http://bura.brunel.ac.uk/handle/2438/32873 2026-04-26T02:08:45Z http://bura.brunel.ac.uk/handle/2438/33179 Title: CAR-mediated release of IL-10 increases the function of regulatory T cells: relevance for future clinical application Authors: Saleem, A; Peng, Q; Tang, Z; Mohseni, YR; Scottà, C; Shangaris, P; Smit, K; Vermeij, WP; Issa, F; Lombardi, G; Fruhwirth, GO Abstract: Regulatory T cell (Treg) therapy emerges for various indications associated with a breakdown of immune tolerance. Antigen-specific chimeric antigen receptor (CAR) Tregs are frontrunners for transplantation and autoimmune diseases and are currently being clinically evaluated. We aimed to link CAR-antigen engagement with immunosuppressive cargo release into the local microenvironment to boost efficacy and reduce side effects. We used our HLA-A∗02 CAR and immunosuppressive interleukin-10 (IL-10) as model components to generate human CAR Tregs that release IL-10 upon CAR engagement. These were compared to CAR Tregs with constitutive or no IL-10 expression by evaluating phenotypes, antigen-specific IL-10 release, and suppression of effector cell proliferation in vitro and performance in vivo in a humanized xenogeneic graft-versus-host disease (xeno-GvHD) model. We demonstrated successful multi-construct engineering of CAR Tregs, which released upon CAR engagement 2.5-fold more IL-10 than CAR Tregs lacking the corresponding antigen-specific IL-10 secretion module. Neither phenotype nor function was affected by expressing this module. In the xeno-GvHD model, we showed the beneficial effect of IL-10 release, particularly evident when compared to constitutive IL-10 expression that impaired CAR-Treg efficacy. We provide first proof-of-principle for engineering human CAR Tregs to release an immunosuppressive cytokine upon CAR engagement. This approach will both enhance the potency of CAR Tregs at the intended target sites and limit their off-target effects. Description: Data and code availability: The data presented here are available on request from the corresponding authors.; Supplemental information is available online at: https://www.sciencedirect.com/science/article/pii/S1525001626000870#appsec2 . 2026-02-06T00:00:00Z http://bura.brunel.ac.uk/handle/2438/33171 Title: Clinical assessment meets laboratory science: adapting OSCE methodology for authentic biosciences evaluation in the age of generative AI Authors: Mann, R; Tosi, S; Tree, D Abstract: The proliferation of generative artificial intelligence (AI) tools has fundamentally challenged traditional written assessments across higher education, with particular implications for laboratory-based disciplines where written work may substitute for demonstration of practical competence, necessitating approaches that prioritise direct performance. This study presents the adaptation of objective structured clinical examination (OSCE) methodology from medical education to laboratory biosciences, demonstrating a practical framework for authentic assessment in the AI era. We describe and evaluate the transformation of a microscopy assessment in FHEQ Level 4 Biomedical Sciences from a traditional laboratory report to a 20-minute OSCE-style practical evaluation. The redesigned assessment maintained grade distributions while eliminating AI vulnerability through real-time performance demonstration and conversational examination. The implementation achieved close alignment between learning outcomes and assessment methods while providing inherent resistance to generative AI exploitation through direct performance requirements. Equity implications are complex and context-dependent, with potential barriers for students with communication differences alongside potential benefits for others, such as those with written communication difficulties, emphasising the importance of balanced assessment portfolios and appropriate reasonable adjustments. The cross-disciplinary adaptation demonstrates that OSCE methodology offers a scalable solution to AI-era assessment challenges, with performance-focused design maintaining academic integrity more effectively than restrictive policies while enhancing authenticity and equity outcomes. Description: Perspective. 2026-03-24T00:00:00Z http://bura.brunel.ac.uk/handle/2438/33116 Title: Single-cell transcriptomics identifies regulatory T cell heterogeneity in gestational diabetes mellitus Authors: Mensah, NE; Efthymiou, A; Mureanu, N; Martín Monreal, MT; Vaikkinen, H; Kannambath, S; Bowman, A; Menon, A; Tree, T; Lombardi, G; Dhami, P; Nicolaides, KH; Scottà, C; Shangaris, P Abstract: Background: Gestational diabetes mellitus (GDM) is a common pregnancy complication associated with hyperglycaemia, chronic inflammation and adverse health outcomes. Regulatory T cells (Tregs) are thought to contribute to GDM due to their role in suppressing inflammation. However, whether specific Treg subsets are transcriptionally dysregulated in patients with GDM remains unclear. Methods: To investigate Treg transcriptional variation in GDM, we applied single-cell RNA sequencing to Tregs and CD4 + T cells isolated from the blood of 13 healthy pregnant women and 10 female patients with GDM. Results: We observed no significant differences in Treg cluster proportions with disease status, however, Memory CD4 + T cells were more abundant in patients diagnosed with GDM, substantiated by mass cytometry. We report Treg subsets altered in GDM, including naive Tregs with reduced expression of AP-1 transcription factor subunits and effector Tregs with increased signalling of genes associated with angiogenesis. Expression levels of genes dysregulated in GDM Tregs were informative of GDM status in pseudobulk, placental and whole blood mRNA from independent cohorts. TXNIP, which regulates glucose levels, emerged as the most significant discriminator of GDM status from bulk mRNA. Conclusions: This study uncovers transcriptional differences of Treg cell subsets from GDM patients and transcriptional markers informative of GDM status. Description: Plain Language Summary: Gestational diabetes mellitus (GDM) is a common pregnancy condition linked to high blood sugar and increased inflammation, which can affect the health of both mother and baby. Immune cells called regulatory T cells (Tregs) help control inflammation, and their activity in a mother’s blood may be linked to GDM. To understand how Tregs behave in patients with GDM, we captured these cells from blood samples of pregnant women diagnosed with GDM and pregnant women without a GDM diagnosis. We profiled the expression of RNA in individual Tregs from these patients. We found that, while overall Treg numbers are similar, the activity of specific genes varies in Tregs from women with GDM. Disrupted RNA levels of one gene related to glucose control (TXNIP) may be an informative marker for GDM in blood. Our findings enhance the understanding of immune changes in GDM and may inform future approaches for early detection and monitoring.; Data availability: Single-cell RNA sequencing data has been submitted to Gene Expression Omnibus (Accession: GSE280975). Bulk RNA sequencing data was downloaded from Gene Expression Omnibus (Accession: GSE154414; GSE92772 - RNA sequencing data of whole blood cells of normal glucose tolerant (NGT) and gestational diabetes (GDM) pregnant women). Data underlying the figures are available on Zenodo [59.] Mensah, N. Single-cell transcriptomics identifies regulatory T cell heterogeneity in Gestational Diabetes Mellitus [Data set]. Zenodo. https://doi.org/10.5281/zenodo.18032075 (2026).; Code availability: R scripts used to perform the analyses are available at GitHub [60.] Mensah, N. E. rutepo_gdm_treg. GitHub repository. https://github.com/NMNS93/rutepo_gdm_treg (2025).; Springer Nature is providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.; Supplementary information is available online at: https://www.nature.com/articles/s43856-026-01563-0#Sec24 . 2026-04-03T00:00:00Z http://bura.brunel.ac.uk/handle/2438/32944 Title: Perfusion microbioreactor for CAR-Treg manufacturing Authors: Edwards, W; Sun, N; Wang, Y; Lu, Y; Wang, C; Mastronicola, D; Scottà, C; Romano, M; Cejas, CM; Espinet, A; Lombardi, G; Chiappini, C Abstract: Summary: Manufacturing cell and gene therapies (CGTs) at scale presents challenges in cost, product consistency, and adaptability to personalised treatments. Traditional large-volume bioreactors are designed to support cell growth through controlled nutrient delivery and gas exchange, but are poorly suited to the decentralised, small-batch production required for personalised therapies like Chimeric Antigen Receptor (CAR) T-cells. To address this, we have developed the KCL-Microbioreactor (K-MBR), a closed microbioreactor platform based on microfluidic principles. Engineered in polydimethylsiloxane (PDMS), the K-MBR combines spatial confinement, semi-continuous perfusion, and integrated viral transduction in a compact footprint enabling efficient gene delivery and robust expansion of therapeutic cells. We demonstrate the platform’s utility by generating functional CAR-Tregs targeting HLA-A2, achieving a 92% increase in yield compared to conventional methods. The K-MBR offers a streamlined, solution for CGT manufacturing, with potential to reduce productions cost and enhance scalability across a broad range of cell therapies. Description: Highlights: • Perfusion microbioreactor achieves Treg expansion comparable to gold standard G-Rex device. • Spatial confinement increases lentiviral transduction efficiency of primary, human cells. • Compact, low-volume platform reduces the physical footprint of cell manufacturing. • Device supports future automation and advances progress toward point-of-care production.; Data and code availability: All data reported in this paper will be shared by Ciro Chiappini upon request; this paper does not report original code. All datasets generated and analysed in this study, including raw flow cytometry files and source data, are available from the lead contact upon reasonable request.; Supplemental Information is available online. 2026-03-05T00:00:00Z