Biology subjects

Sunshine, S.

Publications and source records attributed to Sunshine, S..

10 recordsLinked to original sources

bioRxiv2026

Coordinated immune architecture underlies durable survival in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterised by profound immune dysfunction and limited response to immunotherapy. Although tertiary lymphoid structures (TLSs) are associated with improved outcomes, most studies focus on their presence or density, providing limited insight into how their organisation contributes to tumour control. Here, we analyse a unique cohort of treatment-naive PDAC patients enriched for exceptionally rare long-term survivors (LTS, n = 23; >5-year survival) alongside more common short-term survivors (STS, n = 24), enabling direct interrogation of mechanisms underlying durable tumour control. We develop a multi-scale spatial framework integrating multiplex imaging, computational modelling, and transcriptomics to quantify TLS architecture. By modelling TLSs as higher-order immune assemblies, we define their structural states, spatial organisation, assembly rules and tissue context-dependency within the tumour microenvironment. We show that long-term survival is associated with organised, spatially integrated TLSs exhibiting coordinated B and T cell zoning, whereas short-term survival is characterised by disorganised, Treg-enriched, and spatially isolated TLSs, despite similar immune cell abundance. These architectural differences align with transcriptional programmes: LTS tumours display coordinated lymphoid and NF-{kappa}B-driven chemokine signalling, while STS tumours exhibit inflammation uncoupled from immune organisation. Together, these findings demonstrate that durable anti-tumour immunity in PDAC is defined by coordinated transcriptional, cellular, and spatial organisation, rather than immune presence alone. This study provides a blueprint for structure-informed strategies to reprogram the tumour microenvironment and improve outcomes in PDAC. Statement of significanceIntegrating multiplex imaging, computational modelling and transcriptomics in treatment-naive PDAC enriched for exceptional survivors shows that durable tumour control is associated with organised TLS architecture rather than immune abundance alone, providing a framework for immune-architecture-based biomarkers and therapeutic reprogramming.

cancer biology↗

bioRxiv2025

Defining the host dependencies and the transcriptional landscape of RSV infection and bystander activation

Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life [1]. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells, and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV infected and bystander activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing. ImportanceRespiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.

microbiology↗

bioRxiv2024

Diminished placental Factor XIIIA1 expression associates with long term ART and preterm birth in pregnant people living with HIV.

We previously showed a link between maternal vascular malperfusion and pre-term birth (PTB) in pregnant people living with HIV (PPLH) initiating antiretroviral treatment (ART) before pregnancy, indicating poor placental vascularisation. After measuring antenatal plasma angiogenic factors to seek mechanistic insights, low levels of plasma Factor XIIIA1 (FXIIIA1) and vascular-endothelial-growth-factor (VEGF) was significantly associated with PTB at the time closest to delivery (median 34 weeks) in PPLH initiating ART before pregnancy. Knowing that FXIIIA1 is crucial for haemostasis, angiogenesis, implantation and pregnancy maintenance and that expression is found on placental macrophages (Hofbauer cells), we examined placentae at delivery from matching participants who either initiating ART before pregnancy or during gestation. Highest FXIIIA1 expression was on Hofbauer cells but was significantly lower in PTB regardless of HIV infection, but was significantly lower in PPLH in PTB from women who initiated ART before pregnancy. To test the hypothesis that antiretroviral drugs may disrupt vascularisation in the placenta, we used a human umbilical vein endothelial cell (HUVEC) matrigel angiogenesis assay. We identified that addition of pre-treated FXIIIA1-expressing MCSF-and IL-10-induced placenta-like macrophages with physiological concentrations of tenofovir, 3TC, and efavirenz resulted in significantly inhibited angiogenesis; akin to the inhibition observed with titratable concentrations of ZED1301, an inhibitor of FXIIIA1. Overall, an efavirenz-containing ART combination inhibits vasculogenesis without causing toxicity and likely does so through inhibition of a FXIIIA1-mediated-placental macrophage pathway.

immunology↗

bioRxiv2024

How Mycobacterium tuberculosis builds a home: Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs.

Mycobacterium tuberculosis (MTB) infects and replicates in lung mononuclear phagocytes (MNPs) with astounding ability to evade elimination. ESX-1, a type VII secretion system, acts as a virulence determinant that contributes to MTBs ability to survive within MNPs, but its effect on MNP recruitment and/or differentiation remains unknown. Here, using single-cell RNA sequencing, we studied the role of ESX-1 in MNP heterogeneity and response in mice and murine bone marrow-derived macrophages (BMDM). We found that ESX-1 is required for MTB to recruit diverse MNP subsets with high MTB burden. Further, MTB induces an anti-inflammatory signature in MNPs and BMDM in an ESX-1 dependent manner. Similarly, spatial transcriptomics revealed an upregulation of anti-inflammatory signals in MTB lesions, where monocyte-derived macrophages concentrate near MTB-infected cells. Together, our findings suggest that MTB ESX-1 mediates the recruitment and differentiation of anti-inflammatory MNPs, which MTB can infect and manipulate for survival.

microbiology↗

bioRxiv2023

Global organelle profiling reveals subcellular localization and remodeling at proteome scale

Defining the subcellular distribution of all human proteins and its remodeling across cellular states remains a central goal in cell biology. Here, we present a high-resolution strategy to map subcellular organization using organelle immuno-capture coupled to mass spectrometry. We apply this proteomics workflow to a cell-wide collection of membranous and membrane-less compartments. A graph-based representation of our data reveals the subcellular localization of over 7,600 proteins, defines spatial protein networks, and uncovers interconnections between cellular compartments. We demonstrate that our approach can be deployed to comprehensively profile proteome remodeling during cellular perturbation. By characterizing the cellular landscape following hCoV-OC43 viral infection, we discover that many proteins are regulated by changes in their spatial distribution rather than by changes in their total abundance. Our results establish that proteome-wide analysis of subcellular remodeling provides essential insights for the elucidation of cellular responses. Our dataset can be explored at organelles.czbiohub.org.

cell biology↗

bioRxiv2023

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and MT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

immunology↗

bioRxiv2022

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-{kappa}B inhibitor I{kappa}B (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.

systems biology↗

bioRxiv2022

Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus

Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remain incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses, in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including a N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues, and highlights the importance of CD164, a sialomucin that has previously not been associated with viral infection.

microbiology↗

bioRxiv2020

Persistence of ambigrammatic narnaviruses requires translation of the reverse open reading frame

Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods and nematodes. Though initially described as simple single-gene non-segmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as "ambigrammatic" harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2 kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8 kb "Robin" RNA. We developed a system to launch CxNV1 in a naive mosquito cell line, then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively-translating ribosomes on host mRNA or co-infecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission. IMPORTANCEFundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome, relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses, and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.

microbiology↗

bioRxiv2020

Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by PhIP-Seq

The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from an APS1 cohort and discovered multiple common antibody targets. These novel autoantigens exhibit tissue-restricted expression, including expression in enteroendocrine cells and dental enamel. Using detailed clinical phenotyping, we find novel associations between autoantibodies and organ-restricted autoimmunity, including between anti-KHDC3L autoantibodies and premature ovarian insufficiency, and between anti-RFX6 autoantibodies and diarrheal-type intestinal dysfunction. Our study highlights the utility of PhIP-Seq for interrogating antigenic repertoires in human autoimmunity and the importance of antigen discovery for improved understanding of disease mechanisms.

immunology↗