Biology subjects

Smith, K. N.

Publications and source records attributed to Smith, K. N..

12 recordsLinked to original sources

bioRxiv2026

replicateFest: An R Package and Shiny App for Analysis of T Cell Receptor Repertoire Data from the Functional Expansion

MotivationThe Functional Expansion of Specific T cell (FEST)-based assays combine short-term peptide stimulation with TCR sequencing to identify clonotypes that expand in response to specific antigens. These approaches have proven invaluable for detecting neoantigen-specific T cell responses, guiding vaccine development, and assessing checkpoint blockade efficacy. However, variability introduced by biological and technical replicates poses challenges for reproducibility and interpretation, and existing computational tools do not address replicate-level analysis in these assays. ResultsWe developed replicateFest, a computational framework implemented as an R package and Shiny web application, to analyze FEST-based TCR-seq data with and without replicates. replicateFest applies Fishers exact test for non-replicate datasets and negative binomial modeling for replicate experiments, returning adjusted p-values and odds ratios to identify clonotypes significantly expanded in antigen-stimulated conditions. The framework distinguishes FEST-expanded clonotypes (relative to a no-antigen control) and FEST-positive clonotypes (expanded compared to all other conditions). Validation using synthetic datasets confirmed accurate detection of antigen-specific clonotypes. Application to published HIV-1 epitope stimulation data reproduced original findings and demonstrated replicateFests utility for reproducibility assessment and quality control. Availability and ImplementationreplicateFest is freely available under the Apache-2.0 license as an R package at https://github.com/OncologyQS/replicateFest and as an interactive Shiny application at http://www.stat-apps.onc.jhmi.edu/FEST/.

immunology↗

bioRxiv2026

Tissue Injury and Biomaterial Treatment Modulate Tumor Growth and Response to Immunotherapy

Immunotherapies have transformed cancer care; however, tumor intrinsic and extrinsic factors contribute to high variability in therapeutic responses. While tissue injuries can impact cancer recurrence and metastatic spread, little is known about their potential to effect immune checkpoint blockade (ICB) response. In this study, we reveal that distal traumatic muscle injury accelerated progression and impaired adjuvant ICB response of multiple murine tumors. This injury-induced accelerated tumor growth coincided with decreased intra-tumoral density and effector phenotype of tumor-reactive CD8+ T cells and relied on communication through a shared draining lymph node. Enhancing injury repair using a biological scaffold abrogated the injury-induced accelerated tumor growth in an interleukin-4-dependent manner and improved ICB response. In a retrospective cohort analysis of breast cancer patients undergoing ICB treatment, biological scaffold implantation following mastectomy was associated with increased overall survival. This work suggests that injury-driven immune dysfunction may contribute to cancer progression and ICB resistance, but enhancing wound healing with pro-regenerative biomaterials may offer a viable strategy for mitigating adverse cancer outcomes, particularly in the setting of adjuvant and neoadjuvant ICB.

cancer biology↗

bioRxiv2025

Synergistic role of riboflavin-auxotrophic Enterococcus for MR1 expression and intra-tumoral mucosal associated invariant T (MAIT) cell activation

The role of mucosal invariant T cells (MAITs), in the lung tumor microenvironment re-mains poorly understood, especially in the setting of immune checkpoint inhibitors. We identified intratumoral MAIT cells from paired single cell RNA and TCR sequencing datasets of tumor infil-trating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadju-vant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associ-ated TCR clonotypes predicted to recognize intratumoral bacteria, which we then tested for func-tional recognition using a MAIT TCR capture functional assay. Strikingly, although not directly recognized by MAIT cells, previously identified probiotic Enterococcus spp and detected in the intratumoral microbiome of lung cancer patients, selectively synergized with exogenous riboflavin biosynthesis-derived metabolites to induce expression of MR1 by antigen presenting cells, includ-ing dendritic cells, B cells and mononuclear phagocytes. Boosting of MR1 cell surface expression resulted from perturbation of endo-lysosomal vacuolar pathway by Enterococcus and recycling of early endosomal MR1 to the cytoplasmic membrane. Riboflavin auxotrophic Enterococcus spp may therefore exercise their beneficial immunomodulatory functions upon immune checkpoint blockade treatment, at least in part, by promoting intratumoral MR1 expression and innate like T cell activation. Our results indicate that composition of the intratumoral microbiome during im-mune checkpoint inhibitor treatment has the potential to impact the function of human intratumoral MAIT cells.

immunology↗

bioRxiv2025

Dual checkpoint blockade of glioblastoma with Anti-PD-1 and Anti-LAG-3 promotes expansion of tumor-reactive T cell clones along a unique pathway of differentiation

IDH-wildtype grade IV glioblastoma is the most aggressive adult primary brain tumor and remains refractory to anti-PD-1 monotherapy despite evidence of limited tumor-specific T cell induction. To determine the impact of immune checkpoint inhibitors (ICIs) on glioblastoma T cell transcriptional landscape and repertoire, we conducted paired single-cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (TCR-seq) of tumor-infiltrating lymphocytes (TILs) from patients with untreated, newly diagnosed glioblastoma and from recurrent glioblastoma treated with dual checkpoint blockade targeting PD-1 and LAG-3. Using a validated transcriptional signature, we found that predicted tumor-reactive T cells (TRC) in untreated glioblastomas reside almost exclusively in a clonally expanded GZMKhi population with developmental plasticity, affording them the potential to differentiate into both tissue-resident and terminal effector T cells. Dual ICI therapy induced substantial clonal remodeling, characterized by the recruitment of new TRC from the periphery into the tumor microenvironment (TME) and differentiation into transitional effectors and ultimately terminal effectors along a gradient characterized by simultaneous acquisition of cytotoxic and exhaustion genes, regulated by specific transcriptional, metabolic, and epigenetic programs. Longitudinal clonal tracking in peripheral blood confirmed that with ICI treatment, most TRC expand transiently in circulation prior to tumor infiltration, with peripherally derived clones becoming the major contributor to the GZMKhi TRC that further expand in the tumor. Our study provides the first comprehensive map of T cell clonal dynamics and differentiation in glioblastoma following dual ICIs and highlights a potential mechanism of immune activation and peripheral recruitment of TRC in glioblastoma not previously described. Our results suggest that therapeutic strategies to sustain these GZMKhi early effector and transitional effector T cells may further enhance ICI therapeutic efficacy in glioblastoma.

immunology↗

bioRxiv2025

Novel Predictive Spatial Biomarker in Non-Small Cell Lung Carcinoma: The Diversity of Niches Unlocking Treatment Sensitivity (DONUTS)

Probabilistic spatial modelling techniques developed on large-scale tumor-immune Atlases ([~]35M individually mapped cells; 50,000 high power fields) were used to characterize predictive features of treatment-responsive lung cancer. We identified CD8+FoxP3+ cell density as a robust pre-treatment biomarker for outcomes across disease stages and therapy types. In parallel, single-cell RNAseq studies of CD8+FoxP3+ T-cells revealed an activated, early effector phenotype, substantiating an anti-tumor role, and contrasting with CD4+FoxP3+ T-regulatory cells. A spatial biomarker was developed using an empirical probabilistic model to define the immediate cell neighbors or niche surrounding CD8+FoxP3+ cells and proximity to the tumor-stromal boundary. The resultant Diversity of Niches Unlocking Treatment Sensitivity (DONUTS) are more prevalent than the CD8+FoxP3+ cells themselves, mitigating sampling error in small biopsies. Further, the DONUTS only require four markers, are additive to PD-L1, and associate with tertiary lymphoid structure counts. Taken together, the DONUTS represent a next-generation predictive biomarker poised for clinical implementation. HIGHLIGHTSO_LILarge-scale tumor-immune Atlases drive robust computational biomarker development C_LIO_LICD8+FoxP3+ cells are anti-tumor T-cells and predict response to therapy C_LIO_LIThe niches or spatial donuts around CD8+FoxP3+ cells boost biomarker performance C_LIO_LICD8+FoxP3+ donuts are hallmarks of a larger immune organization that includes TLS C_LI

pathology↗

bioRxiv2025

Longitudinal T cell repertoire analysis reveals dynamic clonal T cell populations in Ulcerative Colitis

BackgroundUlcerative Colitis (UC) is characterized by chronic, relapsing and remitting inflammation in the colon and rectum. Pathogenic T cell activity is thought to play a major role in this process. T cell effector function is determined by the T cell receptor (TCR) and the antigen it recognizes. Examining the TCR repertoire can provide key insights into the adaptive immune response. ObjectiveTo characterize the longitudinal TCR repertoire of patients with UC across disease activity to determine if recurrent antigen(s) are responsible for active inflammation. DesignBulk TCR V{beta} sequencing was done on colon tissue of 20 patients with UC across multiple time points of disease. Corresponding clinical metadata was also obtained over the same time period for each patient to map their clinical disease course. The top ten most highly abundant clones from each time point were longitudinally tracked and correlated with disease phenotype. ResultsSeventy-five percent of patients did not have overlapping abundant TCR clones across multiple time points of disease. The remaining 25% of patients had one to five TCR clones present in high abundance in their tissue during every time point analyzed. ConclusionThese results demonstrate that most patients with UC do not share a similar TCR repertoire over time, indicating that times of inflammation are associated with unique antigen exposures. A smaller group of patients have persistent, private TCR clones with high abundance, 60% of whom had more unremitting, active disease.

immunology↗

bioRxiv2023

Structural basis for VLDLR recognition by eastern equine encephalitis virus

Alphaviruses are arthropod-borne enveloped RNA viruses that include several important human pathogens with outbreak potential. Among them, eastern equine encephalitis virus (EEEV) is the most virulent, and many survivors develop neurological sequelae, including paralysis and intellectual disability. The spike proteins of alphaviruses comprise trimers of heterodimers of their envelope glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), two closely related proteins that are expressed in the brain, as cellular receptors for EEEV and a distantly related alphavirus, Semliki forest virus (SFV)1. The EEEV and SFV spike glycoproteins have low sequence homology, and how they have evolved to bind the same cellular receptors is unknown. Here, we used single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain. The structures reveal that EEEV and SFV use distinct surfaces to bind VLDLR; EEEV uses a cluster of basic residues on the E2 subunit of its spike glycoprotein, while SFV uses two basic residues at a remote site on its E1 glycoprotein. Our studies reveal that different alphaviruses interact with the same cellular receptor through divergent binding modes. They further suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

microbiology↗

bioRxiv2022

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapyinduced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, in particular LAG3. Functionally, the OX40hiGITRhi subset in the most highly suppressive ex vivo and Treg expression of OX40, GITR and LAG3, correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFN{gamma} and certain pro-inflammatory granzymes. Application of a gene score from the murine TAA-specific Th1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1 responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses. One-Sentence SummaryWe define 10 subsets of lung cancer-infiltrating regulatory T cells, one of which is highly suppressive and enriched in anti-PD-1 non-responders and the other is Th1-like and is enriched in PD-1 responders.

immunology↗

bioRxiv2022

Deep Neural Networks Predict MHC-I Epitope Presentation and Transfer Learn Neoepitope Immunogenicity

Identifying neoepitopes that elicit an adaptive immune response is a major bottleneck to developing personalized cancer vaccines. Experimental validation of candidate neoepitopes is extremely resource intensive, and the vast majority of candidates are non-immunogenic, making their identification a needle-in-a-haystack problem. To address this challenge, we present computational methods for predicting MHC-I epitopes and identifying immunogenic neoepitopes with improved precision. The BigMHC method comprises an ensemble of seven pan-allelic deep neural networks trained on peptide-MHC eluted ligand data from mass spectrometry assays and transfer learned on data from assays of antigen-specific immune response. Compared with four state-of-the-art classifiers, BigMHC significantly improves the prediction of epitope presentation on a test set of 45,409 MHC ligands among 900,592 random negatives (AUROC=0.9733, AUPRC=0.8779). After transfer learning on immunogenicity data, BigMHC yields significantly higher precision than seven state-of-the-art models in identifying immunogenic neoepitopes, making BigMHC effective in clinical settings. All data and code are freely available at https://github.com/KarchinLab/bigmhc.

bioinformatics↗

bioRxiv2021

RBBP4 modulates gene activity through acetylation and methylation of histone H3 lysine 27

RBBP4 is a core subunit of polycomb repressive complex 2 (PRC2) and HDAC1/2-containing complexes, which are responsible for histone H3 lysine 27 (H3K27) methylation and deacetylation respectively. However, the mechanisms by which RBBP4 modulates the functions of these complexes remain largely unknown. We generated viable mouse embryonic stem cell lines with RBBP4 mutations that disturbed methylation and acetylation of H3K27 on target chromatin and found that RBBP4 is required for PRC2 assembly and H3K27me3 establishment on target chromatin. Moreover, in the absence of EED and SUZ12, RBBP4 maintained chromatin binding on PRC2 loci, suggesting that the pre-existence of RBBP4 on nucleosomes serves to recruit PRC2 to restore H3K27me3 on newly synthesized histones. As such, disruption of RBBP4 function led to dramatic changes in transcriptional profiles. In spite of the PRC2 association, we found that transcriptional changes were more closely tied to the deregulation of H3K27ac rather than H3K27me3 where increased levels of H3K27ac were found on numerous cis-regulatory elements, especially putative enhancers. These data suggest that RBBP4 controls acetylation levels by adjusting the activity of HDAC complexes. As histone methylation and acetylation have been implicated in cancer and neural disease, RBBP4 could serve as a potential target for disease treatment.

genomics↗

bioRxiv2021

Rhizoliths identified as prehistoric filing tools for fishhook production on San Nicolas Island, California

Chemical analysis of archeological objects can provide important clues about their purpose and function. In this study, we used scanning electron microscopy (SEM) and chemical spectroscopy (SEM-EDS and XRD) to identify a white residue present on cylindrical rhizoliths from a component at an archaeological site (CA-SNI-25) on San Nicolas Island, California, dated ca. AD 1300 to 1700. The residue was found to consist of biogenic calcite and aragonite particles, different in composition and morphology from the CaCO3 particles in the rhizoliths, but identical to marine shell material. These results, together with observations of surface micro-wear patterning on fishhooks and rhizoliths, replicative experiments, in situ spatial analysis, and other archaeological evidence, show that rhizoliths were used as files in a larger tool kit for crafting shell fishhooks. Our findings shed new light on the technological innovations devised by Native Americans to exploit the rich marine resources surrounding the Channel Islands, and provide the first analytical evidence for the use of rhizoliths as a production tool.

paleontology↗

bioRxiv2018

SWI/SNF remains localized to chromatin in the presence of SCHLAP1

SCHLAP1 is a long-noncoding RNA that is prognostic for progression to metastatic prostate cancer and promotes an invasive phenotype. SCHLAP1 is reported to function by depleting the core SWI/SNF subunit, SMARCB1, from the genome. SWI/SNF is a large, multi-subunit, chromatin remodeling complex that can be combinatorially assembled to yield hundreds to thousands of distinct complexes. Here, we investigated the hypothesis that SCHLAP1 affects only specific forms of SWI/SNF and that the remaining SWI/SNF complexes were important for the increased invasion in SCHLAP1 expressing prostate cells. Using several assays we found that SWI/SNF is not depleted from the genome by SCHLAP1 expression. We find that SCHLAP1 induces changes to chromatin openness but is not sufficient to drive changes in histone modifications. Additionally, we show that SWI/SNF binds many coding and non-coding RNAs. Together these results suggest that SCHLAP1 has roles independent of canonical SWI/SNF and that SWI/SNF broadly interacts with RNA.

cancer biology↗