Background
Medulloblastomas cause significant mortality and morbidity, and recurrent tumors are generally considered to be incurable [
1]. Patients that present with high risk features, moderate-risk SHH tumors, and poor prognosis group 3 tumors have survival rates between 50 and 75% [
2], and survivors almost uniformly have significant hearing, cognitive, and endocrinologic impairment as a result of toxic therapies [
3,
4]. The need for alternative therapies is clear, and has led to interest in methods of tumor cell eradication based on immune modulation.
Medulloblastomas express heterogenous antigens [
5] and have variable MHC expression [
6], which make identifying appropriate targets difficult; hence, the use of vaccine or T cell-based strategies can be problematic. Alternatively, natural killer (NK) cells can recognize and eliminate tumor cells with broad specificity without requiring prior antigen identification [
7,
8].
Natural killer cells have documented activity against medulloblastoma [
7,
8]. Lymphokine-activated killer cells, which are mostly composed of NK cells, have shown some clinical efficacy against this disease [
9]. However, complete elimination of tumor by autologous NK cells is unlikely as the inhibitory signals from the tumor generally render their own NK cells incapable of inducing potent cytolytic activity. We propose to overcome the inhibitory signals provided by the expression of MHC Class I tumor cells by using KIR-MHC Class I mismatched allogeneic, rather than autologous, NK cells. Although most NK cell clinical trials have used allogeneic peripheral blood (PB) as a cell source [
11], in vitro studies suggest that umbilical cord blood (CB) NK cells may possess better cytolytic ability [
12,
13]. The use of cord blood as a source of allogeneic NK cells is also advantageous because: (a) they can be ex vivo expanded to clinically useful cell numbers; and (b) they allow a higher chance of identifying HLA-compatible and KIR-mismatched products because of their immediate availability in established cord blood banks. Such a readily available “off-the-shelf” source of NK cells greatly enhances the feasibility of using these cells as therapy for medulloblastoma.
Finally, it has become clear that the immune suppressive environment of cancer in general, and MB in particular, may prevent response from immune therapies like NK cells. Medulloblastomas secrete TGF-β [
14‐
18], which is a potent immune suppressive strategy employed by most human cancers—with negative effects on NK cell function [
19,
20]. We have previously demonstrated the successful use of TGF-β dominant negative receptor-modified cord blood NK cells against glioblastoma [
21], which showed resistance against TGF-β and maintained killing of glioma cells in vitro. Therefore, we propose the same novel immunotherapeutic approach for medulloblastoma, consisting of TGF-β-resistant cord blood-derived NK cells as an “off the shelf” cell therapeutic, and specifically propose to evaluate its application as a treatment to overcome the TGF-β-rich environment in medulloblastoma.
Methods
Cells
Umbilical cord blood (UCB) samples were obtained from Dr. E.J. Shpall at the UT MD Anderson Cancer Center cord blood bank, using an IRB approved protocol (Pro00003896). Cord blood samples were processed within 24 h of receipt (which may be after 3 days of collection), using Ficoll-Paque Plus density gradient media (GE Life Science, Marlborough, US) to obtain cord blood mononuclear cells (CBMC). CBMCs were either frozen for future use or immediately used for natural killer cell selection. Patient samples were obtained at Children’s National Medical Center from patients diagnosed with a malignant brain tumor (EH, IRB Pro00004033). Patient samples were processed within 24 h of blood collection. Deidentified human primary medulloblastoma cell lines were obtained from Dr. Yanxin Pei, and were initially expanded in the brains of NSG mice before culture for 1 week in neurobasal conditioned media.
TGF-β luminex of Daoy and primary medulloblastoma
To measure TGF-β concentrations in Daoy and primary medulloblastoma cell lines, tumor cells were allowed to grow to confluence and supernatant was collected after 24 h. TGF-β concentrations were determined by a TGF-β-1, 2, 3 multiplex assay (Millipore, Burlington MA). Supernatants were frozen at − 80 °C until further analysis. The kit was run according to manufacturer’s protocol and the TGF-β concentration determined using the provided standards.
TGF-β dominant negative receptor
A PG13 cell line expressing the TGF-β dominant negative receptor-2 (TGF-β DNRII) was used [
22]. The PG13 TGF-β DNRII cell line was cultured in complete DMEM with 10% FBS. Transduction efficiency of the PG13 cell lines were tested on a weekly basis by TGF-β cell surface expression as analyzed by flow cytometry. Retroviral supernatants were collected 24 to 48 h after cells were split and once cells reached about 70% confluency. Retroviral supernatants were either used fresh or snap-frozen and stored at − 80 °C.
NK cell manufacture
StemCell EasySep NK Cell Enrichment Kit (StemCell Technologies, Vancouver, Canada) was used to obtain a pure population of NK cells, according to the manufacturer’s protocol. NK cells were activated with IL15 and incubated overnight in Stem Cell Growth Media (CellGenix, Freiburg, Germany) supplemented with 10% FBS and 1% GlutaMax (cSCGM), and expanded for 14 days.
A modified K562 immortalized human myeloid leukemia cell line expressing membrane bound IL15 and 41BB was obtained from Dr. Cliona Rooney at Baylor College of Medicine/Texas Children’s Hospital [
23]. Modified K562s were irradiated at 200 Gy prior to stimulating NK cells. NK cells were stimulated at a 1 to 2 ratio of NK to K562 cells, and fed with 200 U/mL rhIL2 (R&D, Minneapolis, MN) and 15 ng/mL rhIL15 (R&D, Minneapolis, MN).
Three days post-stimulation, NK cells were transduced with retroviral supernatant, using Retronectin (Takara Bio USA, Mountainview, CA) coated plates, according to the manufacturer's protocol. Retrovirus supernatant was spun on the coated plates for 2 h at 2000 G at 30 °C. NK cells were plated at 5 × 105 cells/well with the addition of 200 IU/mL IL2 in complete Stem Cell Growth Media (cSCGM).
Three days post-transduction, NK cells were again stimulated with K562 feeder cells, IL2, and IL15, as previously described [
21]. NK cells were challenged with 5 ng/mL TGF-β cytokine and 2 mL/well of fresh Daoy (ATCC, Manassas, VA) supernatant for 5 days following stimulation. NK cells were then collected for functional assays. Excess cells were cryopreserved in freeze media containing 50% FBS, 40% RPMI, and 10% Dimethyl Sulfoxide (Sigma-Aldrich, St Louis, MO).
Flow cytometry
Cell phenotype, transduction efficiency, activation, and exhaustion of TGF-β DNR transduced cells and their nontransduced counterparts were determined by flow cytometry, using the following cell surface markers: CD3, CD56 (BioLegend, San Diego, CA), TGF-β RII (“wildtype” R&D, Minneapolis, MN), TGF-β RII (“DNR” Cambridge, UK), goat-anti mouse IgG, CD16, NKG2D, DNAM-1, NKp30, NKp46, CCR2, and CX3CR1 (BioLegend, San Diego, CA and BD Biosciencees, Franklin Lakes, NJ). Where reported, MFI was calculated from the geometric mean.
Cytokine luminex
To assess the polyfunctionality of TGF DNR expressing NK cells, cytokine secretion was measured using the Bio-plex Pro Human 17-plex Cytokine Assay Kit (Bio-Rad, Hercules, CA). Supernatants were collected on day 12 of manufacture, 5 days after the second stimulation and TGF-β cytokine and Daoy supernatant challenge. The Cytokine Assay Kit was run according to manufacturer’s protocol. The cytokine concentrations were calculated using the provided standards.
Chromium release cytotoxicity assay
The ability of TGF-β DNR transduced NK cells to kill medulloblastoma was determined by chronium-51 (Cr51) release cytotoxicity assay. Both Daoy (ATCC, Manassas, VA) and primary medulloblastoma lines were used as targets and incubated with chromium 51 for 1 h. Targets were then cocultured with NK cells for 4 h, in 37 °C, at effector to target ratios of 40:1, 20:1, 10:1, 5:1, and 2.5:1. After the 4 h coincubation, plates were spun to allow cells to settle at the bottom and 100 μL of supernatant was collected onto a Lumia plate (Perkin-Elmer, Waltham, MA). The plate was incubated overnight at room temperature to allow for the supernatant to dry. Lumia plates were read on a MicroBeta2 counter. Specific lysis was calculated as the difference of experimental and spontaneous release divided by the difference of the maximum and spontaneous release times 100.
TGF-β luminex of conditioned media
To assess the ability of the TGF-β dominant negative receptor to remove TGF-β from the cell supernatant, TGF-β concentrations were determined by a TGF-β-1, 2, 3 multiplex assay (Millipore, Burlington MA). Supernatants were collected on day 12 of manufacture, 5 days after the second stimulation and TGF-β cytokine and Daoy supernatant challenge. Supernatants were frozen at − 80 °C until kit was run. The kit was run according to the manufacturer’s protocol and the TGF-β concentration determined using the provided standards.
Statistical analysis
Data is reported as mean ± standard error of the mean. Comparisons between cord and patient samples were done using the Mann–Whitney test. Comparisons between transduced and nontransduced cells, grown in medulloblastoma-conditioned and unconditioned media, were analyzed using Wilcoxon signed rank tests. Cytotoxicity comparisons were done using t test (a Shapiro–Wilk test showed the data passed the normality test). A p < 0.05 was considered statistically significant. Statistical analysis was performed using Graphpad PRISM.
Discussion
A few studies [
25‐
29] have documented the immunosuppressive capabilities of medulloblastoma, although we show for the first time that medulloblastoma-conditioned media (which we demonstrate to have high levels of TGF-β1) impairs NK cell activity, which can be restored by a dominant negative receptor against TGF-β. The use of TGF-β DNR to protect cells in other tumor settings has been described by other groups, including our own [
21,
22,
30‐
32]. Hence, we extended this approach as a potential immunotherapy for the treatment of medulloblastoma.
In this study, we looked at the effects of TGF-β-rich medulloblastoma supernatant on DNR-transduced NK cells, and we demonstrated protection from impaired cytotoxicity similar to what other groups [
21,
22,
30‐
32] have reported, maintenance of TGF-β RII receptor expression, and protection from CD16 downregulation (which may suggest maintenance of ADCC in an immune suppressive environment) in line with observations made by Keskin et al. [
33]. It would be interesting to explore the relationship between TGF-β and ADCC further, by looking at the effects of the cytokine on the ability of NK cells to mediate killing through obinutuzumab (CD20), mogamulizumab (CCR4), margetuximab (HER2), and others. This downregulation of CD16 is countered by the dominant negative receptor and to our knowledge, this is the first time that such protection by a DNR has been reported. Of note, we observed lower cytotoxicity against medulloblastoma cell lines compared to those previously reported by Castriconi et al. [
34]. Though our Daoy cell lines express ligands for NK mediated killing (Additional file
1: Figure S4), they also express HLA-class I, which is inhibitory to NK cells (Additional file
1: Figure S4). One major difference between our work and Castriconi’s work is our use of NK cells derived from umbilical cord blood. While some groups do report lower cytolytic activity in NK cells derived from cord blood [
35], this is overcome with ex vivo expansion and ultimately observed differences in cytolytic activity are likely due to the different assays used in different laboratories. It is also worth noting the advantages of cord blood, such as the easy availability for off-the-shelf cell therapies, minimized risks of graft versus host disease, ability to ex vivo expand cord blood as a source of cells which is why we explored cord blood as a donor source for an NK cell therapeutic in the brain tumor setting [
36]. In addition, the use of cord blood as a source of allogeneic NK cells is advantageous because: (a) they can be ex vivo expanded to clinically useful cell numbers; and (b) they allow increased chances of identifying HLA-compatible and KIR-mismatched products because of their immediate availability in established cord blood banks.
Maintenance of TGF-β RII receptor expression likely results from abrogation of negative enrichment that occurs in the untransduced cells. We posit that in the untransduced cells, continued culture of cells in the TGF-β-rich medulloblastoma media selected against cells that expressed the wildtype receptor, and thus, over time, the percentage of cells expressing TGF-β RII receptor decreases. This is not apparent in transduced cells because such negative enrichment does not occur.
Given that we did not observe any correlation between transduction efficiency and immune abrogation efficacy, a minimum effective dose for the therapy was not determined. The expression of wildtype TGF-β RII receptor varied in our samples (Additional file
1: Figure S1B), and might account for the variable results: higher expression of wildtype TGF-β RII receptor would make cells more susceptible to immune suppression.
Our results also suggest that this receptor can potentially restore function to other immune cell subsets by acting as a cytokine sink. We posit that this likely results from increased binding of the cytokine to the DNR, relative to the wildtype receptor. We therefore envision a scenario where the presence of DNR on adoptively transferred immune cells helps to clear the immune suppressive environment in malignancies, improving the efficacy of endogenous immune cells.
Finally, CCR2 expression in TGF-β-protected cells may improve efficacy (although expression is limited to a small subset of the population). Previous studies have shown that this chemokine is sufficient for migration of immune cells [
37], including across the blood brain barrier [
37]. Other studies have shown similar reductions in chemokine receptor expression in the presence of TGF-β: CX3CR1 levels decreased in NK cells when exposed to neuroblastoma-derived TGF-β [
38]. We, however, have not seen similar decreases (Additional file
1: Figure S5). Furthermore, the upregulation in CCR2 does not seem to translate to consistent improvements in migration (Additional file
1: Figure S6), although it would still be interesting in future studies to evaluate whether this effect has functional consequences in optimized in vivo models.
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