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Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies.

Background Neuroblastoma (NBL) is the most common pediatric solid tumor and responsible for about 15% of all pediatric cancer deaths. The majority of high-risk (HR) patients suffers from relapse after intense therapy regimens, resulting in a 5-year survival rate of only 40%. Even though the potential of immune interference in HR-NBL is shown by the additive effect of anti-GD2 monoclonal antibody therapy to the treatment protocol, long-term follow-up studies reveal that the beneficial effect of immunotherapy diminishes over time. We hypothesize that this is a result of inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL and its microenvironment. One of the most remarkable immunomodulatory strategies of NBL tumors is the absence of MHC-I surface expression, thereby preventing cytotoxic T cell recognition and killing. MHC-I lacking cells are known to be subjected to NK cell mediated cytotoxicity, however, we have shown that NBL is able to evade this by temporary upregulating surface expression of MHC-I, thereby becoming temporarily more prone to T cell mediated cytotoxicity. The aim of this project is to identify pharmacological strategies to enhance adaptive immune activation and therewith immunogenicity of HR-NBL. Materials and Methods FDA-approved drug libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cell lines using high-throughput flow cytometry analyses optimized for adherent NBL cells. The effect of positive hits was subsequently confirmed in a panel of NBL patient-derived tumeroids. Alterations in the transcriptome and translatome upon incubation with compounds of interest were further studied to identify potential additional immunomodulatory effects in NBL. Ultimately, compound treated NBL cell lines and tumeroids were co-cultured with PRAME reactive tumor-specific T cells and healthy-donor NK cells to determine the in vitro effect on T- and NK cell cytotoxicity. Results Drug library screening revealed MHC-I upregulation upon treatment of NBL cell lines and patient-derived tumeroids with multiple histon deacetylase inhibitors (HDACi). Further investigation of immunomodulatory effects of HDACi in NBL revealed enhanced expression of several additional players of the antigen presenting machinery, immunoproteasome expression, and MICA/MICB upregulation in NBL cells. We show that in untreated NBL cells, plasticity of MHC-I expression causes evasion of both NK- and T cell mediated cytotoxicity. Intriguingly, co-culture of NBL cells with tumor-specific T cells and healthy-donor NK cells upon treatment with the HDACi Entinostat resulted in enhanced in vitro T- and NK cell activation and cytotoxicity. Conclusions We show pharmacological upregulation of MHC-I, other antigen presenting machinery players, and the NKG2D ligands MICA/MICB upon HDACi in HR-NBL. Pre-treatment of NBL with HDACi resulted in enhanced in vitro T- and NK cell mediated cytotoxicity, substantiating HDACi as a potential strategy to improve adaptive immune engagement and therewith immunogenicity to aid NBL treatment. This work was supported by the Villa Joep Foundation [IWOV-Actief.51391.180034]. Disclosure Information A.M. Cornel: None. D.A.M.H. van den Beemt: None. E. Dunnebach: None. M.P. van Dierselhuis: None. S. Nierkens: None.

Active immunotherapy approaches for neuroblastoma (NB), a pediatric cancer of the sympathetic nervous system, has met with limited success. Especially challenging is the genetic heterogeneity of NB which makes it difficult to identify factors that consistently indicate the likelihood of an effective immune response and thereby select patients who are most likely to benefit from immunotherapy. Hence, we undertook an unbiased analysis of gene expression signatures from >500 well-annotated primary NBs representing diverse clinical and genetic subtypes to identify of predictors of immune response. Using clustering analysis of bulk transcriptomic signatures from these tumors, we identified a subset of NBs that was notable for the high expression of genes associated with anti-tumor immune response. These “immunogenic” tumors showed a predominance of gene expression signatures derived from malignant cells with primitive neural crest-like or mesenchymal properties, one of the two cell states that shape intratumoral heterogeneity in NB. In contrast, tumors that expressed committed, adrenergic neuron-like signatures were less immunogenic. Single-cell (sc) RNA-seq and immunohistochemistry analysis further confirmed that NBs comprise both adrenergic and mesenchymal tumor cells, and that the presence of mesenchymal cells positively associated with immune cell infiltration into the TME. scRNA-seq also revealed that mesenchymal NB cells were enriched for inflammatory gene signature. Gene expression analysis of isogenic pairs of adrenergic and mesenchymal cells showed that mesenchymal NBs differentially upregulate genes involved in regulating antigen processing and presentation, MHC class I expression, type-I interferon and TLR3 signaling, and NK cell activation. This is achieved through a permissive chromatin landscape at the promoters of these immune regulatory genes that support their high expression in mesenchymal cells. By contrast, in adrenergic cells, tumor-intrinsic immune genes are epigenetically silenced by the PRC2 complex and PRC2 inhibition leads to increased immune cell activation. Remarkably, induction of the mesenchymal state in adrenergic cells through transcriptional reprogramming by PRRX1 or therapy resistance is accompanied by the epigenetic activation of innate and adaptive immune response genes. Functionally, the inherent immunogenicity of mesenchymal cells promotes T cell infiltration by secreting inflammatory cytokines, enables efficient targeting by antigen-specific cytotoxic T and NK cells, and imparts responsiveness to immune checkpoint blockade in a syngeneic NB model. In conclusion, our study uncovers an unappreciated link between immunogenicity and tumor lineage state in NB, and rationalizes future interrogations into (i) avenues through which the vulnerability of mesenchymal cells to immune-mediated targeting could be harnessed clinically and (ii) how perturbation of epigenetically-regulated cell states could be harnessed to promote anti-tumor immune response. Citation Format: Satyaki Sengupta, Sanjukta Das, Angela C. Crespo, Annelisa M. Cornel, Anand G. Patel, Navin R. Mahadevan, Marco Campisi, Alaa K. Ali, Bandana Sharma, Jared H. Rowe, Rogier Versteeg, Rudolf Jaenisch, Stefani Spranger, Rizwan Romee, Brian C. Miller, David A. Barbie, Stefan Nierkens, Michael A. Dyer, Judy Lieberman, Rani E. George. Divergent tumor cell states in neuroblastoma possess distinct immunogenic phenotypes [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A08.

BackgroundImmunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to tackle one of the most notable immunomodulatory processes in neuroblastoma (NBL), absence of major histocompatibility complex class I (MHC-I) surface expression, a process greatly limiting cytotoxic T cell engagement. We and others have previously shown that MHC-I expression can be induced by cytokine-driven immune modulation. Here, we aimed to identify tolerable pharmacological repurposing strategies to upregulate MHC-I expression and therewith enhance T cell immunogenicity in NBL.MethodsDrug repurposing libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cells using high-throughput flow cytometry analyses optimized for adherent cells. The effect of positive hits was confirmed in a panel of NBL cell lines and patient-derived organoids. Compound-treated NBL cell lines and organoids were cocultured with preferentially expressed antigen of melanoma (PRAME)-reactive tumor-specific T cells and healthy-donor natural killer (NK) cells to determine the in vitro effect on T cell and NK cell cytotoxicity. Additional immunomodulatory effects of histone deacetylase inhibitors (HDACi) were identified by transcriptome and translatome analysis of treated organoids.ResultsDrug library screening revealed MHC-I upregulation by inhibitor of apoptosis inhibitor (IAPi)- and HDACi drug classes. The effect of IAPi was limited due to repression of nuclear factor kappa B (NFκB) pathway activity in NBL, while the MHC-I-modulating effect of HDACi was widely translatable to a panel of NBL cell lines and patient-derived organoids. Pretreatment of NBL cells with the HDACi entinostat enhanced the cytotoxic capacity of tumor-specific T cells against NBL in vitro, which coincided with increased expression of additional players regulating T cell cytotoxicity (eg, TAP1/2 and immunoproteasome subunits). Moreover, MICA and MICB, important in NK cell cytotoxicity, were also increased by entinostat exposure. Intriguingly, this increase in immunogenicity was accompanied by a shift toward a more mesenchymal NBL cell lineage.ConclusionsThis study indicates the potential of combining (immuno)therapy with HDACi to enhance both T cell-driven and NKcell-driven immune responses in patients with HR-NBL.

Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. However, the limited numbers of autologous tumor-associated antigen (TAA)-specific T cells and the functional aberrancies, due to disease progression or treatment, remain factors that may significantly limit the success of the therapy. The use of allogeneic T cells, such as umbilical cord blood (CB) derived, overcomes these issues but requires gene modification to induce a robust and specific anti-tumor effect. CB T cells are readily available in CB banks and show low toxicity, high proliferation rates, and increased anti-leukemic effect upon transfer. However, the combination of anti-tumor gene modification and preservation of advantageous immunological traits of CB T cells represent major challenges for the harmonized production of T cell therapy products. In this manuscript, we optimized a protocol for expansion and lentiviral vector (LV) transduction of CB CD8+ T cells, achieving a transduction efficiency up to 83%. Timing of LV treatment, selection of culture media, and the use of different promoters were optimized in the transduction protocol. LentiBOOST was confirmed as a non-toxic transduction enhancer of CB CD8+ T cells, with minor effects on the proliferation capacity and cell viability of the T cells. Positively, the use of LentiBOOST does not affect the functionality of the cells, in the context of tumor cell recognition. Finally, CB CD8+ T cells were more amenable to LV transduction than peripheral blood (PB) CD8+ T cells and maintained a more naive phenotype. In conclusion, we show an efficient method to genetically modify CB CD8+ T cells using LV, which is especially useful for off-the-shelf adoptive cell therapy products for cancer treatment., Graphical abstract, The manuscript proposes an efficient method to gene modify cord blood-derived CD8+ T cells using lentiviral vectors and a transduction enhancer (LentiBOOST). Future application of these findings can improve the generation of allogeneic T cell therapies for the treatment of cancer.

Simple Summary Neuroblastoma is a type of childhood cancer accounting for approximately 15% of childhood cancer deaths. Despite intensive treatment, including immunotherapy, prognosis of high-risk neuroblastoma is poor. Increasing amounts of research show that the fighting capacity of the immune system is very important for the outcome of neuroblastoma patients. Therefore, we investigated the fighting capacity of immune cells in blood at diagnosis and during the different phases of therapy. In this study, we observed both processes that stimulate and processes that decrease fighting capacity of immune cells in neuroblastoma patients during therapy. Despite this, we show that overall fighting capacity of the immune system of neuroblastoma patients is impaired at diagnosis as well as during therapy. In addition, we observed a lot of variation between patients, which might explain differences in therapy efficacy between patients. This study provides insight for improvement of therapy timing as well as new therapy strategies enhancing immune cell fighting capacity. Abstract Despite intensive treatment, including consolidation immunotherapy (IT), prognosis of high-risk neuroblastoma (HR-NBL) is poor. Immune status of patients over the course of treatment, and thus immunological features potentially explaining therapy efficacy, are largely unknown. In this study, the dynamics of immune cell subsets and their function were explored in 25 HR-NBL patients at diagnosis, during induction chemotherapy, before high-dose chemotherapy, and during IT. The dynamics of immune cells varied largely between patients. IL-2- and GM-CSF-containing IT cycles resulted in significant expansion of effector cells (NK-cells in IL-2 cycles, neutrophils and monocytes in GM-CSF cycles). Nonetheless, the cytotoxic phenotype of NK-cells was majorly disturbed at the start of IT, and both IL-2 and GM-CSF IT cycles induced preferential expansion of suppressive regulatory T-cells. Interestingly, proliferative capacity of purified patient T-cells was impaired at diagnosis as well as during therapy. This study indicates the presence of both immune-enhancing as well as regulatory responses in HR-NBL patients during (immuno)therapy. Especially the double-edged effects observed in IL-2-containing IT cycles are interesting, as this potentially explains the absence of clinical benefit of IL-2 addition to IT cycles. This suggests that there is a need to combine anti-GD2 with more specific immune-enhancing strategies to improve IT outcome in HR-NBL.

In recent years, major advances have been made in cancer immunotherapy. This has led to significant improvement in prognosis of cancer patients, especially in the hematological setting. Nonetheless, translation of these successes to solid tumors was found difficult. One major mechanism through which solid tumors can avoid anti-tumor immunity is the downregulation of major histocompatibility complex class I (MHC-I), which causes reduced recognition by- and cytotoxicity of CD8+ T-cells. Downregulation of MHC-I has been described in 40–90% of human tumors, often correlating with worse prognosis. Epigenetic and (post-)transcriptional dysregulations relevant in the stabilization of NFkB, IRFs, and NLRC5 are often responsible for MHC-I downregulation in cancer. The intrinsic reversible nature of these dysregulations provides an opportunity to restore MHC-I expression and facilitate adaptive anti-tumor immunity. In this review, we provide an overview of the mechanisms underlying reversible MHC-I downregulation and describe potential strategies to counteract this reduction in MHC-I antigen presentation in cancer.

Background Multiparameter flow cytometry is the preferred method to determine immunophenotypic features of cells present in a wide variety of sample types. Standardization is key to avoid inconsistencies and subjectivity of interpretations between clinical diagnostic laboratories. Among these standardization requirements, synchronization between different flow cytometer instruments is indispensable to obtain comparable results. This study aimed to investigate whether two widely used flow cytometers, the FACSCanto II and LSRFortessa, can be effectively synchronized utilizing calibration bead–based synchronization. Method Two FACSCanto II and two LSRFortessa flow cytometers were synchronized with both multicolor hard‐dyed and single‐fluorochrome–conjugated surface‐dyed beads according to the manufacturer's instructions. Cell staining was performed on five whole‐blood samples obtained from healthy controls and were analyzed upon synchronization with the respective synchronization protocols. Results Comparability criteria (defined as

Neuroblastoma (NBL) is the most common extracranial solid tumor in childhood. Despite intense treatment, children with this high-risk disease have a poor prognosis. Immunotherapy showed a significant improvement in event-free survival in high-risk NBL patients receiving chimeric anti-GD2 in combination with cytokines and isotretinoin after myeloablative consolidation therapy. However, response to immunotherapy varies widely, and often therapy is stopped due to severe toxicities. Objective markers that help to predict which patients will respond or develop toxicity to a certain treatment are lacking. Immunotherapy guided via immune monitoring protocols will help to identify responders as early as possible, to decipher the immune response at play, and to adjust or develop new treatment strategies. In this review, we summarize recent studies investigating frequency and phenotype of immune cells in NBL patients prior and during current treatment protocols and highlight how these findings are related to clinical outcome. In addition, we discuss potential targets to improve immunogenicity and strategies that may help to improve therapy efficacy. We conclude that immune monitoring during therapy of NBL patients is essential to identify predictive biomarkers to guide patients towards effective treatment, with limited toxicities and optimal quality of life.

In the last decade, screening compound libraries on live cells has become an important step in drug discovery. The abundance of compounds in these libraries requires effective high‐throughput (HT) analyzing methods. Although current cell‐based assay protocols are suitable for HT analyses, the analysis itself is often restrained to simple, singular outcomes. Incorporation of HT samplers on flow cytometers has provided an interesting approach to increase the number of measurable parameters and increase the sensitivity and specificity of analyses. Nonetheless, to date, the labor intensive and time‐consuming strategies to detach and stain adherent cells before flow cytometric analysis has restricted use of HT flow cytometry (HTFC) to suspension cells. We have developed a universal “no‐touch” HTFC antibody staining protocol in 384‐well microplates to bypass washing and centrifuging steps of conventional flow cytometry protocols. Optimizing culture conditions, cell‐detachment and staining strategies in 384‐well microplates resulted in an HTFC protocol with an optimal stain index with minimal background staining. The method has been validated using six adherent cell lines and simultaneous staining of four parameters. This HT screening protocol allows for effective monitoring of multiple cellular markers simultaneously, thereby increasing informativity and cost‐effectiveness of drug screening. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.