Glioblastoma is the most common primary malignant brain tumor in adults and is associated with poor survival. The Ivy Foundation Early Phase Clinical Trials Consortium conducted a randomized, multi-institution clinical trial to evaluate immune responses and survival following neoadjuvant and/or adjuvant therapy with pembrolizumab in 35 patients with recurrent, surgically resectable glioblastoma. Patients who were randomized to receive neoadjuvant pembrolizumab, with continued adjuvant therapy following surgery, had significantly extended overall survival compared to patients that were randomized to receive adjuvant, post-surgical programmed cell death protein 1 (PD-1) blockade alone. Neoadjuvant PD-1 blockade was associated with upregulation of T cell- and interferon-γ-related gene expression, but downregulation of cell-cycle-related gene expression within the tumor, which was not seen in patients that received adjuvant therapy alone. Focal induction of programmed death-ligand 1 in the tumor microenvironment, enhanced clonal expansion of T cells, decreased PD-1 expression on peripheral blood T cells and a decreasing monocytic population was observed more frequently in the neoadjuvant group than in patients treated only in the adjuvant setting. These findings suggest that the neoadjuvant administration of PD-1 blockade enhances both the local and systemic antitumor immune response and may represent a more efficacious approach to the treatment of this uniformly lethal brain tumor.

Autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination is a promising immunotherapy for patients with high grade gliomas, but responses have not been demonstrated in all patients. To determine the most effective combination of autologous tumor lysate-pulsed DC vaccination, with or without the adjuvant toll-like receptor (TLR) agonists poly-ICLC or resiquimod, we conducted a Phase 2 clinical trial in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. We then performed deep, high-dimensional immune profiling of these patients to better understand how TLR agonists may influence the systemic immune responses induced by ATL-DC vaccination. Bulk RNAseq data demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant a TLR agonist together with ATL-DC. CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased expression of PD-1 on CD4+ T-cells, decreases in CD38 and CD39 on CD8+ T cells and elevated proportion of monocytes after ATL-DC + TLR agonist administration. In addition, scRNA-seq demonstrated a higher expression fold change of IFN-induced genes with poly-ICLC treatment in both peripheral blood monocytes and T lymphocytes. Patients who had higher expression of interferon response genes lived significantly longer and had longer time to progression compared to those with lower expression. The results suggest that ATL-DC in conjunction with adjuvant poly-ICLC induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population, which may represent an important blood biomarker for immunotherapy in this patient population.

The lack of effective treatments for gliomas makes them a significant health problem and highlights the need for the development of novel and innovative treatment approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. However, the absence of well-characterized, highly immunogenic tumor-rejection antigens (TRA) in gliomas has limited the implementation of targeted immune-based therapies.

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres.

Ets1 is a lymphoid-enriched transcription factor that regulates B- and Tcell functions in development and disease. Mice that lack Ets1 (Ets1 KO) develop spontaneous autoimmune disease with high levels of autoantibodies. Naïve CD4 + T cells isolated from Ets1 KO mice differentiate more readily to Th17 cells that secrete IL-17, a cytokine implicated in autoimmune disease pathogenesis. To determine if increased IL-17 production contributes to the development of autoimmunity in Ets1 KO mice, we crossed Ets1 KO mice to mice lacking the IL-17 receptor A subunit (IL17RA KO) to generate double knockout (DKO) mice.

In comparison with responses in recurrent glioblastoma (rGBM), the intracranial response of brain metastases (BrM) to immune checkpoint blockade (ICB) is less well studied. Here, we present an integrated single-cell RNA-Seq (scRNA-Seq) study of 19 ICB-naive and 9 ICB-treated BrM samples from our own and published data sets. We compared them with our previously published scRNA-Seq data from rGBM and found that ICB led to more prominent T cell infiltration into BrM than rGBM. These BrM-infiltrating T cells exhibited a tumor-specific phenotype and displayed greater activated/exhausted features. We also used multiplex immunofluorescence and spatial transcriptomics to reveal that ICB reduced a distinct CD206+ macrophage population in the perivascular space, which may modulate T cell entry into BrM. Furthermore, we identified a subset of progenitor exhausted T cells that correlated with longer overall survival in BrM patients. Our study provides a comprehensive immune cellular landscape of ICB's effect on metastatic brain tumors and offers insights into potential strategies for improving ICB efficacy for brain tumor patients.

Histone deacetylase (HDAC) inhibitors are a class of agents that have potent antitumor activity with a reported ability to upregulate MHC and costimulatory molecule expression. We hypothesized that epigenetic pharmacological immunomodulation could sensitize tumors to immune mediated cell death with an adoptive T cell therapy.

Despite new additions to the standard of care therapy for high grade primary malignant brain tumors, the prognosis for patients with this disease is still poor. A small contingent of clinical researchers are focusing their efforts on testing the safety, feasibility and efficacy of experimental active and passive immunotherapy approaches for gliomas and are primarily conducting Phase I and II clinical trials. Few trials have advanced to the Phase III arena. Here we provide an overview of the cellular therapies and vaccine trials currently open for patient accrual obtained from a search of http://www.clinicaltrials.gov. The search was refined with terms that would identify the Phase I, II and III immunotherapy trials open for adult glioma patient accrual in the United States. From the list, those that are currently open for patient accrual are discussed in this review. A variety of adoptive immunotherapy trials using ex vivo activated effector cell preparations, cell-based and non-cell-based vaccines, and several combination passive and active immunotherapy approaches are discussed.

Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches.

Loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark of clear cell renal cell carcinoma (ccRCC). The importance of heterogeneity in the loss of this tumor suppressor has been under reported. To study the impact of intratumoral VHL heterogeneity observed in human ccRCC, we engineered VHL gene deletion in four RCC models, including a new primary tumor cell line derived from an aggressive metastatic case. The VHL gene-deleted (VHL-KO) cells underwent epithelial-to-mesenchymal transition (EMT) and exhibited increased motility but diminished proliferation and tumorigenicity compared to the parental VHL-expressing (VHL+) cells. Renal tumors with either VHL+ or VHL-KO cells alone exhibit minimal metastatic potential. Combined tumors displayed rampant lung metastases, highlighting a novel cooperative metastatic mechanism. The poorly proliferative VHL-KO cells stimulated the proliferation, EMT, and motility of neighboring VHL+ cells. Periostin (POSTN), a soluble protein overexpressed and secreted by VHL non-expressing (VHL-) cells, promoted metastasis by enhancing the motility of VHL-WT cells and facilitating tumor cell vascular escape. Genetic deletion or antibody blockade of POSTN dramatically suppressed lung metastases in our preclinical models. This work supports a new strategy to halt the progression of ccRCC by disrupting the critical metastatic crosstalk between heterogeneous cell populations within a tumor.

Cancer immunotherapy critically depends on fitness of cytotoxic and helper T cell responses. Dysfunctional cytotoxic T cell states in the tumor microenvironment (TME) are a major cause of resistance to immunotherapy. Intratumoral myeloid cells, particularly blood-borne myeloids (bbm), are key drivers of T cell dysfunction in the TME. We show here that major histocompatibility complex class II (MHCII)-restricted antigen presentation on bbm is essential to control the growth of brain tumors. Loss of MHCII on bbm drives dysfunctional intratumoral tumor-reactive CD8+ T cell states through increased chromatin accessibility and expression of Tox, a critical regulator of T cell exhaustion. Mechanistically, MHCII-dependent activation of CD4+ T cells restricts myeloid-derived osteopontin that triggers a chronic activation of NFAT2 in tumor-reactive CD8+ T cells. In summary, we provide evidence that MHCII-restricted antigen presentation on bbm is a key mechanism to directly maintain functional cytotoxic T cell states in brain tumors.

The response of patients with recurrent glioblastoma multiforme to neoadjuvant immune checkpoint blockade has been challenging to interpret due to the inter-patient and intra-tumor heterogeneity. We report on a comparative analysis of tumor tissues collected from patients with recurrent glioblastoma and high-risk melanoma, both treated with neoadjuvant checkpoint blockade. We develop a framework that uses multiplex spatial protein profiling, machine learning-based image analysis, and data-driven computational models to investigate the pathophysiological and molecular factors within the tumor microenvironment that influence treatment response. Using melanoma to guide the interpretation of glioblastoma analyses, we interrogate the protein expression in microscopic compartments of tumors, and determine the correlates of cytotoxic CD8+ T cells, tumor growth, treatment response, and immune cell-cell interaction. This work reveals similarities shared between glioblastoma and melanoma, immunosuppressive factors that are unique to the glioblastoma microenvironment, and potential co-targets for enhancing the efficacy of neoadjuvant immune checkpoint blockade.

Increased T cell infiltration and interferon gamma (IFNγ) pathway activation are seen in tumors of melanoma patients who respond to ICI (immune checkpoint inhibitor) or MAPK pathway inhibitor (MAPKi) therapies. Yet, the rate of durable tumor control after ICI is almost twice that of MAPKi, suggesting that additional mechanisms may be present in patients responding to ICI therapy that are beneficial for anti-tumor immunity.

No abstract available

Immunological quiescence in the central nervous system (CNS) is a potential barrier to immune mediated anti-tumor response. One suppressive mechanism results from the interaction of parenchyma-derived CD200 and its receptor on myeloid cells. We suggest that CD200/CD200R interactions on myeloid cells expand the myeloid-derived suppressor cell (MDSC) population and that blocking tumor-derived CD200 will enhance the efficacy of immunotherapy.

To evaluate the association between a vessel size index (VSIMRI) derived from dynamic susceptibility contrast (DSC) perfusion imaging using a custom spin-and-gradient echo echoplanar imaging (SAGE-EPI) sequence and quantitative estimates of vessel morphometry based on immunohistochemistry from image-guided biopsy samples. The current study evaluated both relative cerebral blood volume (rCBV) and VSIMRI in eleven patients with high-grade glioma (7 WHO grade III and 4 WHO grade IV). Following 26 MRI-guided glioma biopsies in these 11 patients, we evaluated tissue morphometry, including vessel density and average radius, using an automated procedure based on the endothelial cell marker CD31 to highlight tumor vasculature. Measures of rCBV and VSIMRI were then compared to histological measures. We demonstrate good agreement between VSI measured by MRI and histology; VSIMRI = 13.67 μm and VSIHistology = 12.60 μm, with slight overestimation of VSIMRI in grade III patients compared to histology. rCBV showed a moderate but significant correlation with vessel density (r = 0.42, p = 0.03), and a correlation was also observed between VSIMRI and VSIHistology (r = 0.49, p = 0.01). The current study supports the hypothesis that vessel size measures using MRI accurately reflect vessel caliber within high-grade gliomas, while traditional measures of rCBV are correlated with vessel density and not vessel caliber.

Dendritic cell (DC) vaccines have recently emerged as an innovative therapeutic option for glioblastoma patients. To identify novel surrogates of anti-tumor immune responsiveness, we studied the dynamic expression of activation and inhibitory markers on peripheral blood lymphocyte (PBL) subsets in glioblastoma patients treated with DC vaccination at UCLA.

Immunotherapeutic approaches, such as dendritic cell (DC) vaccination, have emerged as promising strategies in the treatment of glioblastoma. Despite their promise, however, the absence of objective biomarkers and/or immunological monitoring techniques to assess the clinical efficacy of immunotherapy still remains a primary limitation. To address this, we sought to identify a functional biomarker for anti-tumor immune responsiveness associated with extended survival in glioblastoma patients undergoing DC vaccination.

Brain-infiltrating lymphocytes (BILs) were isolated from resected brain tissue from 10 pediatric epilepsy patients who had undergone surgery for Hemimegalencephaly (HME) (n = 1), Tuberous sclerosis complex (TSC) (n = 2), Focal cortical dysplasia (FCD) (n = 4), and Rasmussen encephalitis (RE) (n = 3). Peripheral blood mononuclear cells (PBMCs) were also isolated from blood collected at the time of the surgery. Cells were immunostained with a panel of 20 antibody markers, and analyzed by mass cytometry. To identify and quantify the immune cell types in the samples, an unbiased clustering method was applied to the entire data set. More than 85 percent of the CD45+ cells isolated from resected RE brain tissue comprised T cells; by contrast NK cells and myeloid cells constituted 80-95 percent of the CD45+ cells isolated from the TSC and the FCD brain specimens. Three populations of myeloid cells made up >50 percent of all of the myeloid cells in all of the samples of which a population of HLA-DR+ CD11b+ CD4- cells comprised the vast majority of myeloid cells in the BIL fractions from the FCD and TSC cases. CD45RA+ HLA-DR- CD11b+ CD16+ NK cells constituted the major population of NK cells in the blood from all of the cases. This subset also comprised the majority of NK cells in BILs from the resected RE and HME brain tissue, whereas NK cells defined as CD45RA- HLA-DR+ CD11b- CD16- cells comprised 86-96 percent of the NK cells isolated from the FCD and TSC brain tissue. Thirteen different subsets of CD4 and CD8 αβ T cells and γδ T cells accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples. At least 90 percent of the T cells in the RE BILs, 80 percent of the T cells in the HME BILs and 40-66 percent in the TSC and FCD BILs comprised activated antigen-experienced (CD45RO+ HLA-DR+ CD69+) T cells. We conclude that even in cases where there is no evidence for an infection or an immune disorder, activated peripheral immune cells may be present in epileptogenic areas of the brain, possibly in response to seizure-driven brain inflammation.