IMMU-14. Focused ultrasound–mediated blood-brain barrier disruption, combined with antigen-presenting cell activation, promotes CNS antigen-specific T cell immunity and bypasses immunotherapy resistance mechanism in malignant brain tumors

Abstract The efficacy of Immunotherapies against malignant brain tumors—characterized by profound heterogeneity—remains limited. The brain’s anatomical barriers and immunosuppressive microenvironment impede immune cell trafficking and antigen presentation, enabling tumors to escape immune surveillance. Innovative strategies are therefore urgently needed to stimulate endogenous immune responses within the central nervous system (CNS). Low-intensity pulsed ultrasound (LIPU) with microbubbles (MB)–mediated blood-brain barrier (BBB) opening represents a promising approach to transiently circumvent these physical constraints. However, its immunological effects remain poorly understood. To investigate whether LIPU+MB-induced BBB disruption can enhance immune responses against CNS-localized antigens, we employed comprehensive immune profiling in multiple models: novel transgenic mice with CNS-restricted antigen expression and intact BBB, bone marrow chimeras, and mice bearing immunosuppressive SB28 brain tumors with heterogeneous EGFRvIII expression. We found that LIPU+MB selectively increased infiltration of CNS antigen-specific—but not unspecific—T cells into the brain via ICAM-1–LFA-1–dependent mechanisms. Notably, BBB disruption alone was insufficient to induce antigen-specific immunity, despite transient T-cell entry. However, combining LIPU+MB with immune adjuvants—a Toll-like receptor 3 agonist and interleukin-2—effectively primed CNS antigen-specific T cells in peripheral lymphoid organs and the brain, leading to their recruitment and differentiation into tissue-resident memory T cells capable of sustaining long-term CNS immunity. Genetic depletion of β2-microglobulin in hematopoietic cells revealed the critical role of antigen presentation by bone marrow-derived cells and CNS-resident microglia for the sustained T-cell immunity in the CNS. Ultimately, LIPU+MB combined with immune-adjuvants significantly enhanced the therapeutic efficacy of CAR T-cell therapy in mice with heterogeneous brain tumors, by promoting endogenous tumor-antigen-specific T-cell responses. Together, these findings demonstrate that LIPU+MB, when combined with adoptive T-cell therapy and antigen-presenting cell activation, can elicit robust CNS-specific immune responses and help overcome key limitations of immunotherapies targeting malignant brain tumors.