Background 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 TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3-specific CAR T-cells.
Methods Using an innovative approach in an immune competent glioma model, we generated a diverse panel of seven fully murine B7-H3 CARs with variations in transmembrane, co-stimulatory, and activation domains. This enabled us to comprehensively investigate their effector functions within the intact immune system. High-dimensional flow cytometry, spatial transcriptomic analysis, and single-cell RNA sequencing were then used to investigate changes in the brain TIME following CAR T-cell therapy.
Results Five out of six B7-H3 CARs with single co-stimulatory domains demonstrated robust functionality in vitro. However, optimizing co-stimulation and signaling did not lead to superior anti-glioma efficacy of B7-H3 CAR T-cells in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 4–1BB and CD28 co-stimulation through transgenic expression of 4–1BBL on CD28-based CAR T-cells. This modification significantly improved the anti-glioma efficacy of B7-H3 CAR T-cells in vitro but did not result in additional improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the brain TIME. Recruitment, activation, and spatial localization of unique inflammatory ‘immune hubs’ with specific subsets of macrophages and endogenous T-cells dictated successful anti-tumor responses.
Conclusions Our study highlights the critical role of CAR structural design and its modulation of the TIME in mediating the efficacy of CAR T-cell therapy for high-grade glioma. Our findings contribute to a broader understanding of the complex interactions within the tumor microenvironment and provide insights for optimizing CAR T-cell immunotherapies for glioma treatment. Further research is warranted to fully elucidate the underlying mechanisms and identify strategies to enhance the therapeutic potential of CAR T-cell therapies in high-grade glioma.
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