Biology subjects

Green, E. W.

Publications and source records attributed to Green, E. W..

10 recordsLinked to original sources

bioRxiv2026

Tissue resident CD4+ memory T-cells mark response to immune checkpoint inhibition in high-grade glioma

Background: Immune checkpoint inhibitors (ICI) are efficacious in many solid tumors, but response in glioma is restricted to a small subgroup. The determinants of response and resistance to ICI remain poorly understood. Methods: Here we exploit a syngeneic hypermutated high-grade glioma model with dichotomous response to combined PD-1 and CTLA-4 inhibition to unravel determinants of tumor-infiltrating T-cells driving response. Tumor-infiltrating T-cells from ICI-responsive and non-responsive tumors were analyzed by single-cell RNA and T-cell receptor sequencing and tumor-reactive T-cell receptor clonotypes were functionally validated to characterize their transcriptional phenotypes. We verify our findings in IDH1 wildtype glioblastoma patients treated with neoadjuvant pembrolizumab. Results: ICI response was associated with intratumoral clonal expansion of tumor-reactive cytotoxic T-cells and increased infiltration of CXCR6+ CD4+ tissue resident memory T-cells (Trm). CD4 stem-like memory T-cells in responding tumors demonstrated elevated interferon responses, following trajectories toward clonally expanded Trm, versus trajectories toward exhaustion in non-responsive tumors. In responsive tumors, CD4+ Trm interacted with infiltrating CXCR3+ tumor-reactive and clonally expanded, yet transcriptionally versatile cytotoxic T-cells. Probing the post neoadjuvant ICI high-grade glioma patient tissue dataset, we confirmed increased CXCR6 expression in CD4+ T cells and the association of CD4+ Trm with prolonged overall survival. Conclusion: These findings identify CD4 tissue-resident memory T-cells as determinants of ICI response in IDH1 wildtype high-grade glioma and warrant their further investigation to improve immunotherapy outcomes.

cancer biology↗

bioRxiv2026

Functional Deorphanization of Tumor-reactive TCRs Uncovers a Landscape of Shared, Immune Dominant Antigens across Cancers

Single-cell sequencing has charted the clonal landscape of tumor-infiltrating T cells, but the antigens that drive these responses remain largely undefined, limiting rational immunotherapy design. Here, we introduce TWISTAR, a platform that systematically deorphanizes single-cell-derived TCRs against tumor transcriptomes. By directly validating TCR engagement, TWISTAR exclusively recovers functionally immunogenic antigens. Applying TWISTAR to melanoma, breast, and hepatocellular carcinoma patients responding to immune checkpoint blockade, we uncover that tumor-reactive TCRs recognize a broad repertoire of immunogenic targets beyond neoantigens, including peptides derived from unannotated coding sequences and novel transcripts. Many of these antigens are shared across patients and cancer types, revealing communal targets. Furthermore, we observe immune dominance, consisting of multiple clones converging on few immune dominant epitopes. Together, these findings reveal an unexpectedly broad landscape of tumor immunogenicity, expanding opportunities for the development of pan-cancer vaccines.

cancer biology↗

bioRxiv2026

A Modular Chromosomally Integrated Yeast Surface Display Platform for Quantitative Serology and Immune Profiling

Precise quantification of antigen-specific antibodies is essential for assessing immune status and fundamental to both clinical and research settings. However, current serological platforms remain limited in their ability to combine quantitative performance, flexibility, and scalability. Here, we present a chromosomally integrated yeast surface display (YSD) system engineered for stable, quantitative, and scalable antibody profiling. Genomic integration of the YSD cassette supports uniform antigen presentation without continuous selective pressure, reducing variability associated with plasmid-based systems. Two distinct cell-wall anchoring strategies are implemented within a modular synthetic framework that enables rapid, cloning-free strain construction. Coupled with flow-cytometric readout, the system provides sensitive detection of antigen-specific antibodies with single-cell normalization for antigen expression. In addition, the combination of barcoded YSD strain libraries with fluorescence-activated cell sorting and next-generation sequencing enables multiplexed serological profiling across multiple samples and antigens. As a proof of concept, we demonstrate quantitative antibody detection against SARS-CoV-2 and extend the approach to hepatitis virus antigens, highlighting the utility of this platform as a flexible framework for serological assay development and immune profiling.

bioengineering↗

bioRxiv2025

Conserved programs and specificities of T cells targeting hematological malignancies

T cell-mediated immune surveillance is critical for cancer control, yet its endogenous effectiveness in hematological malignancies remains limited and poorly understood. Here, we integrate single-cell T cell receptor (TCR) profiling, HLA immunopeptidomics and functional antigen mapping to dissect the specificity landscape of bone marrow lymphocytes (BMLs) in multiple myeloma (MM) and acute myeloid leukemia (AML). We identify a rare subset of tumor-reactive T cells that exhibit a stereotyped transcriptional state distinct from bystander and virus-specific populations. Across both malignancies, immunopeptidomic profiling uncovers a partially conserved antigen repertoire enriched for noncanonical peptides, including products of novel or unannotated open reading frames (nuORFs), pseudogenes, and clonotypic immunoglobulin sequences. Several of these epitopes are recurrently presented and associated with convergent TCR responses across individuals. Based on this immune architecture, we develop a TCR-intrinsic fitness model that infers BML tumor specificity from transcriptional cues and stratifies immunotherapy response across three independent patient cohorts. Together, these findings map the latent potential of endogenous anti-tumor immunity in two biologically distinct diseases and provide a framework for decoding and restoring productive immune surveillance of hematological malignancies. HighlightsO_LISingle-cell resolved TCR profiling maps rare tumor-reactive T cells in the bone marrow of multiple myeloma (MM) and acute myeloid leukemia (AML) reveals conserved transcriptional programs C_LIO_LIA shared immunopeptidome across MM and AML includes noncanonical epitopes from nuORFs and idiotype sequences C_LIO_LIConserved tumor antigens elicit convergent T cell responses across patients C_LI O_LIA TCR fitness model predicts tumor specificity in bone marrow lymphocytes and stratifies immunotherapy response in both hematological malignancies C_LI

immunology↗

bioRxiv2025

makeTCR: a Flexible Platform for Rapid, Scalable, Single-Step T Cell Receptor Synthesis

Personalised cell therapies utilising T cell receptors (TCRs) show tremendous clinical promise, though TCR synthesis and validation techniques lag behind our ability to sequence TCR repertoires. To address this gap, we developed makeTCR: a flexible modular, scalable TCR cloning system that enables single-step, 100% fidelity assembly of {beta} and {gamma}{delta} TCRs into commonly used expression vectors. By implementing cell-free manufacturing, makeTCR enables patient-derived TCRs to be functionally validated within 48 hours. We provide an open-source, easily extensible, web-based graphical platform that integrates existing tools to simplify and standardise the manufacture of TCRs across all scales of synthesis.

immunology↗

bioRxiv2023

T-FINDER: A highly sensitive, pan-HLA platform for functional T cell receptor and ligand discovery

Effective, unbiased, high-throughput methods to functionally identify both class II and class I HLA-presented T cell epitopes and their cognate T cell receptors (TCRs) are essential for and prerequisite to diagnostic and therapeutic applications, yet remain underdeveloped. Addressing this bottleneck, we established T-FINDER (T cell Functional Identification and (Neo)-antigen Discovery of Epitopes and Receptors), a platform that rapidly deconvolutes CD4 and CD8 TCR reactivities to targets physiologically processed and presented by an individuals unmanipulated, complete HLA haplotype. By using a highly sensitive TCR signaling reporter capable of detecting even low-affinity TCR:ligand interactions, T-FINDER not only robustly identifies unknown peptide:HLA ligands from complex antigen libraries, but also rapidly screens and functionally validates the specificity of complex TCR libraries against known or predicted targets. To demonstrate its pan-HLA presentation capacity, we apply the platform to multiple TCR-based applications, including glioma, celiac disease, and rheumatoid arthritis, providing unique biological insights and showcasing T-FINDERs potency and versatility.

immunology↗

bioRxiv2023

Neoepitope-specific vaccination of a patient with diffuse midline glioma targeting H3K27M induces polyclonal B and T cell responses across diverse HLA alleles

H3K27M, a driver mutation with T- and B-cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient who was treated with an H3K27M peptide vaccine and subsequently entered complete remission. The vaccine robustly expanded class II HLA-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their CDR3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ, and -DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.

cancer biology↗

bioRxiv2023

ARDitox: platform for the prediction of TCRs potential off-target binding

Cellular immunotherapies, such as those utilizing T lymphocytes expressing native or engineered T cell receptors (TCRs), have already demonstrated therapeutic efficacy. However, some high-affinity TCRs have also proved to be fatal due to off-target immunotoxicity. This process occurs when the immune system acts against epitopes found on both tumor cells and healthy tissues. Moreover, some TCRs can be cross-reactive to epitopes with highly dissimilar sequences. To address this issue, we developed ARDitox, a novel in silico method based on computational immunology and artificial intelligence (AI) for predicting and analyzing potential off-target binding. We tested the performance of ARDitox in silico on different cases found in the literature where TCRs were used to target cancer-related antigens, as well as on a set of TCRs targeting a viral epitope. ARDitox was able to identify previously reported cross-reactive epitopes in line with the data available in the literature. In addition, we investigated a TCR targeting an HLA-A*02:01-restricted immunodominant epitope from the glioblastoma-associated antigen NLGN4X, identifying a cross-reactive ADH1A epitope that would not be detected in murine models. In conclusion, our in silico approach is a powerful tool that identifies potential off-target epitopes, complementing preclinical studies in developing safer cell therapies targeting tumor(- associated) antigens.

immunology↗

bioRxiv2022

The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal ([~]12.4 hour) or other lunar-mediated cycles ([~]15 day, [~]29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between the positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2. Epcry2 and Epbmal1 knockdown disrupted Eurydices circadian phenotypes as expected but in addition, circatidal behaviour was also sensitive to Epbmal1 knockdown. Thus three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM, do not appear to be required for the expression of circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators BMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.

genetics↗

bioRxiv2020

EGFR transactivates RON to drive oncogenic crosstalk

SUMMARYCrosstalk between disparate membrane receptors is thought to drive oncogenic signaling and allow for therapeutic resistance. EGFR and RON are members of two unique receptor tyrosine kinase (RTK) subfamilies that engage in crosstalk through unknown mechanisms. We combined high resolution imaging with biochemical studies and structural mutants to understand how EGFR and RON communicate. We found that EGF stimulation results in EGFR-dependent RON phosphorylation. Crosstalk is unidirectional, since MSP stimulation of RON does not trigger EGFR phosphorylation. Two-color single particle tracking captured the formation of complexes between RON and EGFR, supporting a role for direct interactions in propagating crosstalk. We further show that RON is a substrate for EGFR kinase, and transactivation of RON requires the formation of a signaling competent EGFR dimer. These results identify critical structural features of EGFR/RON crosstalk and provide new mechanistic insights into therapeutic resistance.

cancer biology↗