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- Material and Methods
- Supporting Information
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. 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. Although neuroblastoma cells express tumor-associated antigens that may be recognized by cytotoxic T cells, they often lack constitutive 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. 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. 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. 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. 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.
Neuroblastoma has the highest rate of spontaneous regression observed among human cancers. Based on the biological characteristics of neuroblastomas, various mechanisms including delayed activation of normal apoptotic pathways, inhibition of angiogenesis and anti-tumor reactions by the immune system 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. Low-stage neuroblastomas are often heavily infiltrated by tumor-infiltrating lymphocytes (TILs), suggesting that the immune system carries out surveillance for these tumors. Furthermore, differentiation of neuroblastoma cells has recently been shown to increase antigenicity of these cells, making them better targets for immune effector cells.
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.
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- Material and Methods
- Supporting Information
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. 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. The presence of TILs in solid tumors has been described as a favorable prognostic factor in numerous tumor entities including neuroblastoma 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. MHC class I expression can be raised by INFγ 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. 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 receptors have previously been shown to have a high capacity for ligand-independent activation particularly in neuroblastoma, presumably via spontaneous interactions.
MHC class I upregulation on neuroblastoma cells has previously been shown to result in increased CTL cytoxicity. Indeed, we observed an increase in T cell activation, proliferation and cytotoxicity in response to TrkA-expressing neuroblastoma cells, with both CD8+ and CD4+ 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. Monoclonal antibodies neutralizing CD58 have been shown to inhibit melanoma cell lysis by allogeneic NK cells and autologous TILs. 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). ISG15 is a ubiquitin-like protein that becomes conjugated to many cellular proteins upon activation by IFN-α. Interestingly, it is located on chromosome 1p36.3 in a region hypothesized to harbor one or more neuroblastoma tumor suppressor genes. ISG15 stimulates IFN-γ production by CD3+ T cells, as well as NK cell proliferation and non-MHC-mediated tumor cell cytotoxicity. 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. 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. 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 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. NKG2D, 2B4 and DNAM-1 are also activating receptors, and recognize a variety of well-defined ligands. 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, 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.