Download Expression of NTRK1/TrkA affects immunogenicity of neuroblastoma

IJC
International Journal of Cancer
Expression of NTRK1/TrkA affects immunogenicity of
neuroblastoma cells
Kristian W. Pajtler1, Vera Rebmann2, Monika Lindemann2, Johannes H. Schulte1,3, Stefanie Schulte1, Michael Stauder1,
€hl6, Alexander Schramm1 and Angelika Eggert1
Ivo Leuschner4, Kurt-Werner Schmid5, Ulrike Ko
1
Department of Pediatric Oncology and Hematology, University Hospital Essen, Germany
Department for Transfusion Medicine, University Hospital Essen, Germany
3
Centre for Medical Biotechnology, University Duisburg-Essen, Essen, Germany
4
Institute of Pediatric Pathology, University Hospital Schleswig-Holstein, Germany
5
Department of Pathology, University Hospital Essen, Germany
6
Institute of Cellular Therapeutics, Medical School Hannover, Germany
High levels of the NTRK1/TrkA receptor are expressed in low-stage neuroblastomas, which are characterized by a good patient
prognosis and often undergo spontaneous regression. In addition to apoptosis, tumor-immune responses might contribute to
this regression. We hypothesized that TrkA expression might enhance the immune response to neuroblastomas. Immunohistochemistry on neuroblastoma tissue microarrays confirmed significantly higher lymphocyte infiltration in low-stage compared
with high-stage tumors. Flow cytometry of human SH-SY5Y cells stably transfected with NTRK1/TrkA cDNA revealed significant
upregulation of major histocompatibility complex (MHC) class I complexes on TrkA-expressing cells. Corresponding to this upregulation, T cell activity and cytoxicity was enhanced in the presence of SY5Y-TrkA cells or by medium conditioned by them,
suggesting the existence of additional soluble factors stimulating the T cell response. Activation of natural killer (NK) cells
was only increased in the presence of SY5Y-TrkA conditioned medium (CM) and not in co-culture assays, suggesting a dominant inhibitory effect of upregulated MHC class I as the primary NK cell escape mechanism of TrkA-expressing neuroblastomas. We reanalyzed gene expression data obtained from the cell culture model to identify additional genes involved in the
TrkA-mediated modulation of immune responses. Upregulation of selected target genes in SY5Y-TrkA cells was confirmed on
transcript and protein levels. However, none of the proteins were detected in medium conditioned by SY5Y-TrkA cells, arguing
against these factors as soluble mediators of the TrkA-induced immune response. We here provide evidence that TrkA expression in neuroblastoma leads to an increased immunogenicity that may contribute to a less malignant phenotype and/or spontaneous regression of neuroblastoma cells.
Neuroblastoma is a common solid tumor of childhood and
originates from primitive cells of the sympathetic nervous
system. It is characterized by a broad biological heterogeneity
comprising oncogene amplification or allelic loss, chromosomal ploidy and expression of neurotrophin receptors correlating to a different degree with clinical outcome.1 The
identification of an IgM natural antibody in sera from neuroblastoma patients and healthy individuals, which exerts
potent antitumor activity, suggests that immune responses
play a role in controlling this tumor.2 Although neuroblastoma cells express tumor-associated antigens that may be recognized by cytotoxic T cells, they often lack constitutive
Key words: neurotrophin receptor, spontaneous regression, cytotoxic lymphocytes, NK cells
Abbreviations: BDNF: brain-derived neurotrophic factor; CM: conditioned medium; CPM: counts per minute; DC: dendritic cell;
IFN-g: interferon-g; MFI: mean fluorescence intensity; MHC: major histocompatibility complex; MICA: MHC class I polypeptide-related
sequences A; MICB: MHC class I polypeptide-related sequences B; NCR: natural cytoxicity receptor; NGF: nerve growth factor; PBMC:
Peripheral blood mononuclear cells; PBS: phosphate buffered saline; PHA: phytohemagglutinin; TIL: tumor-infiltrating lymphocyte;
TMA: Tissue microarrays; ULBP: UL16 binding protein
Additional Supporting Information may be found in the online version of this article.
Grant sponsors: Kind-Philipp-Stiftung f€
ur Leuk€amieforschung and the Faculty of Medicine (IFORES grant), University Duisburg-Essen (to
K.W.P.); Grant sponsor: European Union (European Network for Cancer Research in Children and Adolescents/ENCCA: 7th Framework
Program (to A.E.); Grant number: NoE 261474; Grant sponsor: European Union (Analyzing and Striking the Sensitivities of Embryonal
Tumours/ASSET: 7th Framework Program) (to A.E.); Grant number: CP 259348
DOI: 10.1002/ijc.28096
History: Received 29 Aug 2012; Accepted 24 Jan 2013; Online 7 Feb 2013
Correspondence to: Dr. Kristian W. Pajtler, Department of Pediatric Oncology and Hematology, University Hospital Essen, Hufelandstraße
55, Essen 45147, Germany, Tel.: 149-201-723 85157, Fax: 149-201-723 5386, E-mail: [email protected]
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Tumor Immunology
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NTRK1 affects neuroblastoma immunogenicity
Tumor Immunology
What’s new?
Elevated expression of neurotrophic tyrosine kinase type 1 receptor (NTRK1, or TrkA) previously was found to be associated
with favorable prognosis in neuroblastoma. That effect may be the result of TrkA-induced enhancement of the immune
response toward neuroblastoma cells, as reported here. The findings suggest that TrkA-associated immune activity might
partly explain spontaneous regression of low-stage neuroblastomas. They also have novel translational implications, among
them the possibility for the development of T cell-based immunotherapy approaches.
expression of co-stimulatory molecules and surface HLA class
I and II molecules. This renders them a difficult target for the
host T cell compartment.3 In contrast, it has been shown that
NK cells are able to lyse neuroblastoma cells in vitro and
inhibit neuroblastoma growth in mice and humans.4–6 Neuroblastoma cells display numerous strategies to escape immune
control mechanisms. Nonetheless, immunotherapy with
monoclonal antibodies directed against tumor-associated antigens, such as the GD2 disialoganglioside, has been tested in
advanced clinical trials with promising results.7 Despite the
need to better understand tumor-immune response mechanisms to further improve immunotherapeutic approaches
against neuroblastoma, few reports have investigated the
nature of the tumor-immune effector cell interaction.
High expression of the neurotrophic tyrosine kinase type 1
receptor, NTRK1, also named TrkA, is associated with a favorable prognosis in neuroblastoma.8 TrkA activation by binding
of the specific ligand, nerve growth factor (NGF), inhibits
angiogenesis, induces differentiation and growth arrest and
mediates apoptosis.9,10 In contrast, high tumor expression of
NTRK2/TrkB and its specific ligand, brain-derived neurotrophic factor (BDNF), correlates with progressive disease and
unfavorable prognosis in neuroblastoma patients based on an
autocrine loop leading to enhanced proliferation, metastatic
behavior and chemoresistance of neuroblastoma cells.11 Trk receptor signalling is a contributing factor to the diverse spectrum of clinical presentation in neuroblastoma patients, which
ranges from widespread metastatic disease that is refractory to
aggressive multimodal therapies to complete spontaneous
regression or differentiation in low-stage or stage 4s neuroblastomas, even without therapeutic intervention.1
Neuroblastoma has the highest rate of spontaneous regression observed among human cancers.12 Based on the biological characteristics of neuroblastomas, various mechanisms
including delayed activation of normal apoptotic pathways,13
inhibition of angiogenesis14 and anti-tumor reactions by the
immune system15 have been suggested to contribute to this
phenomenon. Most low-stage neuroblastomas express high
TrkA levels, leading to differentiation in the presence of NGF
or apoptosis in its absence. Therefore, TrkA is likely involved
in the regulation of spontaneous regression as well as differentiation of neuroblastomas with favorable biology.16
Low-stage neuroblastomas are often heavily infiltrated by
tumor-infiltrating lymphocytes (TILs), suggesting that the
immune system carries out surveillance for these tumors.17
Furthermore, differentiation of neuroblastoma cells has
recently been shown to increase antigenicity of these cells,
making them better targets for immune effector cells.18
Based on these observations, we hypothesized that TrkA
expression might enhance immunogenicity of neuroblastoma
cells in addition to its previously described biological effects.
The resulting increased immune response might contribute
to spontaneous neuroblastoma regression and finally lead to
a less malignant tumor phenotype. To further investigate the
potential implications of TrkA expression for the immune
response, we examined the interaction between TrkA- or
TrkB-expressing neuroblastoma cells with immune effector
cells in more detail.
Material and Methods
Cell culture, conditioned medium and cell models
Neuroblastoma cell lines were cultured as described.19 Cell line
identity was authenticated by DSMZ (Braunschweig, Germany).
NTRK1/TrkA or NTRK2/TrkB cDNAs were cloned into the retroviral expression vectors, pLNCX or pLNCX2 (Clontech, Palo
Alto, CA), respectively.20 NTRK1/TrkA was also stably
expressed in NB69 human neuroblastoma cells. Single-cell
clones of transfected cells were obtained by limiting dilution.
The empty pLNCX vector (SY5Yvec, NB68vec) served as negative control. Sequencing confirmed the identity of all transfectants. For generation of conditioned medium (CM),
neuroblastoma cell cultures were washed with phosphate buffered saline (PBS) and incubated 48 hr with serum-free medium. CM was centrifuged after collection and complete
protease inhibitor cocktail (Roche Applied Science, Mannheim,
Germany) was added before 50-fold concentration using Amicon Ultra-15 centrifugal filter units (Millipore, Billerica, MA).
Human NTRK1/TrkA cDNA was also cloned into the pT-RExDEST30 vector (Invitrogen, Carlsbad, CA) for Tet-conditional
expression. The SH-SY5Y cell line was sequentially transfected
with pT-REx-DEST30, then pcDNA6/TR (Invitrogen), harboring the tetracycline repressor gene. Single-cell clones were
selected by limiting dilution in blasticidine-containing medium.
Tissue microarrays and immunohistochemistry
Tissue microarrays (TMAs) were prepared from paraffinembedded tissue of 78 primary neuroblastomas from the
archival files of the Institutes of Pathology, Universities of
Kiel and Essen. Clinical data and informed patient consent
were obtained within the German Neuroblastoma Trial BFMC 2013 UICC
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Pajtler et al.
monitored using “Assay on demand” (Life Technologies,
Darmstadt, Germany) real-time RT-PCR. Expression values
were normalized to the geometric mean of three housekeeping genes (GAPDH, UBC and HPRT).
Western blotting
Western blotting was performed according to standard protocols. Membranes were incubated with anti-IFI16 (1:1000,
Abcam), washed with PBST and incubated with a horseradish
peroxidase-conjugated secondary antibody (1:1000, Dako,
Hamburg, Germany). Immunocomplexes were visualized
using enhanced chemiluminescence (ECL) (Amersham Biosciences, Freiburg, Germany), with b-actin as loading control
(1:5000, Sigma, Munich, Germany).
Figure 1. Low-stage TrkA-expressing neuroblastomas show a higher
degree of leukocyte infiltration (p 5 0.02). (a) Two representative
images for CD45-expressing cells in (I) low-stage neuroblastomas
(45 neuroblastomas of INSS Stages 1 and 2) compared to (II) highstage neuroblastomas (33 neuroblastomas of INSS Stages 3 and
4) on a TMA. (b) Degrees of CD45-positive cells (10–50% and
>50%) are markedly increased in the subset of neuroblastomas
with high TrkA expression on the TMA. (c) Kaplan-Meier analysis
showing significantly longer overall survival of patients bearing
tumors with a moderate (green and blue lines) or high (red line)
degree of leukocyte infiltration (p 5 0.02).
NB-2004. TrkA expression in 24 neuroblastomas represented
on the TMA was previously analyzed.21 Immunohistochemical
staining was performed using a monoclonal antibody against
CD45 (1:1000, Abcam, Cambridge, UK). TMA immunostaining
was independently evaluated by two examiners using a semiquantitative scoring system. Briefly, the number and intensity
of positive cells were counted and scored between 0 and 3 in
relation to the total number of tumor cells (0 5 no CD45 positive cells, 1 5 <10% CD45 positive cells, 2 5 10–50% CD45
positive cells, 3 5 >50% CD45 positive cells).
Real-time and semiquantitative RT–PCR
Semiquantitative RT–PCR was performed by standard techniques using 1 mg of total RNA. GAPDH was co-amplified as
an internal standard control.22 Primer sequences for human
ULBP1–3, MICA, MICB and GAPDH are available on
request. Agrin, IFI16, IFI30, ISG15 and CD58 expression was
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Total cell number was counted in neuroblastoma cell suspensions. A total of 0.5–1 3 106 cells were labeled with monoclonal antibodies against CD58 (LFA-3) conjugated to FITC
(Serotec, Oxford, UK) or major histocompatibility complex
(MHC) class I or II conjugated to phycoerythrine (PE)
(Abcam). Cells were resuspended in PBS and analyzed by flow
cytometry. The cytolytic ability of T and NK cells was assessed
by flow cytometric analysis of CD107a expression after 4 hr
co-incubation with neuroblastoma cell targets at a 1:10 stimulator to responder ratio. Alternatively, T or NK cells were incubated with CM from the same cells. After 1 hr, 5 lL of 2 mM
monensin (Sigma) was added. Cells were washed and labeled
with anti-CD3-PerCP (clone SP34-2), anti-CD8-FITC (clone
G42-8) or anti-CD56-APC (clone B159) and anti-CD107a-PE
(clone H4A3) (Becton Dickinson, San Jose, CA), followed by
analysis of PE-positive cells within the PerCP-CD31, FITCCD81 or APC-CD561 gate. PE positive CD41 cells were
detected as CD31 and CD8- from the CD3-CD8-dot plot.
Isolation of immune effector cells and incubation with
conditioned medium
Peripheral blood mononuclear cells (PBMC) were isolated from
30 mL buffy coat from healthy donors using Ficoll-PaqueTM
(Amersham) density gradient centrifugation. PBMCs were
incubated 24 hr with interleukin-2 for NK cell isolation. T cells
were isolated from third party donors. T cells and NK cells
were purified by positive or negative selection, respectively,
TM
R Untouched
using the DynabeadsV
technique (Life Technolo23
gies). Suspensions were adjusted to 106 cells/mL serum-free
TM
R X-VIVO 20
medium (BioWhittakerV
, Lonza, Walkersville,
MD). Purity was assessed flow cytometrically and was routinely
>90%. NK cells and T cells were incubated 2 hr with medium
conditioned by SY5Y cells and their stable transfectants as indicated. NK and T cells were incubated with full medium for
neuroblastoma cell culture as a control.
ELISpot assays
ELISpot assays detected secreted interferon-g (IFN-g) (10 mg/
mL anti-IFN-g, clone 1-D1K and 2 mg/mL biotinylated clone
Tumor Immunology
Flow cytometry of MHC class I/II, CD58 and CD107a
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NTRK1 affects neuroblastoma immunogenicity
cells were mixed 20:1 and 10:1, plated in triplicates in a 96well plate and incubated for 2 hr at 37 C. Cell supernatants
were then transferred to a plate containing the kit substrate
mix to detect LDH activity. After 60 min at RT, the absorbance at 450 nm was determined. Cell-mediated cytotoxicity
was calculated using: cytotoxicity effector (%) 5 [(target cell
mix 2 spontaneous effector LDH release 2 spontaneous target
LDH release)/(maximum target LDH release2spontaneous
target LDH release)]3100. NK cell cytotoxicity toward neuroblastoma cells in direct contact was assessed flow cytometrically. After 2 hr of NK and neuroblastoma cell co-culture,
dead cells were determined by propidium iodide staining.
MHC depletion of neuroblastoma cells was performed using
antibodies against MHC class I (clone w6/32) and MHC class
II (anti-CD74) as a control (Abcam).
Tumor Immunology
Lymphocyte transformation test
Figure 2. MHC class I molecules are upregulated on TrkA-expressing neuroblastoma cells independently of differentiation and presence of NGF. Flow cytometric analysis of MHC class I expression on
the surface of the indicated neuroblastoma cell lines. Bars indicate
percentages of MHC class I-positive cells, mean 6 SD of three or
more experiments is presented. (a) **P < 0.01 indicates significantly higher expression of MHC class I on SY5Y-TrkA cells compared to SY5Y-TrkB and the empty vector control, SY5Yvec, cells.
(b) Significantly higher expression of MHC class I on TrkA-expressing cells *p < 0.05 was confirmed in a second stably transfected
neuroblastoma cell line (NB69).
7-B6-1) and IL-10 (10 mg/mL anti-IL-10, clone 9D7 and
1 mg/mL biotinylated clone 12G8). All antibodies were
obtained from Mabtech (Nacka, Sweden). ELISpot plates
were incubated with 60 mL of primary antibody overnight at
4 C before NK and T cell isolation, washed three times and
blocked with cell culture medium.24 Three different types of
experiments were performed: (i) using nitrocellulose ELISpot
plates, duplicates of 160,000 NK cells were incubated with
10,000 K562 erythroleukemic cells for 48 hr after activation
with neuroblastoma cell CM for 2 hr, (ii) using nitrocellulose
ELISpot plates, 400,000 T cells were incubated with neuroblastoma cell CM for 2 hr, then stimulated by PHA (1 mg/
mL) for 24 hr and (iii) using PVDF ELISpot plates, 100,000
allogeneic T cells were stimulated by co-culture with 10,000
neuroblastoma cells for a total of 72 hr. After co-culture/
stimulation at 37 C, plates were washed six times. Captured
cytokines were detected by incubation for 2 hr with 60 mL of
the second antibody and color reactions.24,25
Cytotoxicity assays
NK cell cytotoxicity was quantified colorimetrically using the
LDH-Cytotoxicity Assay kit (Biovision). Effector and target
Proliferative T cell responses to the mitogen PHA (1 mg/mL)
were measured using 200,000 T cells in 200 mL of BioR X-VIVO 20 culture medium/well in microtitre
WhittakerV
plates (37 C, 5% CO2). Tests were conducted in triplicate.
Cells were cultured 3 days and 1 mCi 3H-thymidine
(TRA.120, specific activity 5 Ci/mmol, Amersham) was added
per well for the last 16 hr of culture. Cells were harvested
onto filter pads, and the incorporated radioactivity was quantified by liquid scintillation counting.26
Microarray data and statistical analyses
Correlation of gene expression and Kaplan-Meier analyses
was carried out using microarray data previously obtained for
the same cell culture model on R2 (http://r2.amc.nl).19 Data
analyses in R2 were performed using “Time Series” tools,
including “Correlation analyses” and “Gene Filtering” using
KEGG pathways on all arrays deployed in this study.
SPSS18.0 was used for further statistical analysis. GO term
analysis was conducted for genes showing a >3-fold expression difference between SY5Y-TrkA and SY5Y-TrkB cells. GO
terms resulting in a p < 0.01 corrected for multiple testing
were considered. GO categories were first ranked according to
the corresponding UniGene entries of differentially expressed
genes, solely considering terms having more than ten entries,
then the resulting GO categories were sorted by p-value corrected for multiple testing. A Student’s two-sided t-test was
used to compare all interval variables, and a chi-square test
was used to compare all categorical variables. Graph Pad
Prism 5.0 was used to perform Kaplan-Meier survival analysis
with log-rank statistics. Microarray data have been deposited
in the GEO database, accession no. GSE18409.
Results
Low-stage neuroblastomas expressing NTRK1/TrkA show a
higher degree of leukocyte infiltration
To gain first insights about interactions between neuroblastoma cells and the immune system, we assessed the degree
that primary neuroblastomas were infiltrated by immune
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Figure 3. TrkA expression of neuroblastoma cells evokes an enhanced activity of T cells. (a) Assessment of IFN-g production of T cells by ELISpot assay following co-culture of T cells with the indicated neuroblastoma cell lines. Data from three or more experiments are presented for
each cell line as mean 6 SEM. Co-culture of T cells with SY5Y-TrkA cells showed significant higher activation of T cells compared to co-cultures with SY5Y-TrkA 1 the MHC class I neutralizing antibody, w6/32 (p 5 0.001) and with SY5Y-TrkB (p 5 0.03). IFN-g production of T cells
was normalized to IFN-g production in response to the empty vector control (SY5Yvec). (b) Assessment of IFN-g production by T cells in ELISpot assay following incubation with CM from the indicated cell lines and stimulation by PHA. Data from three or more experiments are presented for each cell line as mean 6 SEM. Asterisks indicate significant higher activation of T cells by CM from SY5Y-TrkA cells compared to
SY5Y-TrkB (p 5 0.02) and SY5Yvec (p 5 0.03) cells. (c) Proliferative T cell responses toward the mitogen, PHA, were measured in a lymphocyte transformation test after incubation with the indicated CM. Incorporated radioactivity was quantified in counts per minute (CPM). Significantly higher T cell proliferation (indicated by *) was stimulated by CM derived from SY5Y-TrkA cells in comparison to SY5Y-TrkB (p 5 0.02)
and SY5Yvec (p 5 0.02) cells. (d) IL-10 secretion by T cells, indicating the immunosuppressive potential of factors secreted into the CM from
neuroblastoma cell lines. (e)–(g) MFI of CD107a was determined on CD31, CD41 and CD81 T cell populations from two or more donors cocultured with SY5Y cells and their stable transfectants or incubated with CM derived from the respective cell lines.
Tumor Immunology
6
effector cells. TMAs of 78 tumors representing all stages were
immunohistochemically stained using an unrestricted
antibody against the CD45 leukocyte tyrosine phosphatase.
High-stage neuroblastomas (Stages 3 and 4, n 5 33) had significantly less leukocyte infiltration compared to low-stage
neuroblastomas (Stages 1 and 2, n 5 45) (p 5 0.02; Fig. 1a).
Neuroblastoma infiltrating leukocytes have previously been
shown to predominantly originate from the T cell lineage,
characterized by CD4 or CD8 expression, with some CD56positive cells being of NK cell origin or representing NK-like
T cells.27 A specific TrkA antibody, not cross-reacting with
TrkB and suitable for immunohistochemistry, is currently not
commercially available. Thus, TrkA expression in the arrayed
tumors was assessed at the mRNA level from existing expression microarray data. A substantial part of tumors with high
TrkA expression contained moderate and high levels of
CD45-positive cells (defined as >10% and >50% infiltration,
respectively), whereas all tumors with low TrkA expression
showed <10% or no CD45-positive cells (Fig. 1b). The presence of TILs in the tumor mass has previously been reported
to correlate with favorable prognosis of neuroblastoma
patients.17 Accordingly, the degree of leukocyte infiltration in
our tumor cohort was predictive for overall patient survival
(log-rank test, p 5 0.02) in Kaplan-Meier analysis (Fig. 1c).
Multivariate analysis revealed only stage and CD45 positivity
as independent prognostic markers (p 5 0.0007 and p 5 0.04,
respectively). However, the important prognostic marker,
MYCN amplification, could not be included in the analysis
due to the low number of MYCN-amplified tumors on the
TMA, limiting the significance of the multivariate analysis.
Taken together, low-stage neuroblastomas with high TrkA
expression were characterized by a higher percentage of leukocyte infiltration, suggesting a more active tumor response
from the immune system, which is likely to contribute to a
better patient outcome.
MHC class I molecules are upregulated on TrkA-expressing
neuroblastoma cells independently of differentiation and
the presence of NGF
Since the mechanisms underlying recruitment of lymphoid
infiltrates to low-stage primary neuroblastomas are poorly
understood to date, we aimed to further assess a potential
relationship between TrkA receptor expression and neuroblastoma cell immunogenicity in vitro. We first focussed on
MHC class I molecules, as (i) they are major players in regulating immune effector cell interactions with target cells and
(ii) have previously been implicated in TrkA-mediated differentiation of neuroblastoma cells.18 It has been shown that the
cells in most primary neuroblastomas express very low MHC
class I levels on their surfaces, thereby, generating a mechanism for immune escape.28 However, neuroblastoma cell lines
were previously demonstrated to upregulate expression of
MHC class I molecules during differentiation, regardless of
TrkA expression.18 To discriminate between the effect of differentiation and TrkA expression on MHC class I molecule
NTRK1 affects neuroblastoma immunogenicity
expression, the percentage of cells expressing MHC class I
complexes was analyzed by flow cytometry in an established
human neuroblastoma cell culture model stably expressing
full-length constructs for NTRK1/TrkA, NTRK2/TrkB or the
empty vector in the SH-SY5Y cell line (designated SY5YTrkA, SY5Y-TrkB and SY5Yvec, respectively).10,29 Interestingly, twice as many SY5Y-TrkA cells expressed MHC class I
molecules even in an undifferentiated state and in the absence of exogenous NGF compared to the parental cell line,
and ten-fold more SY5Y-TrkA cells expressed MHC class I
molecules than SY5Y-TrkB cells (Fig. 2a). These findings
were confirmed in a second human neuroblastoma cell culture model, NB69 cells stably transfected with NTRK1/TrkA
(NB69-TrkA) or empty vector (NB69vec) (Fig. 2b).30 These
results demonstrate that TrkA expression and basal activation
in neuroblastoma cells is sufficient to upregulate MHC class I
expression independently of differentiation or the presence of
exogenous NGF. In addition, cytokine expression profiles of
neuroblastoma cell lysates and their CM did not display significant overexpression of cytokines known to be involved in
MHC class I upregulation (data not shown). Thus, MHC
class I upregulation in TrkA transfectants is (i) not artificially
induced by the transfection process itself and (ii) presumably
independent of the expression of major cellular cytokines.
TrkA expression in neuroblastoma cells enhances T cell
activity and cytotoxicity
Since the expression level of MHC class I complexes on the
target cells is a crucial parameter for T cell activation, the
higher MHC class I expression by SY5Y-TrkA cells is likely
to affect, for example, the T cell alloresponse to these cells.
To test this hypothesis, SY5Y-TrkA cells were co-cultured
with human allogeneic T cells from healthy, MHC class I
and II disparate donors. After 2 days in co-culture on ELISpot plates, T cell activation was analyzed by measuring
IFN-g production as a surrogate marker for functional activity in the T cells. As neuroblastoma cell lines only
express MHC class I, but no MHC class II, as determined
by flow cytometric analysis (data not shown), the IFN-g
production can be attributed to MHC class I disparity. Our
previous experiments indicate that after 3 days in culture (1
day pre-incubation and 2 days in ELISpot plates) MHC
class I differences lead to IFN-g production. Co-culture of
T cells with SY5Y-TrkA cells enhanced IFN-g production
by the T cells in comparison to co-culture with the parental
cell line or TrkB-expressing cells (Fig. 3a). IFN-g production also was slightly elevated in T cells co-cultured with
SY5Yvec cells, suggesting some unspecific activation of
immune effector cells by the vector system itself or following stable transfection. Enhanced IFN-g production by T
cells was reduced by approximately 15%, when MHC class I
molecules on SY5Y-TrkA cells were blocked using a specific
antibody against MHC class I (w6/32) (Fig. 3a). This suggests additional factors to be involved in increased T cell
activation.
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Figure 4. TrkA expression increases NK cell activation, but inhibits direct NK cell cytotoxicity. (a) NK cell-mediated lysis of indicated cells in
co-culture experiments. NK cell cytotoxicity against SY5Y-TrkA cells significantly increased after depletion of MHC class I (p 5 0.01). An antibody against MHC class II (anti-CD74) was used as control. (b), (c) NK cells from three or more healthy donors were incubated with CM
from the stably transfected NB cells, SY5Yvec, SY5Y-TrkA or SY5Y-TrkB. NK cell activity was measured by their IFN-g production in an ELISpot assay, NK cell-mediated lysis by colorimetric quantification of LDH-release. Medium without supplements was used as control. (d)
CD107a expression in NK cells as a read out for their cytotoxic capability measured as MFI was comparably low in response to direct cell
contact with SY5Y cells and their stable transfectants. (e) Expression of activating NKG2D ligands was analyzed on mRNA levels by semiquantitative RT-PCR in the indicated cell lines.
8
NTRK1 affects neuroblastoma immunogenicity
Tumor Immunology
To assess a potential additional contribution of secreted
soluble factors to the observed effect of SY5Y-TrkA cells on
T cell function, we repeated the IFN-g production assay
using CM collected after 48 hr of neuroblastoma cell culture.
Figure 5.
T cells were incubated for 2 hr with CM, then stimulated by
phytohemagglutinin (PHA). Interestingly, CM from SY5YTrkA cells showed a significant higher ability to induce
IFN-g production in T cells in comparison to SY5Y-TrkB,
SY5Yvec and SY5Ywt cells (Fig. 3b). In addition, lymphocyte
proliferation measured by lymphocyte transformation test
was higher in T cells incubated with SY5Y-TrkA CM than
with SY5Y-TrkB, SY5Yvec or SY5Ywt CM (Fig. 3c). In contrast, T cells incubated with SY5Y-TrkA CM secreted less
interleukin-10 (IL-10) than T cells incubated with medium
conditioned by SY5Y-TrkB, SY5Ywt or SY5Yvec cells (Fig.
3d), indicating lymphocyte suppression. These results suggest
that SY5Y-TrkA cells harbor a significantly higher potential
to activate T cells by both membrane-adherent and soluble
secreted factors. Direct cell contact is likely to contribute to
the tumor-immune cell interaction, but is not solely responsible for the TrkA-mediated effect on T cells.
To further resolve the functional properties of different T
cell subgroups during TrkA-mediated T cell effects, we
assessed the degranulation capacity of the subgroups by flow
cytometric analysis of CD107a/LAMP-1 surface expression.31
CD107a is a vesicle membrane protein in immune effector
cells that becomes transiently mobilized to the cell surface during the degranulation process, leading to the release of cytotoxic mediators such as perforin or granzymes. Its surface
expression has previously been shown to correlate with
immune effector cell cytotoxic activity, and can be used as a
marker for the capability of T or NK cells to kill tumor targets.31 The mean fluorescence intensity (MFI) of CD107a was
determined on CD31, CD41 and CD81 T cell subpopulations co-cultured with SY5Y cells or their stable transfectants
or incubated with medium conditioned by the respective cell
lines. CD107a expression was markedly elevated in all T cell
subpopulations co-cultured with SY5Y-TrkA cells or CM
derived from these cells (Figs. 3e–3g), demonstrating a TrkAmediated stimulation of both cytotoxic CD81 T cells as well
as CD41 T helper cells, by membrane-adherent and soluble
factors. CD81 cytotoxic T cells, but not CD31 and CD41 T
cells, also exhibited a limited amount of degranulation activity
after co-culture with SY5Yvec cells or incubation with their
CM. These results corroborate the results from our cytokine
Figure 5. Immune modulating genes are differentially expressed in
SY5Y-TrkA versus SY5Y-TrkB expressing cells. (a) Reanalysis of
microarray expression profiles obtained in 68 primary neuroblastomas shows a correlation between the mRNA expression levels of
TrkA and agrin in these tumors (p 5 4 3 1028). (b) Real-time PCR
validation of gene expression in SY5Y cells and their stable transfectants confirmed upregulation of all five target genes in SY5YTrkA cells. (c) Target gene expression in a SY5Y cell line with tetracycline-inducible expression of TrkA. Induction of TrkA led to
increased expression of all five selected target genes, with markedly enhanced expression after addition of the TrkA-specific ligand,
NGF. (d), (e) Upregulation of the selected target proteins on the
surface of neuroblastoma cells was confirmed by flow cytometry
for CD58 or western blot analysis for IFI16.
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Pajtler et al.
TrkA expression increases NK cell activation, but inhibits
direct NK cell cytotoxicity
Potential interactions between TrkA-expressing neuroblastoma cells and NK cells are also of interest for neuroblastoma
immunotherapy development. We, therefore, also assessed
the effects of TrkA expression on NK cell-mediated lysis of
tumor cells in co-culture experiments. Co-culture of SY5YTrkA cells with NK cells did not significantly enhance NK
cell cytotoxicity in comparison to co-culture with SY5Y,
SY5Yvec and SY5Y-TrkB cells (Fig. 4a). The absence of a significant difference could be explained by MHC class I upregulation in TrkA-expressing neuroblastoma cells counteracting
other potentially positive TrkA-mediated effects on the NK
cells. To test whether MHC class I expression inhibited NK
cell cytotoxicity toward SY5Y-TrkA cells, MHC class I molecules were depleted in cell co-cultures by adding an MHC
class I antibody to culture media. MHC class I depletion
restored NK cell cytotoxicity toward SY5Y-TrkA cells, whereas
lysis of SY5Yvec and SY5Y-TrkB cells remained low (Fig. 4a).
NK cells were significantly less cytotoxic toward SY5Y cells and
their stable transfectants than toward K562 cells, an erythroleukemia cell line lacking MHC class I expression and known to
be an NK cell-sensitive target. These results suggest an inhibition of NK cell cytotoxicity in TrkA-expressing neuroblastoma
cells via upregulation of MHC class I as the primary immune
escape mechanism, whereas different immune escape mechanisms seem to be in place in SY5Yvec and SY5Y-TrkB cells.
To assess the need for direct cell–cell contact for the
observed effects on tumor-immune cell interaction, NK cell
activation and cytoxicity were measured after exposure to
medium conditioned by SY5Y-TrkA, SY5Y-TrkB, SY5Yvec or
SY5Ywt cells. NK cell activity measured by IFN-g production
increased only in response to SY5Y-TrkA CM (Fig. 4b).
SY5Y-TrkA CM also enhanced NK cell cytotoxicity toward
K562 cells in comparison to medium conditioned by SY5Y,
SY5Yvec or SY5Y-TrkB cells (Fig. 4c). In contrast to the
results for T cells, SY5Y-TrkA cells or their CM did not
enhance degranulation in NK cells assessed by analysis of
CD107a expression (Fig. 4d). CD107a expression in NK cells
was comparably low in response to SY5Y cells and all their
stable transfectants, whereas K562 cells demonstrated a
higher capability to induce CD107a. These results suggest
that humoral factors secreted by SY5Y-TrkA cells can stimulate NK cell cytotoxicity toward non-neuroblastoma target
cells, such as K562 cells, to some extent. However, the
induced NK cell cytotoxicity is insufficient to kill neuroblastoma target cells, which are likely protected by membraneassociated molecules on the tumor cell surface. MHC class I
expression is only one possible immune escape mechanism,
as SY5Yvec and SY5Y-TrkB cells also proved to be protected from NK cell lysis independently of MHC class I
expression.
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Tumor cells may also express ligands of NK cell activating
receptors, such as the UL16 binding proteins (ULBPs), MHC
class I polypeptide-related sequences A (MICA) or B (MICB)
or natural ligands of the NKG2D receptor, to regulate the
interaction with immune effector cells. We analyzed mRNA
expression of these activating ligands in the SY5Y cell culture
model. ULBP2, ULBP3, MICA and MICB were indeed significantly upregulated on the surface of SY5Y-TrkA cells in
comparison to SY5Y, SY5Yvec and SY5Y-TrkB cells (Fig. 4e).
ULBP1 expression was upregulated on the surfaces of both
SY5Y-TrkA and SY5Y-TrkB cells in comparison to SY5Yvec
and the parental SY5Y cells. These results suggest that the
tumor-protecting effects of membrane-associated factors,
including MHC class I molecules, cancel out the activating
effects of expressing NKG2D ligands toward NK cells.
Immune modulating genes are differentially expressed in
TrkA- and TrkB-expressing neuroblastoma cells
To search for additional underlying molecular mechanisms
for TrkA-mediated immune responses, we reanalyzed Affymetrix U95A expression data previously obtained for the same
SH-SY5Y cell culture model to identify further TrkA-regulated genes capable of enhancing tumor immunogenicity,
apart from MHC class I.19 Comparison of SY5Y-TrkA and
SY5Y-TrkB cell transcriptomes revealed multiple differentially
expressed genes, even in the absence of the respective exogenous ligands, NGF or BDNF. An automated search in the R2
molecular database32 for a distinct subgroup of genes relevant
for tumor immunology based on gene ontology annotations
identified 17 differentially expressed genes (p < 0.002). We
selected five TrkA-induced candidate genes with the highest
potential relevance for immune modulations for further analyses (Supporting Information Table 1). To assess the potential
relevance of these five candidate genes in vivo, we reanalyzed
microarray expression profiles obtained from 68 primary neuroblastomas for correlations with TrkA expression levels in
these tumors. Agrin expression significantly correlated with
TrkA expression (Fig. 5a), whereas only a trend toward positive correlation with TrkA expression was observed for the
other four candidates. We next investigated this connection in
the SH-SY5Y cell culture model using real-time RT-PCR. All
five candidate genes were upregulated in SY5Y-TrkA cells
compared with parental cells and SY5Y-TrkB cells (Fig. 5b).
However, the five candidate genes were also slightly upregulated in SY5Yvec cells compared with the parental cell line.
As an inducible model can better control for artificial and
unspecific effects induced by stable transfection of the empty
vector, we repeated the experiments using a vector system for
conditional TrkA expression operating under a tetracycline
repressor. Induction of NTRK1/TrkA resulted in reproducible
upregulation of CD58, IFI16 and ISG15 expression, which was
further enhanced by addition of NGF to the culture medium
(Fig. 5c). Enhanced CD58 expression on the cell surface was
confirmed by flow cytometry, and enhanced IFI16 expression
was detected in western blots of whole-cell lysates (Fig. 5d).
Tumor Immunology
response assay, and together demonstrate that TrkA expression
on neuroblastoma cells enhances T cell activity and cytoxicity.
10
However, western blotting of CM conditioned by the stably
transfected SY5Y-TrkA cells or the inducible TrkA model
cells could not detect soluble CD58 and IFI16 (data not
shown), arguing against these factors as the underlying molecular mechanism of immune effector cell activation mediated
by secreted factors. High-throughput proteomic analyses are
warranted to identify additional molecular players of the
TrkA-mediated immune response to neuroblastoma cells.
Tumor Immunology
Discussion
The molecular mechanisms regulating neuroblastoma cell immunogenicity are not yet completely understood. We show
here that TrkA overexpression in human neuroblastoma cells
affects tumor cell antigenicity and markedly enhances activation of immune effector cells. We demonstrate (i) increased
leukocyte infiltration of TrkA-expressing low-stage neuroblastomas, (ii) an enhanced ability of TrkA-expressing neuroblastoma cells to stimulate T cell activation, proliferation and
cytotoxicity in vitro, (iii) a corresponding upregulation of
MHC class I molecules as major players in the regulation of
immune responses by TrkA-expressing neuroblastoma cells
and (iv) an increased ability of TrkA-expressing neuroblastoma cells to stimulate NK cell activation, but not cytotoxicity toward neuroblastoma cells.
We show that the percentage of TILs is significantly higher
in low-stage neuroblastomas expressing high levels of TrkA.
This observation is in agreement with a previous report demonstrating a correlation between TILs and neuroblastoma
tumor stage.27 TILs infiltrating low-stage neuroblastomas have
previously been characterized to be predominantly cells of the
T cell lineage with a smaller fraction of CD56-positive cells
being of NK cell origin or representing NK-like T cells.27 The
presence of TILs in solid tumors has been described as a
favorable prognostic factor in numerous tumor entities
including neuroblastoma33 and our results verify this for a
further cohort of primary neuroblastomas.
A successful T cell-mediated immune response requires
expression of MHC classes I and II. It has been shown that
neuroblastoma cells express antigens suitable for recognition
by cytotoxic T cells, however, they display little to no surface
MHC class I.28,34,35 Defects in the antigen-processing
machinery have been described as a potential underlying
mechanism.36 MHC class I expression can be raised by INFg
or retinoid treatment.3,37,38 T cell-based immunotherapy cannot be successful unless MHC class I expression is upregulated on the neuroblastoma cell surface. We show here that
TrkA overexpression or induction in a neuroblastoma cell
model upregulated MHC class I expression. TrkA-expressing
neuroblastoma cells induced to differentiate in vitro by NGF
treatment have previously been shown to upregulate MHC
class I expression, however, MHC class I upregulation was
attributed to differentiation rather than a direct effect of
TrkA upregulation.18 We demonstrate here that TrkA expression in the absence of NGF is sufficient to upregulate MHC
class I expression at the neuroblastoma cell surface. Trk
NTRK1 affects neuroblastoma immunogenicity
receptors have previously been shown to have a high capacity
for ligand-independent activation particularly in neuroblastoma, presumably via spontaneous interactions.19
MHC class I upregulation on neuroblastoma cells has
previously been shown to result in increased CTL cytoxicity.18 Indeed, we observed an increase in T cell activation,
proliferation and cytotoxicity in response to TrkA-expressing neuroblastoma cells, with both CD81 and CD41 T
cells demonstrating enhanced responses. Interestingly, these
effects were not only restricted to cell–cell contacts and
increased MHC class I expression, but were also mediated
by CM, suggesting that at least some effector molecules are
soluble factors. Our first attempts to identify these soluble
factors based on differentially expressed mRNAs were
unsuccessful. An unbiased proteomic approach with mass
spectrometric identification of differentially expressed proteins in conditioned media derived from the SY5Y and
NB69 cell culture models should be a more promising
approach.
Although not detected as soluble proteins in CM of the
neuroblastoma cell culture models, the TrkA-upregulated and
validated candidate genes, CD58, IFI16, ISG15 and agrin
might nonetheless play a role in the modulation of neuroblastoma immunogenicity. CD58 is a ligand of the T lymphocyte CD2 protein that functions in adhesion and
T lymphocyte activation by advancing the contact between
cytolytic effector cells and target cells.39 Monoclonal antibodies neutralizing CD58 have been shown to inhibit melanoma
cell lysis by allogeneic NK cells and autologous TILs.40 IFI16
has recently been identified as a double-strand DNA
(dsDNA) receptor involved in T cell effector functions, as intracellular dsDNA is a potent activator of human dendritic
cells (DCs).41 ISG15 is a ubiquitin-like protein that becomes
conjugated to many cellular proteins upon activation by
IFN-a.42 Interestingly, it is located on chromosome 1p36.3 in
a region hypothesized to harbor one or more neuroblastoma
tumor suppressor genes. ISG15 stimulates IFN-g production
by CD31 T cells, as well as NK cell proliferation and nonMHC-mediated tumor cell cytotoxicity.43 Overexpression of
agrin was very recently shown to occur in activated T cells,
leading to further activation of these immune effecor cells by
synergizing with the T cell receptor.44 If and how the here
identified increased expression of CD58, IFI16, ISG15 and
agrin, in TrkA-expressing neuroblastoma cells contributes to
the increased immunogenicity of neuroblastoma cells remains
to be elucidated in further analyses.
The “missing self” recognition theory describes malignantly transformed cells lacking MHC class I expression as
triggering NK cell-dependent activation.45 Vice versa, upregulation of class I MHC expression in TrkA-expressing neuroblastoma cells inhibited NK cell cytoxicity as expected, and
neutralizing MHC class I using an antibody restored NK cell
cytotoxicity toward SY5Y-TrkA cells. In contrast, medium
conditioned by SY5Y-TrkA cells activated NK cells and
stimulated cytotoxicity against K562 cells, which do not
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11
Pajtler et al.
express MHC class I. As a balance of activating and inhibitory signals mediates NK cell recognition and killing of target cells,46,47 we also assessed the presence of NK cell
activating signals in the SY5Y cell culture model. The natural cytoxicity receptors (NCRs), NKp30, NKp44 and
NKp46, are the main activating receptors, for which only
few ligands are known and remain poorly characterized on
the molecular level.48 NKG2D, 2B4 and DNAM-1 are also
activating receptors, and recognize a variety of well-defined
ligands.46 MICA, MICB and ULBPs are among the best
studied ligands, and bind NKG2D. Upregulation of these
ligands has been detected in solid tumors of multiple origins, including neuroblastomas,49 and might be a cell intrinsic protective mechanism to render transformed cells
susceptible to killing. NK cells expressing high NKG2D levels, especially following IL-2 stimulation, have previously
been reported to lyse MHC class I-positive target cells.5,50
Although TrkA-expressing neuroblastoma cells express high
NKG2D ligand levels, inhibitory signals mediated by MHC
class I expression appear to override the effect, since we
observed NK cell activation but no significant cytotoxicity
toward SY5Y-TrkA cells unless MHC class I expression was
depleted.
Taken together, we demonstrate that TrkA-expression
enhances the immune response toward neuroblastoma cells
by regulating immune-modulating surface molecules including MHC class I molecules as well as secreted factors yet to
be identified. These results point to three potentially important clinical implications: (i) the TrkA-induced immune
response might explain a part of the molecular mechanisms
resulting in spontaneous regression of low-stage neuroblastomas; (ii) therapeutic induction of TrkA might be a promising
approach to create a less malignant and more treatable tumor
phenotype and (iii) the subgroup of neuroblastomas with
high TrkA expression might not be a suitable target for NK
cell-based immunotherapy, but would rather benefit from T
cell-based immunotherapy approaches.
Acknowledgements
The authors thank E. Mahlow and A. Rieb for excellent technical assistance,
K. Astrahantseff for article proofreading and H. Stephan for figure
preparation.
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