MAPK/ERK signaling in gliomas modulates interferon responses, T cell recruitment, microglia phenotype, and immune checkpoint blockade efficacy.

Background: Glioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. Method: We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single cell RNA-sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Results: CRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. Conclusion: Our data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.

A covalent creatine kinase inhibitor ablates glioblastoma migration and sensitizes tumors to oxidative stress.

Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

B cell-based therapy produces antibodies that inhibit glioblastoma growth.

Glioblastoma (GBM) is a highly aggressive and malignant brain tumor with limited therapeutic options and a poor prognosis. Despite current treatments, the invasive nature of GBM often leads to recurrence. A promising alternative strategy is to harness the potential of the immune system against tumor cells. Our previous data showed that the Bvax (B-cell-based vaccine) can induce therapeutic responses in preclinical models of GBM. In this study, we aim to characterize the antigenic reactivity of BVax-derived antibodies and evaluate their therapeutic potential. We performed immunoproteomics and functional assays in murine models and human GBM patient samples. Our investigations revealed that BVax distributes throughout the GBM tumor microenvironment (TME) and then differentiates into antibody-producing plasmablasts. Proteomic analyses indicate that the antibodies produced by BVax display unique reactivity, predominantly targeting factors associated with cell motility and the extracellular matrix. Crucially, these antibodies inhibit critical processes such as GBM cell migration and invasion. These findings provide valuable insights into the therapeutic potential of BVax-derived antibodies for GBM patients, pointing towards a novel direction in GBM immunotherapy.

Fc-enhanced anti-CTLA-4, anti-PD-1, doxorubicin, and ultrasound-mediated BBB opening: A novel combinatorial immunotherapy regimen for gliomas.

BACKGROUND: Glioblastoma is a highly aggressive brain cancer that is resistant to conventional immunotherapy strategies. Botensilimab, an Fc-enhanced anti-CTLA-4 antibody (FcE-aCTLA-4), has shown durable activity in "cold" and immunotherapy-refractory cancers. METHOD: We evaluated the efficacy and immune microenvironment phenotype of a mouse analogue of FcE-aCTLA-4 in treatment-refractory preclinical models of glioblastoma, both as a monotherapy and in combination with doxorubicin delivered via low-intensity pulsed ultrasound and microbubbles (LIPU/MB). Additionally, we studied 4 glioblastoma patients treated with doxorubicin, anti-PD-1 with concomitant LIPU/MB to investigate the novel effect of doxorubicin modulating FcγR expressions in tumor associated macrophages/microglia (TAMs). RESULTS: FcE-aCTLA-4 demonstrated high-affinity binding to FcγRIV, the mouse ortholog of human FcγRIIIA, which was highly expressed in TAMs in human glioblastoma, most robustly at diagnosis. Notably, FcE-aCTLA-4 mediated selective depletion of intra-tumoral regulatory T cells (Tregs) via TAM-mediated phagocytosis, while sparing peripheral Tregs. Doxorubicin, a chemotherapeutic drug with immunomodulatory functions, was found to upregulate FcγRIIIA on TAMs in glioblastoma patients who received doxorubicin and anti-PD-1 with concomitant LIPU/MB. In murine models of immunotherapy-resistant gliomas, a combinatorial regimen of FcE-aCTLA-4, anti-PD-1, and doxorubicin with LIPU/MB, achieved a 90% cure rate, that was associated robust infiltration of activated CD8+ T cells and establishment of immunological memory as evidenced by rejection upon tumor rechallenge. CONCLUSION: Our findings demonstrate that FcE-aCTLA-4 promotes robust immunomodulatory and anti-tumor effects in murine gliomas and is significantly enhanced when combined with anti-PD-1, doxorubicin, and LIPU/MB. We are currently investigating this combinatory strategy in a clinical trial (clinicaltrials.gov NCT05864534).

Ultrasound-mediated delivery of doxorubicin to the brain results in immune modulation and improved responses to PD-1 blockade in gliomas.

Given the marginal penetration of most drugs across the blood-brain barrier, the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication, respectively. Similarly, LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab, respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice, which rely on myeloid cells and lymphocytes for their efficacy. Overall, this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM.

STING agonist 8803 reprograms the immune microenvironment and increases survival in preclinical models of glioblastoma.

STING agonists can reprogram the tumor microenvironment to induce immunological clearance within the central nervous system. Using multiplexed sequential immunofluorescence (SeqIF) and the Ivy Glioblastoma Atlas, STING expression was found in myeloid populations and in the perivascular space. The STING agonist 8803 increased median survival in multiple preclinical models of glioblastoma, including QPP8, an immune checkpoint blockade-resistant model, where 100% of mice were cured. Ex vivo flow cytometry profiling during the therapeutic window demonstrated increases in myeloid tumor trafficking and activation, alongside enhancement of CD8+ T cell and NK effector responses. Treatment with 8803 reprogrammed microglia to express costimulatory CD80/CD86 and iNOS, while decreasing immunosuppressive CD206 and arginase. In humanized mice, where tumor cell STING is epigenetically silenced, 8803 therapeutic activity was maintained, further attesting to myeloid dependency and reprogramming. Although the combination with a STAT3 inhibitor did not further enhance STING agonist activity, the addition of anti-PD-1 antibodies to 8803 treatment enhanced survival in an immune checkpoint blockade-responsive glioma model. In summary, 8803 as a monotherapy demonstrates marked in vivo therapeutic activity, meriting consideration for clinical translation.

Prohibitin Expression in Antigen-Presenting Cells: Implications for Inciting Trigger in CNS IgG4-Related Disease.

Immunoglobulin G4-related disease (IgG4-RD) is a rare autoimmune disorder with an unknown etiology. Using orthogonal immune profiling and automated sequential multiplexing, we found an enhanced frequency of activated circulating B cells, antigen-presenting myeloid cells in peripheral blood, and a distinct distribution of immune cells within the CNS lesions. Prohibitin-expressing CD138+ plasma B cells and CD11c+ dendritic cells have been found interacting with T cells resulting in irmnune cell activation within the lesion. The data implicate prohibitin as a potential triggering antigen in the pathogenesis of IgG4-RD and shed light on the cellular dynamics and interactions driving IgG4-RD in the central nervous system, emphasizing the need for further studies corroborating these findings.

Understanding the glioblastoma tumor microenvironment: leveraging the extracellular matrix to increase immunotherapy efficacy.

Glioblastoma (GBM) accounts for approximately half of all malignant brain tumors, and it remains lethal with a five-year survival of less than 10%. Despite the immense advancements in the field, it has managed to evade even the most promising therapeutics: immunotherapies. The main reason is the highly spatiotemporally heterogeneous and immunosuppressive GBM tumor microenvironment (TME). Accounting for this complex interplay of TME-driven immunosuppression is key to developing effective therapeutics. This review will explore the immunomodulatory role of the extracellular matrix (ECM) by establishing its contribution to the TME as a key mediator of immune responses in GBM. This relationship will help us elucidate therapeutic targets that can be leveraged to develop and deliver more effective immunotherapies.

ERK1/2 Phosphorylation Predicts Survival in Recurrent Glioblastoma Following Intracerebral and Adjuvant PD-1/CTLA-4 Immunotherapy: A REMARK-guided Analysis.

PURPOSE: Evidence suggests that MAPK pathway activation, as measured by ERK1/2 phosphorylation (p-ERK), predicts overall survival (OS) in patients with recurrent glioblastoma receiving anti-PD-1 therapy. We aimed to validate these findings in independent cohorts. EXPERIMENTAL DESIGN: In a 24-patient clinical trial on recurrent glioblastoma and high-grade gliomas, we examined the link between p-ERK levels and OS. Patients received intravenous nivolumab, followed by maximal safe resection and an intracerebral injection of either ipilimumab alone or combined with nivolumab. Biweekly adjuvant nivolumab was then administered up to five times (NCT03233152). Using REporting recommendations for tumor MARKER prognostic studies (REMARK) criteria, we conducted independent analyses for p-ERK quantification and statistical evaluations. Additional comparative analysis included prior cohorts, totaling 65 patients. Cox proportional hazards models and meta-analysis were employed to assess p-ERK as a predictive biomarker after immunotherapy. RESULTS: Lower median p-ERK+ cell density was observed compared with prior studies, likely due to variable tissue processing across cohorts. Nonetheless, high p-ERK was associated with prolonged OS, particularly in isocitrate dehydrogenase wild-type glioblastomas (P = 0.036). Median OS for high and low p-ERK patients were 55.6 and 30 weeks, respectively. Multivariable analysis reinforced p-ERK's significance in survival prediction (P = 0.011). Upon p-ERK normalization across cohorts (n = 65), meta-analysis supported the survival benefit of elevated tumor p-ERK levels (P = 0.0424). CONCLUSIONS: This study strengthens the role of p-ERK as a predictive biomarker for OS in patients with glioblastoma on immune checkpoint blockade. Future research should focus on further validation in prospective trials and the standardization of preanalytical variables influencing p-ERK quantification.

Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth.

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

High-sensitive spatially resolved T cell receptor sequencing with SPTCR-seq.

Spatial resolution of the T cell repertoire is essential for deciphering cancer-associated immune dysfunction. Current spatially resolved transcriptomic technologies are unable to directly annotate T cell receptors (TCR). We present spatially resolved T cell receptor sequencing (SPTCR-seq), which integrates optimized target enrichment and long-read sequencing for highly sensitive TCR sequencing. The SPTCR computational pipeline achieves yield and coverage per TCR comparable to alternative single-cell TCR technologies. Our comparison of PCR-based and SPTCR-seq methods underscores SPTCR-seq's superior ability to reconstruct the entire TCR architecture, including V, D, J regions and the complementarity-determining region 3 (CDR3). Employing SPTCR-seq, we assess local T cell diversity and clonal expansion across spatially discrete niches. Exploration of the reciprocal interaction of the tumor microenvironmental and T cells discloses the critical involvement of NK and B cells in T cell exhaustion. Integrating spatially resolved omics and TCR sequencing provides as a robust tool for exploring T cell dysfunction in cancers and beyond.

Generation of B-cell-based cellular vaccine for cancer in murine models.

A B-cell-based cellular vaccine (BVax), produced from 4-1BBL+ B cells, can select tumor-specific B cells that, upon ex vivo culture, can generate tumor-specific antibodies and activate T cells. Here, we present a protocol to generate a B-cell-based vaccine in a CT2A orthotopic glioma murine model. We describe steps for BVax production involving glioma cell implantation, tissue harvesting, 4-1BBL+ B cell isolation, and activation. We also describe assessing BVax phenotype in vitro and in vivo functional status. For complete details on the use and execution of this protocol, please refer to Lee-Chang et al. (2021).1.

STING agonist-loaded, CD47/PD-L1-targeting nanoparticles potentiate antitumor immunity and radiotherapy for glioblastoma.

As a key component of the standard of care for glioblastoma, radiotherapy induces several immune resistance mechanisms, such as upregulation of CD47 and PD-L1. Here, leveraging these radiotherapy-elicited processes, we generate a bridging-lipid nanoparticle (B-LNP) that engages tumor-associated myeloid cells (TAMCs) to glioblastoma cells via anti-CD47/PD-L1 dual ligation. We show that the engager B-LNPs block CD47 and PD-L1 and promote TAMC phagocytic activity. To enhance subsequent T cell recruitment and antitumor responses after tumor engulfment, the B-LNP was encapsulated with diABZI, a non-nucleotidyl agonist for stimulator of interferon genes. In vivo treatment with diABZI-loaded B-LNPs induced a transcriptomic and metabolic switch in TAMCs, turning these immunosuppressive cells into antitumor effectors, which induced T cell infiltration and activation in brain tumors. In preclinical murine models, B-LNP/diABZI administration synergized with radiotherapy to promote brain tumor regression and induce immunological memory against glioma. In summary, our study describes a nanotechnology-based approach that hijacks irradiation-triggered immune checkpoint molecules to boost potent and long-lasting antitumor immunity against glioblastoma.

Checkpoint kinase 1/2 inhibition potentiates anti-tumoral immune response and sensitizes gliomas to immune checkpoint blockade.

Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cell mediated killing are less understood. Kinases are potentially druggable targets that drive tumor progression and might influence immune response. Here, we perform an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cell recognition. The screen reveals checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are currently being evaluated in clinical trials, in combination with PD-1 or PD-L1 blockade, lead to survival benefit in multiple preclinical glioma models. Mechanistically, loss of Chek2 enhances antigen presentation, STING pathway activation and PD-L1 expression in mouse gliomas. Analysis of human GBMs demonstrates that Chek2 expression is inversely associated with antigen presentation and T-cell activation. Collectively, these results support Chek2 as a promising target for enhancement of response to immune checkpoint blockade therapy in GBM.

IL15 modification enables CAR T cells to act as a dual targeting agent against tumor cells and myeloid-derived suppressor cells in GBM.

INTRODUCTION: The immunosuppressive tumor microenvironment (TME) is a major barrier to the efficacy of chimeric antigen receptor T cells (CAR-T cells) in glioblastoma (GBM). Transgenic expression of IL15 is one attractive strategy to modulate the TME. However, at present, it is unclear if IL15 could be used to directly target myeloid-derived suppressor cells (MDSCs), a major cellular component of the GBM TME. Here, we explored if MDSC express IL15Rα and the feasibility of exploiting its expression as an immunotherapeutic target. METHODS: RNA-seq, RT-qPCR, and flow cytometry were used to determine IL15Rα expression in paired peripheral and tumor-infiltrating immune cells of GBM patients and two syngeneic murine GBM models. We generated murine T cells expressing IL13Rα2-CARs and secretory IL15 (CAR.IL15s) or IL13Rα2-CARs in which IL15 was fused to the CAR to serve as an IL15Rα-targeting moiety (CAR.IL15f), and characterized their effector function in vitro and in syngeneic IL13Rα2+glioma models. RESULTS: IL15Rα was preferentially expressed in myeloid, B, and dendritic cells in patients' and syngeneic GBMs. In vitro, CAR.IL15s and CAR.IL15f T cells depleted MDSC and decreased their secretion of immunosuppressive molecules with CAR.IL15f T cells being more efficacious. Similarly, CAR.IL15f T cells significantly improved the survival of mice in two GBM models. TME analysis showed that treatment with CAR.IL15f T cells resulted in higher frequencies of CD8+T cells, NK, and B cells, but a decrease in CD11b+cells in tumors compared with therapy with CAR T cells. CONCLUSIONS: We demonstrate that MDSC of the glioma TME express IL15Ra and that these cells can be targeted with secretory IL15 or an IL15Rα-targeting moiety incorporated into the CAR. Thus, IL15-modified CAR T cells act as a dual targeting agent against tumor cells and MDSC in GBM, warranting their future evaluation in early-phase clinical studies.

Next-generation antigen-presenting cell immune therapeutics for gliomas.

Antigen presentation machinery and professional antigen-presenting cells (APCs) are fundamental for an efficacious immune response against cancers, especially in the context of T cell-centric immunotherapy. Dendritic cells (DCs), the gold standard APCs, play a crucial role in initiating and maintaining a productive antigen-specific adaptive immunity. In recent decades, ex vivo-differentiated DCs from circulating CD14+ monocytes have become the reference for APC-based immunotherapy. DCs loaded with tumor-associated antigens, synthetic peptides, or RNA activate T cells with antitumor properties. This strategy has paved the way for the development of alternative antigen-presenting vaccination strategies, such as monocytes, B cells, and artificial APCs, that have shown effective therapeutic outcomes in preclinical cancer models. The search for alternative APC platforms was initiated by the overall limited clinical impact of DC vaccines, especially in indications such as gliomas, a primary brain tumor known for resistance to any immune intervention. In this Review, we navigate the APC immune therapeutics' past, present, and future in the context of primary brain tumors.

Immune checkpoint blockade in glioblastoma: from tumor heterogeneity to personalized treatment.

Immune checkpoint blockade (ICB) has revolutionized modern cancer therapy, arousing great interest in the neuro-oncology community. While several reports show that subsets of patients with glioma exhibit durable responses to immunotherapy, the efficacy of this treatment has not been observed for unselected patient populations, preventing its broad clinical implementation for gliomas and glioblastoma (GBM). To exploit the maximum therapeutic potential of ICB for patients with glioma, understanding the different aspects of glioma-related tumor immune responses is of critical importance. In this Review, we discuss contributing factors that distinguish subsets of patients with glioma who may benefit from ICB. Specifically, we discuss (a) the complex interaction between the tumor immune microenvironment and glioma cells as a potential influence on immunotherapy responses; (b) promising biomarkers for responses to immune checkpoint inhibitors; and (c) the potential contributions of peripheral immune cells to therapeutic responses.

Endoplasmic Reticulum Stress in the Brain Tumor Immune Microenvironment.

Immunotherapy has emerged as a powerful strategy for halting cancer progression. However, primary malignancies affecting the brain have been exempt to this success. Indeed, brain tumors continue to portend severe morbidity and remain a globally lethal disease. Extensive efforts have been directed at understanding how tumor cells survive and propagate within the unique microenvironment of the central nervous system (CNS). Cancer genetic aberrations and metabolic abnormalities provoke a state of persistent endoplasmic reticulum (ER) stress that in turn promotes tumor growth, invasion, therapeutic resistance, and the dynamic reprogramming of the infiltrating immune cells. Consequently, targeting ER stress is a potential therapeutic approach. In this work, we provide an overview of how ER stress response is advantageous to brain tumor development, discuss the significance of ER stress in governing antitumor immunity, and put forth therapeutic strategies of regulating ER stress to augment the effect of immunotherapy for primary CNS tumors.

B-cells Drive Response to PD-1 Blockade in Glioblastoma Upon Neutralization of TGFß-mediated Immunosuppression.

Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in other solid tumors suggest a central role of B-cell immunity in driving immune-checkpoint-blockade efficacy. Using single-cell and single-nuclei transcriptomics of human glioblastoma and melanoma brain metastasis, we found that tumor-associated B-cells have high expression of checkpoint molecules, known to block B-cell-receptor downstream effector function such as plasmablast differentiation and antigen-presentation. We also identified TGFß-1/TGFß receptor-2 interaction as a crucial modulator of B-cell suppression. Treatment of glioblastoma patients with pembrolizumab induced expression of B-cell checkpoint molecules and TGFß-receptor-2. Abrogation of TGFß using different conditional knockouts expanded germinal-center-like intratumoral B-cells, enhancing immune-checkpoint-blockade efficacy. Finally, blocking αVß8 integrin (which controls the release of active TGFß) and PD-1 significantly increased B-cell-dependent animal survival and immunological memory. Our study highlights the importance of intratumoral B-cell immunity and a remodeled approach to boost the effects of immunotherapy against brain tumors.

The CXCL16-CXCR6 axis in glioblastoma modulates T-cell activity in a spatiotemporal context.

Introduction: Glioblastoma multiforme (GBM) pathobiology is characterized by its significant induction of immunosuppression within the tumor microenvironment, predominantly mediated by immunosuppressive tumor-associated myeloid cells (TAMCs). Myeloid cells play a pivotal role in shaping the GBM microenvironment and influencing immune responses, with direct interactions with effector immune cells critically impacting these processes. Methods: Our study investigates the role of the CXCR6/CXCL16 axis in T-cell myeloid interactions within GBM tissues. We examined the surface expression of CXCL16, revealing its limitation to TAMCs, while microglia release CXCL16 as a cytokine. The study explores how these distinct expression patterns affect T-cell engagement, focusing on the consequences for T-cell function within the tumor environment. Additionally, we assessed the significance of CXCR6 expression in T-cell activation and the initial migration to tumor tissues. Results: Our data demonstrates that CXCL16 surface expression on TAMCs results in predominant T-cell engagement with these cells, leading to impaired T-cell function within the tumor environment. Conversely, our findings highlight the essential role of CXCR6 expression in facilitating T-cell activation and initial migration to tumor tissues. The CXCL16-CXCR6 axis exhibits dualistic characteristics, facilitating the early stages of the T-cell immune response and promoting T-cell infiltration into tumors. However, once inside the tumor, this axis contributes to immunosuppression. Discussion: The dual nature of the CXCL16-CXCR6 axis underscores its potential as a therapeutic target in GBM. However, our results emphasize the importance of carefully considering the timing and context of intervention. While targeting this axis holds promise in combating GBM, the complex interplay between TAMCs, microglia, and T cells suggests that intervention strategies need to be tailored to optimize the balance between promoting antitumor immunity and preventing immunosuppression within the dynamic tumor microenvironment.