Biology subjects

Gallus, M.

Publications and source records attributed to Gallus, M..

4 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↗

bioRxiv2023

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma

BackgroundDespite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter-and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of neoantigens. ResultsIn this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface neoantigens that could be targeted by antibodies and chimeric antigen receptor (CAR)-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas [TCGA]) and 9,166 normal tissue samples (from the Genotype-Tissue Expression project [GTEx]), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative neoantigens. ConclusionsOur investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

cancer biology↗

bioRxiv2023

Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22, respectively. TCR-engineered CD8+ T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.

cancer biology↗