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.

Dynamic Multiplex Tissue Imaging in Inflammation Research.

Inflammation is a highly dynamic process with immune cells that continuously interact with each other and parenchymal components as they migrate through tissue. The dynamic cellular responses and interaction patterns are a function of the complex tissue environment that cannot be fully reconstructed ex vivo, making it necessary to assess cell dynamics and changing spatial patterning in vivo. These dynamics often play out deep within tissues, requiring the optical focus to be placed far below the surface of an opaque organ. With the emergence of commercially available two-photon excitation lasers that can be combined with existing imaging systems, new avenues for imaging deep tissues over long periods of time have become available. We discuss a selected subset of studies illustrating how two-photon microscopy (2PM) has helped to relate the dynamics of immune cells to their in situ function and to understand the molecular patterns that govern their behavior in vivo. We also review some key practical aspects of 2PM methods and point out issues that can confound the results, so that readers can better evaluate the reliability of conclusions drawn using this technology.

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.

TCR ligand potency differentially impacts PD-1 inhibitory effects on diverse signaling pathways.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.

Multiplex imaging in immuno-oncology.

Multiplex imaging has emerged as an invaluable tool for immune-oncologists and translational researchers, enabling them to examine intricate interactions among immune cells, stroma, matrix, and malignant cells within the tumor microenvironment (TME). It holds significant promise in the quest to discover improved biomarkers for treatment stratification and identify novel therapeutic targets. Nonetheless, several challenges exist in the realms of study design, experiment optimization, and data analysis. In this review, our aim is to present an overview of the utilization of multiplex imaging in immuno-oncology studies and inform novice researchers about the fundamental principles at each stage of the imaging and analysis process.

The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

CD8+ T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion1. Although it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the ß1-adrenergic receptor ADRB1. We identify that exhausted CD8+ T cells increase ADRB1 expression and that exposure of ADRB1+ T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8+ T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of ß1-adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, ß-blockers and ICB synergize to boost CD8+ T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients2,3, and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

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.

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.

Spatiotemporal and cell-state control of antigen presentation during tolerance and immunity.

Effective adaptive immunity is rendered possible by highly organized tissue architecture and coordinated cellular crosstalk. While detailed spatiotemporal analyses of antigen presentation and adaptive immune activation in secondary lymphoid tissues have been a major focus of study, it is clear that antigen presentation in other tissues also plays a critical role in shaping the immune response. In this article, we concentrate on two opposing aspects of adaptive immunity: tolerance and antitumor immunity, to illustrate how a complex set of antigen presentation mechanisms contributes to maintaining a delicate balance between robust immunity and avoidance of autoimmune pathology. We emphasize the importance of how immune cell identity, state, and location collectively determine the nature of adaptive immune responses.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

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.

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 entities 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 entities in complex arrangements. To address this challenge, we present Spatial Patterning Analysis of Cellular Ensembles (SPACE), an R package for analysis of high-plex tissue images generated using any collection modality. Unlike existing platforms, SPACE detects context-dependent associations, quantitative gradients and orientations, and other organizational complexities. Via a robust information theoretic framework, SPACE explores all possible ensembles - single entities, pairs, triplets, and so on - and ranks the strongest patterns of tissue organization. Using lymph node images for which ground truth has been defined, we validate SPACE and demonstrate its advantages. We then use SPACE to reanalyze a public dataset of human tuberculosis granulomas, verifying known patterns and discovering new patterns with possible insights into disease progression.

How can systems approaches help us understand and treat infectious disease?

Leading researchers at the intersection of infectious disease and systems biology speak about how systems approaches have influenced modern infectious disease research and what these tools can offer for the future of the field.

Double knockin mice show NF-κB trajectories in immune signaling and aging.

In vitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins in vivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled. Overcoming hurdles in simultaneous live-cell imaging of RelA and c-Rel, we show that quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells, and shift toward c-Rel in microglia from aged brains. RelA:c-Rel heterodimer is unexpectedly depleted in the nuclei of stimulated cells. Trajectories of subunit co-expression in immune lineages reveal a reduction at key cell maturation stages. These results demonstrate the power of these reporters in gaining deeper insights into NF-κB biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications.

Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways.

The Toll-like receptor (TLR) and chemotaxis pathways are key components of the innate immune system. Subtle variation in the concentration, timing, and molecular structure of the ligands are known to affect downstream signaling and the resulting immune response. Computational modeling and simulation at the molecular interaction level can be used to study complex biological pathways, but such simulations require protein concentration values as model parameters. Here we report the development and application of targeted mass spectrometry assays to measure the absolute abundance of proteins of the mouse macrophage Toll-like receptor 4 (TLR4) and chemotaxis pathways. Two peptides per protein were quantified, if possible. The protein abundance values ranged from 1,332 to 227,000,000 copies per cell. They moderately correlated with transcript abundance values from a previously published mouse macrophage RNA-seq dataset, and these two datasets were combined to make proteome-wide abundance estimates. The datasets produced during this investigation can be used for pathway modeling and simulation, as well as for other studies of the TLR and chemotaxis pathways.

TGF-ß regulates the stem-like state of PD-1+ TCF-1+ virus-specific CD8 T cells during chronic infection.

Recent studies have defined a novel population of PD-1+ TCF-1+ stem-like CD8 T cells in chronic infections and cancer. These quiescent cells reside in lymphoid tissues, are critical for maintaining the CD8 T cell response under conditions of persistent antigen, and provide the proliferative burst after PD-1 blockade. Here we examined the role of TGF-ß in regulating the differentiation of virus-specific CD8 T cells during chronic LCMV infection of mice. We found that TGF-ß signaling was not essential for the generation of the stem-like CD8 T cells but was critical for maintaining the stem-like state and quiescence of these cells. TGF-ß regulated the unique transcriptional program of the stem-like subset, including upregulation of inhibitory receptors specifically expressed on these cells. TGF-ß also promoted the terminal differentiation of exhausted CD8 T cells by suppressing the effector-associated program. Together, the absence of TGF-ß signaling resulted in significantly increased accumulation of effector-like CD8 T cells. These findings have implications for immunotherapies in general and especially for T cell therapy against chronic infections and cancer.

Spatial meta-transcriptomics reveal associations of intratumor bacteria burden with lung cancer cells showing a distinct oncogenic signature.

BACKGROUND: The lung intratumor microbiome influences lung cancer tumorigenesis and treatment responses, but detailed data on the extent, location, and effects of microbes within lung tumors are missing, information needed for improved prognosis and treatment. METHODS: To address this gap, we developed a novel spatial meta-transcriptomic method simultaneously detecting the expression level of 1,811 host genes and 3 microbe targets (bacteria, fungi, and cytomegalovirus). After rigorous validation, we analyzed the spatial meta-transcriptomic profiles of tumor cells, T cells, macrophages, other immune cells, and stroma in surgically resected tumor samples from 12 patients with early-stage lung cancer. RESULTS: Bacterial burden was significantly higher in tumor cells compared with T cells, macrophages, other immune cells, and stroma. This burden increased from tumor-adjacent normal lung and tertiary lymphoid structures to tumor cells to the airways, suggesting that lung intratumor bacteria derive from the latter route of entry. Expression of oncogenic ß-catenin was strongly correlated with bacterial burden, as were tumor histological subtypes and environmental factors. CONCLUSIONS: Intratumor bacteria were enriched with tumor cells and associated with multiple oncogenic pathways, supporting a rationale for reducing the local intratumor microbiome in lung cancer for patient benefit. TRIAL REGISTRATION NUMBER: NCT00242723, NCT02146170.

Tuning T cell receptor sensitivity through catch bond engineering.

Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3-specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.