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IMMUNOBIOLOGY
From the Département de Pédiatrie, the
Unité d'Immunologie, the Unité de Thérapie
Cellulaire, and the Service de Génétique, Département
de Biologie Clinique, Institut Gustave Roussy, Villejuif, France; and
the Etablissement Français du sang, Créteil, France.
Natural killer (NK)/lymphokine-activated killer
(LAK) cell-based immunotherapy could be beneficial against
major histocompatibility complex class I-negative tumor
residual disease such as neuroblastoma (NB), provided that
interleukin 2 (IL-2) or surrogate nontoxic NK cell stimulatory
factors could sustain NK cell activation and survival in vivo. Here we
show that human monocyte-derived dendritic cells (MD-DCs) promote
potent NK/LAK effector functions and long-term survival, circumventing
the need for IL-2. This study demonstrates (1) the feasibility of
differentiating granulocyte colony-stimulating factor-mobilized hematopoietic peripheral blood stem cells
(PBSCs) into high numbers of functional MD-DCs and NK/LAK
cells in a series of 12 children with stage 4 neuroblastoma
(NB); (2) potent DC-mediated NK cell activation in autologous
settings; (3) the reciprocal capacity of NK/LAK cells to turn immature
DCs into maturing cells electively capable of triggering NK
cell functions; and (4) the unique capacity of maturing DCs to sustain
NK cell survival, superior to that achieved in IL-2. These data show a
reciprocal interaction between DCs and NK/LAK cells, leading to the
amplification of NK cell effector functions, and support the
implementation of DC/NK cell-based immunotherapy for purging the graft
and/or controlling minimal residual disease after autologous stem cell transplantation.
(Blood. 2002;100:2554-2561) Cell therapy based on autologous stem cell
transplantation (ASCT) following myeloablative treatments significantly
improves the prognosis of certain types of solid tumors. High-dose
chemotherapy (HDC) regimen and ASCT are indicated in children with
stage 4 neuroblastoma (NB).1,2 Nonetheless, tumor
relapses frequently occur, either from clinically occult residual
disease or from contaminating tumor cells in autologous grafts.
Postgraft immunointerventions consisting of adoptive transfer
of ex vivo-activated and/or antitumor effector cells derived from
peripheral blood stem cells (PBSCs) in conjunction with
lymphokines remain promising.
In the neuroblastoma model, several lines of evidence support the
notion that antitumor immune responses might be clinically beneficial
in that (1) spontaneous regressions are observed in Pepper
syndrome3; (2) tumor-infiltrating lymphocytes exhibited, in
some cases, oligoclonal T-cell repertoire4; and (3)
objective responses were reported following interleukin-2 (IL-2)
systemic administration.5 However, many in vitro studies
pointed to a critical susceptibility of neuroblastoma cell lines to
innate effectors, that is, natural killer (NK)/lymphokine-activated
killer (LAK) lysis.6,7 In the 1990s, attempts to
develop clinical trials using systemic high-dose IL-2 administration
with or without LAK cells after HDC plus ASCT have been
hampered by severe IL-2-related toxicities reported in such
children.8 Alternative strategies are clearly required to
boost innate effector functions against residual neuroblastoma.
Besides their pivotal role in promoting naive T-cell activation,
dendritic cells (DCs) are capable of triggering innate immune responses
in vitro and in vivo. We have initially demonstrated that murine bone
marrow-derived DCs (BM-DCs) promote resting NK cell activation, that
is, interferon- Here we analyzed the functionality and relevance of the reciprocal
DC/NK cell cross-talk in a preclinical study conducted in children with
NB. In a series of 12 children with stage 4 NB, we show that
granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs
can be differentiated into high numbers of functional MD-DCs and NK/LAK
cells and that coculture of autologous DCs/LAK cells allows reciprocal
activation of both subsets, long-term IL-2 deprivation, and sustained
NK/LAK antitumor effector functions. These data show that in
NK/LAK-based immunotherapy, DCs can substitute for NK cell stimulatory
soluble factors, which were not devoid of toxicity.
Patients and sample collection
Culture procedures
Monocyte-derived dendritic cell (MD-DC) derivation.
Total leukocytes were incubated at 5 × 106/mL in
75-cm2 culture flasks in AIM-V medium (Gibco-BRL, Paisley,
United Kingdom) supplemented with 10% fetal calf serum (FCS; Pan
Biotech, Aidenbach, Germany) for 2 hours at 37°C.
Nonadherent cells were removed; adherent cells were washed 3 times in
1 × phosphate-buffered saline (PBS; Gibco, Paisley, United
Kingdom) and cultured until day 7 in AIM-V with 10% FCS supplemented
with recombinant human IL-4 (rhuIL-4, 1000 U/mL; Schering
Plough, Kenilworth, NJ) and recombinant human
granulocyte-macrophage CSF (rhuGM-CSF, 1000 U/mL; Leucomax, Schering Plough) in order to derive MD-DCs at an immature stage (iDCs).
In some experiments, MD-DCs were induced to maturation (mDCs) with the
use of lipopolysaccharide (LPS, 20 µg/mL; Sigma Chemical, St Louis,
MO) added in cultures on day 5 for 48 hours.
IL-2 activation for NK/LAK generation.
Mononuclear cells were plated in 24-well culture plates at
1 × 106 CD3+ and CD56+
lymphocytes per milliliter in RPMI 1640 (Gibco-BRL) supplemented with
10% human AB serum (Institut Jacques Boy, Reims, France). The
rhuIL-2 (1000 IU/mL, Proleukin; Chiron, Ratingen,
Germany) was added only once, at day 0 of the culture.
Coculture of MD-DCs with NK/LAK.
At day 7 or 8, IL-2-activated bulk NK/LAK cells (or immunopurified
CD3 Immunophenotyping
Functional assays Cytotoxicity assays.
The cytolytic activity of NK/LAK cell cultures was measured in a
standard 4-hour 51Cr-release assay against K562 and Daudi
cell line targets. In addition, the lytic activity was also tested
against 2 established NB cell lines, IGR-NB1 (NB-91) and IGR-NB2 (GAU).
Furthermore, the susceptibility of freshly isolated NB cells to
autologous NK/LAK killing was assessed with the use of short-term NB
cell lines (fewer than 3 passages and referred as AUTO NB) established from invaded bone marrow samples. Briefly, target cells
(106) were labeled with 150 µCi (5.55 MBq)
Na2 51CrO4 (1 mCi/mL [37
MBq/mL]; Dupont, Boston, MA) and 2 × 103
target cells per well were incubated with effector cells at different effector-target (E/T) ratios for 4 hours at 37°C. Experimental, spontaneous, and maximal releases of 2 × 103 labeled
target cells were counted on a microplate scintillation counter
(TopCount-NXT; United Technologies, Packard Laguna Hills, CA). For each
E/T ratio, the percentage of lysis was calculated conventionally as
follows: % lysis = (experimental cpm Mixed lymphocyte reactions. T-cell allostimulatory functions of immature or LPS-treated MD-DCs were assessed in triplicate cocultures of 2 × 105 allogeneic mononuclear cells with a decreased number of MD-DCs. Proliferative capacity was determined after overnight pulsing with [3H]thymidine (1 µCi [0.037 MBq] per well; NEN, Paris, France) at day 5 of the coculture. Cells were harvested onto 96-well Unifilter microplates and dried overnight, and the radioactivity was counted on a microplate scintillation counter (Topcount-NXT). All determinations were made in triplicate wells, and data were calculated as mean ± SEM.
PBSCs from 12 children with a stage 4 NB were collected after
mobilization by G-CSF with or without chemotherapy and thawed for the
study with the clinical procedures used for ASCT. After the gating of
mononuclear cells to exclude contaminating granulocytes, CD45+ PBSCs contained 49.6% ± 25.3%
CD14+CD15 ± cells, 18.7% ± 12.6% CD3+ T
lymphocytes, and 6.1% ± 3.0% CD3 PBSCs from children with NB are a source of functional MD-DCs MD-DCs were derived from the adherent fractions of PBSCs by culture in AIM-V medium with 10% FCS and IL-4 plus GM-CSF for 1 week. It is noteworthy that starting CD14+ cells in PBSCs were distributed in 2 phenotypes according to CD15 expression: CD14+CD15 (21.6% ± 6.8% true monocytes)
and CD14+CD15+ (78.4% ± 6.8%).
From day 5, cultures contained a majority of floating veiled cells with
a typical morphology of MD-DCs. At day 7, the mean MD-DC yield (mean
recovery of 42.6 ± 35.1 × 106 MD-DCs) was
10.1% ± 2.1% of the starting PBSCs (367.0 ±
284.0 × 106 cells).
Differentiation, assessed by immunophenotyping at day 7, revealed that
MD-DCs exhibit an immature phenotype (iDCs; CD14
NK/LAK cells from IL-2-activated PBSCs display high cytotoxicity against NB tumor cells PBSCs and paired PBMCs were cultured in the presence of 1000 IU/mL rIL-2 at a concentration of 1 × 106/mL effectors CD3+/CD56+ lymphocytes. As shown in
Figure 3A, phenotypic analysis displayed a significant and gradual expansion of
CD56brightCD3 NK cells between day 0 and 7 (ie, 4- to 9.4-fold), representing 6.1% ± 3.0% to
31.8% ± 12.6% of CD45+cells, respectively
(P < .001). The same range of expansion was observed in
paired PBMC samples collected before (13.0% ± 5.2% to 65.2% ±
21.5%) and during (10.1% ± 7.3% to 49.0% ± 25.6%)
mobilization. All together, starting with PBSCs or
PBMCs, the percentage of all NK cells was increased by 5-fold.
At day 7, the early and late activation markers, CD69 and HLA-DR, were
significantly up-regulated on CD56brightCD3
NK cells in a mean percentage range of 58.6% ± 2.3% to
22.3% ± 10.8% of these cells, respectively (data not
shown).
Lytic activity of cultures was tested at days 2, 4, and 7 against the
K562 and Daudi cell lines, as well as against the 2 NB cell lines. As
early as day 2, rIL-2-treated PBSCs displayed a strong cytotoxicity
against K562 and Daudi (42.3% ± 14.7% and 34 % ± 15.6%,
respectively, at a 25:1 E/T ratio). This NK/LAK activity progressively
increased with culture duration with up to 50.2 % ± 15.3% and 49.8 % ± 14.2% lysis, respectively, at day 7, and remained
high, even at a low E/T ratio (30.2 % ± 9.8% and 35.8 % ± 13.2% against K562 and Daudi, respectively, at a 1.5:1 E/T
ratio). Interestingly, the 2 NB cell lines were also sensitive to
lysis by activated effectors. Although lower at day 2 and 4, cytotoxicity against NB-1 and NB-2 was comparable to that achieved against K562 and Daudi at day 7 (Figure 3B). Interestingly, we confirmed the susceptibility of freshly isolated NB cells to autologous NK/LAK killing. As shown in Figure 3C, day-7 immunopurified
CD3 Taken together, PBSCs displayed a significant cytotoxicity increase on the 4 target cells lines between day 2 and day 7, (P = .005 and P = .007, respectively, with the use of a paired t test), but not between day 2 and day 4. In addition, we did not observe major significant differences related to mobilization (data not shown), except against the Daudi target at day 4 (mean ± SD percentage lysis at 6.25:1 ratio): PBMCs before mobilization were 64.5% ± 17.0% versus PBMCs during mobilization (40.7% ± 17.7%) versus PBSCs (35.0 % ± 12.0%) (PBMCs before mobilization versus PBMCs during mobilization, P = .010; PBMCs before mobilization versus PBSCs, P < .001; PBMCs during mobilization versus PBSCs; nonsignificant); this suggests a potential impact of G-CSF on NK functions, but this impact was not evidenced at day 7 or on other cell targets. Overall, these results suggest that potent non-MHC-restricted
cytolytic activity (against autologous and allogeneic NB tumor cells)
is optimally displayed by PBSCs after 7 days of rIL-2 stimulation that
is mediated mostly by activated CD56brightCD3 MD-DCs sustain survival and activation of NK/LAK cells Because IL-2 was associated with toxicity in children but was required to maintain NK/LAK cell survival in vivo, we tested whether cocultures of MD-DCs with NK/LAK cells in the absence of exogenous rIL-2 would maintain and/or enhance LAK activity in autologous systems. Therefore, day-7 rIL-2-deprived NK/LAK cells were cocultured with day-7 immature MD-DCs derived from autologous PBSCs in 10 independent experiments from 7 NB patient samples. These cocultures, as well as control cultures of NK/LAK alone with or without IL-2, were evaluated for cell viability, immunophenotype, and cytolytic activity. Survival of activated CD56brightCD3 NK/LAK cells
cocultured with MD-DCs was dramatically enhanced compared with control
cultures (NK/LAK cells with or without IL-2) as assessed by trypan blue
exclusion and flow cytometry analysis counting (Figure
4A). In addition to survival, NK/LAK cell
cytotoxic activity was also significantly maintained until day 7 of
coculture in the absence of IL-2. Very high levels of
cytotoxicity were obtained with median percentages of lysis at
a 6.25:1 E/T ratio of 56% (range, 52%-99%); 56%
(range, 53%-72%); 12% (range, 9%-26%); 3% (range,
1%-23%) against K562, Daudi, NB1, and NB2 respectively (Figure 4B). Cytolytic activity was not detected when the cells were
separated by a porous membrane (transwells), arguing in favor of a
direct cell-to-cell contact for the DC-mediated NK cell survival and
activation (data not shown). The MD-DC-mediated NK cell activation could not be accounted for by the T/LAK component. Indeed, coculture of
MD-DCs with CD3 and CD3+ cell subsets
immunopurified from NK/LAK bulk suspensions demonstrated that the
responding effectors were indeed CD56+CD3 and
not CD3+ cells (Figure 4B, inset). Therefore, MD-DCs
selectively activate NK/LAK cells in vitro in a cell-to-cell
contact- dependent manner.
The phenotypic characterization of NK/LAK cells cocultured with
MD-DCs included CD16/CD56/CD3/CD45 immunostainings. Before coculture,
on the basis of CD16 density, 3 CD56bright NK/LAK
subsets were identified, CD56brightCD16 LPS-activated MD-DCs are the most potent antigen-presenting cells (APCs) for NK/LAK activation To further dissect the dynamic of the DC/NK cell cross-talk, cocultures of CD56+CD3 immunopurified day-7
NK/LAK cells were performed with MD-DCs at various stages of
maturation, that is, immature MD-DCs (iDCs; CD86dim/CD83), maturing DCs (iDCs with LPS
stimulation), or mature MD-DCs (mDCs;
CD86bright/CD83+) after 2 days of LPS
stimulation, at various NK/DC ratios (1:1, 5:1, 10:1). NK cell
functions were assessed (cytotoxicity, IFN- production) following 3- to 7-day cocultures. Interestingly, at all differentiation stages, DCs
can maintain (day 3; Figure 5A) and
promote (day 7; Figure 5B) NK cell activation. This DC-mediated NK cell
activation even occurred at a 10:1 NK/DC ratio and, most strikingly,
for maturing DCs. Importantly, at late time points (day 7), maturing
DCs were more potent than rhuIL-2 at triggering IFN-
production from NK cells (Figure 5C). Immature DCs could promote NK
cell activation only at early time points (day 3 compared with NK
alone) for both killing and IFN- secretion. In contrast, mature DCs
promoted only cytolytic activity but not IFN- production, an
activation pattern resembling IL-2 (Figure 5C). As mentioned above,
DC-mediated NK cell cytolytic activity required cell-to-cell contact
(not shown). However, at a ratio of 1 DC to 5 or 10 NK cells, the
capacity of maturing DCs to trigger IFN- production was not
dependent on IL-12 (Figure 5D). Overall, immature DCs stressed
with an inflammatory stimulus electively induced and/or maintained
NK/LAK effector functions.
Reciprocal cross-talk between MD-DCs and activated NK/LAK cells Because immature DCs were also capable of maintaining long-term activation of NK/LAK cells, we addressed the question of whether NK/LAK cells could activate such iDCs. Indeed, a significant up-regulation of CD86 and CD83 molecules was observed in the whole CD45+HLA-DR+ population (Figure 6A, center panels), as compared with iDCs without NK cells (Figure 2A). Up to 33% of iDCs became CD83/CD86bright in the presence of NK/LAK cells, a percentage that was further enhanced (56%) in the maturing DCs (iDCs with LPS in the coculture). As already reported, it is likely that iDCs represented NK cell targets, since we observed 26%, 27.8%, and 41.8% of CD45+HLA-DR+ cells representing iDCs, iDCs with LPS, and mDCs, respectively (DC1 + DC2 in left panels, Figure 5A) alive at day 3 of cocultures. Recruitment of multiple NK/LAK cells onto single iDCs could be observed at day 7 (Figure 7A).
Interestingly, the most activating DCs for NK/LAK cells, that is, maturing DCs (iDCs with LPS), were also those that significantly promoted NK cell long-term survival without exogenous IL-2 (up to 25%; Figure 6B, left panels). Morphologic analyses highlighted clustering of DCs with NK/LAK cells, mostly in the maturing DCs (Figure 7B,E). In contrast, mature DCs did not promote NK cell survival (27% and 2% survival at days 3 and 7, respectively). As expected, mDCs seemed to be engaged toward apoptotic process (Figure 7C). It is noteworthy that most NK cells alive at day 7 exhibited a mature phenotype, CD56brightCD16bright, that is associated with effector functions. All together, NK/LAK cells can directly activate immature DCs with or without additional inflammatory stimulus, and such activated DCs, in return, are empowered to maintain not only NK cell effector functions but also long-term survival.
The purpose of this study was to investigate the potential
therapeutic relevance of the newly described DC/NK cell cross-talk on
the basis of our previous results demonstrating that mouse DCs enhance
NK cell functions in vitro and in vivo,9 and on recent
similar reports in human in vitro model systems demonstrating that DCs
can act on the priming phase of NK cell activation.10-14 The dynamics of such DC/NK cell cross-talk was dissected in a feasibility study aimed at generating autologous DC-NK/LAK cell cocultures designed from G-CSF-mobilized PBSCs in a series of 12 children with metastatic NB. In this poor-prognosis tumor model, high
therapeutic numbers of functional MD-DCs and NK/LAK cells could be
propagated and further cocultured without exogenous IL-2 to allow
expansion of CD56bright/CD16+ NK cells with
enhanced survival and effector functions (cytotoxicity, IFN- G-CSF is currently used in ASCT to mobilize hematopoietic precursors, and several studies in adults have suggested that this growth factor may have a negative impact on NK cell functions and recovery15-17 and, more recently, on the derivation of fully competent MD-DCs,18 but little is known about this in children. Impaired NK activity of freshly isolated G-CSF-mobilized PBSCs has been documented mainly in series of healthy donors and, moreover, in short-term IL-2 culture (24 hours), while NK cells in prolonged culture with or without feeder cells restored normal cytolytic function.17,19 In accordance with these results, we previously observed that the generation of cytotoxic effectors from PBSCs obtained in short-term cultures in IL-2 (fewer than 4 days) was significantly less efficient in children with tumors compared with adults.20 In the present study, in which we compared expansion and cytolytic functions of NK/LAK from samples collected before (PBMCs) or during (PBMCs and PBSCs) mobilization, we did not, owing to interpatient variations, observe major significant differences imputable to the G-CSF conditoning, except against the Daudi target at day 4 (but not at day 7). One can postulate that children with advanced NB, frequently heavily pretreated with chemotherapy, indeed displayed impaired NK functions compared with healthy children (whom we have not assessed), but, in this situation, it seems difficult to find evidence for an additional impact of G-SCF. Here we show that day-7 LAK cytolytic activity was potent not only against K562 and Daudi but also against relevant targets, that is, human autologous short-term neuroblastoma cells derived from invaded bone marrow as well as allogeneic neuroblastoma cell lines. It is noteworthy that the NB tumor cells used in this study as target cells for NK/LAK killing do not express detectable levels of MHC class I molecules (eg, with the use of W6.32 mAbs; data not shown), a common feature of NB tumors as reported by others.21 Therefore, the lack of MHC class I molecules, described as major KIR ligands,22 on these tumor cells may account for the high sensitivity of neuroblastoma cells to NK/LAK lysis and constitute a strong rationale for implementing LAK cell-based therapy in this refractory tumor model. Overall, we conclude that a day-7 culture duration is required for optimal expansion of cytotoxic NK/LAK cells. Although it was shown that in adults G-CSF-mobilized stem cells are a
more productive source of DCs than bone marrow-derived CD34+ progenitors,23 few studies have reported
the feasibility of generating MD-DC cultures from PBSCs in
children. Indeed, in this series of 12 children with NB, up to
10.1% ± 2.1% of total leukocytes from PBSCs could be
differentiated into MD-DCs with the use of IL-4 and GM-CSF. The
percentage of CD14+ cells contained in the PBSCs was
49.6% ± 25.3%, and this proportion was found to be enriched when
G-CSF, rather than chemotherapy alone, was used for
mobilization. With respect to the starting monocytic
population, one can extrapolate the yield of MD-DC recovery to 89.7%
if one considers the starting CD14+CD15 IL-2 has been administered in vivo to expand and/or sustain LAK
activity of NK/LAK cells propagated at high numbers ex vivo. However,
the selective expansion of human NK cells during continuous in vivo
infusion of low-dose IL-2 probably results from enhanced NK cell
differentiation from CD34+ hematopoietic progenitors
combined with an IL-2-dependent delay in NK cell death, rather than
from proliferation of mature NK cells in the
periphery.24 A similar observation has been recently reported with the newly identified IL-21 cytokine.25 Other
NK cell-stimulatory factors, IL-12, tumor necrosis factor- We further dissected the dynamics of the DC/NK cell cross-talk in these
autologous settings by studying the effects of DC-differentiation stage
on NK cell effector functions and survival. Strikingly, immature DCs
maturing under inflammatory stimulation or NK/LAK signaling were the
most potent antigen-presenting cells triggering cytolysis, IFN- Regulatory pathways of recruitment and migration of DCs and NK cells
remain largely unknown. In mice infused with tumor cells, it has been
recently shown that tumor cells lacking appropriate MHC class I
expression induced NK cell infiltration, cytotoxic activation, and
induction of transcription of These results support the use of PBSCs not only as a hematopoietic support but also as a source of effector cells with additive or synergistic functions. The ability to generate both NK/LAK effectors and MD-DCs in short-term cultures dramatically extends the therapeutic field of autologous PBSCs. The administration of pluripotent grafts might improve antitumor activity in vivo and lessen IL-2-related morbidity. NB constitutes an ideal tumor model to demonstrate the efficacy of such a combined multicellular adoptive therapy.
We are endebted to Dr Chantal Le Forestier from ETS Créteil for providing PBSC samples and to Gwenaëlle Leroux from Institut Gustave Roussy for expert technical assistance in recovery and culture of neuroblastoma cells. We also thank Dr Eric Vivier for helpful discussions.
Submitted January 4, 2001; accepted April 5, 2002.
Supported by a PHRC96 grant from the French Health Ministry; K.M. was a recipient of grant from Ligue Française contre le Cancer.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Eric Angevin, Unité d'Immunologie, Département de Biologie Clinique, Institut Gustave Roussy, 39 Rue Camille Desmoulins, 94805 Villejuif Cedex, France; e-mail: angevin{at}igr.fr.
1.
Matthay KK, Villablanca JG, Seeger RC, et al.
Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children's Cancer Group.
N Engl J Med.
1999;341:1165-1173 2. Hartmann O, Valteau-Couanet D, Vassal G, et al. Prognostic factors in metastatic neuroblastoma in patients over 1 year of age treated with high-dose chemotherapy and stem cell transplantation: a multivariate analysis in 218 patients treated in a single institution. Bone Marrow Transplant. 1999;23:789-795[CrossRef][Medline] [Order article via Infotrieve]. 3. D'Angio GJ, Evans AE, Koop CE. Special pattern of widespread neuroblastoma with a favourable prognosis. Lancet. 1971;1:1046-1049[Medline] [Order article via Infotrieve].
4.
Valteau D, Scott V, Carcelain G, et al.
T-cell receptor repertoire in neuroblastoma patients.
Cancer Res.
1996;56:362-369 5. Negrier S, Michon J, Floret D, et al. Interleukin-2 and lymphokine-activated killer cells in 15 children with advanced metastatic neuroblastoma. J Clin Oncol. 1991;9:1363-1370[Abstract].
6.
Rossi AR, Pericle F, Rashleigh S, Janiec J, Djeu JY.
Lysis of neuroblastoma cell lines by human natural killer cells activated by interleukin-2 and interleukin-12.
Blood.
1994;83:1323-1328 7. Foreman NK, Rill DR, Coustan-Smith E, Douglass EC, Brenner MK. Mechanisms of selective killing of neuroblastoma cells by natural killer cells and lymphokine activated killer cells: potential for residual disease eradication. Br J Cancer. 1993;67:933-938[Medline] [Order article via Infotrieve]. 8. Valteau-Couanet D, Rubie H, Meresse V, Farace F, Brandely M, Hartmann O. Phase I-II study of interleukin-2 after high-dose chemotherapy and autologous bone marrow transplantation in poorly responding neuroblastoma. Bone Marrow Transplant. 1995;16:515-520[Medline] [Order article via Infotrieve]. 9. Fernandez NC, Lozier A, Flament C, et al. Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo. Nat Med. 1999;5:405-411[CrossRef][Medline] [Order article via Infotrieve].
10.
Zitvogel L.
Dendritic and natural killer cells cooperate in the control/switch of innate immunity.
J Exp Med.
2002;195:F9-F14 11. Gerosa F, Baldani-Guerra B, Nisii C, Marchesini V, Carra G, Trinchieri G. Reciprocal activating interactions between natural killer cells and dendritic cells. J Exp Med. 2002;195:327-333 |
Top
Abstract
Introduction
spontaneous lysis
cpm)/(maximal lysis cpm 
), Daudi (
), IGR-NB1 (
), and
IGR-NB2 (
). ELISA assay for IFN-
(TNF-
, IL-10, or cytokines known to use -IL-2
receptor-