Biology subjects

Crawford, J. C.

Publications and source records attributed to Crawford, J. C..

13 recordsLinked to original sources

bioRxiv2026

MakeMyFigure: An Interactive Platform for Reproducible Quantitative Data Visualization, Analysis, and Scientific Figure Construction

Reproducible data analysis and visualization are essential for reliable biological and biomedical research, yet creating high-quality, publication-ready figures often requires moving data and results between multiple analysis, visualization, and graphics tools. This fragmented, multi-layered process can make it difficult to trace how individual figure panels were generated, preserve the underlying analytical decisions, and reproduce them later. Here, we introduce MakeMyFigure, a free and open-source platform for data visualization, analysis, and creation of multi-panel scientific figures. MakeMyFigure integrates data processing, statistical analysis, visualization, and figure assembly within a single workflow. It supports a range of quantitative data formats and structures, including feature-by-sample matrices and precomputed statistical results, and provides data-aware visualization recommendations to help users select appropriate plots from a library of 38 visualization types. These capabilities allow researchers to move directly from experimental measurements to commonly used statistical analyses and graphical representations without requiring programming expertise. Eighteen statistical procedures are implemented, and their results are drawn directly onto the plot families that support statistical annotation, keeping analytical results linked to the panels they generate. Importantly, MakeMyFigure uses machine-readable JSON specifications to record the identity and checksum of the source data, the processing steps, visualization settings, and statistical parameters used to generate each figure panel. It can also save a figure as a portable package, freezing the specifications together with the data. This allows figures to be regenerated from their recorded specifications and source data, or from the package alone on another computer, rather than relying on manually reconstructed workflows. Using published datasets from several biological fields, independent statistical validation in R, and a comparison with 15 representative analysis, visualization, and figure-generation tools, we show that MakeMyFigure combines accessible, code-free figure creation with panel-level computational reproducibility. Overall, MakeMyFigure provides a unified approach for creating, documenting, and reproducing scientific figures. Source code and documentation are available at https://github.com/surPoudel/make-my-figure, https://github.com/surPoudel/make-my-figure/releases/tag/v1.1.0.

bioinformatics↗

bioRxiv2026

High-dimensional multiomics reveals perturbations to IL-6/IL-6R axis and RUNX3 in CD4+ T cells during third trimester pregnancy

ObjectivesCD4+ T cells play key roles in regulating immune responses during pregnancy, therefore we aimed to understand the CD4+ T cell surface proteome and transcriptome during pregnancy. MethodsCD4+ T cells were analysed in blood and decidua from term-pregnancies (>37 weeks), and non-pregnant blood. >350 surface proteins were screened via flow cytometry, and transcriptomes were analysed using single-cell RNA sequencing with >130 CITE-seq barcoded antibodies. ResultsSurface protein screening identified changes to ILT4/CD85d, CD9, IFN-{gamma} receptor {beta}-chain, CX3CR1 and CCR5 in the pregnant blood and decidual CD4+ T cells. CX3CR1 and CCR5 had the highest expression on the effector-memory T cell (TEM) subset in the blood, with expression consistent across subsets in decidua. CD126/IL-6R was lower in pregnant blood and decidual CD4+ T cells, while scRNAseq identified enrichment in the IL-6R signalling pathway in naive CD4+ T cells in pregnant blood. Both sIL-6R and IL-6 concentrations were increased in plasma during pregnancy, suggesting perturbations to the IL-6/IL-6R signalling axis. Meanwhile, decidual CD4+ T cells had increased expression of transcription factor RUNX3 in the CD69+ tissue-resident-like subset. ConclusionsOur findings demonstrate altered molecular expression in CD4+ T cells during pregnancy. This provides important mechanistic insight of their adaptation and regulation during placental development, which may drive placental dysfunction or pregnancy complications including preeclampsia, fetal growth restriction and stillbirth. These new data may inform future studies that focus on determining the significance of differentially- expressed immune features in pregnancy to identify potential targets for immune modulation to treat pregnancy complications and infections.

immunology↗

bioRxiv2025

Single-cell profiling of CAR-T CD19 cell phenotypes and immune system dynamics in pediatric BCP-ALL

BackgroundChimeric Antigen Receptor T-cell (CAR-T) therapy targeting CD19 has transformed the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the phenotypic heterogeneity of CAR+ T-cells and their interactions with the immune system remain poorly understood. Here, we characterize CAR+ T-cell subsets and persistence in patients receiving standard-of-care tisagenlecleucel in an effort to optimize therapeutic efficacy in pediatric BCP-ALL. MethodsNineteen pediatric patients with relapsed/refractory BCP-ALL treated with tisagenlecleucel in Poland were included in the study. CAR+ T-cell composition and the peripheral blood mononuclear cell (PBMC) immune system were assessed using mass cytometry, with qPCR validation of CAR+ T-cells. Machine learning algorithms in R classified phenotypes. Statistical analyses examined associations between CAR+ T-cell subsets, immune system changes, and CAR-T therapy outcomes, including cytokine release syndrome (CRS). ResultsInfusion product analysis showed a predominance of CD4+ Central Memory (16.6%), MAIT/NKT (7.04%), and Treg memory (63.3%) cells, with high consistency between paired products. Post-infusion, CAR+ Treg memory cells declined to <1%, while CD8+ subsets expanded. PBMC immune system analysis revealed increased monocyte and NK cell counts post-infusion. Patients who experienced CRS had fewer classical monocytes at day 7 and fewer transitional monocytes at day 28. Early and late NK cells increased post-infusion in all patients. However, pre-infusion levels were lower in patients experiencing CRS. CRS correlated with higher CAR+ CD8+ Terminal Effector and MAIT/NKT frequencies in infusion products. While CAR+ composition was not linked to disease burden, prior treatments, or therapy outcomes, changes in monocyte and NK cell dynamics were associated with disease burden, relapse, and CRS. ConclusionsThis study provides a detailed characterization of CAR-T CD19 cell composition and post-infusion dynamics in pediatric BCP-ALL patients treated with tisagenlecleucel. Infusion products were predominantly CAR+ CD4+ T cells with a Central Memory phenotype, including a notable Treg memory subset that declined post-infusion. CAR+ CD8+ subsets expanded, coinciding with immune system shifts. These changes correlated with clinical responses, including CRS, highlighting the role of the immune system in CAR-T therapy outcomes and informing strategies to optimize treatment. Summary boxO_ST_ABSWHAT IS ALREADY KNOWN ON THIS TOPICC_ST_ABSCD19-targeted CAR-T therapy has revolutionized the treatment of relapsed/refractory pediatric BCP-ALL. While studies have examined the diverse composition of CAR-T infusion products in adult large B-cell lymphoma (LBCL), there is limited data in the pediatric BCP-ALL setting. WHAT THIS STUDY ADDSThis study demonstrates that standard-of-care CAR-T infusion products (tisagenlecleucel) are primarily composed of CAR+ CD4+ Central Memory and regulatory T cells, which decline post-infusion as CAR+ CD8+ subsets expand. Additionally, monocyte and NK cell dynamics are closely linked to cytokine release syndrome (CRS) and treatment outcomes, emphasizing the critical role of immune system interactions in CAR-T therapy efficacy. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICYThese findings indicate that monitoring immune system shifts could enhance CRS risk assessment and improve patient stratification, leading to more personalized CAR-T therapy. Additionally, insights into CAR+ T-cell composition may guide the development of strategies to optimize CAR-T efficacy, potentially improving long-term treatment outcomes in pediatric BCP-ALL.

cancer biology↗

bioRxiv2023

Spatiotemporal development of the human T follicular helper cell response to Influenza vaccination

We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.

immunology↗

bioRxiv2023

The beta 6 Integrin Negatively Regulates TLR7-Mediated Epithelial Immunity via Autophagy During Influenza A Virus Infection

Integrins are essential surface receptors that sense extracellular changes to initiate various intracellular signaling cascades. The rapid activation of the epithelial-intrinsic {beta}6 integrin during influenza A virus (IAV) infection has been linked to innate immune impairments. Yet, how {beta}6 regulates epithelial immunity remains undefined. Here, we identify the role of {beta}6 in mediating the Toll-like receptor 7 (TLR7) through the regulation of intracellular trafficking. We demonstrate that deletion of the {beta}6 integrin in lung epithelial cells significantly enhances the TLR7-mediated activation of the type I interferon (IFN) response during homeostasis and respiratory infection. IAV-induced {beta}6 facilitates TLR7 trafficking to lysosome-associated membrane protein (LAMP2a) components, leading to a reduction in endosomal compartments and associated TLR7 signaling. Our findings reveal an unappreciated role of {beta}6-induced autophagy in influencing epithelial immune responses during influenza virus infection.

immunology↗

bioRxiv2023

Establishing thresholds for cytokine storm and defining their relationship to disease severity in respiratory viral infections

Previous studies have identified cytokines associated with respiratory virus infection illness outcome. However, few studies have included comprehensive cytokine panels, longitudinal analyses, and/or simultaneous assessment across the severity spectrum. This, coupled with subjective definitions of cytokine storm syndrome (CSS), have contributed to inconsistent findings of cytokine signatures, particularly with COVID severity. Here, we measured 38 plasma cytokines and compared profiles in healthy, SARS-CoV-2 infected, and multisystem inflammatory syndrome in children (MIS-C) patients (n = 169). Infected patients spanned the severity spectrum and were classified as Asymptomatic, Mild, Moderate or Severe. Our results showed acute cytokine profiles and longitudinal dynamics of IL1Ra, IL10, MIP1b, and IP10 can differentiate COVID severity groups. Only 4% of acutely infected patients exhibited hypercytokinemia. Of these subjects, 3 were Mild, 3 Moderate, and 1 Severe, highlighting the lack of association between CSS and COVID severity. Additionally, we identified IL1Ra and TNFa as potential biomarkers for patients at high risk for long COVID. Lastly, we compare hypercytokinemia profiles across COVID and influenza patients and show distinct elevated cytokine signatures, wherein influenza induces the most elevated cytokine profile. Together, these results identify key analytes that, if obtained at early time points, can predict COVID illness outcome and/or risk of complications, and provide novel insight for improving the conceptual framework of hypercytokinemia, wherein CSS is a subgroup that requires concomitant severe clinical manifestations, and including a list of cytokines that can distinguish between subtypes of hypercytokinemia.

immunology↗

bioRxiv2023

Astrovirus replication is dependent on induction of double membrane vesicles through a PI3K-dependent, LC3-independent pathway

Human astrovirus is a positive sense, single stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double membrane vesicles (DMVs). Here we show that astrovirus infection leads to an increase in DMV formation, and this process is replication-dependent. Our data suggest that astrovirus infection induces rearrangement of endoplasmic reticulum fragments, which may become the origin for DMV formation. Transcriptional data suggested that formation of DMVs during astrovirus infection requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Inhibition of the PI3K complex leads to significant reduction in viral replication and release from cells. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. ImportanceThese studies provide critical new evidence that astrovirus replication requires formation of double membrane vesicles, which utilize class III PI3K, but not LC3 conjugation autophagy machinery for biogenesis. These results are consistent with replication mechanisms for other positive sense RNA viruses. This suggests that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive sense RNA virus infections.

microbiology↗

bioRxiv2022

Latent human herpesvirus 6 is reactivated in chimeric antigen receptor T cells

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are still being understood. For example, we currently lack a comprehensive understanding of the mechanisms of neurotoxicity observed in patients receiving T cell therapies, including recent reports of encephalitis caused by human herpesvirus 6 (HHV-6) reactivation1. Here, via petabase-scale viral RNA data mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in human CD4+ T cells in standard in vitro cultures. Using single-cell sequencing, we identify a rare population of HHV-6 super-expressors (~1 in 300-10,000 cells) that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture before spreading to infect other cells in vitro. Through the analysis of single-cell sequencing data from patients receiving cell therapy products that are FDA-approved2 or used in clinical studies3,4, we identify the presence of CAR+, HHV-6 super-expressor T cells in vivo. Together, our study implicates cell therapy products as a source of lytic HHV-6 reported in clinical trials1,5-7 and has broad implications for the design, production, and monitoring of cell therapies.

immunology↗

bioRxiv2021

Fetal neural progenitors process TLR signals from bacterial components to enhance proliferation and rework brain development

Bacterial cell wall, a universal pathogen-associated molecular pattern (PAMP), crosses the placenta into the fetal brain. We determined that PAMPs interact with TLR2/6 on murine fetal neural progenitor cells (NPCs) to induce overexpansion of all neocortical layers leading to a larger, folded cortex and abnormal postnatal behavior. The NPC overexpansion originated at E10 and targeted ventricular radial glia (vRG), the primary NPC, by shortening cell cycle and increasing self-renewal. The mechanism involved two novel signaling pathways in NPCs mediated by recognition of bacterial PAMPs by TLR2/6 including: a) loss of primary cilia, activation of hedgehog signaling, and increased FOXG1 and b) increased PI3K/AKT activity. These findings reveal PAMP/TLR2/6 acts as a morphogen in fetal neurodevelopment. In addition, the loss of Tlr2 or Tlr6 without pathogenic challenge, increased the number of neurons, establishing the requirement for an endogenous TLR2 signal for normal neurodevelopment in the embryo.

neuroscience↗

bioRxiv2021

SARS-CoV-2 mRNA vaccination elicits robust and persistent T follicular helper cell response in humans

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we show that frequency of TFH correlates with that of S-binding germinal center B cells. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLADPB1* 04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

immunology↗

bioRxiv2021

Neuroblastoma formation requires unconventional CD4 T cells and myeloid amino acid metabolism

By mirroring their function as tissue repair organizers in normal tissues, immune cells regulate tumor growth. To understand the different facets of immune-tumor collaboration through genetics, spatial transcriptomics, and immunological manipulation with non-invasive, longitudinal imaging, we generated a penetrant double oncogene-driven autochthonous model of neuroblastoma. Using spatial transcriptomic analysis, we co-localized CD4+ and myeloid populations within the tumor parenchyma, while CD8+ T cells and B cells were peripherally dispersed. Depletion of CD4+ T cells or CCR2+ macrophages, but not B cells, CD8+, or NK cells, prevented tumor formation. Tumor CD4+ T cells displayed unconventional phenotypes, were clonotypically diverse, and antigen-independent. Within the myeloid fraction, tumor growth required myeloid cells expressing Arginase-1. Overall, our results suggest that arginine-metabolizing myeloid cells conspire with pathogenic CD4+ T cells to create permissive conditions for tumor formation, and therefore suggest that these pro-tumorigenic pathways can be disabled by targeting myeloid amino acid metabolism.

cancer biology↗

bioRxiv2020

TCR meta-clonotypes for biomarker discovery with tcrdist3: quantification of public, HLA-restricted TCR biomarkers of SARS-CoV-2 infection

As the mechanistic basis of adaptive cellular antigen recognition, T cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages antigen-enriched repertoires to form meta-clonotypes - groups of biochemically similar TCRs - that can be used to robustly identify and quantify functionally similar TCRs in bulk repertoires. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the 17 SARS-CoV-2 antigen-enriched repertoires with the strongest evidence of HLA-restriction. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (FDR < 0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.

immunology↗

bioRxiv2020

Linking T cell receptor sequence to transcriptional profiles with clonotype neighbor graph analysis (CoNGA)

Multi-modal single-cell technologies capable of simultaneously assaying gene expression and surface phenotype across large numbers of immune cells have described extensive heterogeneity within these complex populations, in healthy and diseased states. In the case of T cells, these technologies have made it possible to profile clonotype, defined by T cell receptor (TCR) sequence, and phenotype, as reflected in gene expression (GEX) profile, surface protein expression, and peptide:MHC (pMHC) binding, across large and diverse cell populations. These rich, high-dimensional datasets have the potential to reveal new relationships between TCR sequence and T cell phenotype that go beyond identification of features shared by clonally related cells. In order to uncover these connections in an unbiased way, we developed a graph-theoretic approach---clonotype neighbor-graph analysis or "CoNGA"---that identifies correlations between GEX profile and TCR sequence through statistical analysis of a pair of T cell similarity graphs, one in which cells are linked based on gene expression similarity and another in which cells are linked by similarity of TCR sequence. Applying CoNGA across diverse human and mouse T cell datasets uncovered known and novel associations between TCR sequence features and cellular phenotype including the classical invariant T cell subsets; a novel defined population of human blood CD8+ T cells expressing the transcription factors HOBIT and HELIOS, NK-associated receptors, and a biased TCR repertoire, representing a potential previously undescribed lineage of "natural lymphocytes"; a striking association between usage of a specific V-beta gene segment and expression of the EPHB6 gene that is conserved between mouse and human; and TCR sequence determinants of differentiation in developing thymocytes. As the size and scale of single-cell datasets continue to grow, we expect that CoNGA will prove to be a useful tool for deconvolving complex relationships between TCR sequence and cellular state in single-cell applications.

immunology↗