Identification of tumor rejection antigens and the immunologic landscape of medulloblastoma.

BACKGROUND: The current standard of care treatments for medulloblastoma are insufficient as these do not take tumor heterogeneity into account. Newer, safer, patient-specific treatment approaches are required to treat high-risk medulloblastoma patients who are not cured by the standard therapies. Immunotherapy is a promising treatment modality that could be key to improving survival and avoiding morbidity. For an effective immune response, appropriate tumor antigens must be targeted. While medulloblastoma patients with subgroup-specific genetic substitutions have been previously reported, the immunogenicity of these genetic alterations remains unknown. The aim of this study is to identify potential tumor rejection antigens for the development of antigen-directed cellular therapies for medulloblastoma. METHODS: We developed a cancer immunogenomics pipeline and performed a comprehensive analysis of medulloblastoma subgroup-specific transcription profiles (n = 170, 18 WNT, 46 SHH, 41 Group 3, and 65 Group 4 patient tumors) available through International Cancer Genome Consortium (ICGC) and European Genome-Phenome Archive (EGA). We performed in silico antigen prediction across a broad array of antigen classes including neoantigens, tumor-associated antigens (TAAs), and fusion proteins. Furthermore, we evaluated the antigen processing and presentation pathway in tumor cells and the immune infiltrating cell landscape using the latest computational deconvolution methods. RESULTS: Medulloblastoma patients were found to express multiple private and shared immunogenic antigens. The proportion of predicted TAAs was higher than neoantigens and gene fusions for all molecular subgroups, except for sonic hedgehog (SHH), which had a higher neoantigen burden. Importantly, cancer-testis antigens, as well as previously unappreciated neurodevelopmental antigens, were found to be expressed by most patients across all medulloblastoma subgroups. Despite being immunologically cold, medulloblastoma subgroups were found to have distinct immune cell gene signatures. CONCLUSIONS: Using a custom antigen prediction pipeline, we identified potential tumor rejection antigens with important implications for the development of immunotherapy for medulloblastoma.

Toxoplasma gondii infection supports the infiltration of T cells into brain tumors.

Few T cells infiltrate into primary brain tumors, fundamentally hampering the effectiveness of immunotherapy. We hypothesized that Toxoplasma gondii, a microorganism that naturally elicits a Th1 response in the brain, can promote T cell infiltration into brain tumors despite their immune suppressive microenvironment. Using a mouse genetic model for medulloblastoma, we found that T. gondii infection induced the infiltration of activatable T cells into the tumor mass and led to myeloid cell reprogramming toward a T cell-supportive state, without causing severe health issues in mice. The study provides a concrete foundation for future studies to take advantage of the immune modulatory capacity of T. gondii to facilitate brain tumor immunotherapy.

[Clinicopathological characteristics of the CD8+ T lymphocytes infiltration and its mechanism in distinct molecular subtype of medulloblastoma].

OBJECTIVE: To investigate the characteristics of the CD8+ T cells infiltration from the 4 subtypes in medulloblastoma (MB), to analyze the relationship between CD8+ T cells infiltration and prognosis, to study the function of C-X-C motif chemokine ligand 11 (CXCL11) and its receptor in CD8+ T cells infiltration into tumors and to explore the potential mechanism, and to provide the necessary clinicopathological basis for exploring the immunotherapy of MB. METHODS: In the study, 48 clinical MB samples (12 cases in each of 4 subtypes) were selected from the multiple medical center from 2012 to 2019. The transcriptomics analysis for the tumor of 48 clinical samples was conducted on the NanoString PanCancer IO360TM Panel (NanoString Technologies). Immunohistochemistry (IHC) staining of formalin-fixed, paraffin-embedded sections from MB was carried out using CD8 primary antibody to analyze diffe-rential quantities of CD8+ T cells in the MB four subtypes. Through bioinformatics analysis, the relationship between CD8+T cells infiltration and prognosis of the patients and the expression differences of various chemokines in the different subtypes of MB were investigated. The expression of CXCR3 receptor on the surface of CD8+T cells in MB was verified by double immunofluorescence staining, and the underlying molecular mechanism of CD8+T cells infiltration into the tumor was explored. RESULTS: The characteristic index of CD8+T cells in the WNT subtype of MB was relatively high, suggesting that the number of CD8+T cells in the WNT subtype was significantly higher than that in the other three subtypes, which was confirmed by CD8 immunohistochemical staining and Gene Expression Omnibus (GEO) database analysis by using R2 online data analysis platform. And the increase of CD8+T cells infiltration was positively correlated with the patient survival. The expression level of CXCL11 in the WNT subtype MB was significantly higher than that of the other three subtypes. Immunofluorescence staining showed the presence of CXCL11 receptor, CXCR3, on the surface of CD8+T cells, suggesting that the CD8+T cells might be attracted to the MB microenvironment by CXCL11 through CXCR3. CONCLUSION: The CD8+T cells infiltrate more in the WNT subtype MB than other subtypes. The mechanism may be related to the activation of CXCL11-CXCR3 chemokine system, and the patients with more infiltration of CD8+T cells in tumor have better prognosis. This finding may provide the necessary clinicopathological basis for the regulatory mechanism of CD8+T cells infiltration in MB, and give a new potential therapeutic target for the future immunotherapy of MB.

GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

PURPOSE: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. EXPERIMENTAL DESIGN: GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. RESULTS: GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. CONCLUSIONS: Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Molecular profile reveals immune-associated markers of medulloblastoma for different subtypes.

Medulloblastoma, a common pediatric malignant tumor, has been recognized to have four molecular subgroups [wingless (WNT), sonic hedgehog (SHH), group 3, group 4], which are defined by the characteristic gene transcriptomic and DNA methylomic profiles, and has distinct clinical features within each subgroup. The tumor immune microenvironment is integral in tumor initiation and progression and might be associated with therapeutic responses. However, to date, the immune infiltrative landscape of medulloblastoma has not yet been elucidated. Thus, we proposed MethylCIBERSORT to estimate the degree of immune cell infiltration and weighted correlation network analysis (WGCNA) to find modules of highly correlated genes. Synthesizing the hub genes in the protein-protein interaction (PPI) network and modules of the co-expression network, we identify three candidate biomarkers [GRB2-associated-binding protein 1 (GAB1), Abelson 1 (ABL1), and CXC motif chemokine receptor type 4 (CXCR4)] via the molecular profiles of medulloblastoma. Given this, we investigated the correlation between these three immune hub genes and immune checkpoint blockade response and the potential of drug prediction further. In addition, this study demonstrated a higher presence of endothelial cells and infiltrating immune cells in Group 3 tumor bulk. The above results will be conducive to better comprehending the immune-related pathogenesis and treatment of medulloblastoma.

Changes in Peripheral Blood Regulatory T Cells and IL-6 and IL-10 Levels Predict Response of Pediatric Medulloblastoma and Germ Cell Tumors With Residual or Disseminated Disease to Craniospinal Irradiation.

PURPOSE: Radiation therapy (RT) modulates immune cells and cytokines, resulting in both clinically beneficial and detrimental effects. The changes in peripheral blood T lymphocyte subsets and cytokines during RT for pediatric brain tumors and the association of these changes with therapeutic outcomes have not been well described. METHODS AND MATERIALS: The study population consisted of children (n = 83, aged 3~18) with primary brain tumors (medulloblastoma, glioma, germ cell tumors (GCT), and central nervous system embryonal tumor-not otherwise specified), with or without residual or disseminated (R/D) diseases who were starting standard postoperative focal or craniospinal irradiation (CSI). Peripheral blood T lymphocyte subsets collected before and 4 weeks after RT were enumerated by flow cytometry. Plasma levels of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-α, interferon-γ, and IL-17A were measured by cytometric bead array. RESULTS: Patients with R/D lesions receiving CSI (n = 32) had a post-RT increase in the frequency of CD3+T and CD8+T cells, a decrease in CD4+T cells, and an increase in regulatory T cells (Tregs) and CD8+CD28- suppressor cells, which was more predominantly seen in these patients than in other groups. In the CSI group with such R/D lesions, consisting of patients with medulloblastoma and germ cell tumors, 19 experienced a complete response (CR) and 13 experienced a partial response (PR) on imaging at 4 weeks after RT. The post/pre-RT ratio of Tregs (P = .0493), IL-6 (P = .0111), and IL-10 (P = .0070) was lower in the CR group than in the PR group. Multivariate analysis revealed that the post/pre-RT ratios of Treg, IL-6, and IL-10 were independent predictors of CR (P < .0001, P = .018, P < .0001, respectively). The areas under the receiver operating curves and confidence intervals were 0.7652 (0.5831-0.8964), 0.7794 (0.5980-0.9067), and 0.7085 (0.5223-0.8552) for IL-6, IL-10, and Treg, respectively. The sensitivities of IL-6, IL-10, and Treg to predict radiotherapeutic responses were 100%, 92.3%, and 61.5%, and specificity was 52.6%, 57.9%, and 84.2%, respectively. CONCLUSIONS: CSI treatment to those with R/D lesions predominantly exerted an effect on antitumor immune response compared with both R/D lesion-free but exposed to focal or CSI RT and with R/D lesions and exposed to focal RT. Such CSI with R/D lesions group experiencing CR is more likely to have a decrease in immunoinhibitory molecules and cells than patients who only achieve PR. Measuring peripheral blood Treg, IL-6, and IL-10 levels could be valuable for predicting radiotherapeutic responses of pediatric brain tumors with R/D lesions to CSI for medulloblastoma and intracranial germ cell tumors.

Macrophages in SHH subgroup medulloblastoma display dynamic heterogeneity that varies with treatment modality.

Tumor-associated macrophages (TAMs) play an important role in tumor immunity and comprise of subsets that have distinct phenotype, function, and ontology. Transcriptomic analyses of human medulloblastoma, the most common malignant pediatric brain cancer, showed that medulloblastomas (MBs) with activated sonic hedgehog signaling (SHH-MB) have significantly more TAMs than other MB subtypes. Therefore, we examined MB-associated TAMs by single-cell RNA sequencing of autochthonous murine SHH-MB at steady state and under two distinct treatment modalities: molecular-targeted inhibitor and radiation. Our analyses reveal significant TAM heterogeneity, identify markers of ontologically distinct TAM subsets, and show the impact of brain microenvironment on the differentiation of tumor-infiltrating monocytes. TAM composition undergoes dramatic changes with treatment and differs significantly between molecular-targeted and radiation therapy. We identify an immunosuppressive monocyte-derived TAM subset that emerges with radiation therapy and demonstrate its role in regulating T cell and neutrophil infiltration in MB.

Development of a prognostic model based on an immunogenomic landscape analysis of medulloblastoma.

Medulloblastoma (MB) is one of the most common central nervous system tumors in children. At present, the vital role of immune abnormalities has been proved in tumorigenesis and progression. However, the immune mechanism in MB is still poorly understood. In the present study, 51 differentially expressed immune-related genes (DE-IRGs) and 226 survival associated immune-related genes (Sur-IRGs) were screened by an integrated analysis of multi-array. Moreover, the potential pathways were enriched by functional analysis, such as 'cytokine-cytokine receptor interaction', 'Ras signaling pathway', 'PI3K-Akt signaling pathway' and 'pathways in cancer'. Furthermore, 10 core IRGs were identified from DE-IRGs and Sur-IRGs. And the potential regulatory mechanisms of core IRGs were also explored. Additionally, a new prognostic model, including 7 genes (HDGF, CSK, PNOC, S100A13, RORB, FPR1, and ICAM2) based on IRGs, was established by multivariable COX analysis. In summary, our study revealed the underlying immune mechanism of MB. Moreover, we developed a prognostic model associated with clinical characteristics and could reflect the infiltration of immune cells.

Histone demethylase UTX/KDM6A enhances tumor immune cell recruitment, promotes differentiation and suppresses medulloblastoma.

The deregulation of epigenetic pathways has been implicated as a critical step in tumorigenesis including in childhood brain tumor medulloblastoma. The H3K27me3 demethylase UTX/KDM6A plays important roles in development and is frequently mutated in various types of cancer. However, how UTX regulates tumor development remains largely unclear. Here, we report the generation of a UTX-deleted mouse model of SHH medulloblastoma that demonstrates the tumor suppressor functions of UTX, which could be antagonized by the deletion of another H3K27me3 demethylase JMJD3/KDM6B. Intriguingly, UTX deletion in cancerous cerebellar granule neuron precursors (CGNPs) resulted in the impaired recruitment of host CD8+ T cells to the tumor microenvironment through a non-cell autonomous mechanism. In both mouse medulloblastoma models and in human medulloblastoma cells, we showed that UTX activates Th1-type chemokines, which are responsible for T cell migration. Surprisingly, our results showed that the depletion of cytotoxic CD8+ T cells did not affect mouse medulloblastoma growth. Nevertheless, the UTX/chemokine/T cell recruitment pathway we identified may be applied to many other cancers and may be important for improving cancer immunotherapy. In addition, UTX is required for the expression of NeuroD2 in precancerous progenitors, which encodes a potent proneural transcription factor. Overexpression of NEUROD2 in CGNPs decreased cell proliferation and increased neuron differentiation. We showed that UTX deletion led to impaired neural differentiation, which could coordinate with active SHH signaling to accelerate medulloblastoma development. Thus, UTX regulates both cell-intrinsic oncogenic processes and the tumor microenvironment in medulloblastoma. Our study provides insights into both medulloblastoma development and context dependent functions of UTX in tumorigenesis.

Pediatric pan-central nervous system tumor analysis of immune-cell infiltration identifies correlates of antitumor immunity.

Immune-therapy is an attractive alternative therapeutic approach for targeting central nervous system (CNS) tumors and the constituency of the Tumor Immune Microenvironment (TIME) likely to predict patient response. Here, we describe the TIME of >6000 primarily pediatric CNS tumors using a deconvolution approach (methylCIBERSORT). We produce and validate a custom reference signature defining 11 non-cancer cell types to estimate relative proportions of infiltration in a panCNS tumor cohort spanning 80 subtypes. We group patients into three broad immune clusters associated with CNS tumor types/subtypes. In cohorts of medulloblastomas (n = 2325), malignant rhabdoid tumors (n = 229) and pediatric high-grade gliomas (n = 401), we show significant associations with molecular subgroups/subtypes, mutations, and prognosis. We further identify tumor-specific immune clusters with phenotypic characteristics relevant to immunotherapy response (i.e. Cytolytic score, PDL1 expression). Our analysis provides an indication of the potential future therapeutic and prognostic possibilities of immuno-methylomic profiling in pediatric CNS tumor patients that may ultimately inform approach to immune-therapy.

YBX1 Indirectly Targets Heterochromatin-Repressed Inflammatory Response-Related Apoptosis Genes through Regulating CBX5 mRNA.

Medulloblastomas arise from undifferentiated precursor cells in the cerebellum and account for about 20% of all solid brain tumors during childhood; standard therapies include radiation and chemotherapy, which oftentimes come with severe impairment of the cognitive development of the young patients. Here, we show that the posttranscriptional regulator Y-box binding protein 1 (YBX1), a DNA- and RNA-binding protein, acts as an oncogene in medulloblastomas by regulating cellular survival and apoptosis. We observed different cellular responses upon YBX1 knockdown in several medulloblastoma cell lines, with significantly altered transcription and subsequent apoptosis rates. Mechanistically, PAR-CLIP for YBX1 and integration with RNA-Seq data uncovered direct posttranscriptional control of the heterochromatin-associated gene CBX5; upon YBX1 knockdown and subsequent CBX5 mRNA instability, heterochromatin-regulated genes involved in inflammatory response, apoptosis and death receptor signaling were de-repressed. Thus, YBX1 acts as an oncogene in medulloblastoma through indirect transcriptional regulation of inflammatory genes regulating apoptosis and represents a promising novel therapeutic target in this tumor entity.

Tumor necrosis factor overcomes immune evasion in p53-mutant medulloblastoma.

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-ß receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood-brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.

Safety and efficacy of oncolytic HSV-1 G207 inoculated into the cerebellum of mice.

Primary malignant central nervous system (CNS) tumors are the leading cause of childhood cancer-related death and morbidity. While advances in surgery, radiation, and chemotherapy have improved the survival rates in children with malignant brain tumors, mortality persists in certain subpopulations and current therapies are associated with extreme morbidity. This is especially true for children with malignant infratentorial tumors. Accordingly, G207, a genetically engineered herpes simplex virus (HSV-1) capable of selectively targeting cancer cells has emerged as a promising therapeutic option for this patient population. Herein, we demonstrate that cerebellar inoculation of G207 was systemically non-toxic in an immunocompetent, HSV-1 sensitive mouse strain (CBA/J). Mice had neither abnormal brain/organ pathology nor evidence of G207 replication by immunohistochemistry at days 7 and 30 after cerebellar G207 inoculation. While a minute amount viral DNA was recovered in the cerebellum and brainstem of mice at day 7, no viral DNA persisted at day 30. Critically, G207 delivered to the cerebellum was able to target/treat the highly aggressive MYC-overexpressed group 3 murine medulloblastoma increasing survival vs controls. These results provide critical safety and efficacy data to support the translation of G207 for pediatric clinical trials in intractable cerebellar malignancies.

Massive clonal expansion of medulloblastoma-specific T cells during adoptive cellular therapy.

In both human and murine systems, we have developed an adoptive cellular therapy platform against medulloblastoma and glioblastoma that uses dendritic cells pulsed with a tumor RNA transcriptome to expand polyclonal tumor-reactive T cells against a plurality of antigens within heterogeneous brain tumors. We demonstrate that peripheral TCR Vß repertoire analysis after adoptive cellular therapy reveals that effective response to adoptive cellular therapy is concordant with massive in vivo expansion and persistence of tumor-specific T cell clones within the peripheral blood. In preclinical models of medulloblastoma and glioblastoma, and in a patient with relapsed medulloblastoma receiving adoptive cellular therapy, an early and massive expansion of tumor-reactive lymphocytes, coupled with prolonged persistence in the peripheral blood, is observed during effective therapeutic response to immunotherapy treatment.

Medulloblastoma rendered susceptible to NK-cell attack by TGFß neutralization.

BACKGROUND: Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor ß (TGF-ß). Here, we address this challenge in in vitro models of MB. METHODS: CB-derived NK cells were modified to express a dominant negative TGF-ß receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. RESULTS: We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-ß-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-ß (decreased TGF-ß levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). CONCLUSIONS: CB NK cells expressing a TGF-ß DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-ß-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

Low mutational load in pediatric medulloblastoma still translates into neoantigens as targets for specific T-cell immunotherapy.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in childhood and adolescence. Although some patients present with distinct genetic alterations, such as mutated TP53 or MYC amplification, pediatric medulloblastoma is a tumor entity with minimal mutational load and low immunogenicity. METHODS: We identified tumor-specific mutations using next-generation sequencing of medulloblastoma DNA and RNA derived from primary tumor samples from pediatric patients. Tumor-specific mutations were confirmed using deep sequencing and in silico analyses predicted high binding affinity of the neoantigen-derived peptides to the patients' human leukocyte antigen molecules. Tumor-specific peptides were synthesized and used to induce a de novo T-cell response characterized by interferon gamma and tumor necrosis factor alpha release of CD8+ cytotoxic T cells in vitro. RESULTS: Despite low mutational tumor burden, at least two immunogenic tumor-specific peptides were identified in each patient. T cells showed a balanced CD4/CD8 ratio and mostly effector memory phenotype. Induction of a CD8-specific T-cell response was achieved for the neoepitopes derived from Histidine Ammonia-Lyase (HAL), Neuraminidase 2 (NEU2), Proprotein Convertase Subtilisin (PCSK9), Programmed Cell Death 10 (PDCD10), Supervillin (SVIL) and tRNA Splicing Endonuclease Subunit 54 (TSEN54) variants. CONCLUSION: Detection of patient-specific, tumor-derived neoantigens confirms that even in tumors with low mutational load a molecular design of targets for specific T-cell immunotherapy is possible. The identified neoantigens may guide future approaches of adoptive T-cell transfer, transgenic T-cell receptor transfer or tumor vaccination.

Tumour-associated macrophages exhibit anti-tumoural properties in Sonic Hedgehog medulloblastoma.

Medulloblastoma, which is the most common malignant paediatric brain tumour, has a 70% survival rate, but standard treatments often lead to devastating life-long side effects and recurrence is fatal. One of the emerging strategies in the search for treatments is to determine the roles of tumour microenvironment cells in the growth and maintenance of tumours. The most attractive target is tumour-associated macrophages (TAMs), which are abundantly present in the Sonic Hedgehog (SHH) subgroup of medulloblastoma. Here, we report an unexpected beneficial role of TAMs in SHH medulloblastoma. In human patients, decreased macrophage number is correlated with significantly poorer outcome. We confirm macrophage anti-tumoural behaviour in both ex vivo and in vivo murine models of SHH medulloblastoma. Taken together, our findings suggest that macrophages play a positive role by impairing tumour growth in medulloblastoma, in contrast to the pro-tumoural role played by TAMs in glioblastoma, another common brain tumour.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

Lin-CCR2+ hematopoietic stem and progenitor cells overcome resistance to PD-1 blockade.

Immune checkpoint blockade using anti-PD-1 monoclonal antibodies has shown considerable promise in the treatment of solid tumors, but brain tumors remain notoriously refractory to treatment. In CNS malignancies that are completely resistant to PD-1 blockade, we found that bone marrow-derived, lineage-negative hematopoietic stem and progenitor cells (HSCs) that express C-C chemokine receptor type 2 (CCR2+) reverses treatment resistance and sensitizes mice to curative immunotherapy. HSC transfer with PD-1 blockade increases T-cell frequency and activation within tumors in preclinical models of glioblastoma and medulloblastoma. CCR2+HSCs preferentially migrate to intracranial brain tumors and differentiate into antigen-presenting cells within the tumor microenvironment and cross-present tumor-derived antigens to CD8+ T cells. HSC transfer also rescues tumor resistance to adoptive cellular therapy in medulloblastoma and glioblastoma. Our studies demonstrate a novel role for CCR2+HSCs in overcoming brain tumor resistance to PD-1 checkpoint blockade and adoptive cellular therapy in multiple invasive brain tumor models.