Biology subjects

Sykes, M.

Publications and source records attributed to Sykes, M..

11 recordsLinked to original sources

bioRxiv2026

A Donor T-Cell Receptor Structural Signature Determines Alloreactive Potential and Predicts Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (GVHD) remains a lethal barrier to successful allogeneic hematopoietic cell transplantation, yet pre-transplant donor selection entirely ignores hypervariable T-cell receptor (TCR) architecture. Here, by characterizing over 64,000 alloreactive clonotypes, we demonstrate that human alloreactivity is dictated by a constrained, predictable baseline structural signature. Pathogenic, tissue-infiltrating alloreactive T cells exhibit significantly shortened CDR3{beta} regions, altered antigen-facing biophysical features, biased VJ gene usage and extensive inter-donor sharing originating from public anti-pathogen memory reservoirs. These potent clones natively cluster within the high-frequency fraction of the unstimulated baseline donor repertoire. We introduce R50, an assay-independent metric quantifying this clonal dominance, which independently predicted a six-fold increased risk of acute GVHD in a cross-institutional cohort. This scalable in silico platform shifts pre-transplant risk stratification from HLA typing and demographic surrogates to precision immune-receptor modeling.

immunology↗

bioRxiv2026

A novel model demonstrating that human immune cells promote multiorgan SARS-CoV-2 dissemination and human T cells limit anti-viral innate immunity

Despite extensive studies, many questions remain regarding the pathology and mechanisms behind Coronavirus disease 2019 (COVID-19), and particularly on post-acute sequelae of COVID-19 (PASC). Because existing mouse models cannot recapitulate human immune responses, we developed a human immune system (HIS) mouse model with physiologic expression of hACE2. After intranasal infection, persistent viral RNA was observed in multiple organs for 8 weeks, despite the generation of human SARS-CoV-2-specific T cell responses. Human immune cells increased viral infection in the lung and non-pulmonary tissues. COVID-19-related pathology was recapitulated in the lungs, with fibrosis peaking at 14 days post-infection. Immune activation was detected in the lungs, hearts, intestines and brains of acutely infected mice and persisted at 8 weeks in the heart and lungs. The presence of human T cells correlated with attenuated innate antiviral transcriptional signatures in the lung, where a persisting cytotoxic mature CD4+ T cell population with JAK-STAT activation was enriched in infected mice. Thus, human T cells mount an antigen-specific but ultimately dysfunctional response, while paradoxically suppressing the innate interferon response, permitting chronic interferon activation and long-term viral persistence, recapitulating several immunologic and histopathologic features associated with PASC. This model will uniquely facilitate understanding of virus-human immune dynamics and therapeutic approaches to PASC.

immunology↗

bioRxiv2026

A universal platform for simultaneous TCRα/β removal enables safer and more potent TCR therapies and autoimmune modeling

Adoptive T-cell therapies using tumour-specific T-cell receptors (TCRs) are limited by competition with endogenous receptors, which impairs efficacy and poses risks of off-target autoreactivity. Here we present a CRISPR-based platform that completely and selectively eliminates both endogenous TCR- and -{beta} chains without affecting introduced transgenic TCRs, irrespective of codon optimization. This approach achieves >90% deletion efficiency in Jurkat and primary human T cells, markedly enhancing the expression, pairing fidelity, and functional potency of transgenic receptors. Using a clinically relevant HLA-A*02:01-restricted DMF5 TCR, we show that dual TCR ablation boosts antigen-specific activation and cytotoxicity in vitro and significantly enhances tumor clearance in vivo in human immune system (HIS) mice, while preventing graft-versus-host disease (GVHD). Targeted locus amplification revealed that CRISPR-induced double-strand breaks did not alter lentiviral integration profiles, confirming genomic safety. Extending this approach to four insulin-reactive TCRs demonstrated that removal of endogenous receptors increased transduction efficiency and functional activity, with one (1E6) showing selective activation and infiltration of stem cell-derived islet grafts (SC-islets) in vivo. This study establishes a universal, safe, and scalable genome-editing platform for generating functionally precise human T cells. By integrating cancer immunotherapy and autoimmune disease modelling within a single framework, it provides a strong preclinical rationale for dual endogenous TCR removal as a route to improved specificity, safety, and therapeutic efficacy in TCR-based cell therapies.

immunology↗

bioRxiv2025

Coordinated circulating and tissue-based T cell responses precede xenograft rejection

Despite the life-saving successes of solid organ transplantation, the number of individuals needing organ transplant far exceeds the number of organs available for use each year. Porcine xenotransplantation, or the use of pig organs for transplantation in people, holds substantial promise but xenograft rejection in humans is poorly understood. T cell rejection by the host immune system is a major challenge for human allografts and may limit the longevity of porcine xenografts. To study the xenograft rejection, we evaluated T cell responses and repertoire dynamics across tissues following porcine thymokidney transplantation in a decedent model over 61 days after bilateral native kidney nephrectomy. Despite induction with anti-thymocyte globulin and ongoing immune suppression consisting of rituximab, corticosteroids, calcineurin inhibition, and mycophenolate mofetil, human T cell infiltration of the xenograft was observed and was associated with xenograft dysfunction. Longitudinal analysis of T cell clonotypes in biopsies of thymokidney revealed accumulation of clonal human CD4 and CD8 T cell responses. Moreover, circulating activated T cells, including circulating T follicular helper (cTfh), were xeno-reactive and increased in frequency around rejection events. We confirmed clonal dominance of a single CD8 clonotype - identified as donor-reactive in a mixed lymphocyte reaction - in the circulation leading up to the acute cellular rejection event. Following re-treatment with anti-thymocyte globulin and intensification of corticosteroids, the T cell clonotypes were dramatically diminished in frequency in thymokidney and lymph nodes, though not eliminated. Over time of observation, the T cell clonotypes were shared across multiple compartments, including xenograft, circulation and lymph nodes and formed clonal families with known xeno-reactive clonotypes, suggesting a coordinated immune response against a limited pool of antigenic targets. Together, these data demonstrate T cell repertoire dynamics across tissues in the setting of xenograft rejection and highlight opportunities for early surveillance, prediction and potential intervention. One-sentence summaryAfter pig-to-human kidney xenotransplantation, xenoreactive T cells form clonotypic families across blood, graft, and lymphoid tissues, indicating a coordinated and targeted response.

immunology↗

bioRxiv2025

Form Follows Function: A New Paradigm of Pancreatic Cancer Progression

Pancreatic ductal adenocarcinoma (PDAC) exhibits a distinctive propensity to invade nearby organs and infiltrate large blood vessels, even in the absence of distant metastasis. While the genetic and transcriptomic drivers of PDAC progression have been well studied, the mechanisms by which these molecular changes translate into functional, invasive behavior remain largely unknown. Here, we uncover a striking level of tissue organization, characterized by previously unrecognized spatial and geometric properties within and among tumor structures. Leveraging the first large-scale, AI-assisted, human-curated PDAC atlas from hematoxylin and eosin (H&E) images, we annotated, classified, and characterized 144,474 malignant and normal structures from treatment-naive (n=118) and neoadjuvant-treated PDAC patients (n=50). Additionally, we developed a new computational tool, SHAPE, to investigate PDAC aggressiveness through a comprehensive "geometrization" of cancer progression. Using traditional H&E-stained slides and three-dimensional (3D) tissue reconstruction experiments, we observed that invading tumor structures display an eccentric morphology with pronounced local angular coherence. These geometric and spatial properties revealed coherent architectural patterns, with invasive structures closely tracking vessels and nerves as they infiltrate surrounding tissue. Mechanistically, integration of morphological features from 39,045 annotated tumor structures with whole-genome and RNA sequencing data revealed that PDACs with numerous eccentric structures exhibit increased copy number alterations (CNAs), loss of heterozygosity (LoH) on the p-arm of chromosome 17, and a quasi-mesenchymal/basal-like molecular subtype. Spatial transcriptomic analysis of 1,650 tumor structures from six additional PDAC patients further confirmed upregulation of invasive cellular programs within highly eccentric structures, such as epithelial-to-mesenchymal transition (EMT), angiogenesis, coagulation, and complement pathways, underscoring their infiltrative nature. Finally, cross-validation of our AI-based method enabled a fully automated, highly interpretable computational approach to assist pathologists and clinicians in evaluating neoadjuvant chemotherapy response, predicting patient survival, and guiding chemotherapy in adjuvant settings. Collectively, these findings deepen our understanding of PDAC progression, identify a new hallmark of tumor architecture, and pave the way for full integration of AI-driven morphology-based approaches into clinical workflows to improve the management of PDAC patients.

cancer biology↗

bioRxiv2025

Spatiotemporal Single-Cell Analysis Reveals T Cell Clonal Dynamics and Phenotypic Plasticity in Human Graft-versus-Host Disease

Allogeneic hematopoietic cell transplantation (alloHCT) is curative for various hematologic diseases but often leads to acute graft-versus-host disease (GVHD), a potentially life-threatening complication. We leverage GVHD as a uniquely tractable disease model to dissect complex T-cell-mediated pathology in 27 alloHCT recipients. We integrate pre-transplant identification of alloreactive T-cells with longitudinal tracking across blood and gut, using mixed lymphocyte reaction-based clonal "fingerprinting", TCR clonotyping, single-cell RNA/TCR sequencing, and spatial transcriptomics. Using DecompTCR, a novel computational tool for longitudinal TCR analysis, we uncover clonal expansion programs linked to GVHD severity and TCR features. Multi-omics profiling of gut biopsies reveals enrichment and clonal expansion of CD8 effector and ZNF683(Hobit) resident memory T-cells, cytolytic remodeling of regulatory and unconventional T-cells, and localization of CD8 effector T-cells near intestinal stem cells in crypt loss regions. This framework defines dynamic immune circuit rewiring and phenotypic plasticity with implications for biomarkers and therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/655962v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@178b77dorg.highwire.dtl.DTLVardef@569226org.highwire.dtl.DTLVardef@1951281org.highwire.dtl.DTLVardef@1f1e046_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPersistent expansion of diverse alloreactive T cell clones is a hallmark of severe GVHD C_LIO_LIDecompTCR reveals dynamic clonal expansion programs linked to GVHD severity and clinical outcome C_LIO_LICD8+ T cell clones exhibit phenotypic plasticity in vivo across intestinal tissue compartments in GVHD C_LIO_LIHigh-resolution spatial profiling shows CD8+ effector T cells localize near intestinal stem cell niches and drive epithelial injury in GVHD C_LI

systems biology↗

bioRxiv2024

Follicular helper- and peripheral helper-like T cells drive autoimmune disease in human immune system mice

Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdcscid Il2rgtm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1+CD4+ peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh- like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. Tfh/Tph cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies and lymphopenia-induced proliferation (LIP) have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.

immunology↗

bioRxiv2023

The prospect of universal coronavirus immunity: a characterization of reciprocal and non-reciprocal T cell responses against SARS-CoV2 and common human coronaviruses

T cell immunity plays a central role in clinical outcomes of Coronavirus Infectious Disease 2019 (COVID-19). Therefore, T cell-focused vaccination or cellular immunotherapy might provide enhanced protection for immunocompromised patients. Pre-existing T cell memory recognizing SARS-CoV2 antigens antedating COVID-19 infection or vaccination, may have developed as an imprint of prior infections with endemic non-SARS human coronaviruses (hCoVs) OC43, HKU1, 229E, NL63, pathogens of "common cold". In turn, SARS-CoV2-primed T cells may recognize emerging variants or other hCoV viruses and modulate the course of subsequent hCoV infections. Cross-immunity between hCoVs and SARS-CoV2 has not been well characterized. Here, we systematically investigated T cell responses against the immunodominant SARS-CoV2 spike, nucleocapsid and membrane proteins and corresponding antigens from - and {beta}-hCoVs among vaccinated, convalescent, and unexposed subjects. Broad T cell immunity against all tested SARS-CoV2 antigens emerged in COVID-19 survivors. In convalescent and in vaccinated individuals, SARS-CoV2 spike-specific T cells reliably recognized most SARS-CoV2 variants, however cross-reactivity against the omicron variant was reduced by approximately 50%. Responses against spike, nucleocapsid and membrane antigens from endemic hCoVs were more extensive in COVID-19 survivors than in unexposed subjects and displayed cross-reactivity between - and {beta}-hCoVs. In some, non-SARS hCoV-specific T cells demonstrated a prominent non-reciprocal cross-reactivity with SARS-CoV2 antigens, whereas a distinct anti-SARS-CoV2 immunological repertoire emerged post-COVID-19, with relatively limited cross-recognition of non-SARS hCoVs. Based on this cross-reactivity pattern, we established a strategy for in-vitro expansion of universal anti-hCoV T cells for adoptive immunotherapy. Overall, these results have implications for the future design of universal vaccines and cell-based immune therapies against SARS- and non-SARS-CoVs.

immunology↗

bioRxiv2021

CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals

Transgenic CD47 overexpression is an encouraging approach to ameliorating xenograft rejection and alloresponses to pluripotent stem cells, and the efficacy correlates with the level of CD47 expression. However, CD47, upon ligation, also transmits signals leading to cell dysfunction or death, raising a concern that overexpressing CD47 could be harmful. Here, we unveiled an alternative source of cell surface CD47. We showed that extracellular vesicles (EVs), including exosomes (Exos), released from normal or tumor cells overexpressing CD47 (transgenic or native) can induce efficient CD47 cross-dressing on pig or human cells. Like the autogenous CD47, CD47 cross-dressed on cell surfaces is capable of interacting with SIRP to inhibit phagocytosis. However, ligation of the autogenous, but not cross-dressed, CD47 induced cell death. Thus, CD47 cross-dressing provides an alternative source of cell surface CD47 that may elicit its anti-phagocytic function without transmitting harmful signals to the cells. CD47 cross-dressing also suggests a previously unidentified mechanism for tumor-induced immunosuppression. Our findings should help to further optimize the CD47 transgenic approach that may improve outcomes by minimizing the harmful effects of CD47 overexpression.

immunology↗

bioRxiv2021

High-Yield Monocyte, Macrophage, and Dendritic Cell Differentiation from Induced Pluripotent Stem Cells

Differentiation of induced pluripotent stem cells (iPSC) into monocytes, monocyte-derived macrophages (MDM), and monocyte-derived dendritic cells (moDC) represents a powerful tool for studying human innate immunology and developing novel iPSC-derived immune therapies. Challenges include inefficiencies in iPSC-derived cell cultures, labor-intensive culture conditions, low purity of desired cell types, and feeder cell requirements. Here, a highly efficient method for differentiating monocytes, MDMs, and moDCs that overcomes these challenges is described. The process utilizes commercially-available materials to derive CD34+ progenitor cells that are apically released from a hemogenic endothelium. Subsequently, the hemogenic endothelium gives rise to highly pure (>95%), CD34-CD14+ monocytes in 19-23 days and yields 13.5-fold more monocytes by day 35 when compared to previous methods. These iPSC-monocytes are analogous to human blood-derived monocytes and readily differentiate into MDM and moDC. The efficient workflow and increase in monocyte output heightens feasibility for high throughput studies and enables clinical-scale iPSC-derived manufacturing processes.

immunology↗

bioRxiv2021

Interleukin-15 Receptor α Chain Knockout NOD-SCID mice have reduced numbers of NK cells, develop thymic lymphomas and fail to engraft human hematopoietic cells

NOD-SCID IL2r{gamma}cnull (NSG) mice are widely used to model human immune cell development because they are more permissive for human hematopoietic cell engraftment and reconstitution than NOD-SCID mice. While increased human reconstitution in the blood of NSG mice has been attributed to the absence of mouse NK cells, deletion of the common gamma chain ({gamma}c) limits development of lymphoid tissue inducer cells and precludes development of normal secondary lymphoid structures. The disorganized lymphoid tissue leads to compromised human T cell-B cell interactions and results in variable human immune cell function in human immune system (HIS) NSG compared to NOD-SCID mice. We attempted to remove mouse NK cells from NOD-SCID mice while retaining other {gamma}c-dependent cytokine responses by targeted disruption of the mouse genomic IL15RA locus with CRISPR/Cas9. IL15R is required for the development, function and survival of NK cells. NOD-SCID IL15R-/- mice showed reductions in NK cells and NK cell function. However, NOD-SCID IL15R-/- mice demonstrated accelerated thymic lymphomagenesis and showed earlier mortality compared to NOD-SCID mice. This result suggests that mouse NK cells are important to delay lymphoma development in NOD-SCID mice. We transplanted thymectomized NOD-SCID ILl5R-/- mice with human fetal liver CD34+ cells and thymus to determine if these mice supported engraftment and development of a transplanted HIS. Surprisingly, we found that peripheral human engraftment was inferior (mean 0.05% of lymphocytes) to that in both NOD-SCID (mean, 10.5% of lymphocytes) and NSG (mean, 54% of lymphocytes) mice. These results indicate that NOD-SCID IL15R-/- mice are not permissive for human CD34+ cell engraftment.

immunology↗