KS05.6.A T-CELL THERAPY COMBINED WITH LOW-INTENSITY PULSED FOCUSED ULTRASOUND, MICROBUBBLES AND POLY-ICLC PROMOTES THE PRESENTATION OF BRAIN-DERIVED ANTIGENS, BREAKING CNS TOLERANCE AND INDUCING ACTIVE T-CELL RESPONSES
Authors: Gallus M , Yamamichi A , Andres Arrieta V , Saijo A , Chuntova P , Haegelin J , Phyu S , Phung L , Long J , Lakshmanachetty S , Nejo T , Okada K , Chen L , Habashy K , Gould A , Amidei C , Chang C , Stupp R , Canney M , Sonabend A , Okada H 
Published: October 2024

Abstract

BACKGROUND
Immunotherapies targeting glioma, such as chimeric antigen receptor (CAR) T cell therapies, offer new therapeutic opportunities; however, their application has yielded limited success. Due to the antigenic heterogeneity of gliomas, targeting a few or several antigens may still result in the recurrence with “antigen-loss” tumors, indicating a need to enhance the “epitope spreading” process where additional tumor antigens not targeted by the therapy are effectively presented to the immune system. However, due to the immune-privileged status of the brain parenchyma, attributed to natural anatomical barriers and the immunosuppression of the CNS parenchyma, neoplasms within the brain often evade detection by the peripheral immune system..Low-intensity pulsed focused ultrasound combined with microbubbles (LIPU/MB) has been recently shown to transiently open the blood-brain barrier (BBB), enabling penetration of cells and drug into the CNS.

MATERIAL AND METHODS
Using intravital two-photon imaging, two photon-microscopy, multiparametric spectral flow cytometry, and bone marrow chimera mice, alongside blood samples from glioblastoma patients treated with LIPU/MB, we examined whether LIPU/MB-induced transient BBB- opening promotes migration of CNS antigen-presenting cells into the periphery. Additionally, we developed new transgenic mice (GFAP-minigene mice) with intact BBB expressing four immunogenic peptides under the CNS-specific GFAP promoter, enabling assessment of the immunological relevance of LIPU/MB-mediated CNS antigen presentation to the peripheral immune system.

RESULTS
Within one hour after LIPU/MB treatment in colony-stimulating factor receptor 1 (CSFR1) reporter mice, we observed the migration of CSFR1+ immune cells from the brain parenchyma and perivascular space into the bloodstream, a phenomenon absent prior to LIPU/MB treatment. Spectral flow cytometry of bone marrow-chimera mice with CNS-restricted CX3CR1-GFP expression confirmed CNS-derived antigen-presenting cells in the blood following LIPU/MB. In GFAP-minigene mice, LIPU/MB combined with adoptive T cell transfer targeting CNS (minigene peptides) and toll-like receptor 3 agonist poly-ICLC, promoted CNS antigen-spreading, mirrored by the presence of antigen-specific T cells in cervical lymph nodes and brain, resulting in clinically symptomatic cytotoxic T cell responses in the CNS parenchyma. Furthermore, blood samples of glioblastoma patients undergoing LIPU/MB demonstrated increased CD11b+CD14+P2YR12+ cells that were absent in healthy donor samples.

CONCLUSION
Our findings collectively suggest that LIPU/MB combined with adoptive T cell transfer and poly-ICLC overcomes inherent limitations of CAR T cell therapy by enhancing epitope spreading within the context of intact blood-brain barrier.