cBAF complex components and MYC cooperate early in CD8+ T cell fate.

The identification of mechanisms to promote memory T (Tmem) cells has important implications for vaccination and anti-cancer immunotherapy1-4. Using a CRISPR-based screen for negative regulators of Tmem cell generation in vivo5, here we identify multiple components of the mammalian canonical BRG1/BRM-associated factor (cBAF)6,7. Several components of the cBAF complex are essential for the differentiation of activated CD8+ T cells into T effector (Teff) cells, and their loss promotes Tmem cell formation in vivo. During the first division of activated CD8+ T cells, cBAF and MYC8 frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high MYC and high cBAF display a cell fate trajectory towards Teff cells, whereas those with low MYC and low cBAF preferentially differentiate towards Tmem cells. The cBAF complex and MYC physically interact to establish the chromatin landscape in activated CD8+ T cells. Treatment of naive CD8+ T cells with a putative cBAF inhibitor during the first 48 h of activation, before the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a mouse solid tumour model. Our results establish cBAF as a negative determinant of Tmem cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Therapeutic advantage of combinatorial CAR T cell and chemo-therapies.

Chimeric antigen receptor (CAR) T cell therapies have transformed outcomes for many patients with hematological malignancies. However, some patients do not respond to CAR T cell treatment, and adapting CAR T cells for solid and brain tumors has been met with many challenges including a hostile tumor microenvironment and poor CAR T cell persistence. Thus, it is unlikely that CAR T cell therapy alone will be sufficient for consistent, complete tumor clearance across cancer patients. Combinatorial therapies of CAR T cells and chemotherapeutics are a promising approach for overcoming this as chemotherapeutics could augment CAR T cells for improved anti-tumor activity or work in tandem with CAR T cells to clear tumors. Herein, we review efforts towards achieving successful CAR T cell and chemical drug combination therapies. We focus on combination therapies with approved chemotherapeutics as these will be more easily translated to the clinic, but also review non-approved chemotherapeutics and drug screens designed to reveal promising new CAR T cell and chemical drug combinations. Together, this review highlights the promise of CAR T cell and chemotherapy combinations with specific focus on how combinatorial therapy overcomes challenges faced by either monotherapy and supports the potential of this therapeutic strategy to improve outcomes for cancer patients. Significance Statement Improving currently available CAR T cell products via combinatorial therapy with chemotherapeutics has the potential to drastically expand the types of cancers and number of patients that could benefit from these therapies when neither alone has been sufficient to achieve tumor clearance. Herein, we provide a thorough review of the current efforts towards studying CAR T and chemotherapy combinatorial therapies and provide perspectives on optimal ways to identify new and effective combinations moving forward.

Analysis of the human endogenous coregulator complexome.

Elucidation of endogenous cellular protein-protein interactions and their networks is most desirable for biological studies. Here we report our study of endogenous human coregulator protein complex networks obtained from integrative mass spectrometry-based analysis of 3290 affinity purifications. By preserving weak protein interactions during complex isolation and utilizing high levels of reciprocity in the large dataset, we identified many unreported protein associations, such as a transcriptional network formed by ZMYND8, ZNF687, and ZNF592. Furthermore, our work revealed a tiered interplay within networks that share common proteins, providing a conceptual organization of a cellular proteome composed of minimal endogenous modules (MEMOs), complex isoforms (uniCOREs), and regulatory complex-complex interaction networks (CCIs). This resource will effectively fill a void in linking correlative genomic studies with an understanding of transcriptional regulatory protein functions within the proteome for formulation and testing of future hypotheses.

Engineering naturally occurring CD7- T cells for the immunotherapy of hematological malignancies.

Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.

The Mechanisms of Altered Blood-Brain Barrier Permeability in CD19 CAR T-Cell Recipients.

Cluster of differentiation 19 (CD19) chimeric antigen receptor (CAR) T cells are a highly effective immunotherapy for relapsed and refractory B-cell malignancies, but their utility can be limited by the development of immune effector cell-associated neurotoxicity syndrome (ICANS). The recent discovery of CD19 expression on the pericytes in the blood-brain barrier (BBB) suggests an important off-target mechanism for ICANS development. In addition, the release of systemic cytokines stimulated by the engagement of CD19 with the CAR T cells can cause endothelial activation and decreased expression of tight junction molecules, further damaging the integrity of the BBB. Once within the brain microenvironment, cytokines trigger a cytokine-specific cascade of neuroinflammatory responses, which manifest clinically as a spectrum of neurological changes. Brain imaging is frequently negative or nonspecific, and treatment involves close neurologic monitoring, supportive care, interleukin antagonists, and steroids. The goal of this review is to inform readers about the normal development and microstructure of the BBB, its unique susceptibility to CD19 CAR T cells, the role of individual cytokines on specific elements of the brain's microstructural environment, and the clinical and imaging manifestations of ICANS. Our review will link cellular pathophysiology with the clinical and radiological manifestations of a complex clinical entity.

Tyrosine kinase signaling-independent MET-targeting with CAR-T cells.

BACKGROUND: Recent progress in cancer immunotherapy encourages the expansion of chimeric antigen receptor (CAR) T cell therapy in solid tumors including hepatocellular carcinoma (HCC). Overexpression of MET receptor tyrosine kinase is common in HCC; however, MET inhibitors are effective only when MET is in an active form, making patient stratification difficult. Specific MET-targeting CAR-T cells hold the promise of targeting HCC with MET overexpression regardless of signaling pathway activity. METHODS: MET-specific CARs with CD28ζ or 4-1BBζ as co-stimulation domains were constructed. MET-CAR-T cells derived from healthy subjects (HS) and HCC patients were evaluated for their killing activity and cytokine release against HCC cells with various MET activations in vitro, and for their tumor growth inhibition in orthotopic xenograft models in vivo. RESULTS: MET-CAR.CD28ζ and MET-CAR.4-1BBζ T cells derived from both HS and HCC patients specifically killed MET-positive HCC cells. When stimulated with MET-positive HCC cells in vitro, MET-CAR.CD28ζ T cells demonstrated a higher level of cytokine release and expression of programmed cell death protein 1 (PD-1) than MET-CAR.4-1BBζ T cells. When analyzed in vivo, MET-CAR.CD28ζ T cells more effectively inhibited HCC orthotopic tumor growth in mice when compared to MET-CAR.4-1BBζ T cells. CONCLUSION: We generated and characterized MET-specific CAR-T cells for targeting HCC with MET overexpression regardless of MET activation. Compared with MET-CAR.4-1BBζ, MET-CAR.CD28ζ T cells showed a higher anti-HCC potency but also a higher level of T cell exhaustion. While MET-CAR.CD28ζ is preferred for further development, overcoming the exhaustion of MET-CAR-T cells is necessary to improve their therapeutic efficacy in vivo.

Chimeric antigen receptor T-cell therapy in glioblastoma: charging the T cells to fight.

Glioblastoma multiforme (GBM) is the most common malignant brain cancer that invades normal brain tissue and impedes surgical eradication, resulting in early local recurrence and high mortality. In addition, most therapeutic agents lack permeability across the blood brain barrier (BBB), further reducing the efficacy of chemotherapy. Thus, effective treatment against GBM requires tumor specific targets and efficient intracranial drug delivery. With the most recent advances in immunotherapy, genetically engineered T cells with chimeric antigen receptors (CARs) are becoming a promising approach for treating cancer. By transducing T lymphocytes with CAR constructs containing a tumor-associated antigen (TAA) recognition domain linked to the constant regions of a signaling T cell receptor, CAR T cells may recognize a predefined TAA with high specificity in a non-MHC restricted manner, and is independent of antigen processing. Active T cells can travel across the BBB, providing additional advantage for drug delivery and tumor targeting. Here we review the CAR design and technical innovations, the major targets that are in pre-clinical and clinical development with a focus on GBM, and multiple strategies developed to improve CAR T cell efficacy.

Adoptive Transfer of IL13Rα2-Specific Chimeric Antigen Receptor T Cells Creates a Pro-inflammatory Environment in Glioblastoma.

In order to fully harness the potential of immunotherapy with chimeric antigen receptor (CAR)-modified T cells, pre-clinical studies must be conducted in immunocompetent animal models that closely mimic the immunosuppressive malignant glioma (MG) microenvironment. Thus, the goal of this project was to study the in vivo fate of T cells expressing CARs specific for the MG antigen IL13Rα2 (IL13Rα2-CARs) in immunocompetent MG models. Murine T cells expressing IL13Rα2-CARs with a CD28.ζ (IL13Rα2-CAR.CD28.ζ) or truncated signaling domain (IL13Rα2-CAR.Δ) were generated by retroviral transduction, and their effector function was evaluated both in vitro and in vivo. IL13Rα2-CAR.CD28.ζ T cells' specificity toward IL13Rα2 was confirmed through cytokine production and cytolytic activity. In vivo, a single intratumoral injection of IL13Rα2-CAR.CD28.ζ T cells significantly extended the survival of IL13Rα2-expressing GL261 and SMA560 glioma-bearing mice; long-term survivors were resistant to re-challenge with IL13Rα2-negative and IL13Rα2-positive tumors. IL13Rα2-CAR.CD28.ζ T cells proliferated, produced cytokines (IFNγ, TNF-α), and promoted a phenotypically pro-inflammatory glioma microenvironment by inducing a significant increase in the number of CD4+ and CD8+ T cells and CD8α+ dendritic cells and a decrease in Ly6G+ myeloid-derived suppressor cells (MDSCs). Our data underline the significance of CAR T cell studies in immunocompetent hosts and further validate IL13Rα2-CAR T cells as an efficacious therapeutic strategy for MG.

Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma.

Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies.

Nuclear BAG6-UBL4A-GET4 complex mediates DNA damage signaling and cell death.

BCL2-associated athanogene 6 (BAG6) is a member of the BAG protein family, which is implicated in diverse cellular processes including apoptosis, co-chaperone, and DNA damage response (DDR). Recently, it has been shown that BAG6 forms a stable complex with UBL4A and GET4 and functions in membrane protein targeting and protein quality control. The BAG6 sequence contains a canonical nuclear localization signal and is localized predominantly in the nucleus. However, GET4 and UBL4A are found mainly in cytoplasm. Whether GET4 and UBL4A are also involved in DDR in the context of the BAG6 complex remains unknown. Here, we provide evidence that nuclear BAG6-UBL4A-GET4 complex mediates DDR signaling and damage-induced cell death. BAG6 appears to be the central component for the process, as depletion of BAG6 leads to the loss of both UBL4A and GET4 proteins and resistance to cell killing by DNA-damaging agents. In addition, nuclear localization of BAG6 and phosphorylation of BAG6 by ATM/ATR are also required for cell killing. UBL4A and GET4 translocate to the nucleus upon DNA damage and appear to play redundant roles in cell killing, as depletion of either one has no effect but co-depletion leads to resistance. All three components of the BAG6 complex are required for optimal DDR signaling, as BAG6, and to a lesser extent, GET4 and UBL4A, regulate the recruitment of BRCA1 to sites of DNA damage. Together our results suggest that the nuclear BAG6 complex is an effector in DNA damage response pathway and its phosphorylation and nuclear localization are important determinants for its function.

Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion.

T cell exhaustion is linked to persistent antigen exposure and perturbed activation events, correlating with poor disease prognosis. Tumor-mediated T cell exhaustion is well documented; however, how the nutrient-deprived tumor niche affects T cell receptor (TCR) activation is largely unclear. We show that methionine metabolism licenses optimal TCR signaling by regulating the protein arginine methylome, and limiting methionine availability during early TCR signaling promotes subsequent T cell exhaustion. We discovered a novel arginine methylation of a Ca 2+ -activated potassium transporter, KCa3.1, prevention of which results in increased Ca 2+ -mediated NFAT1 activation, NFAT1 promoter occupancy, and T cell exhaustion. Furthermore, methionine supplementation reduces nuclear NFAT1 in tumor-infiltrating T cells and augments their anti-tumor activity. These findings demonstrate metabolic regulation of T cell exhaustion determined during TCR engagement.

Peptide-scFv antigen recognition domains effectively confer CAR T cell multiantigen specificity.

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.

Steering CAR T cell epigenetic programs by tweaking manufacturing protocol.

CAR T cell persistence remains a significant roadblock to effective clinical translation of CAR T cells for solid and brain tumors. Jung et al.1 demonstrate enrichment of resident memory phenotypes through simple changes to the CAR T cell manufacturing process.

Synapse-tuned CARs enhance immune cell anti-tumor activity.

Chimeric antigen receptor (CAR) technologies have been clinically implemented for the treatment of hematological malignancies; however, solid tumors remain resilient to CAR therapeutics. Natural killer (NK) cells may provide an optimal class of immune cells for CAR-based approaches due to their inherent anti-tumor functionality. In this study, we sought to tune CAR immune synapses by adding an intracellular scaffolding protein binding site to the CAR. We employ a PDZ binding motif (PDZbm) that enables additional scaffolding crosslinks that enhance synapse formation and NK CAR cell polarization. Combined effects of this CAR design result in increased effector cell functionality in vitro and in vivo. Additionally, we used T cells and observed similar global enhancements in effector function. Synapse-tuned CAR immune cells exhibit amplified synaptic strength, number and abundance of secreted cytokines, enhanced killing of tumor cells and prolonged survival in numerous different tumor models, including solid tumors.

Overcoming on-target, off-tumour toxicity of CAR T cell therapy for solid tumours.

Therapies with genetically modified T cells that express chimeric antigen receptors (CARs) specific for CD19 or B cell maturation antigen (BCMA) are approved to treat certain B cell malignancies. However, translating these successes into treatments for patients with solid tumours presents various challenges, including the risk of clinically serious on-target, off-tumour toxicity (OTOT) owing to CAR T cell-mediated cytotoxicity against non-malignant tissues expressing the target antigen. Indeed, severe OTOT has been observed in various CAR T cell clinical trials involving patients with solid tumours, highlighting the importance of establishing strategies to predict, mitigate and control the onset of this effect. In this Review, we summarize current clinical evidence of OTOT with CAR T cells in the treatment of solid tumours and discuss the utility of preclinical mouse models in predicting clinical OTOT. We then describe novel strategies being developed to improve the specificity of CAR T cells in solid tumours, particularly the role of affinity tuning of target binders, logic circuits and synthetic biology. Furthermore, we highlight control strategies that can be used to mitigate clinical OTOT following cell infusion such as regulating or eliminating CAR T cell activity, exogenous control of CAR expression, and local administration of CAR T cells.

CD47 expression is critical for CAR T-cell survival in vivo.

BACKGROUND: CD47 is an attractive immunotherapeutic target because it is highly expressed on multiple solid tumors. However, CD47 is also expressed on T cells. Limited studies have evaluated CD47-chimeric antigen receptor (CAR) T cells, and the role of CD47 in CAR T-cell function remains largely unknown. METHODS: Here, we describe the development of CD47-CAR T cells derived from a high affinity signal regulatory protein α variant CV1, which binds CD47. CV1-CAR T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo. The role of CD47 in CAR T-cell function was examined by knocking out CD47 in T cells followed by downstream functional analyses. RESULTS: While CV1-CAR T cells are specific and exhibit potent activity in vitro they lacked antitumor activity in xenograft models. Mechanistic studies revealed CV1-CAR T cells downregulate CD47 to overcome fratricide, but CD47 loss resulted in their failure to expand and persist in vivo. This effect was not limited to CV1-CAR T cells, since CD47 knockout CAR T cells targeting another solid tumor antigen exhibited the same in vivo fate. Further, CD47 knockout T cells were sensitive to macrophage-mediated phagocytosis. CONCLUSIONS: These findings highlight that CD47 expression is critical for CAR T-cell survival in vivo and is a 'sine qua non' for successful adoptive T-cell therapy.

GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells.

Lack of targetable antigens is a key limitation for developing successful T cell-based immunotherapies. Members of the unfolded protein response (UPR) represent ideal immunotherapy targets because the UPR regulates the ability of cancer cells to resist cell death, sustain proliferation, and metastasize. Glucose-regulated protein 78 (GRP78) is a key UPR regulator that is overexpressed and translocated to the cell surface of a wide variety of cancers in response to elevated endoplasmic reticulum (ER) stress. We show that GRP78 is highly expressed on the cell surface of multiple solid and brain tumors, making cell surface GRP78 a promising chimeric antigen receptor (CAR) T cell target. We demonstrate that GRP78-CAR T cells can recognize and kill GRP78+ brain and solid tumors in vitro and in vivo. Additionally, our findings demonstrate that GRP78 is upregulated on CAR T cells upon T cell activation; however, this expression is tumor-cell-line specific and results in heterogeneous GRP78-CAR T cell therapeutic response.

CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity.

Understanding interactions between adoptively transferred immune cells and the tumor immune microenvironment (TIME) is critical for developing successful T-cell based immunotherapies. Here we investigated the impact of the TIME and chimeric antigen receptor (CAR) design on anti-glioma activity of B7-H3-specific CAR T-cells. We show that five out of six B7-H3 CARs with varying transmembrane, co-stimulatory, and activation domains, exhibit robust functionality in vitro. However, in an immunocompetent glioma model, these CAR T-cells demonstrated significantly varied levels of anti-tumor activity. We used single-cell RNA sequencing to examine the brain TIME after CAR T-cell therapy. We show that the TIME composition was influenced by CAR T-cell treatment. We also found that successful anti-tumor responses were supported by the presence and activity of macrophages and endogenous T-cells. Together, our study demonstrates that efficacy of CAR T-cell therapy in high-grade glioma is dependent on CAR structural design and its capacity to modulate the TIME.

CAR T-cell Design-dependent Remodeling of the Brain Tumor Immune Microenvironment Modulates Tumor-associated Macrophages and Anti-glioma Activity.

Understanding the intricate dynamics between adoptively transferred immune cells and the brain tumor immune microenvironment (TIME) is crucial for the development of effective T cell-based immunotherapies. In this study, we investigated the influence of the TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3-specific CAR T-cells. Using an immunocompetent glioma model, we evaluated a panel of seven fully murine B7-H3 CARs with variations in transmembrane, costimulatory, and activation domains. We then investigated changes in the TIME following CAR T-cell therapy using high-dimensional flow cytometry and single-cell RNA sequencing. Our results show that five out of six B7-H3 CARs with single costimulatory domains demonstrated robust functionality in vitro. However, these CARs had significantly varied levels of antitumor activity in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 41BB and CD28 costimulation through transgenic expression of 41BBL on CD28-based CAR T-cells. This CAR design was associated with significantly improved anti-glioma efficacy in vitro but did not result in similar improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the TIME, with the recruitment and activation of distinct macrophage and endogenous T-cell subsets crucial for successful antitumor responses. Indeed, complete brain macrophage depletion using a CSF1R inhibitor abrogated CAR T-cell antitumor activity. In sum, our study highlights the critical role of CAR design and its modulation of the TIME in mediating the efficacy of adoptive immunotherapy for high-grade glioma. SIGNIFICANCE: CAR T-cell immunotherapies hold great potential for treating brain cancers; however, they are hindered by a challenging immune environment that dampens their effectiveness. In this study, we show that the CAR design influences the makeup of the immune environment in brain tumors, underscoring the need to target specific immune components to improve CAR T-cell performance, and highlighting the significance of using models with functional immune systems to optimize this therapy.