed ATCs by DCs yields epitopes that can access the MHC class I pathway via TAP (transporter of antigen-processing)-dependent mechanisms and are ultimately presented to and recognized by antigen-specific CTLs. This type of effective cross-presentation of murine TAAs or viral antigens by macrophages and DCs has been described previously in ex vivo experiments (7, 8, 9, 10) .
In the second in vitro model (cross-priming), we used DCs,
which had internalized ATCs or other tumor-derived epitopes, to
generate effector T cells able to recognize and kill PCI-13 targets. In
the recent in vivo experiments in rodents, Henry et
al. (11)
and Ronchetti et al.
(12)
described generation of tumor-specific CTLs, which
were able to mediate tumor rejection and induce long-term memory, when
using DCs + ATCs but not DCs pulsed with tumor extracts.
These studies in rodents indicated a superior ability of DCs, which had
internalized ATCs, to stimulate antitumor responses. Using human DCs
and autologous T cells obtained from the circulation of normal
HLA-A2+ donors, we demonstrated in the
cross-priming model that stimulation with DCs + ATCs yielded
T-cell lines strongly responsive to the tumor. To reach this
conclusion, we performed several cycles of IVS, under conditions
designed to compare -irradiated viable tumor cells, non-ATCs, ATCs
alone, DCs + ATCs, DCs ± non-ATCs, DCs + lysate, or DCs alone for the ability to prime naïve T
cells. This type of cross-priming was successful in generating
tumor-specific CTLs in all four attempts using
HLA-A2+ PBMCs of normal donors. Lymphocytes
cross-primed with DCs + ATCs contained the highest frequency
of IFN--producing T cells specific for the tumor. In only one of
four experiments (donor 4) -irradiated (viable) PCI-13 cells were as
stimulatory as DCs + ATCs. ATCs alone did not induce the
outgrowth of tumor-reactive T cells in our experiments. Interestingly,
DCs pulsed with tumor lysates showed lower, if any, cross-priming
capacity compared with DCs + ATCs. This finding was somewhat
surprising in view of the widespread practice of using tumor lysates
pulsed on DCs as a potentially effective antigen-delivery procedure for
CTL generation. On the other hand, it has been shown that the
administration of exogenous class I-restricted antigens in the form
that requires phagocytosis is essential for their effective
presentation to T cells (7
, 8)
. Moreover, Inaba et
al. (33)
have shown that phagocytosed cellular
fragments are 3000 times more efficient in forming MHC-peptide
complexes than the preprocessed peptide. Because Herr et
al.5
observed the induction of a strong CD4 response, but only a weak CD8
response, when loading DCs with lysates in an EBV/viral model, it is
possible that tumor-derived lysates primarily induce CD4 responses.
This possibility is currently under investigation in our
laboratory.
Overall, this study demonstrates that human monoctye-derived DCs that internalize and process ATCs can cross-prime T cells and generate more effective antitumor-specific T cells in vitro than viable tumor cells or tumor cell lysates pulsed on these DCs. Although these studies were performed in a semi-allogeneic setting, which could enhance T-cell activation and proliferation of both HLA-A2-restricted tumor-specific T cells as well as nonspecific T cells, the antitumor responses we measured in ELISPOT and cytotoxicity assays were mediated by tumor-specific, HLA-A2-restricted T cells. The results available from vaccination experiments in tumor-bearing rodents indicate that a similar approach may be successful in vivo (11 , 12) . Therefore, vaccination of cancer patients with ATCs + autologous DCs should be considered in the future as a reasonable therapeutic strategy, especially applicable when immunogenic tumor epitopes are not available.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant PO1-DE-12321 (to
T. L. W.). T. K. H. is funded by a postdoctoral training fellowship
from the Dr. Mildred Scheel Stiftung fur Krebsforschung (Grant
D/99/08916). 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at University of Pittsburgh Cancer Institute, W 1041
Biomedical Science Tower, 211 Lothrop Street, Pittsburgh, PA
15213-2582. Phone: (412) 624-0096; Fax: (412) 624-0264; E-mail: whitesidetl{at}msx.upmc.edu
4 The abbreviations used are: CTL, cytolytic
T lymphocytes; APC, antigen-presenting cell; Ab, antibody; ATC,
apoptotic tumor cell; DC, dendritic cell; DCimm, immature
DC; DCmat, mature DC; ELISPOT, enzyme-linked immunospot;
IL, interleukin; IVS, in vitro stimulation; mAb,
monoclonal Ab; PBMC, peripheral blood mononuclear cell; PE,
phycoerythrin; PG, prostaglandin; SCCHN, squamous cell carcinoma of the
head and neck; TAA, tumor-associated antigen; TNF, tumor necrosis
factor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling; DiOC6, 3,3'-dihexadecyloxacarbocyanine
perchlorate.
5 W. Herr, E. Ranieri, W. Olson, H. Zarour,
L. Gesualdo, and W. J. Storkus. Mature dendritic cells pulsed with
freeze-thaw cell lysates define an effective in vitro
vaccine designed to elicit EBV-specific CD4+ and
CD8+ T lymphocyte responses, submitted for publication.
Received 10/15/99. Accepted 5/ 4/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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V. S. Zimmermann, A. Bondanza, A. Monno, P. Rovere-Querini, A. Corti, and A. A. Manfredi TNF-{alpha} Coupled to Membrane of Apoptotic Cells Favors the Cross-Priming to Melanoma Antigens J. Immunol., February 15, 2004; 172(4): 2643 - 2650. [Abstract] [Full Text] [PDF]
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R. S. Goldszmid, J. Idoyaga, A. I. Bravo, R. Steinman, J. Mordoh, and R. Wainstok Dendritic Cells Charged with Apoptotic Tumor Cells Induce Long-Lived Protective CD4+ and CD8+ T Cell Immunity against B16 Melanoma J. Immunol., December 1, 2003; 171(11): 5940 - 5947. [Abstract] [Full Text] [PDF]
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T. Hayashi, T. Hideshima, M. Akiyama, N. Raje, P. Richardson, D. Chauhan, and K. C. Anderson Ex vivo induction of multiple myeloma-specific cytotoxic T lymphocytes Blood, August 15, 2003; 102(4): 1435 - 1442. [Abstract] [Full Text] [PDF]
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F. Moschella, B. Bisikirska, A. Maffei, K. P. Papadopoulos, D. Skerrett, Z. Liu, C. S. Hesdorffer, and P. E. Harris Gene Expression Profiling and Functional Activity of Human Dendritic Cells Induced with IFN-{alpha}-2b: Implications for Cancer Immunotherapy Clin. Cancer Res., June 1, 2003; 9(6): 2022 - 2031. [Abstract] [Full Text] [PDF]
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A. K. Nowak, R. A. Lake, A. L. Marzo, B. Scott, W. R. Heath, E. J. Collins, J. A. Frelinger, and B. W. S. Robinson Induction of Tumor Cell Apoptosis In Vivo Increases Tumor Antigen Cross-Presentation, Cross-Priming Rather than Cross-Tolerizing Host Tumor-Specific CD8 T Cells J. Immunol., May 15, 2003; 170(10): 4905 - 4913. [Abstract] [Full Text] [PDF]
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A. Bondanza, V. S. Zimmermann, G. Dell'Antonio, E. Dal Cin, A. Capobianco, M. G. Sabbadini, A. A. Manfredi, and P. Rovere-Querini Cutting Edge: Dissociation Between Autoimmune Response and Clinical Disease After Vaccination with Dendritic Cells J. Immunol., January 1, 2003; 170(1): 24 - 27. [Abstract] [Full Text] [PDF]
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J. Hu, N. M. Cladel, M. D. Pickel, and N. D. Christensen Amino Acid Residues in the Carboxy-Terminal Region of Cottontail Rabbit Papillomavirus E6 Influence Spontaneous Regression of Cutaneous Papillomas J. Virol., October 25, 2002; 76(23): 11801 - 11808. [Abstract] [Full Text] [PDF]
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G. Dotti, B. Savoldo, P. Yotnda, D. Rill, and M. K. Brenner Transgenic expression of CD40 ligand produces an in vivo antitumor immune response against both CD40+ and CD40- plasmacytoma cells Blood, June 17, 2002; 100(1): 200 - 207. [Abstract] [Full Text] [PDF]
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T. K. Hoffmann, J. Muller-Berghaus, R. L. Ferris, J. T. Johnson, W. J. Storkus, and T. L. Whiteside Alterations in the Frequency of Dendritic Cell Subsets in the Peripheral Circulation of Patients with Squamous Cell Carcinomas of the Head and Neck Clin. Cancer Res., June 1, 2002; 8(6): 1787 - 1793. [Abstract] [Full Text] [PDF]
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T. Yang, T. F. Witham, L. Villa, M. Erff, J. Attanucci, S. Watkins, D. Kondziolka, H. Okada, I. F. Pollack, and W. H. Chambers Glioma-associated Hyaluronan Induces Apoptosis in Dendritic Cells via Inducible Nitric Oxide Synthase: Implications for the Use of Dendritic Cells for Therapy of Gliomas Cancer Res., May 1, 2002; 62(9): 2583 - 2591. [Abstract] [Full Text] [PDF]
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M. Schnurr, C. Scholz, S. Rothenfusser, P. Galambos, M. Dauer, J. Robe, S. Endres, and A. Eigler Apoptotic Pancreatic Tumor Cells Are Superior to Cell Lysates in Promoting Cross-Priming of Cytotoxic T Cells and Activate NK and {gamma}{delta} T Cells Cancer Res., April 1, 2002; 62(8): 2347 - 2352. [Abstract] [Full Text] [PDF]
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X.-Q. Zhao, X.-L. Huang, P. Gupta, L. Borowski, Z. Fan, S. C. Watkins, E. K. Thomas, and C. R. Rinaldo Jr. Induction of Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+ and CD4+ T-Cell Reactivity by Dendritic Cells Loaded with HIV-1 X4-Infected Apoptotic Cells J. Virol., February 22, 2002; 76(6): 3007 - 3014. [Abstract] [Full Text] [PDF]
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B. M. Janjic, G. Lu, A. Pimenov, T. L. Whiteside, W. J. Storkus, and N. L. Vujanovic Innate Direct Anticancer Effector Function of Human Immature Dendritic Cells. I. Involvement of an Apoptosis-Inducing Pathway J. Immunol., February 15, 2002; 168(4): 1823 - 1830. [Abstract] [Full Text] [PDF]
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G. Lu, B. M. Janjic, J. Janjic, T. L. Whiteside, W. J. Storkus, and N. L. Vujanovic Innate Direct Anticancer Effector Function of Human Immature Dendritic Cells. II. Role of TNF, Lymphotoxin-{alpha}1{beta}2, Fas Ligand, and TNF-Related Apoptosis-Inducing Ligand J. Immunol., February 15, 2002; 168(4): 1831 - 1839. [Abstract] [Full Text] [PDF]
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P. Castiglioni, A. Martin-Fontecha, G. Milan, V. Tomajer, F. Magni, J. Michaelsson, C. Rugarli, A. Rosato, and M. Bellone Apoptosis-dependent Subversion of the T-Lymphocyte Epitope Hierarchy in Lymphoma Cells Cancer Res., February 1, 2002; 62(4): 1116 - 1122. [Abstract] [Full Text] [PDF]
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T. K. Hoffmann, D. J. Loftus, K. Nakano, M. J. Maeurer, K. Chikamatsu, E. Appella, T. L. Whiteside, and A. B. DeLeo The Ability of Variant Peptides to Reverse the Nonresponsiveness of T Lymphocytes to the Wild-Type Sequence p53264-272 Epitope J. Immunol., February 1, 2002; 168(3): 1338 - 1347. [Abstract] [Full Text] [PDF]
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 I. Renard, D. Mezzanzanica, S. Canevari, S. Ferrini, J. Boniver, P. Delvenne, and N. Jacobs Anti-CD3/Anti-Epidermal Growth Factor Receptor-Bispecific Antibody Retargeting of Lymphocytes against Human Neoplastic Keratinocytes in an Autologous Organotypic Culture Model Am. J. Pathol., January 1, 2002; 160(1): 113 - 122. [Abstract] [Full Text] [PDF]
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G. Pietra, R. Mortarini, G. Parmiani, and A. Anichini Phases of Apoptosis of Melanoma Cells, but not of Normal Melanocytes, Differently Affect Maturation of Myeloid Dendritic Cells Cancer Res., November 1, 2001; 61(22): 8218 - 8226. [Abstract] [Full Text] [PDF]
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M. J. Gough, A. A. Melcher, A. Ahmed, M. R. Crittenden, D. S. Riddle, E. Linardakis, A. N. Ruchatz, L. M. Emiliusen, and R. G. Vile Macrophages Orchestrate the Immune Response to Tumor Cell Death Cancer Res., October 1, 2001; 61(19): 7240 - 7247. [Abstract] [Full Text] [PDF]
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S. Liu, Y. Yu, M. Zhang, W. Wang, and X. Cao The Involvement of TNF-{{alpha}}-Related Apoptosis-Inducing Ligand in the Enhanced Cytotoxicity of IFN-{{beta}}-Stimulated Human Dendritic Cells to Tumor Cells J. Immunol., May 1, 2001; 166(9): 5407 - 5415. [Abstract] [Full Text] [PDF]
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S. Paczesny, S. Beranger, J.-L. Salzmann, D. Klatzmann, and B. M. Colombo Protection of Mice against Leukemia after Vaccination with Bone Marrow-derived Dendritic Cells Loaded with Apoptotic Leukemia Cells Cancer Res., March 1, 2001; 61(6): 2386 - 2389. [Abstract] [Full Text]
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T. K. Hoffmann, K. Nakano, E. M. Elder, G. Dworacki, S. D. Finkelstein, E. Appella, T. L. Whiteside, and A. B. DeLeo Generation of T Cells Specific for the Wild-Type Sequence p53264-272 Peptide in Cancer Patients: Implications for Immunoselection of Epitope Loss Variants J. Immunol., November 15, 2000; 165(10): 5938 - 5944. [Abstract] [Full Text] [PDF]
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