http://bura.brunel.ac.uk/handle/2438/32871 Sun, 05 Apr 2026 21:01:51 GMT 2026-04-05T21:01:51Z 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.; This is a PDF of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability. This version will undergo additional copyediting, typesetting and review before it is published in its final form. As such, this version is no longer the Accepted Manuscript, but it is not yet the definitive Version of Record; we are providing this early version to give early visibility of the article. Please note that Elsevier’s sharing policy for the Published Journal Article applies to this version, see: https://www.elsevier.com/about/policies-andstandards/sharing#4-published-journal-article. Please also note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Thu, 05 Mar 2026 00:00:00 GMT http://bura.brunel.ac.uk/handle/2438/32944 2026-03-05T00:00:00Z http://bura.brunel.ac.uk/handle/2438/32769 Title: Novel memory phenotype Tfh cells arise without overt antigen stimulation and are important for adaptive immune responses against viral infection Authors: Busharat, Zabreen Abstract: Pathogen-induced memory Tfh cells exert a Tfh effector response during reinfection, regulating the generation of high-affinity antibodies. Here, we define novel memory-phenotype Tfh cells which are generated from naïve T cells under homeostatic conditions. These MP Tfh cells are phenotypically and functionally similar to pathogen-induced Tfh cells. MP Tfh cells can be defined by Tfh cell specific markers, CXCR5, BCL6, and PD-1, and markers of pathogen-induced long lived Tfh cells, FR4. T-bethigh MP T cells exert an innate-like Th1 response against viral infections. The transcription factor EGR2 is a repressor of T-bet function, and we found that MP Tfh cells are distinct from T-bethigh MP T cells but express EGR2 highly. Previously, we found Egr2 is required for MP T cell homeostasis and inflammation. Here, we observed that, in Egr2/3-/- CD4+ MP T cells, MP Tfh cell development is impaired. FR4+ EGR2 + MP T cells upregulate genes related to homeostatic proliferation, Tfh cell development and metabolic pathways of pathogen-induced memory Tfh cells. MP Tfh cells can exert an adaptive function by regulating B cell-mediated IgG production in vitro whereas MP Tfr cells are involved in suppressing MP Tfh cell function, thereby preventing excessive inflammation. In vivo, MP Tfh cells support germinal centre formation and induce neutralising antibody production after infection with vaccinia virus. Thus, MP Tfh cells with similar characteristics to pathogen-induced memory Tfh cells are developed in absence of environmental antigens and to date are the only CD4+ MP T cell subset associated with an adaptive immune response against viral infection. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London Wed, 01 Jan 2025 00:00:00 GMT http://bura.brunel.ac.uk/handle/2438/32769 2025-01-01T00:00:00Z