Biology subjects

Dovedi, S.

Publications and source records attributed to Dovedi, S..

7 recordsLinked to original sources

bioRxiv2026

A low-matrix organoid-T cell co-culture platform for functional evaluation of T cell engagers in patient-derived colorectal cancer organoids

Background Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, and emerging T cell engager (TCE) immunotherapies require predictive preclinical models that capture patient-specific tumour biology and tumour-immune interactions. Although patient-derived organoid (PDO)-immune co-culture systems show promise for evaluating immunotherapy responses, many rely on matrix-embedded cultures that limit scalability and reproducibility. We therefore developed and validated a low-matrix organoid-T cell co-culture platform for functional assessment of TCE activity in patient-derived CRC models. Methods Patient-derived CRC organoids representing diverse molecular and genetic backgrounds were co-cultured with activated allogeneic CD3+ T cells in a suspension low-matrix format. Matrix concentration, medium composition, T cell activation status, and assay duration were optimized. Tumour killing, T cells activation, cytokine secretion, apoptosis, and motility were assessed using flow cytometry, live-cell imaging, cytokine profiling, and immunofluorescence. The platform was evaluated using EGFR- and HER2-targeting bispecific T cell engagers across 21 CRC organoid models and multiple healthy donor-derived T cell populations. Results Optimization identified 1% matrix and a 1:1 organoid: T cell medium that preserved organoid integrity while maintaining T cell viability, activation, and motility. The final workflow enabled reproducible co-culture of 5-day mature organoids with 7-day activated T cells for 72 hours. EGFR-targeting TCEs induced tumour killing, T cell activation, and IFN-{gamma} secretion across donor-organoid combinations. Screening of 21 CRC organoid models revealed substantial inter-patient heterogeneity, with 12 models maintaining [≥]50% baseline viability and supporting functional TCE evaluation. EGFR- and HER2-targeting TCEs produced potent dose-dependent cytotoxicity, with IC50 values ranging from 0.0188 to 54.77 nM across responsive models and up to 75%-85% tumour killing in the most sensitive organoids. These responses were accompanied by increased effector cytokine secretion. Real-time imaging confirmed dynamic T cell engagement and apoptosis-driven organoid destruction following TCE treatment. Conclusions We establish a robust, scalable, human-relevant low-matrix organoid-T cell co-culture platform for functional screening of T cell engagers in patient-derived CRC models. By enabling integrated assessment of tumour killing, immune activation, cytokine responses, and tumour-immune interactions while preserving inter-patient heterogeneity, this system provides a translational framework for immunotherapy development. The platform may support candidate prioritisation, biomarker discovery, patient stratification, and preclinical evaluation of immune-engaging therapeutics.

cancer biology↗

bioRxiv2026

PD-1 blockade enhances T cell activation by reorchestrating CD28 and CTLA4 ligand interactions

The network of interactions comprising CD28, CTLA4 and PD-1 and their ligands CD80, CD86, and PD-L1 have profound implications for T cell activation. Despite their critical role, factors that determine their receptor occupancy when all three ligands are present remains incompletely understood. Using a supported lipid bilayer to replicate the antigen-presenting cell membrane facilitated a quantitative analysis of CD80, CD86, and PD-L1 interactions. Our observations reveal that PD-1 blockade operates through a previously unrecognized competitive mechanism beyond conventional checkpoint inhibition: liberated PD-L1 molecules redistribute to form CD80-PD-L1 heterodimers that strengthen CD28-CD80 interactions and outcompete CD86 for CD28 binding. This preferential binding enhances T cell activation through superior CD80 co-stimulation. Furthermore, the redistribution by PD-1 blockade reduces CD80 homodimer availability, limiting CTLA4 binding to CD80 and enhancing CD28 signaling. These findings provide vital insights into the competitive principles that allow B7-CD28 family receptors to regulate T cell activation through network-level effects.

immunology↗

bioRxiv2024

Rigid, bivalent CTLA-4 binding to CD80 is required to disrupt the cis CD80 / PD-L1 interaction

The CTLA-4 and PD-1 checkpoints control immune responses to self-antigens and are key targets in cancer immunotherapy. Both pathways are connected via a cis interaction between CD80 and PD-L1, the ligands for CTLA-4 and PD-1 respectively. This cis interaction prevents PD-1 binding to PD-L1 but is reversed by CTLA-4 trans-endocytosis of CD80. However, the mechanism by which CTLA-4 selectively removes CD80 but not PD-L1 is unclear. Here we show that CTLA-4 - CD80 interactions are unimpeded by PD-L1 and that CTLA-4 binding with CD80 does not displace PD-L1 per se. Rather, both the rigidity and bivalency of the WT CTLA-4 molecule is required to orientate CD80 such that PD-L1 interactions are no longer permissible. Moreover, soluble CTLA-4 released PD-L1 only at specific expression levels of CD80 and PD-L1, whereas CTLA-4 trans-endocytosis released PD-L1 in all conditions. These data show that PD-L1 release from CD80 is driven by biophysical factors associated with orientation and bivalent cross-linking of proteins in the cell membrane and that trans-endocytosis of CD80 efficiently promotes PD-L1 availability.

immunology↗

bioRxiv2023

Rapid establishment of a tumor-retained state curtails the contribution of conventional NK cells to anti-tumor immunity in solid cancers

Immune cell dysfunction within the tumor microenvironment undermines the control of cancer progression. NK cells play critical roles in limiting early tumor growth and metastatic disease, however, established cancers contain a phenotypically distinct, tumor-specific NK cell compartment. The temporal dynamics, mechanistic underpinning and functional significance of this tumor NK pool remains incompletely understood. To address this, we exploited photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of NK cells after tumor entry. In multiple murine cancer models we reveal that conventional NK cells are continuously recruited into tumors, but rapidly adopt a distinct phenotypic state with features associated with tissue-residency and complete loss of effector functions (including chemokine and cytokine production and cytotoxicity), within 48-72 hrs of entering the tumor. Depletion of NK cells from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti- tumor responses. Furthermore, comparable NK populations were identified in human colorectal cancers, confirming translational relevance and raising the possibility that interventions to reactivate NK cells within tissues may boost anti-tumor immunity in established cancers. Indeed, administration of IL-15:IL-15Ra complexes prevented the loss of NK cell function and improved tumor control, generating intratumoral NK cells with both enhanced tissue-residency characteristics and effector function. Collectively, our data reveals the fate of cNK cells after recruitment into tumors and provides insight into how intratumoral NK cell functions may be revived. SummaryConventional NK cells recruited from the circulation rapidly establish a tissue-resident phenotype defined by impaired cytotoxicity and chemokine production after tumor entry; administration of IL- 15:IL-15R complexes further promotes this tissue-residency programme but maintains core NK cell effector functions within the tumor.

immunology↗

bioRxiv2023

Time-, tissue- and treatment-associated heterogeneity in tumour-residing migratory DCs

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to a maturation programme enriched in regulatory molecule expression, including PD-L1, termed mRegDC. However, the spatio- temporal dynamics and role of mRegDCs in anti-tumour immune responses remain unclear. Using photoconvertible mice to precisely track DC migration, we found that mRegDCs were the dominant DC population arriving in the dLN, but a subset remained tumour-resident despite CCR7 expression. These tumour-retained mRegDCs were phenotypically and transcriptionally distinct from their dLN counterparts and were heterogeneous. Specifically, they demonstrated a progressive reduction in the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour mRegDCs spatially co-localised with PD-1+CD8+ T cells in human and murine solid tumours. Following anti-PD-L1 treatment, tumour-residing mRegDCs adopted a state enriched in lymphocyte stimulatory molecules, including OX40L, which was capable of augmenting anti- tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in mRegDCs that may underpin a variable capacity to support intratumoural cytotoxic T cells, and provide insights into their role in cancer immunotherapy.

immunology↗

bioRxiv2022

Small gene networks can delineate immune cell states and characterize immunotherapy response in melanoma

BackgroundSingle-cell sequencing studies have elucidated some of the underlying mechanisms responsible for immune checkpoint inhibitor (ICI) response, but are difficult to implement as a general strategy or in a clinical diagnostic setting. In contrast, bulk RNAseq is now routine for both research and clinical applications. Therefore, our analysis extracts small transcription factor-directed co-expression networks (regulons) from single-cell RNA-seq data and uses them to deconvolute immune functional states from bulk RNA-seq data to characterize patient responses. MethodsRegulons were inferred in pre-treatment CD45+ cells from metastatic melanoma samples (n=19) treated with first-line ICI therapy (discovery dataset). A logistic regression-based classifier identified immune cell states associated with response, which were characterized according to differentially active, cell-state specific regulons. The complexity of these regulons was reduced and scored in bulk RNAseq melanoma samples from four independent studies (n=209, validation dataset). Patients were clustered according to their regulon scores, and the associations between cluster assignment, response, and survival were determined. Intercellular communication analysis of cell states was performed, and the resulting effector genes were analyzed by trajectory inference. ResultsRegulons preserved the information of gene expression data and accurately delineated immune cell phenotypes, despite reducing dimensionality by > 100-fold. Four cell states, termed exhausted T cells, monocyte lineage cells, memory T cells, and B cells, were associated with therapeutic responses in the discovery dataset. The cell states were characterized by seven differentially active and specific regulons that showed low specificity in non-immune cells. Four clusters with significantly different response outcomes (P <0.001) were identified in the bulk RNAseq validation cohort. An intercellular link between exhausted T cells and monocyte lineage cells was established, whereby their cell numbers were correlated, and exhausted T cells predicted prognosis as a function of monocyte lineage cell number. Analysis of ligand - receptor expression suggested that monocyte lineage cells drive exhausted T cells into terminal exhaustion through programs that regulate antigen presentation, chronic inflammation, and negative co-stimulation. ConclusionsRegulon-based characterization of cell states provides robust and functionally informative markers that can deconvolve bulk RNA-seq data to identify ICI responders.

immunology↗

bioRxiv2022

CTLA-4 regulates PD-L1-PD-1 interactions via transendocytosis of CD80

CTLA-4 and PD-1 are key immune checkpoints that are targeted in the treatment of cancer. Recently it has emerged that there is a physical interaction between the ligands of these pathways (CD80 and PD-L1), which can prevent PD-L1 inhibitory functions. Since CTLA-4 captures and degrades its ligands via transendocytosis, we investigated how transendocytosis of CD80 is impacted by PD-L1 interaction. We find that transendocytosis of CD80 results in a time-dependent recovery of PD-L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is removed, and its heterodimeric PD-L1 partner remains on the APC. We found no evidence that CTLA-4 interactions with CD80 were inhibited by PD-L1, however efficient removal of CD80 required an intact CTLA-4 cytoplasmic domain, distinguishing this process from more general trogocytosis. We also show that simple binding of CTLA-4 to the CD80-PD-L1 heterodimer is insufficient to liberate PD-L1-PD-1 interactions, suggesting that transendocytosis of CD80 is required to effectively control PD-L1-PD-1 interactions.

immunology↗