Biology subjects

Windell, D.

Publications and source records attributed to Windell, D..

4 recordsLinked to original sources

bioRxiv2025

An end-to-end framework for Cell DIVE multiplexed imaging and spatial immune microenvironment analysis

This paper describes an end-to-end workflow for highly multiplexed fluorescence imaging with the Cell DIVE platform, allowing simultaneous detection of 40+ markers at single-cell resolution. Combining whole-slide multiplexed imaging with a dedicated analysis pipeline provides a powerful approach to investigate immune cell interactions with stromal and vascular networks within human tissue microenvironments. With a focus on spatial investigation of human immune niches, here we provide a complete framework for tissue preparation, autofluorescence reduction, multiplex panel design and whole-slide image analysis. For complete details on the use and execution of this protocol, please refer to Korsunsky et al. (Med, 2022) [1]. HighlightsO_LIComplete workflow for Cell DIVE multiplex imaging and quantitative image analysis. C_LIO_LIHuman FFPE tissue preparation, LED-based reduction of tissue autofluorescence. C_LIO_LIAntibody panel design for 3-40 marker multiplexing, in-house antibody conjugation. C_LIO_LIQuPath and DeepCell based analysis workflows for whole-slide multi-marker images. C_LIO_LIAdaptable code templates to accelerate cell segmentation and spatial niche analysis. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/656440v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1ef708dorg.highwire.dtl.DTLVardef@c6422dorg.highwire.dtl.DTLVardef@22d961org.highwire.dtl.DTLVardef@1ed7479_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Tissue resident memory T cells populate the human uveal tract

The current concept is that the eye is an immune privileged site endowed with innate immune regulatory networks to maintain organ function. We now have evidence that resident T cells occupy intraocular tissues. In immune-mediated inflammatory diseases, such as psoriasis and rheumatoid arthritis, tissue resident T cells trigger disease flares in the skin and joints. This suggests resident T cells in the uvea may have similar functions in non-infectious immune-mediated uveitis, a collective term for autoinflammatory and autoimmune diseases of the uveal tract causing intraocular inflammation. Here, we demonstrate by spectral cytometry and immunofluorescence imaging that non-inflamed uveal tissue contains multiple T cell subtypes including CD8+ CD103+ tissue resident memory T (TRM) cells. Using single cell RNA & T cell receptor (TCR) sequencing to profile aqueous humour cells from donors with acute, active uveitis, we identify clonally expanded T cells which are enriched for TRM -associated genes. We further show that in donors with active uveitis, CD8+ CD103+ T cells persist within tissue in the uveal tract. Using bulk RNA sequencing and weighted gene co-expression network analysis (WGCNA) we show that quiescent iris tissue from donors with a history of uveitis are enriched for genes associated with T cell activation and antigen presentation. Finally, we demonstrate that TRM cells persist in the anterior uvea in mice following resolution of experimental autoimmune uveoretinitis (EAU). Our results show that the human eye contains T cells both in health and during active inflammation. Our findings challenge the dogma that the eye is devoid of lymphocytes and supports the concept of resident T cell involvement in the pathogenesis of non-infectious immune-mediated uveitis and as promising targets for therapy. One Sentence SummaryT cells infiltrate aqueous humour during intraocular inflammation and have capacity to migrate into uveal tissue where they remain long-lived.

immunology↗

bioRxiv2023

Primed to resolve: A single cell atlas of the shoulder capsule reveals a cellular basis for resolving inflammatory fibrosis

Fibrotic conditions are a significant global disease burden. While some therapies delay disease progression, none reverse fibrosis. To gain insights into how fibrosis might resolve, we developed a comparative single cell atlas of frozen shoulder capsule tissue; a chronic inflammatory fibrotic human disease that resolves spontaneously. We identified both a population of pro-inflammatory MERTKlowCD48+ macrophages (M{varphi}) and a population of MERTK+LYVE1+MRC1+M{varphi} enriched for negative regulators of inflammation. Micro-cultures of patient-derived cells identified cell-matrix interactions between MERTK+M{varphi} and DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in the resolution of frozen shoulder. Cross-tissue analysis revealed a shared gene expression cassette between MERTK+M{varphi} in the shoulder capsule and a similar cell population enriched in synovial tissues from rheumatoid arthritis patients in disease remi ssion, supporting the concept that MERTK+M{varphi} provide a cellular basis for the resolution of inflammation and fibrosis. Single-cell transcriptomic profiling and spatial analysis of human foetal shoulder tissues identified MERTK+LYVE1+MRC1+M{varphi} and DKK3+ and POSTN+ fibroblast populations analogous to those identified in adult shoulder capsule, suggesting that the template to resolve fibrosis is established during development. Therapeutic enhancement of crosstalk between MerTK+M{varphi} and pro-resolving DKK3+ and POSTN+ fibroblasts could accelerate resolution of frozen shoulder and resolve persistent inflammatory fibrotic disease in other tissues.

cell biology↗

bioRxiv2022

LAT1 enables T cell activation under inflammatory conditions: a new therapeutic target for rheumatoid arthritis

ObjectiveTo assess the L-type amino acid transporter-1 (LAT1) as a possible therapeutic target for rheumatoid arthritis (RA). MethodsSynovial LAT1 expression was monitored by immunohistochemistry and transcriptomic datasets. The contribution of LAT1 to gene expression and immune synapse formation was assessed by RNA-sequencing and total internal reflection fluorescent (TIRF) microscopy, respectively. Mouse models of RA were used to assess the impact of therapeutic targeting of LAT1. ResultsLAT1 was strongly expressed by CD4+ T cells in the synovial membrane of patients with active RA and the level of expression correlated with levels of ESR and CRP as well as DAS-28 scores. Deletion of LAT1 in murine CD4+ T cells inhibited the development of experimental arthritis and prevented the differentiation of CD4+ T cells expressing IFN-{gamma} and TNF-, without affecting regulatory T cells. LAT1 deficient CD4+ T cells demonstrated reduced transcription of genes associated with TCR/CD28 signalling, including Akt1, Akt2, Nfatc2, Nfkb1 and Nfkb2. Functional studies using TIRF microscopy revealed a significant impairment of immune synapse formation with reduced recruitment of CD3{zeta} and phospho-tyrosine signalling molecules in LAT1 deficient CD4+ T cells from the inflamed joints but not the draining lymph nodes of arthritic mice. Finally, it was shown that a small molecule LAT1 inhibitor, currently undergoing clinical trials in man, was highly effective in treating experimental arthritis in mice. ConclusionsIt was concluded that LAT1 plays a critical role in activation of pathogenic T cell subsets under inflammatory conditions and represents a promising new therapeutic target for RA. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LILAT1 is an amino acid transporter that has previously been shown to play a role in T cell activation. C_LI What does this study add?O_LILAT1 is expressed by synovial T cells in human rheumatoid arthritis and the level of expression correlates with disease severity. C_LIO_LILAT1 expression by T cells is necessary for development of severe arthritis in animal models. C_LIO_LILAT1 is required for immune synapse formation and activation of pathogenic CD4+ T cell subsets in the inflamed joint, but not the lymph nodes. C_LIO_LIA small molecular weight LAT1 inhibitor, currently in clinical trials for cancer, is highly effective in animal models of rheumatoid arthritis. C_LI How might this impact on clinical practice of future developments?O_LIThe context-specific nature of LAT1 involvement in T cell activation positions it as an ideal therapeutic target to distinguish between pathogenic and protective T cell responses and this study provides the scientific rationale for clinical evaluation of LAT1 inhibitors in the treatment of rheumatoid arthritis. C_LI

immunology↗