| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,¶,
*
University of Pittsburgh Cancer Institute, and Departments of
Pathology,
Otolaryngology, and
Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15231; and
¶ Institute for Oncology and Radiology, Belgrade, Yugoslavia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has been generally presumed that the majority of tumor debris acquired by cross-presenting DCs is the result of either spontaneous tumor necrosis or specific T cell- or nonspecific NK cell-mediated tumor cell death. A far more efficient process can be envisioned that involves direct DC-mediated killing of tumor cells, with the subsequent ingestion of resulting dead cancer cells and associated tumor Ags. Indeed, it has been shown that human activated DCs acquire the ability to directly kill rare tumor cell lines (11, 12). However, it is unlikely that activated DCs are the physiological mediators of the presumed activity, because they are induced by cytokines produced in an immune response (1, 2, 3) and therefore could not be involved in the initiation of cross-priming. More likely, physiological mediators of the antitumor cytotoxicity might be immature DCs, as they are produced by normal hematopoiesis, reside in the periphery (1, 2, 3), and are able to both populate cancer tissues (13) and uptake cellular debris (5, 6, 7, 8, 9). This hypothesis has remained untested.
In this study, we determined that both in vitro and in vivo generated human blood-derived immature DCs are potent and promiscuous anticancer cytotoxic cells capable of inducing efficient and selective apoptotic death in a variety of human cancer cell lines and freshly isolated tumor cells, but not in normal cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following Abs were applied in this study: FITC-conjugated
anti-human CD3 (IgG1) mouse mAb, PE-conjugated anti-human CD56
(IgG1) mouse mAb, FITC anti-human CD56 (IgG2b) mouse mAb, FITC
anti-human CD16 (IgG1) mouse mAb, PE anti-human CD14 (IgG2a)
mouse mAb, FITC anti-human CD14 (IgG2a) mouse mAb, FITC
anti-human CD19 (IgG1) mouse mAb (all from BD Biosciences, San
Jose, CA); FITC anti-human CD80 (IgM) mouse mAb and PE
anti-human CD86 (IgG1) mouse mAb (Ancell, Bayport, MN); FITC
anti-human CD11c (IgG1) mouse mAb and PE anti-human CD33
(IgG2b) mouse mAb (Caltag Laboratories, Burlingame, CA); FITC
anti-human CD40 (IgG1) mouse mAb (Ancell); PE anti-human CD83
(IgG2b) mouse mAb (Immunotech, Marseille, France); FITC anti-human
HLA-ABC class I (IgG2a) mouse mAb (Serotec, Burlington, Ontario,
Canada); PE anti-human HLA-DR (IgG2a) mouse mAb and
biotin-conjugated anti-human IL-3R
(IgG2a) mouse mAb (BD
Biosciences); FITC anti-human TCR
(IgGb) mouse mAb, FITC H130
anti-human CD45 (IgG1) mouse mAb, PE H130 human DC45 (IgG1) mouse
mAb, and conjugated isotype control mAbs (Caltag Laboratories);
anti-human CD3 (IgG1) mouse mAb, anti-human CD5 (IgG1) mouse
mAb, anti-human CD19 (IgG1) mouse mAb, anti-human CD56 (IgG2a)
mouse mAb, anti-human CD16 (IgG1) mouse mAb, and
anti-glycophorin A (IgG1) mouse mAb (DAKO, Carpinteria,
CA).
The following cytokines and ligands were used: recombinant human GM-CSF and recombinant human IL-4 (both kindly provided by Schering-Plough Research Institute, Kenilworth, NJ), and recombinant human trimeric CD40 ligand (CD40L; kindly provided by Immunex, Seattle, WA).
The following inhibitors of apoptosis were used: caspase-8 inhibitor IETD-fluoromethyl ketone (fmk), caspase-3 inhibitor DEVD-CHO, caspase-9 inhibitor Ac-LEHD-CHO (Calbiochem, San Diego, CA), pancaspase inhibitor Z-Val-Ala-Asp(OMe)-fmk (Z-VAD-fmk; Enzyme Systems Products, Livermore, CA), and adenine nucleotide translocase inhibitor bongkrekic acid (Biomol, Plymouth Meeting, PA).
Isolation of monocytes
Monocytes
(CD5-CD3-TCR-CD19-CD56-CD16-CD14+)
were purified from normal human peripheral blood mononuclear leukocytes
(PBMNL) to 95% purity using a modification of the previously
described negative immunoselection technique with Ab-coated magnetic
beads (14).
Isolation of DCs
DCs were purified from PBMNL as 90% pure populations of
CD3-TCR-CD14-CD19-CD16-CD56-CD11b-CD4+HLA-DR+
cells by the use of a MACS Blood Dendritic Cell Isolation kit (Miltenyi
Biotec, Auburn, CA), as described in the manufacturer’s protocol. To
obtain highly purified (i.e., 95%) populations of DCs, the enriched
DC populations were simultaneously labeled with FITC-conjugated
anti-TCR, anti-CD14, anti-CD19, anti-CD56, and
PE-conjugated anti-HLA-DR mAbs and sorted as
TCR-CD14-CD19-CD56-HLA-DR+
cells using a FACStarPlus cell sorter (all mAbs
and the cell sorter were from BD Biosciences).
DC cultures
Immature DCs were generated using a slight modification of the previously described technique (15). Briefly, freshly purified monocytes were resuspended (12.5 x 106 cells/2.5 ml) in AIM-V medium containing 2.5% FCS (both from Life Technologies, Long Island, NY), placed into T25 tissue culture flasks (Falcon, BD Labware, Franklin Lakes, NJ) and incubated at 37°C for 2 h. Following this incubation, the nonadherent cells were removed and the adherent cells were washed carefully five times with warm (37°C) RPMI 1640 (Life Technologies) medium. After the last washing, the adherent cells were supplemented with AIM-V 2.5% FCS medium containing 1000 U of IL-4 and 1000 U of GM-CSF and cultured for 5–7 days.
Mature DCs were generated from day 5 immature DCs by an additional 2-day incubation in the presence of IL-4, GM-CSF (1000 U/ml), and CD40L (2 µg/ml) (16). Following the above-described two-step purification of monocytes, including negative selection on immunomagnetic beads and adherence, the isolated adherent DC precursors (monocytes) as well as monocyte-derived DCs were consistently 98% pure populations of CD14+ and CD3-CD14-CD19-CD56-CD16-MHC-ABC+MHC-DR+ cells, respectively.
Cell lines and freshly isolated tumor cells
All cell lines were of human origin and mycoplasma free. Normal cell lines included skin keratinocytes (NHEK-Ad), dermal fibroblasts (NHDF-Ad), epidermal melanocytes (HMEM-Neo), mammary epithelial cells (Clonetics, BioWhittaker, Walkersville, MD), HUVEC (Cell Systems, Kirkland, CA), and T cell blasts (PBMNL stimulated for 7 days with 10 µg/ml Con A). Leukemia cell lines used in this study were K562 myeloid leukemia, Daudi Burkitt’s B cell lymphoma, MOLT-4 acute lymphoblastic leukemia, and Jurkat T cell leukemia (American Type Culture Collection (ATCC), Manassas, VA). Solid tumor-derived cell lines included gliomas P303, P319 (University of Pittsburgh Cancer Institute (UPCI), Pittsburgh, PA), and SNB19 (National Institutes of Health, Bethesda, MD); melanomas FemX (ATCC), Pmel 136.34, and Pmel 255.1 (UPCI); breast carcinomas BT-20, MCF-7, and SK-BR-3 (ATCC); lung squamous cell carcinomas LC 226, LC 358 and LC 596 (UPCI); lung small cell carcinomas LC H69 and LC H345 (UPCI); HR gastric carcinoma (UPCI); colon carcinomas LS-174 and LS-180 (NeoRx, Seattle, WA); SK-OV-3, Caov-3, and OVCAR-3 ovarian carcinomas (ATCC); renal cell carcinoma (UPCI); and squamous cell carcinomas of the head and neck (SCCHN) PCI-1, PCI-4A, PCI-4B, PCI-6A, PCI-6B, PCI-13, PCI-15A, PCI-15B, PCI-22A, PCI-22B, PCI-30, and PCI-37A (UPCI). The cell lines were grown under standard cell culture conditions, using the optimal culture conditions, as previously described (17). Fresh viable tumor cells were isolated directly from surgical specimens of SCCHN primary tumors by collagenase digestion and purification with annexin V magnetic beads (MACS; Miltenyi Biotec).
Cytotoxicity assays
51Cr release and [3H]thymidine release assays. 51Cr release and [3H]thymidine release assays were performed as previously described (17).
MTT assay. MTT assay, previously used to measure antitumor cell-mediated cytotoxicity (18), was modified for adherent effector cells. In addition to the previously used controls background of medium alone and total viability/spontaneous death of untreated target cells, we used a new control, background of effector cells. Experimental wells contained medium and both DCs and tumor cells. The percentage of cytotoxicity was calculated using the following formula: % cytotoxicity = (TS - BG0) - (E - BGEC)/(TS - BG0) x 100, where BG0 is background of medium alone, TS is total viability/spontaneous death of untreated target cells, BGEC is background of effector cells, and E is experimental well.
NuMA release assay. Nuclear matrix protein (NuMA) release assay was used to measure decay of the essential cellular protein and thus to demonstrate cell death using the NMP 41/7 ELISA kit (Oncogene Research Products, Boston, MA), according to the manufacturer’s specifications. DCs and tumor cells were coincubated for 72 h. Spontaneous release of NuMA from target cells alone, spontaneous release of NuMa from effector cells alone, and total release of NuMA from target cells induced by irradiation with ultraviolet C rays represented control samples. Release of NuMA following coincubation of DCs and tumor cells at various DC:tumor (E:T) ratios was experimental sample. Cytotoxicity was calculated according to the following formula: % cytotoxicity = E - (S1 + S2)/(T - S1) x 100, where S1 is spontaneous release of NuMA from target cells alone, S2 is spontaneous release of NuMa from effector cells alone, T is target cells induced by irradiation with ultraviolet C rays, and E is experimental sample.
Annexin V assays. Annexin V assays were used to detect target cells showing externalization of the phosphatidylserine on the outer leaflet of cell membrane, which represents an early feature of apoptosis (19). Target cells were exposed for 4 h to DCs or DC culture-conditioned medium. After removal of the effectors, target cells were maintained in culture for an additional 20 h to allow for apoptosis to occur. Tumor cells were harvested by gentle trypsinization and were then stained with 1 µg/ml FITC-conjugated Annexin V (Molecular Probes, Eugene, OR) or 10 µg/ml propidium iodide (PI) and PE- or FITC-conjugated anti-CD45 mAb, respectively. Cells were analyzed by flow cytometry as previously outlined (17).
DiOC6(3) assessment. 3,3'-Dihexyloxacarbocyanine iodide (DiOC6(3)) assessment of changes of the mitochondrial transmembrane potential, which characterize apoptosis, was performed as previously described (20). Following the induction of killing, as described above for the annexin V assay, but before flow cytometry analysis, target cells were stained with 40 nM DiOC6(3) solution for 15 min at 37°C and then with PE-conjugated anti-CD45 mAb.
TUNEL assays. TUNEL assays were performed by staining nuclear DNA in situ with the death detection kit (Roche Diagnostic Systems, Indianapolis, IN), followed by flow cytometry analysis, as per the manufacturer’s instructions. Tumor cell death was induced by DCs as described above for the annexin V assay, and the resulting cells were first stained with PE-conjugated anti-CD45 mAb and then fixed with 4% paraformaldehyde, labeled with TUNEL, and analyzed by flow cytometry (17).
Internucleosomal DNA fragmentation. The internucleosomal DNA fragmentation in target cells exposed to DCs was assessed by a modified DNA laddering assay. Nuclear DNA was labeled in viable target cells by 5-bromo-2'-deoxyuridine (BrdU; Boehringer Mannheim-Roche, Indianapolis, IN). Apoptosis and internucleosomal DNA fragmentation in the BrdU-labeled target cells were induced by their coincubation with DCs. E:T ratios and the time of the coincubation were as indicated. After the coculture, the cells were harvested and their DNA was extracted and purified using the manufacturer’s procedure for the TACS DNA Laddering Apoptosis Detection kit (R&D Systems, Minneapolis, MN). Samples of purified DNA (0.75 µg) were then loaded on 1% agarose gel, and horizontal gel electrophoresis was performed for 4 h at room temperature. DNA was then transferred from the gel onto uncharged nylon membrane (Millipore, Bedford, MA) and BrdU-labeled DNA was detected by standard Western blotting procedures using sequential incubations of the membrane with primary anti-BrdU mAb (Boehringer Mannheim-Roche), biotin-conjugated secondary goat anti-mouse Ig polyclonal Ab (Jackson ImmunoResearch Laboratories, West Grove, PA), streptavidin-HRP, HRP substrate, and ECL reagent (Trevigen, Gaithersburg, MD).
Flow cytometry
All analyses were performed using a FACScan (BD Biosciences) flow cytometer. Phenotypes of monocytes and DCs were determined by direct two-color staining with fluorochrome-conjugated mAbs specific for the lineage markers of T cells, B cells, NK cells, monocytes, and DCs, as previously described (21).
Statistical analysis
LU20/107 effector cells were determined using the formula 107/(T x X20), where T is the number of target cells and X20 is the estimated E:T ratio at which 20% of the target cells were killed. Statistical analyses of results were performed using the Wilcoxon’s signed-rank pair tests. To determine whether the data provide evidence for differences in profiles of percent killing as a function of E:T ratio, multivariate permutation methods were used. Differences were considered significant when the value of p was < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
First, we examined whether in vitro generated, monocyte-derived
DCs could mediate death of cancer cells and, if they could do so,
whether death was the result of cell necrosis or apoptosis. We produced
highly pure populations (95%) of
CD3-CD14-CD19-CD56-
immature and mature DCs from purified CD14+ blood
monocytes by in vitro culture and stimulation with GM-CSF plus IL-4
and/or GM-CSF plus IL-4 plus CD40L, respectively. The two DC
populations displayed predictably distinct morphology (data not shown),
distinct phenotypes (i.e., immature DCs were
CD83- and
CD80lowCD86lowCD40lowMHC
class IintMHC class IIint,
while mature DCs were CD83+ and
CD80highCD86highCD40highMHC
class IhighMHC class
IIhigh) (Fig. 1
),
and distinct functions (i.e., immature DCs exhibited high phagocytic
and intermediate MLR-inducing activities, while mature DCs had low
phagocytic and high MLR-inducing activities) (Ref. 3 and
data not shown).
|
). In all the assays applied, DCs
mediated significant anticancer cytotoxic activity, effective at low
E:T cell ratios (i.e., 0.1:1–3:1). Thus, DCs were capable of inducing
in cancer cells substantial fragmentation and release of
[3H]thymidine-labeled DNA (16–24%) within
1 h (Fig. 2A), the formation of typical DNA ladders
over 2–8 h of coculture (Fig. 2B), and a significant
increase of TUNEL incorporation into nuclear DNA following 24 h of
the cellular interaction (Fig. 2C). These results
unequivocally demonstrated that DCs effectively induced
apoptosis-specific internucleosomal fragmentation of DNA into 200-bp
fragments and their oligomers in the nuclei of cancer cells. Next, we
demonstrated that DCs induced 
30% of cancer cells to become
reactive with annexin V (Fig. 2D) but only 5% of cancer
cells to be stained with PI (data not shown), after 4 or 24 h of
incubation. Therefore, this interaction provoked apoptosis-specific
phosphatidylserine externalization on the tumor cell membrane. DCs also
induced a large proportion of cancer cells to become nonreactive with
MTT (Fig. 2E), DiOC6(3) (Fig. 2F), or rhodamine 123 (data not shown) within 3–24 h of
their coincubation. These results showed that DCs effectively promoted
apoptosis-specific mitochondrial damage and dysfunction in cancer cells
by affecting mitochondrial dehydrogenases (assessed in MTT assay)
and/or mitochondrial transmembrane potential (18), as
measured in DiOC6(3) and rhodamine 123 assays.
DCs also lysed 20–30% of cancer cells in either 24- or 72-h
51Cr release (Fig. 2G) or NuMA release
(Fig. 2H) assays, respectively. These results indicated that
DCs mediated disruption of cancer cell membrane and disintegration of
nuclear proteins, respectively, at later time points of the
interaction. Notably, no killing of tumor target cells was detected in
assays measuring necrosis, such as 4-h 51Cr
release (17) or PI uptake (data not shown). Therefore, a
substantial proportion of cancer cells that are exposed to DCs appears
to be killed, and this killing is mediated by apoptotic
mechanism(s).
|
The cell stress-induced mitochondrial pathway and the death
receptor-transduced pathway are main signaling mechanisms of
apoptosis (22, 23). The former involves an early
activation of the caspase-9 (22), while the
latter involves an early activation of the caspase-8
(23). Each of two pathways is able to activate the
caspase-3 and other common effector caspases. In addition, they can
activate and amplify each other. To assess the potential role of two
apoptotic signaling pathways and activation of caspases in DC-mediated
killing of cancer cells, we examined the use of relevant caspases and
involvement of mitochondria by applying the specific inhibitors (Table I). We found that pharmacological
inhibitors of the caspase-8 (IETD-fmk), caspase-9 (Ac-LEHD-CHO),
mitochondrial permeability transition pore (bongkrekic acid), caspase-3
(DEVD-CHO), and pan-caspases (Z-VAD-fmk) inhibited a notable proportion
of DC-mediated killing of cancer cells (49, 41, 52, 57, and 67%,
respectively). These data show that the two main apoptotic pathways and
activation of caspases have critical roles in DC induction of apoptosis
in cancer cells. The findings also indicate that in the course of
DC-mediated killing of cancer cells either of two pathways is primarily
and independently activated or one of the pathways is primarily
activated while the other is secondarily activated.
|
To determine the time dependency of killing of cancer cells by
DCs, we performed kinetic experiments. In MTT assays, we observed
substantial killing of cancer cells by 30 min and an increase of the
activity for up to 3 h of coincubation (Fig. 3). After 3 h, no further increase
was evident. Similarly, in DNA laddering assays, the bands of 200-bp
DNA fragments and their oligomers were readily visible after 2 h,
reaching peak levels after 8 h of coincubation (Fig. 2
B). Therefore, the killing of tumor cells mediated by DCs
was very rapid.
|
To evaluate the in vivo relevance of our in vitro findings, we
examined whether freshly isolated, noncultured, normal blood donor DCs
could kill cancer cells. The DCs directly obtained from PBMNL had a
typical immature DC phenotype. Thus, they were
CD3-TCR-CD14-CD19-CD16-CD56-CD11b-CD4+HLA-DR+
(Fig. 4A and data not shown). They expressed low levels of
CD80, CD86, and CD40, and moderate levels of MHC class I and class II
molecules, but did not express the CD83 marker of mature DCs (Fig. 4A). Assessments of the
anticancer cytotoxic activity of these DCs in 3- (Fig. 4B)
and 24-h (data not shown) MTT assays revealed that they were also able
to induce death in cancer cell targets. Therefore, in vivo and in vitro
generated immature DCs were similar in their ability to mediate death
of cancer cells. These observations suggest that this DC effector
function might be physiologically relevant.
|
To assess the range of target cells that are susceptible to
killing by DCs, we next tested cytotoxicity of in vitro generated
immature DCs against several normal and cancer cell lines and freshly
isolated cancer cells, using 3-h MTT, 1-h
[3H]thymidine release, and 24-h
51Cr release assays. Six normal cell lines, 36
malignant cell lines, and four different samples of fresh SCCHN cells,
directly obtained from surgical specimens of primary tumors of head and
neck cancer patients, were evaluated in total (Fig. 5). Normal proliferating endothelial
cells, 35 of 36 tested leukemia and cancer cell lines, and four of four
freshly isolated cancer cell samples were efficiently killed by DCs, in
marked contrast to normal fibroblasts, keratinocytes, melanocytes,
mammary epithelial cells, and autologous T cell blasts, which were not
harmed by immature DCs. The only cancer cell line found to be
relatively resistant to killing by DCs was PCI-30 SCCHN. These data
demonstrate that immature DCs are promiscuous anticancer cytotoxic
cells, which selectively kill cancer cells and spare normal cells.
|
In contrast to immature DCs, their direct precursors, freshly
isolated blood monocytes, did not show cytotoxic activity against
cancer cells in our experiments (data not shown). Therefore, the
anticancer killing ability appears to be acquired by immature DCs.
Next, we tested the anticancer cytotoxic activity of mature DCs and
compared it with that of immature DCs. In all three cytotoxicity assays
used, including the 1-h [3H]thymidine release,
24-h MTT, and 24-h 51Cr release assays, mature
DCs exhibited significant cytotoxic reactivity against cancer cells
(Table II). However, the levels of
killing by mature DCs were 4- to 4.9-fold lower than those observed for
donor-matched immature DCs. This indicates that maturation of DCs,
known to be accompanied by their multiple phenotypic and functional
changes, including that of Ag-presenting activities, is also
accompanied by a significant decrease of their tumoricidal
function.
|
To examine the mechanisms underlying the cytotoxic activity of DCs
against cancer cells, we evaluated the cytotoxic activity associated
with paraformaldehyde-fixed DCs and DC culture-conditioned medium. It
has been previously shown that cell fixation eliminates secretory
activity and preserves cell membrane-bound cytotoxic ligands
(17), while conditioned cell culture media may contain
secreted soluble forms of cytotoxic ligands (24). We
observed that both fixed DCs and DC culture-conditioned medium were
cytotoxic for cancer cells (Table III).
However, these cytotoxic activities were lower than those of control
unmanipulated DCs. Similarly, inhibition of cellular secretion by
elimination of extracellular Ca2+, using the
Ca2+ chelation with EGTA or EDTA, only partially
suppressed DC-mediated killing of cancer cells (data not shown). In
contrast, the apoptotic activity of DC supernatants was efficiently
inhibited by the proteases papain or trypsin (Fig. 6). The inhibition showed a dose
dependency and was complete in the presence of 
250 µg of the
proteases per 1 ml of DC supernatant, whereas it gradually decreased at
lower concentrations of the enzymes. These data suggest that the
mediators of immature DC-associated tumoricidal activity are both cell
membrane-bound and secreted proteins.
|
|
To determine whether the observed anticancer apoptosis-inducing
activity is a general function of DCs, we analyzed the killing ability
of in vitro generated, monocyte-derived, immature DCs obtained from the
peripheral blood of 25 different normal blood donors. We found that DCs
isolated from all individuals evaluated were cytotoxic against cancer
cell targets (Fig. 7). These data show
that tumoricidal activity is an essential function of normal human
immature DCs. We also observed a significant individual variability in
the tested population of effectors obtained from different donors. This
suggests that DC-mediated killing is genetically and/or environmentally
determined.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies have indicated that certain subsets of rat DCs (26) and activated human DCs might be cytotoxic for rare tumor cell targets (11, 12). From these studies it was not clear whether the killing of cancer cells mediated by DCs represented an infrequent in vitro artifact, a rudimentary activity, or a major activity of DCs. Furthermore, the mechanisms and biological significance of such activity were not clear. Our current data and those presented in a companion manuscript (25) support the conclusion that human nonactivated immature DCs are cytotoxic in vitro in low E:T ratios for the large majority of cancer cell lines and freshly isolated cancer cells, but not for normal cells. In addition, our data demonstrate that anticancer activity of DCs is a constitutive function of these effector cells in normal individuals. Therefore, cytotoxic activity mediated by DCs is an efficient, largely promiscuous (i.e., with regard to tumor target range), selective, and essential anticancer effector function. Our finding that DCs also kill proliferating "normal" endothelial cells may suggest that DCs are also capable of mediating antitumor activity by destroying newly forming tumor blood vessels.
The finding that immature DCs are more effective at mediating tumor cell apoptosis than mature DCs is intriguing. Coupled with the known ability of immature, but not mature, DCs to readily ingest apoptotic bodies, this provides a logical and economical paradigm for effective cross-presentation of tumor-derived epitopes important for the induction of effective antitumor cellular immunity (5, 6, 7, 8, 9). The observed DC-mediated killing is consistent with an innate immune effector function of these cells and may represent a missing link between innate and adaptive anticancer immunity.
Considering the in vivo distribution of DCs in normal tissues as well as their increased numbers in cancer tissues of patients exhibiting better clinical prognoses (1, 2, 3, 13), our findings strongly indicate that DC-mediated anticancer cytotoxic activity might be critically and directly involved in elimination of newly generated cancer cells and in control of tumor progression in vivo. Furthermore, this mechanism may prove to be important in the periodic restimulation of memory CD4 and CD8 antitumor T cell responses in patients who evolve micrometastatic disease but who remain clinically tumor free.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Nikola L. Vujanovic, University of Pittsburgh Cancer Institute, Biomedical Science Tower W1046, 211 Lothrop Street, Pittsburgh, PA 15213. E-mail address: vujanovicnl{at}msx.upmc.edu
3 Abbreviations used in this paper: DC, dendritic cell; PBMNL, peripheral blood mononuclear leukocyte; SCCHN, squamous cell carcinoma of the head and neck; fmk, fluoromethyl ketone; Z-VAD-Fmk, Z-Val-Ala-Asp(OMe)-fmk; NuMA, nuclear matrix protein; PI, propidium iodide; BrdU, 5-bromo-2'-deoxyuridine; CD40L, CD40 ligand; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide.
Received for publication July 16, 2001. Accepted for publication December 5, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
v5 and CD36 and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.-related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN--stimulated human dendritic cells to tumor cells. J. Immunol. 166:5407.. J. Exp. Med. 179:1109.12, Fas ligand, and TNF-related apoptosis-inducing ligand. J. Immunol. 168:1823.This article has been cited by other articles:
|
|
 |
|
  M. Terme, G. Mignot, E. Ullrich, M. Bonmort, V. Minard-Colin, A. Jacquet, J. L. Schultze, G. Kroemer, C. Leclerc, N. Chaput, et al. The Dendritic Cell-like Functions of IFN-Producing Killer Dendritic Cells Reside in the CD11b+ Subset and Are Licensed by Tumor Cells Cancer Res., August 15, 2009; 69(16): 6590 - 6597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Himoudi, M. Yan, G. Bouma, D. Morgenstern, R. Wallace, B. Seddon, J. Buddle, A. Eddaoudi, S. J. Howe, N. Cooper, et al. Migratory and Antigen Presentation Functions of IFN-Producing Killer Dendritic Cells Cancer Res., August 15, 2009; 69(16): 6598 - 6606. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Burt, G. Plitas, J. A. Stableford, H. M. Nguyen, Z. M. Bamboat, V. G. Pillarisetty, and R. P. DeMatteo CD11c identifies a subset of murine liver natural killer cells that responds to adenoviral hepatitis J. Leukoc. Biol., October 1, 2008; 84(4): 1039 - 1046. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chauvin and R. Josien Dendritic Cells as Killers: Mechanistic Aspects and Potential Roles J. Immunol., July 1, 2008; 181(1): 11 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wei, H. Wang, B. Lu, B. Li, S. Hou, W. Qian, K. Fan, J. Dai, J. Zhao, and Y. Guo Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells Cancer Res., May 15, 2008; 68(10): 3854 - 3862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Papewalis, B. Jacobs, M. Wuttke, E. Ullrich, T. Baehring, R. Fenk, H. S. Willenberg, S. Schinner, M. Cohnen, J. Seissler, et al. IFN-{alpha} Skews Monocytes into CD56+-Expressing Dendritic Cells with Potent Functional Activities In Vitro and In Vivo J. Immunol., February 1, 2008; 180(3): 1462 - 1470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Srivastava, C. Varalakshmi, and A. Khar The Ischemia-Responsive Protein 94 (Irp94) Activates Dendritic Cells through NK Cell Receptor Protein-2/NK Group 2 Member D (NKR-P2/NKG2D) Leading to Their Maturation J. Immunol., January 15, 2008; 180(2): 1117 - 1130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Stary, C. Bangert, M. Tauber, R. Strohal, T. Kopp, and G. Stingl Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells J. Exp. Med., June 11, 2007; 204(6): 1441 - 1451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chen, E. Calomeni, J. Wen, K. Ozato, R. Shen, and J.-X. Gao Natural killer dendritic cells are an intermediate of developing dendritic cells J. Leukoc. Biol., June 1, 2007; 81(6): 1422 - 1433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Berhanu, J. Huang, S. M. Alber, S. C. Watkins, and W. J. Storkus Combinational FLt3 Ligand and Granulocyte Macrophage Colony-Stimulating Factor Treatment Promotes Enhanced Tumor Infiltration by Dendritic Cells and Antitumor CD8+ T-Cell Cross-priming but Is Ineffective as a Therapy. Cancer Res., May 1, 2006; 66(9): 4895 - 4903. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Stasiolek, A. Bayas, N. Kruse, A. Wieczarkowiecz, K. V. Toyka, R. Gold, and K. Selmaj Impaired maturation and altered regulatory function of plasmacytoid dendritic cells in multiple sclerosis Brain, May 1, 2006; 129(5): 1293 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Huang, T. Tatsumi, E. Pizzoferrato, N. Vujanovic, and W. J. Storkus Nitric Oxide Sensitizes Tumor Cells to Dendritic Cell-Mediated Apoptosis, Uptake, and Cross-Presentation Cancer Res., September 15, 2005; 65(18): 8461 - 8470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Trinite, C. Chauvin, H. Peche, C. Voisine, M. Heslan, and R. Josien Immature CD4-CD103+ Rat Dendritic Cells Induce Rapid Caspase-Independent Apoptosis-Like Cell Death in Various Tumor and Nontumor Cells and Phagocytose Their Victims J. Immunol., August 15, 2005; 175(4): 2408 - 2417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. V. Shurin, R. Ferris, I. L. Tourkova, L. Perez, A. Lokshin, L. Balkir, B. Collins, G. S. Chatta, and M. R. Shurin Loss of New Chemokine CXCL14 in Tumor Tissue Is Associated with Low Infiltration by Dendritic Cells (DC), while Restoration of Human CXCL14 Expression in Tumor Cells Causes Attraction of DC Both In Vitro and In Vivo J. Immunol., May 1, 2005; 174(9): 5490 - 5498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schmitz, S. Zhao, Y. Deuse, K. Schakel, R. Wehner, H. Wohner, K. Holig, F. Wienforth, A. Kiessling, M. Bornhauser, et al. Tumoricidal Potential of Native Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity J. Immunol., April 1, 2005; 174(7): 4127 - 4134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Tatsumi, J. Huang, W. E. Gooding, A. Gambotto, P. D. Robbins, N. L. Vujanovic, S. M. Alber, S. C. Watkins, H. Okada, and W. J. Storkus Intratumoral Delivery of Dendritic Cells Engineered to Secrete Both Interleukin (IL)-12 and IL-18 Effectively Treats Local and Distant Disease in Association with Broadly Reactive Tc1-type Immunity Cancer Res., October 1, 2003; 63(19): 6378 - 6386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
