Biology subjects

Germain, R. N.

Publications and source records attributed to Germain, R. N..

15 recordsLinked to original sources

bioRxiv2026

Quantitative Modeling of TLR Signaling Reveals Missing Negative Feedback Guiding Identification of TANK-IKKε Checkpoint

Toll-like receptor (TLR) signaling must be activated rapidly and then terminated to support host defense without sustained inflammation. We developed a rule-based model of mouse macrophage TLR4 signaling at the molecular-interaction level using measured protein copy numbers, RNA-seq-based abundance estimates, literature- and structure-informed reaction rates, and 979 dynamic experimental constraints. The trained model reproduced much of the TLR4-induced NF-{kappa}B and MAP kinase response but consistently failed to capture deactivation of MyD88, TRAF6-associated species, and IKK/{beta}. The recurrent model failure conveyed important biological information, localizing missing regulation to the proximal MyD88-IRAK-TRAF6 module and guiding experimental evaluation of IKK{varepsilon} and its scaffold TANK. Loss of IKK{varepsilon} enhanced transcriptional, cytokine, MAP kinase, and NF-{kappa}B responses to MyD88-specific TLR ligands. TANK deficiency produced a similar cellular phenotype and abolished stimulus-induced IKK{varepsilon} phosphorylation. Deficiency of either protein increased IRAK1 and TRAF6 ubiquitination without increasing MyD88 ubiquitination, placing the inhibitory checkpoint at or immediately downstream of the IRAK1-TRAF6 ubiquitin-signaling node. Overlapping but non-identical in vivo phenotypes further supported a shared regulatory axis with additional protein-specific functions. Our study presents a model-experiment discovery cycle where quantitative pathway discordance identifies missing biology and reveals a TANK-dependent IKK{varepsilon} checkpoint that restrains MyD88-driven inflammation.

systems biology↗

bioRxiv2024

PD-1 controls differentiation, survival, and TCR affinity evolution of stem-like CD8+ T cells

Stem-like progenitors are a critical subset of cytotoxic T cells that self-renew and give rise to expanded populations of effector cells critical for successful checkpoint blockade immunotherapy. Emerging evidence suggests that the tumor-draining lymph nodes can support the continuous generation of these stem-like cells that replenish the tumor sites and act as a critical source of expanded effector populations, underlining the importance of understanding what factors promote and maintain activated T cells in the stem-like state. Using advanced 3D multiplex immunofluorescence imaging, here we identified antigen-presentation niches in tumor-draining lymph nodes that support the expansion, maintenance, and affinity evolution of a unique population of TCF-1+PD-1+SLAMF6hi stem-like CD8+ T cells. Our results show that contrary to the prevailing view that persistent TCR signaling drives terminal effector differentiation, prolonged antigen engagement well beyond the initial priming phase sustained the proliferation and self-renewal of these stem-like T cells in vivo. The inhibitory PD-1 pathway plays a central role in this process by mediating the fine-tuning of TCR and co-stimulatory signal input that enables selective expansion of high affinity TCR stem-like clones, enabling them to act as a renewable source of high affinity effector cells. PD-1 checkpoint blockade disrupts this fine tuning of input signaling, leading to terminal differentiation to the effector state or death of the most avid anti-tumor stem-like cells. Our results thus reveal an unexpected relationship between TCR ligand affinity recognition, a key negative feedback regulatory loop, and T cell stemness programming. Furthermore, these findings raise questions about whether anti-PD-1 checkpoint blockade during cancer immunotherapy provides a short-term anti-tumor effect that comes at the cost of diminishing efficacy due to progressive loss of these critical high affinity stem-like precursors.

immunology↗

bioRxiv2024

Rebalancing Viral and Immune Damage versus Tissue Repair Prevents Death from Lethal Influenza Infection

Maintaining tissue function while eliminating infected cells is fundamental to host defense. Innate inflammatory damage contributes to lethal influenza and COVID-19, yet other than steroids, immunomodulatory drugs have modest effects. Among more than 50 immunomodulatory regimes tested in mouse lethal influenza infection, only the previously reported early depletion of neutrophils showed efficacy, suggesting that the infected host passes an early tipping point in which limiting innate immune damage alone cannot rescue physiological function. To re-balance the system late in infection, we investigated whether partial limitation of viral spread using oseltamivir (Tamiflu) together with enhancement of epithelial repair by blockade of interferon signaling or the limitation of further epithelial cell loss mediated by cytotoxic CD8+ T cells would prevent death. These treatments salvaged a large fraction of infected animals, providing new insight into the importance of repair processes and the timing of adaptive immune responses in survival of pulmonary infections.

immunology↗

bioRxiv2024

Optimal CXCR5 Expression during Tfh Maturation Involves the Bhlhe40-Pou2af1 Axis

The pair of transcription factors Bcl6-Blimp1 is well-known for follicular T helper (Tfh) cell fate determination, however, the mechanism(s) for Bcl6-independent regulation of CXCR5 during Tfh migration into germinal center (GC) is still unclear. In this study, we uncovered another pair of transcription factors, Bhlhe40-Pou2af1, that regulates CXCR5 expression. Pou2af1 was specifically expressed in Tfh cells whereas Bhlhe40 expression was found high in non-Tfh cells. Pou2af1 promoted Tfh formation and migration into GC by upregulating CXCR5 but not Bcl6, while Bhlhe40 repressed this process by inhibiting Pou2af1 expression. RNA-Seq analysis of antigen-specific Tfh cells generated in vivo confirmed the role of Bhlhe40-Pou2af1 axis in regulating optimal CXCR5 expression. Thus, the regulation of CXCR5 expression and migration of Tfh cells into GC involves a transcriptional regulatory circuit consisting of Bhlhe40 and Pou2af1, which operates independent of the Bcl6-Blimp1 circuit that determines the Tfh cell fate.

immunology↗

bioRxiv2023

Spatial Patterning Analysis of Cellular Ensembles (SPACE) enables statistically robust discovery of complex spatial organization at the cell and tissue level

Spatial patterns of cells and other biological elements drive both physiologic and pathologic processes within tissues. While many imaging and transcriptomic methods document tissue organization, discerning these patterns is challenging, especially when they involve multiple elements in complex arrangements. To address this challenge, we present Spatial Patterning Analysis of Cellular Ensembles (SPACE), an R package for analysis of high-plex spatial data. SPACE is compatible with any data collection modality that records values (i.e., categorical cell/structure types or quantitative expression levels) at fixed spatial coordinates (i.e., 2d pixels or 3d voxels). SPACE detects not only broad patterns of co-occurrence but also context-dependent associations, quantitative gradients and orientations, and other organizational complexities. Via a robust information theoretic framework, SPACE explores all possible ensembles of tissue elements - single elements, pairs, triplets, and so on - and ranks the most strongly patterned ensembles. For single images, rankings reflect patterns that differ from random assortment. For sets of images, rankings reflect patterns that differ across sample groups (e.g., genotypes, treatments, timepoints, etc.). Further tools then thoroughly characterize the nature of each pattern for intuitive interpretation. We validate SPACE and demonstrate its advantages using murine lymph node images for which ground truth has been defined. We then use SPACE to detect new patterns across varied datasets, including tumors and tuberculosis granulomas.

bioinformatics↗

bioRxiv2023

A spatial human thymus cell atlas mapped to a continuous tissue axis

T cells develop from circulating precursors, which enter the thymus and migrate throughout specialised sub-compartments to support maturation and selection. This process starts already in early fetal development and is highly active until the involution of the thymus in adolescence. To map the micro-anatomical underpinnings of this process in pre- vs. post-natal states, we undertook a spatially resolved analysis and established a new quantitative morphological framework for the thymus, the Cortico-Medullary Axis. Using this axis in conjunction with the curation of a multimodal single-cell, spatial transcriptomics and high-resolution multiplex imaging atlas, we show that canonical thymocyte trajectories and thymic epithelial cells are highly organised and fully established by post-conception week 12, pinpoint TEC progenitor states, find that TEC subsets and peripheral tissue genes are associated with Hassalls Corpuscles and uncover divergence in the pace and drivers of medullary entry between CD4 vs. CD8 T cell lineages. These findings are complemented with a holistic toolkit for spatial analysis and annotation, providing a basis for a detailed understanding of T lymphocyte development.

immunology↗

bioRxiv2023

Non-coding sequence variation reveals fragility within interleukin 2 feedback circuitry and shapes autoimmune disease risk

Genetic variants associated with human autoimmune diseases commonly map to non-coding control regions, particularly enhancers that function selectively in immune cells and fine-tune gene expression within a relatively narrow range of values. How such modest, cell-type-selective changes can meaningfully shape organismal disease risk remains unclear. To explore this issue, we experimentally manipulated species-conserved enhancers within the disease-associated IL2RA locus and studied accompanying changes in the progression of autoimmunity. Perturbing distinct enhancers with restricted activity in conventional T cells (Tconvs) or regulatory T cells (Tregs)--two functionally antagonistic T cell subsets--caused only modest, cell-type-selective decreases in IL2ra expression parameters. However, these same perturbations had striking and opposing effects in vivo, completely preventing or severely accelerating disease in a murine model of type 1 diabetes. Quantitative tissue imaging and computational modelling revealed that each enhancer manipulation impinged on distinct IL-2-dependent feedback circuits. These imbalances altered the intracellular signaling and intercellular communication dynamics of activated Tregs and Tconvs, producing opposing spatial domains that amplified or constrained ongoing autoimmune responses. These findings demonstrate how subtle changes in gene regulation stemming from non-coding variation can propagate across biological scales due to non-linearities in intra- and intercellular feedback circuitry, dramatically shaping disease risk at the organismal level.

immunology↗

bioRxiv2022

Agonistic anti-CD40 converts Tregs into Type 1 effectors within the tumor micro-environment

Multiple cell types, molecules, and processes contribute to inhibition of anti-tumor effector responses, often frustrating effective immunotherapy. Among these, Foxp3+ CD4+ cells (Tregs) are well-recognized to play an immunosuppressive role in the tumor microenvironment. The first clinically successful checkpoint inhibitor, anti-CTLA-4 antibody, may deplete Tregs at least in part by antibody-dependent cellular cytotoxicity (ADCC), but this effect is unreliable in mice, including in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC). In contrast, agonistic CD40 antibody, which serves as an effective therapy, is associated with notable Treg disappearance in the PDAC model. The mechanism of CD40-mediated Treg loss is poorly understood, as Tregs are CD40-negative. Here we have explored the mechanistic basis for the loss of Foxp3 T cells upon anti-CD40 treatment and find, using tissue-level multiplex immunostaining and orthogonal dissociated cell analyses, that Tregs are not depleted but converted into interferon-{gamma} (IFN-{gamma}) producing, Type I CD4+ T effector cells. This process depends on IL-12 and IFN-{gamma} signaling evoked by action of the anti-CD40 antibody on dendritic cells (DCs), especially BATF3-dependent cDC1s. These findings provide insight into a previously unappreciated mechanism of CD40 agonism as a potent anti-tumor intervention that promotes the re-programming of Tregs into tumor-reactive CD4+ effector T cells, markedly augmenting the anti-tumor response.

immunology↗

bioRxiv2022

A Multi-scale, Multiomic Atlas of Human Normal and Follicular Lymphoma Lymph Nodes

Reference atlases, molecular and spatial maps of mammalian tissues, are critical resources for discovery efforts and translational research. Their utility is dependent on operationalizing the resulting data by identifying cell types, histological patterns, and predictive biomarkers underlying health and disease. The human lymph node (LN) offers a compelling use case because of its importance in immunity, structural and cellular diversity, and neoplastic involvement. One hematological malignancy, follicular lymphoma (FL), evolves from developmentally blocked germinal center B cells residing in and trafficking through these tissues. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Here, we present an integrated portrait of healthy and FL LNs using multiple genomic and advanced imaging technologies. By leveraging the strengths of each platform, we identified several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk of FL patients.

cancer biology↗

bioRxiv2021

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation

T cell receptor clustering plays a key role in triggering cell activation, but the relationship between the spatial configuration of clusters and elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by non-stimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands and translates that information into distinct signaling dynamics, and provide insights into engineering new immunotherapies.

cell biology↗

bioRxiv2020

IBEX: A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

The diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only 2-6 molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a new method that overcomes major impediments to highly multi-plex tissue imaging. Iterative Bleaching Extends multi-pleXity (IBEX) uses an iterative staining and chemical bleaching method to enable high resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an open system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and IBEX analysis of the same tissue. To facilitate data processing, we provide an open source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high content imaging to reveal the complex cellular landscape of diverse organs and tissues. Significance StatementSingle cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveal how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multi-plex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard to image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies.

immunology↗

bioRxiv2020

Blood gene expression-based prediction of lethality after respiratory infection by influenza A virus in mice

Lethality after respiratory infection with influenza A virus (IAV) is associated with potent immune activation and lung tissue damage. In a well-controlled animal model of infection, we sought to determine if one could predict lethality using transcriptional information obtained from whole blood early after influenza virus exposure. We started with publicly available transcriptomic data from the lung, which is the primary site of the infection and pathology, to derive a multigene transcriptional signature of death reflective of innate inflammation associated with tissue damage. We refined this affected tissue signature with data from infected mouse and human blood to develop and validate a machine learning model that can robustly predict survival in mice after IAV challenge using data obtained from as little as 10 l of blood from early time points post infection. Furthermore, in genetically identical, cohoused mice infected with the same viral bolus, the same model can predict the lethality of individual animals but, intriguingly, only within a specific time window that overlapped with the early effector phase of adaptive immunity. These findings raise the possibility of predicting disease outcome in respiratory virus infections with blood transcriptional data and pave the way for translating such approaches to humans.

immunology↗

bioRxiv2020

T cell subset-selective IL2RA enhancers shape autoimmune diabetes risk

The majority of genetic variants associated with complex human autoimmune diseases reside in enhancers1-3, non-coding regulatory elements that control gene expression. In contrast with variants that directly alter protein-coding sequences, enhancer variants are predicted to tune gene expression modestly and function in specific cellular contexts4, suggesting that small alterations in the functions of key immune cell populations are sufficient to shape disease risk. Here we tested this concept by experimentally perturbing distinct enhancers governing the high affinity IL-2 receptor alpha chain (IL2RA; also known as CD25). IL2RA is an immune regulator that promotes the pro- and anti-inflammatory functions of conventional T cells (Tconvs) and regulatory T cells (Tregs), respectively, and non-coding genetic variants in IL2RA have been linked to multiple autoimmune disorders4. We previously tiled across the IL2RA locus using CRISPR-activation and identified a stimulation-responsive element (CaRE4) with an enhancer that modestly affects the kinetics of IL2RA expression in Tconvs5. This enhancer is conserved across species and harbors a common human SNP associated with protection from Type 1 Diabetes (T1D)5,6. We now identified an additional conserved enhancer, termed CaRE3 enhancer, which modestly affected steady state IL2RA expression in regulatory T cells (Tregs). Despite their seemingly subtle impact on gene expression, the CaRE3 and CaRE4 enhancers had pronounced yet divergent effects on the incidence of diabetes in autoimmune prone animals. Deletion of the conserved CaRE4 enhancer completely protected against autoimmune diabetes even in animals treated with an immunostimulating anti-PD1 checkpoint inhibitor, whereas deletion of the CaRE3 enhancer accelerated spontaneous disease progression. Quantitative multiplexed imaging of the pancreatic lymph nodes (panLNs) revealed that each enhancer deletion preferentially affected the protein expression levels of IL2RA in activated Tconvs or Tregs, reciprocally tuning local competition for IL-2 input signals. In animals lacking the CaRE4 enhancer, skewed IL-2 signaling favored Tregs, increasing their local density around activated Tconvs to strongly suppress emergence of autoimmune effectors. By contrast, in animals lacking the CaRE3 enhancer, IL-2 signals were skewed towards activated Tconvs, promoting their escape from Treg control. Collectively, this work illustrates how subtle changes in gene regulation due to non-coding variation can significantly alter disease progression and how distinct enhancers controlling the same gene can have opposing effects on disease outcomes through cell type-selective activity.

immunology↗

bioRxiv2020

Efficient immune cell genome engineering with improved CRISPR editing tools

CRISPR (clustered regularly interspaced short palindromic repeats)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of sensitive intracellular sensors. Likewise, lymphocytes of the adaptive immune system are largely refractory to CRISPR-enhanced homology-directed repair (HDR) due to inefficient or toxic delivery of donor templates via transient transfection methods. To overcome these challenges and limitations, we developed three improved methods for CRISPR-based genome editing using a hit-and-run transient expression strategy to minimize off-target effects and generate more precise genome editing. Overall, our enhanced CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering are expected to be widely applicable not only in hematopoietic cells but also other mammalian cell types of interest. All animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional Animal Care and Use Committee.

genomics↗

bioRxiv2019

A sustained small increase in NOD1 expression promotes ligand-independent oncogenic activity

Small genetically-determined differences in transcription (eQTLs) are implicated in complex disease but the mechanisms by which small changes in gene expression impact complex disease are unknown. Here we show that a persistent small increase in expression of the innate sensor NOD1 precipitates large cancer-promoting changes in cell state. A ~1.2-1.4 fold increase in NOD1 protein concentration by loss of miR-15b/16 regulation sensitizes cells to ligand-induced inflammation, with an additional slight increase leading to ligand-independent NOD1 activation that is linked to poor prognosis in gastric cancer. Our data show that tight expression regulation of NOD1 prevents this sensor from exceeding a physiological switching checkpoint that promotes persistent inflammation and oncogene expression and reveal the impact of a single small quantitative change in cell state on cancer.\n\nOne Sentence SummaryA small change in NOD1 expression has a large cancer-promoting impact on cell state.

systems biology↗