Biology subjects

Watchmaker, P. B.

Publications and source records attributed to Watchmaker, P. B..

3 recordsLinked to original sources

bioRxiv2026

Ultrasound-mediated blood-brain barrier modulation enhances T-cell access but requires immune activation for effective CNS immunity

Immunotherapy shows limited efficacy in brain tumours, where restricted immune access, antigenic heterogeneity and local immunosuppression constrain durable responses. Low-intensity pulsed ultrasound with microbubbles (LIPU+MB) transiently modulates the blood-brain barrier (BBB) and is widely assumed to enhance immunotherapy by facilitating drug and immune cell penetration into the central nervous system (CNS). However, whether increased anatomical access alone is sufficient to generate effective CNS immunity remains unclear. Here, using a transgenic mouse model with astrocyte-restricted antigen expression, we showed that BBB modulation alone is insufficient to generate functional T-cell immunity in the CNS. Although LIPU+MB enabled rapid T-cell entry, accumulation required prior T-cell activation and integrin-dependent mechanisms, indicating that entry remains governed by canonical immune processes. Moreover, T-cells failed to persist owing to insufficient activation of antigen-presenting cells (APCs) within the CNS. Systemic immune adjuvants (poly-ICLC and IL-2; PI) induced APC activation, promoted tissue-resident-memory-like differentiation and supported durable T-cell responses. LIPU+MB further enhanced these responses by increasing T-cell recruitment, resulting in greater accumulation than with PI alone. Mechanistically, antigen presentation by bone marrow-derived APCs was more critical than that by microglia for the accumulation and persistence of antigen-specifc T-cells in the CNS. In antigenically heterogeneous glioma models resistant to CAR T-cell therapy, combining PI with BBB modulation enhanced the efficacy of immunotherapy, which was mirrored by prolonged survival and endogenous tumour-specific T-cell responses, consistent with epitope spreading. Together, these findings define key limitations of LIPU+MB in enabling effective T-cell therapy and establish that BBB modulation must be coupled to systemic immune activation to support T-cell-mediated antitumour immunity in the CNS.

immunology↗

bioRxiv2026

Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of "prime-and-kill" synNotch-CAR-T cells

We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (NCT06186401). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro, integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. One Sentence SummaryOur spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.

cancer biology↗

bioRxiv2021

Multi-antigen recognition circuits overcome challenges of specificity, heterogeneity, and durability in T cell therapy for glioblastoma

Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of target antigens that are both tumor-specific and homogeneously expressed. We show that multiantigen prime-and-kill recognition circuits have the flexibility and precision to overcome these challenges in attacking glioblastoma. A synNotch receptor that recognizes a specific priming antigen - the heterogeneous glioblastoma neoantigen EGFRvIII or a brain tissue-specific antigen - is used to locally induce expression of a CAR, enabling thorough but controlled tumor killing by targeting of homogeneous antigens that are not absolutely tumor specific. Moreover, regulated CAR expression maintains a higher fraction of the T cells in the naive-like state which is associated with higher durability in vivo. In summary, using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.

immunology↗