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263 Unveiling the role of macrophages in CAR T-cell induced remodeling of the brain tumor immune microenvironment: implications for anti-glioma adoptive immunotherapy
  1. Dalia Haydar1,
  2. Jorge Ibanez2,
  3. Jeremy Crawford2,
  4. Zhongzhen Yi3,
  5. Christopher DeRenzo2,
  6. Stephen Gottschalk2,
  7. Martine Roussel2,
  8. Paul Thomas2 and
  9. Giedre Krenciute2
  1. 1Children’s National Hospital, Washington, DC, USA
  2. 2St. Jude Children’s Research Hospital, Memphis, TN, USA
  3. 3Children’s National Hospital, Houston, TX, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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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