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.

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.

Antigen-presenting B cells promote TCF-1+ PD1- stem-like CD8+ T-cell proliferation in glioblastoma.

Understanding the spatial relationship and functional interaction of immune cells in glioblastoma (GBM) is critical for developing new therapeutics that overcome the highly immunosuppressive tumor microenvironment. Our study showed that B and T cells form clusters within the GBM microenvironment within a 15-µm radius, suggesting that B and T cells could form immune synapses within the GBM. However, GBM-infiltrating B cells suppress the activation of CD8+ T cells. To overcome this immunosuppression, we leveraged B-cell functions by activating them with CD40 agonism, IFNγ, and BAFF to generate a potent antigen-presenting B cells named BVax. BVax had improved antigen cross-presentation potential compared to naïve B cells and were primed to use the IL15-IL15Ra mechanism to enhance T cell activation. Compared to naïve B cells, BVax could improve CD8 T cell activation and proliferation. Compared to dendritic cells (DCs), which are the current gold standard professional antigen-presenting cell, BVax promoted highly proliferative T cells in-vitro that had a stem-like memory T cell phenotype characterized by CD62L+CD44- expression, high TCF-1 expression, and low PD-1 and granzyme B expression. Adoptive transfer of BVax-activated CD8+ T cells into tumor-bearing brains led to T cell reactivation with higher TCF-1 expression and elevated granzyme B production compared to DC-activated CD8+ T cells. Adoptive transfer of BVax into an irradiated immunocompetent tumor-bearing host promoted more CD8+ T cell proliferation than adoptive transfer of DCs. Moreover, highly proliferative CD8+ T cells in the BVax group had less PD-1 expression than those highly proliferative CD8+ T cells in the DC group. The findings of this study suggest that BVax and DC could generate distinctive CD8+ T cells, which potentially serve multiple purposes in cellular vaccine development.

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.