The IBEX Knowledge-Base: Achieving more together with open science.

Iterative Bleaching Extends multipleXity (IBEX) is a versatile method for highly multiplexed imaging of diverse tissues. Based on open science principles, we created the IBEX Knowledge-Base, a resource for reagents, protocols and more, to empower innovation.

Blunting specific T-dependent antibody responses with engineered "decoy" B cells.

Antibody inhibitors pose an ongoing challenge to the treatment of subjects with inherited protein deficiency disorders, limiting the efficacy of both protein replacement therapy and corrective gene therapy. Beyond their central role as producers of serum antibody, B cells also exhibit many unique properties that could be exploited in cell therapy applications, notably including antigen-specific recognition and the linked capacity for antigen presentation. Here we employed CRISPR-Cas9 to demonstrate that ex vivo antigen-primed Blimp1-knockout "decoy" B cells, incapable of differentiation into plasma cells, participated in and downregulated host antigen-specific humoral responses after adoptive transfer.

Host and pathogen genetic diversity shape vaccine-mediated protection to .

To investigate how host and pathogen diversity govern immunity against (Mtb), we performed a large-scale screen of vaccine-mediated protection against aerosol Mtb infection using three inbred mouse strains [C57BL/6 (B6), C3HeB/FeJ (C3H), Balb/c x 129/SvJ (C129F1)] and three Mtb strains (H37Rv, CDC1551, SA161) representing two lineages and distinct virulence properties. We compared three protective modalities, all of which involve inoculation with live mycobacteria: Bacillus Calmette-Guérin (BCG), the only approved TB vaccine, delivered either subcutaneously or intravenously, and concomitant Mtb infection (CoMtb), a model of pre-existing immunity in which a low-level Mtb infection is established in the cervical lymph node following intradermal inoculation. We examined lung bacterial burdens at early (Day 28) and late (Day 98) time points after aerosol Mtb challenge and histopathology at Day 98.

Distal Immunization and Systemic Cytokines Establish a Transient Immune Alert State in the Intestine.

Conventionally, immune responses are studied in the context of inflamed tissues and their corresponding draining lymph nodes (LNs). However, little is known about the effects of systemic inflammatory signals generated during local inflammation on distal tissues and nondraining LNs. Using a mouse model of cutaneous immunization, we found that systemic inflammatory stimuli triggered a rapid and selective distal response in the small intestine and the mesenteric LN (mesLN).

CD4-mediated immunity shapes neutrophil-driven tuberculous pathology.

Pulmonary (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity.

Site-specific regulation of Th2 differentiation within lymph node microenvironments.

T helper 2 (Th2) responses protect against pathogens while also driving allergic inflammation, yet how large-scale Th2 responses are generated in tissue context remains unclear. Here, we used quantitative imaging to investigate early Th2 differentiation within lymph nodes (LNs) following cutaneous allergen administration. Contrary to current models, we observed extensive activation and "macro-clustering" of early Th2 cells with migratory type-2 dendritic cells (cDC2s), generating specialized Th2-promoting microenvironments.

Prolonged T cell - DC macro-clustering within lymph node microenvironments initiates Th2 cell differentiation in a site-specific manner.

Formation of T helper 2 (Th2) responses has been attributed to low-grade T cell stimulation, yet how large-scale polyclonal Th2 responses are generated remains unclear. Here, we used quantitative imaging to investigate early Th2 differentiation within lymph nodes (LNs) following cutaneous allergen administration. Contrary to current models, Th2 differentiation was associated with enhanced T cell activation and extensive integrin-dependent 'macro-clustering' at the T-B border, which also contrasted clustering behavior seen during Th1 differentiation.

A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging.

Information flow in the spatiotemporal organization of immune responses.

Immune responses must be rapid, tightly orchestrated, and tailored to the encountered stimulus. Lymphatic vessels facilitate this process by continuously collecting immunological information (ie, antigens, immune cells, and soluble mediators) about the current state of peripheral tissues, and transporting these via the lymph across the lymphatic system. Lymph nodes (LNs), which are critical meeting points for innate and adaptive immune cells, are strategically located along the lymphatic network to intercept this information.

Multi-Parameter Quantitative Imaging of Tumor Microenvironments Reveals Perivascular Immune Niches Associated With Anti-Tumor Immunity.

Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6.

TGFβ restricts expansion, survival, and function of T cells within the tuberculous granuloma.

CD4 T cell effector function is required for optimal containment of Mycobacterium tuberculosis (Mtb) infection. IFNɣ produced by CD4 T cells is a key cytokine that contributes to protection. However, lung-infiltrating CD4 T cells have a limited ability to produce IFNɣ, and IFNɣ plays a lesser protective role within the lung than at sites of Mtb dissemination.

Innate cell microenvironments in lymph nodes shape the generation of T cell responses during type I inflammation.

Microanatomical organization of innate immune cells within lymph nodes (LNs) is critical for the generation of adaptive responses. In particular, steady-state LN-resident dendritic cells (Res cDCs) are strategically localized to intercept lymph-draining antigens. Whether myeloid cell organization changes during inflammation and how that might affect the generation of immune responses are unknown.

Ultra-low Dose Aerosol Infection of Mice with Mycobacterium tuberculosis More Closely Models Human Tuberculosis.

Tuberculosis (TB) is a heterogeneous disease manifesting in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). Unlike human TB, murine infection results in uniformly high lung bacterial burdens and poorly organized granulomas. To develop a TB model that more closely resembles human disease, we infected mice with an ultra-low dose (ULD) of between 1-3 founding bacteria, reflecting a physiologic inoculum.

CytoMAP: A Spatial Analysis Toolbox Reveals Features of Myeloid Cell Organization in Lymphoid Tissues.

Recently developed approaches for highly multiplexed imaging have revealed complex patterns of cellular positioning and cell-cell interactions with important roles in both cellular- and tissue-level physiology. However, tools to quantitatively study cellular patterning and tissue architecture are currently lacking. Here, we develop a spatial analysis toolbox, the histo-cytometric multidimensional analysis pipeline (CytoMAP), which incorporates data clustering, positional correlation, dimensionality reduction, and 2D/3D region reconstruction to identify localized cellular networks and reveal features of tissue organization.

Cutting Edge: Bacillus Calmette-Guérin-Induced T Cells Shape Infection before Reducing the Bacterial Burden.

Growing evidence suggests the outcome of infection is established rapidly after exposure, but how the current tuberculosis vaccine, bacillus Calmette-Guérin (BCG), impacts early immunity is poorly understood. In this study, we found that murine BCG immunization promotes a dramatic shift in infected cell types. Although alveolar macrophages are the major infected cell for the first 2 weeks in unimmunized animals, BCG promotes the accelerated recruitment and infection of lung-infiltrating phagocytes.

Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues.

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories.

The Chemoattractant Receptor Ebi2 Drives Intranodal Naive CD4 T Cell Peripheralization to Promote Effective Adaptive Immunity.

Lymph nodes (LNs) play critical roles in adaptive immunity by concentrating in one location the antigens, antigen-presenting cells, and antigen-responsive lymphocytes involved in such responses. Recent studies have revealed nonrandom localization of innate and adaptive immune cells within these organs, suggesting that microanatomical positioning optimizes responses involving sparse cooperating cells. Here, we report that the peripheral localization of LN cDC2 dendritic cells specialized for MHC-II antigen presentation is matched by a similarly biased paracortical distribution of CD4 T cells directed by the chemoattractant receptor Ebi2.

High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging.

Understanding the structure-function relationships between diverse cell types in a complex organ environment requires detailed in situ reconstruction of cell-associated molecular properties in the context of 3D, macro-scale tissue architecture. We recently developed clearing-enhanced 3D (Ce3D), a simple and effective method for tissue clearing that achieves excellent transparency; preserves cell morphology, tissue architecture, and reporter molecule fluorescence; and is robustly compatible with direct immunolabeling. These characteristics permit high-quality multiplex fluorescence microscopy of large tissue volumes, as well as image analysis using advanced platforms such as volumetric histocytometry, collectively allowing quantitative characterization of cells with respect to their spatial positioning within tissues on the basis of phenotypic and functional markers.

Tissue clonality of dendritic cell subsets and emergency DCpoiesis revealed by multicolor fate mapping of DC progenitors.

Conventional dendritic cells (cDCs) are found in all tissues and play a key role in immune surveillance. They comprise two major subsets, cDC1 and cDC2, both derived from circulating precursors of cDCs (pre-cDCs), which exited the bone marrow. We show that, in the steady-state mouse, pre-cDCs entering tissues proliferate to give rise to differentiated cDCs, which themselves have residual proliferative capacity.

Impact of Polymer-TLR-7/8 Agonist (Adjuvant) Morphology on the Potency and Mechanism of CD8 T Cell Induction.

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (∼300 nm submicrometer particles, ∼10 nm micelles and ∼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity).

Alveolar Macrophages Provide an Early Mycobacterium tuberculosis Niche and Initiate Dissemination.

Mycobacterium tuberculosis (Mtb) infection is initiated in the distal airways, but the bacteria ultimately disseminate to the lung interstitium. Although various cell types, including alveolar macrophages (AM), neutrophils, and permissive monocytes, are known to be infected with Mtb, the initially infected cells as well as those that mediate dissemination from the alveoli to the lung interstitium are unknown. In this study, using a murine infection model, we reveal that early, productive Mtb infection occurs almost exclusively within airway-resident AM.

Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism.

The mammalian gut is colonized by numerous microorganisms collectively termed the microbiota, which have a mutually beneficial relationship with their host. Normally, the gut microbiota matures during ontogeny to a state of balanced commensalism marked by the absence of adverse inflammation. Subsets of innate lymphoid cells (ILCs) and conventional T cells are considered to have redundant functions in containment and clearance of microbial pathogens, but how these two major lymphoid-cell populations each contribute to shaping the mature commensal microbiome and help to maintain tissue homeostasis has not been determined.

Dendritic cell and antigen dispersal landscapes regulate T cell immunity.

Dendritic cell (DC) subsets with biased capacity for CD4 and CD8 T cell activation are asymmetrically distributed in lymph nodes (LNs), but how this affects adaptive responses has not been extensively studied. Here we used quantitative imaging to examine the relationships among antigen dispersal, DC positioning, and T cell activation after protein immunization. Antigens rapidly drained into LNs and formed gradients extending from the lymphatic sinuses, with reduced abundance in the deep LN paracortex.