Multi-omic profiling of follicular lymphoma reveals changes in tissue architecture and enhanced stromal remodeling in high-risk patients.

Follicular lymphoma (FL) is a generally incurable malignancy that evolves from developmentally blocked germinal center (GC) B cells. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Dynamic interactions between tumor B cells and the tumor microenvironment (TME) are hypothesized to contribute to the broad spectrum of clinical behaviors observed among FL patients. Despite the urgent need, existing clinical tools do not reliably predict disease behavior. Using a multi-modal strategy, we examined cell-intrinsic and -extrinsic factors governing progression and therapeutic outcomes in FL patients enrolled onto a prospective clinical trial. By leveraging the strengths of each platform, we identify several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk FL patients. These features include stromal desmoplasia and changes to the follicular growth pattern present 20 months before first progression and first relapse.

Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

The follicular lymphoma tumor microenvironment at single-cell and spatial resolution.

ABSTRACT: Follicular lymphoma (FL) is a generally incurable malignancy that originates from developmentally blocked germinal center B cells residing, primarily, within lymph nodes (LNs). During the long natural history of FL, malignant B cells often disseminate to multiple LNs and can affect virtually any organ. Nonmalignant LNs are highly organized structures distributed throughout the body, in which they perform functions critical for host defense. In FL, the malignant B cells "re-educate" the lymphoid environment by altering the phenotype, distribution, and abundance of other cells such as T cells, macrophages, and subsets of stromal cells. Consequently, dramatic anatomical changes occur and include alterations in the number, shape, and size of neoplastic follicles with an accompanying attenuation of the T-cell zone. Ongoing and dynamic interactions between FL B cells and the tumor microenvironment (TME) result in significant clinical heterogeneity observed both within and across patients. Over time, FL evolves into pathological variants associated with distinct outcomes, ranging from an indolent disease to more aggressive clinical courses with early death. Given the importance of both cell-intrinsic and -extrinsic factors in shaping disease progression and patient survival, comprehensive examination of FL tumors is critical. Here, we describe the cellular composition and architecture of normal and malignant human LNs and provide a broad overview of emerging technologies for deconstructing the FL TME at single-cell and spatial resolution. We additionally discuss the importance of capturing samples at landmark time points as well as longitudinally for clinical decision-making.

Spatial Patterning Analysis of Cellular Ensembles (SPACE) discovers complex spatial organization at the cell and tissue levels.

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.

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 Hassall's 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.

The emerging importance of lymphatics in health and disease: an NIH workshop report.

The lymphatic system (LS) is composed of lymphoid organs and a network of vessels that transport interstitial fluid, antigens, lipids, cholesterol, immune cells, and other materials in the body. Abnormal development or malfunction of the LS has been shown to play a key role in the pathophysiology of many disease states. Thus, improved understanding of the anatomical and molecular characteristics of the LS may provide approaches for disease prevention or treatment. Recent advances harnessing single-cell technologies, clinical imaging, discovery of biomarkers, and computational tools have led to the development of strategies to study the LS. This Review summarizes the outcomes of the NIH workshop entitled "Yet to be Charted: Lymphatic System in Health and Disease," held in September 2022, with emphasis on major areas for advancement. International experts showcased the current state of knowledge regarding the LS and highlighted remaining challenges and opportunities to advance the field.

Organ Mapping Antibody Panels: a community resource for standardized multiplexed tissue imaging.

Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.

Mapping the lymphatic system across body scales and expertise domains: A report from the 2021 National Heart, Lung, and Blood Institute workshop at the Boston Lymphatic Symposium.

Enhancing our understanding of lymphatic anatomy from the microscopic to the anatomical scale is essential to discern how the structure and function of the lymphatic system interacts with different tissues and organs within the body and contributes to health and disease. The knowledge of molecular aspects of the lymphatic network is fundamental to understand the mechanisms of disease progression and prevention. Recent advances in mapping components of the lymphatic system using state of the art single cell technologies, the identification of novel biomarkers, new clinical imaging efforts, and computational tools which attempt to identify connections between these diverse technologies hold the potential to catalyze new strategies to address lymphatic diseases such as lymphedema and lipedema. This manuscript summarizes current knowledge of the lymphatic system and identifies prevailing challenges and opportunities to advance the field of lymphatic research as discussed by the experts in the workshop.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children.

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues.

High-content imaging is needed to catalog the variety of cellular phenotypes and multicellular ecosystems present in metazoan tissues. We recently developed iterative bleaching extends multiplexity (IBEX), an iterative immunolabeling and chemical bleaching method that enables multiplexed imaging (>65 parameters) in diverse tissues, including human organs relevant for international consortia efforts. IBEX is compatible with >250 commercially available antibodies and 16 unique fluorophores, and can be easily adopted to different imaging platforms using slides and nonproprietary imaging chambers. The overall protocol consists of iterative cycles of antibody labeling, imaging and chemical bleaching that can be completed at relatively low cost in 2-5 d by biologists with basic laboratory skills. To support widespread adoption, we provide extensive details on tissue processing, curated lists of validated antibodies and tissue-specific panels for multiplex imaging. Furthermore, instructions are included on how to automate the method using competitively priced instruments and reagents. Finally, we present a software solution for image alignment that can be executed by individuals without programming experience using open-source software and freeware. In summary, IBEX is a noncommercial method that can be readily implemented by academic laboratories and scaled to achieve high-content mapping of diverse tissues in support of a Human Reference Atlas or other such applications.

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.

Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

Understanding immunity in a tissue-centric context: Combining novel imaging methods and mathematics to extract new insights into function and dysfunction.

A central question in immunology is what features allow the immune system to respond in a timely manner to a variety of pathogens encountered at unanticipated times and diverse body sites. Two decades of advanced and static dynamic imaging methods have now revealed several major principles facilitating host defense. Suborgan spatial prepositioning of distinct cells promotes time-efficient interactions upon pathogen sensing. Such pre-organization also provides an effective barrier to movement of pathogens from parenchymal tissues into the blood circulation. Various molecular mechanisms maintain effective intercellular communication among otherwise rapidly moving cells. These and related discoveries have benefited from recent increases in the number of parameters that can be measured simultaneously in a single tissue section and the extension of such multiplex analyses to 3D tissue volumes. The application of new computational methods to such imaging data has provided a quantitative, in vivo context for cell trafficking and signaling pathways traditionally explored in vitro or with dissociated cell preparations. Here, we summarize our efforts to devise and employ diverse imaging tools to probe immune system organization and function, concluding with a commentary on future developments, which we believe will reveal even more about how the immune system operates in health and disease.

Anatomical structures, cell types and biomarkers of the Human Reference Atlas.

The Human Reference Atlas (HRA) aims to map all of the cells of the human body to advance biomedical research and clinical practice. This Perspective presents collaborative work by members of 16 international consortia on two essential and interlinked parts of the HRA: (1) three-dimensional representations of anatomy that are linked to (2) tables that name and interlink major anatomical structures, cell types, plus biomarkers (ASCT+B). We discuss four examples that demonstrate the practical utility of the HRA.

Author Correction: Commensal-driven immune zonation of the liver promotes host defence.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03346-0.

Commensal-driven immune zonation of the liver promotes host defence.

The liver connects the intestinal portal vasculature with the general circulation, using a diverse array of immune cells to protect from pathogens that translocate from the gut1. In liver lobules, blood flows from portal triads that are situated in periportal lobular regions to the central vein via a polarized sinusoidal network. Despite this asymmetry, resident immune cells in the liver are considered to be broadly dispersed across the lobule. This differs from lymphoid organs, in which immune cells adopt spatially biased positions to promote effective host defence2,3. Here we used quantitative multiplex imaging, genetic perturbations, transcriptomics, infection-based assays and mathematical modelling to reassess the relationship between the localization of immune cells in the liver and host protection. We found that myeloid and lymphoid resident immune cells concentrate around periportal regions. This asymmetric localization was not developmentally controlled, but resulted from sustained MYD88-dependent signalling induced by commensal bacteria in liver sinusoidal endothelial cells, which in turn regulated the composition of the pericellular matrix involved in the formation of chemokine gradients. In vivo experiments and modelling showed that this immune spatial polarization was more efficient than a uniform distribution in protecting against systemic bacterial dissemination. Together, these data reveal that liver sinusoidal endothelial cells sense the microbiome, actively orchestrating the localization of immune cells, to optimize host defence.

IBEX: A versatile multiplex 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 two to six 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 method that overcomes major impediments to highly multiplex tissue imaging. "Iterative bleaching extends multiplexity" (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, 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.

The lymph node at a glance - how spatial organization optimizes the immune response.

A hallmark of the mammalian immune system is its ability to respond efficiently to foreign antigens without eliciting an inappropriate response to self-antigens. Furthermore, a robust immune response requires the coordination of a diverse range of cells present at low frequencies within the host. This problem is solved, in part, by concentrating antigens, antigen-presenting cells and antigen-responsive cells in lymph nodes (LNs). Beyond housing these cell types in one location, LNs are highly organized structures consisting of pre-positioned cells within well-defined microanatomical niches. In this Cell Science at a Glance article and accompanying poster, we outline the key cellular populations and components of the LN microenvironment that are present at steady state and chronicle the dynamic changes in these elements following an immune response. This review highlights the LN as a staging ground for both innate and adaptive immune responses, while providing an elegant example of how structure informs function.