Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
These findings suggest CAR spatial organization correlates with functional states and demonstrate the power of integrating advanced flow and imaging cytometry to resolve CAR T cell phenotypes and spatial dynamics with high precision.
Compared to the standard 7-day method, the 3-day approach resulted in expression of genes associated with a more stem-like phenotype while reducing manufacturing time and cost, and may provide a practical alternative for decentralized CAR T-cell manufacturing, particularly in resource-limited settings.
Isabella Vignola, M. Procházková, L. Shao et al.· Journal of Translational Med...· 1 citation
Accurate flow cytometric detection of CD19 CAR-expressing cells is critical across CAR-T cell discovery and translational research workflows. However, variability in surface CD19 CAR protein expression presents challenges in reliably identifying CAR+ cells. Additionally, detection using recombinant CD19 protein is hindered by its instability in soluble form.
To address these limitations, BD® has developed a novel, bright, and stable one-step CD19 CAR Detection Reagent, available in multiple fluorochrome formats to support diverse panel designs and instrument configurations.
We demonstrate that this reagent specifically stains CD19 CAR-expressing CHO cells, Jurkat CAR-T cells, and primary human CD19 CAR-T cells. Furthermore, it performs effectively in lysed whole blood, multi-color panels, and workflows involving intracellular staining. Notably, CAR-T cells spiked into donor PBMCs were detected at frequencies as low as < 0.04% of live cells, highlighting its potential utility in studies of CAR-T cell persistence. Minimal background staining was observed in CAR-negative and mismatched CAR populations, and low-expressing CAR cells (CARlo) were distinguishable from negative cells
Overall, these findings highlight the BD® CD19 CAR Detection Reagent as a robust solution for overcoming key challenges in CD19 CAR+ cell detection, enabling progressive CAR-T cell research.
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Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Joseph Cantor, Omar Bazirgan, Christian Carson et al.· Journal of Immunology· 0 citations
Summary CD19-directed chimeric antigen receptor (CAR)-engineered T cells have transformed cellular therapy for hematologic malignancies but have also raised concerns about secondary malignancies. A spatial profiling method was used to analyze the tumor microenvironment and define native and CAR T cell association with a pleomorphic sarcoma arising 12 months post-CAR T therapy for B cell lymphoma. Although CAR T sequences were absent in the secondary tumor, our approach enabled profiling of the stroma, tumor, and infiltrating T cells. Spatial analysis incorporated proteomics via serial antibody staining and transcriptomics using RNA hybridization on an automated MACSima imaging cyclic staining (MICS) system, which allows for in situ detection of CAR T cells. We report here the prevalence of CAR-positive T cells in patient-derived tissue sections, observed metabolic and proliferative shifts in the tumor and its microenvironment, and immune checkpoint analysis. These findings provide insights into post-CAR T secondary cancers and highlight tools that may guide future targeted therapies.
Jia-Jye Lee, Peirong Hu, Kun Luo et al.· iScience· 0 citations
The addition of cellular imaging to high-speed, single-cell flow cytometry has revolutionized simultaneous immunophenotyping with morphological and spatial insights.
We have pioneered the utilization of imaging-enabled platforms such as the CytPix and FACSDiscover to characterize cell biology. The CytPix combines standard flow cytometry with a 20X equivalent brightfield camera to provide detailed morphological information, while the FACSDiscover enables cellular marker localization using three fluorescence imaging detectors in addition to a full spectral cytometer.
Both platforms process images at ultra-high throughput, with FACSDiscover reaching up to 10,000 cells per second. Imaging-derived features and AI-based analysis provide morphological and spatial information, uncovering insights not possible with traditional flow cytometry. This includes enhanced identification of immune cell subsets with unique functionalities, improved resolution of senescence, and more detailed signaling and metabolic characterization of mitochondrial activity and distribution. Moreover, imaging cytometry allows to characterize immunological synapses, which was previously impossible with flow cytometry.
Current developments include leveraging AI to identify cellular states and activation label-free, simplifying cellular characterization and profiling, and bridging phenotype with mechanism–thereby driving innovation in immune cell research across multiple therapeutic areas.
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Technological Innovations in Immunology (TECH)
Nicola Heller, Viji Premkumar, Nicholas G. Battaglia et al.· Journal of Immunology· 0 citations
CAR T cell therapy has shown success in treating hematologic malignancies but faces challenges in solid tumors due to antigen heterogeneity and the complex tumor microenvironment (TME). CAFs expressing fibroblast activation protein (FAP) contribute to immune suppression by creating physical barriers and immune-suppressive environment that blocks T cell infiltration and persistence. Overcoming these barriers is crucial for enhancing CAR T cell therapy in solid tumors.
We engineered a modular CAR T cell platform using anti-FLAG CAR T cells conjugated with FLAG-tagged monoclonal antibodies, allowing flexible targeting of both tumor cells and CAFs. This platform was tested in B-cell lymphoma and pancreatic cancer models, both in vitro and in vivo. Cytotoxicity was evaluated through flow cytometry. TME remodeling and cytokine production were assessed using immunostaining, multiplex cytokine assays, and qPCR. In vivo efficacy was evaluated in subcutaneous and orthotopic models.
In B-cell lymphoma, FLAG CAR T cells, combined with FLAG-tagged CD19 antibodies, exhibited strong anti-tumor activity, significantly increasing tumor cell lysis and achieving cytotoxicity comparable to traditional CD19 CAR T cell therapies. In solid tumor models, FLAG CAR T cells, in combination with FLAG-tagged antibodies targeting FAP+ CAFs and Claudin18.2+ tumor cells, enhanced immune cell infiltration and significantly suppressed tumor growth. This dual-targeting strategy disrupted the TME-induced immune suppression, reduced collagen deposition, and boosted T cell activity, resulting in notable tumor growth inhibition and prolonged survival in both subcutaneous and orthotopic pancreatic cancer models.
This CAR T cell platform enables dual targeting of tumor cells and CAFs, overcoming immune suppression in solid tumors. This approach enhances immune activation, reshapes the TME, and provides a promising strategy for improving CAR T cell therapy in solid tumors.
This study was funded by the National Natural Foundation of China (No. 81970632), Guangdong Science and Technology Department (Nos. 2020B1212060018 and 2020B1212030004), Guangdong Basic and Applied Basic Research Foundation 2025A1515011234.
Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Summary Pancreatic cancer resists immunotherapy due to a suppressive immune microenvironment where CD8+ T cells play a key role. Using single-cell RNA sequencing and spatial transcriptomics, we characterized CD8+ exhausted T (Tex) cells in pancreatic ductal adenocarcinoma (PDAC). We generated single-cell profiles from PDAC tumors and matched peripheral blood mononuclear cells, and performed T cell sub-analysis. We found CXCL13 upregulated and GZMK downregulated in CD8+ Tex cells. Cell-cell interaction analysis showed that T cells most frequently interacted with myeloid cells and cancer cells via ligand-receptor pairs; INHBA+ macrophages and cancer cells communicated most with CD8+ Tex cells. Two key LR pairs (SPP1-integrin α4β1 and PLAUR-integrin α4β1) mediated crosstalk between cancer cells and CD8+ Tex cells, confirmed by immunofluorescence, spatial mapping, and protein docking. High SPP1 and PLAUR expression correlated with poor prognosis in TCGA-PAAD. These findings provide a resource for understanding CD8+ T cell exhaustion in PDAC.
Jing Mao, Chenxin Yan, Ying Mei et al.· iScience· 0 citations