Summary

The lymphatic network that transports interstitial fluid and antigens to lymph nodes constitutes a conduit system that can be hijacked by invading pathogens to achieve systemic spread unless dissemination is blocked in the lymph node itself. Here we show that a network of diverse lymphoid cells (NK cells, γδ T cells, NKT cells, and innate-like CD8+ T cells) are spatially pre-positioned close to lymphatic sinus-lining sentinel macrophages where they can rapidly and efficiently receive inflammasome-generated IL-18 and additional cytokine signals from the pathogen-sensing phagocytes. This leads to rapid IFNγ secretion by the strategically positioned innate lymphocytes, fostering anti-microbial resistance in the macrophage population. Interference with this innate immune response loop allows systemic spread of lymph-borne bacteria. These findings extend our understanding of the functional significance of cellular positioning and local intercellular communication within lymph nodes, while emphasizing the role of these organs as highly active locations of innate host defense.

Dendritic cells (DCs) regulate T cell function by promoting either tolerance or activation, and in the latter case, by directing response quality. New imaging tools now permit direct visualization of the relevant DC-T cell interactions in vivo and have provided a new perspective on the dynamics of these crucial cellular contacts. Here we discuss the insights generated by these analyses and the controversies/unanswered questions that need to be addressed in future work.

Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static – bone resorptive (R) to moving – nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.

Systems biology is an emerging discipline that combines high-content, multiplexed measurements with informatic and computational modeling methods to better understand biological function at various scales. Here we present a detailed review of the methods used to create computational models and conduct simulations of immune function, We provide descriptions of the key data gathering techniques employed to generate the quantitative and qualitative data required for such modeling and simulation and summarize the progress to date in applying these tools and techniques to questions of immunological interest, including infectious disease. We include comments on what insights modeling can provide that complement information obtained from the more familiar experimental discovery methods used by most investigators and why quantitative methods are needed to eventually produce a better understanding of immune system operation in health and disease.

Summary

The immune system, like all biological systems, operates based on a set of check and balances that strive for homeostasis of the organism. Because of its potent effector mechanisms, the potential for self-destructive immune responses is especially high and, whether to prevent autoreactivity or chronic inflammation, many negative regulatory modalities exist to prevent excessive tissue damage. This Commentary places such regulatory mechanisms in the larger context of system organization at multiple scales. The sometimes counter-intuitive nature of feedback control is discussed and a case is made for greater attention to quantitative spatiotemporal aspects of regulation, rather than limiting ourselves to the qualitative descriptions of pathways that dominate at present.

Summary

Flow cytometry allows highly quantitative analysis of complex dissociated populations at the cost of neglecting their tissue localization. In contrast, conventional microscopy methods provide spatial information, but visualization and quantification of cellular subsets defined by complex phenotypic marker combinations is challenging. Here we describe an analytical microscopy method, "Histo-Cytometry," for visualizing and quantifying phenotypically complex cell populations directly in tissue sections. This technology is based on multiplexed antibody staining, tiled high-resolution confocal microscopy, voxel gating, volumetric cell rendering, and quantitative analysis. We have tested this technology on various innate and adaptive immune populations in murine lymph nodes (LN) and were able to identify complex cellular subsets and phenotypes, achieving quantitatively similar results to flow cytometry, while also gathering cellular positional information. Here, we employ Histo-Cytometry to describe the spatial segregation of resident and migratory dendritic cell subsets into specialized micro-anatomical domains, suggesting an unexpected LN demarcation into discrete functional compartments.

Purpose

To develop and characterize the trafficking of a dual-modal agent that identifies primary draining or sentinel lymph node (LN).

Procedure

Herein, a dual-reporting silica-coated iron oxide nanoparticle (SCION) is developed. Nude mice were imaged by magnetic resonance (MR) and optical imaging and axillary LNs were harvested for histological analysis. Trafficking through lymphatics was observed with intravital and ex vivo confocal microscopy of popliteal LNs in B6-albino, CD11c-EYFP, and lys-EGFP transgenic mice.

Results

In vivo, SCION allows visualization of LNs. The particle’s size and surface functionality play a role in its passive migration from the intradermal injection site and its minimal uptake by CD11c+ dendritic cells and CD169+ and lys+ macrophages.

Conclusions

After injection, SCION passively migrates to LNs without macrophage uptake and then can be used to image LN(s) by MRI and fluorescence. Thus, SCION can potentially be developed for use in sentinel node resections or for intralymphatic drug delivery.

We present a method for computational reconstruction of the 3-D morphology of biological objects, such as cells, cell conjugates or 3-D arrangements of tissue structures, using data from high-resolution microscopy modalities. The method is based on the iterative optimization of Voronoi representations of the spatial structures. The reconstructions of biological surfaces automatically adapt to morphological features of varying complexity with flexible degrees of resolution. We show how 3-D confocal images of single cells can be used to generate numerical representations of cellular membranes that may serve as the basis for realistic, spatially resolved computational models of membrane processes or intracellular signaling. Another example shows how the protocol can be used to reconstruct tissue boundaries from segmented two-photon image data that facilitate the quantitative analysis of lymphocyte migration behavior in relation to microanatomical structures. Processing time is of the order of minutes depending on data features and reconstruction parameters.

Cellular signaling processes depend on specific spatiotemporal distributions of their molecular components. Multi-color high-resolution microscopy now permits detailed assessment of such distributions, providing the input for fine-grained computational models that explore the mechanisms governing dynamic assembly of multi-molecular complexes and their role in shaping cellular behavior. However, incorporating into such models both complex molecular reaction cascades and the spatial localization of signaling components within dynamic cellular morphologies presents substantial challenges. Here we introduce an approach that addresses these challenges by automatically generating computational representations of complex reaction networks based on simple bi-molecular interaction rules embedded into detailed, adaptive models of cellular morphology. Using examples of receptor-mediated cellular adhesion and signal-induced localized MAPK activation in yeast, we illustrate the capacity of this simulation technique to provide insights into cell biological processes. The modeling algorithms, implemented in a version of the Simmune tool set, are accessible through intuitive graphical interfaces as well as programming libraries.

The wealth of information available from advanced fluorescence imaging techniques used to analyze biological processes with high spatial and temporal resolution calls for high-throughput image analysis methods. Here, we describe a fully automated approach to analyzing cellular interaction behavior in 3-D fluorescence microscopy images. As example application we present the analysis of drug-induced and S1P1-knock-out-related changes in bone-osteoclast interactions. Moreover, we apply our approach to images showing the spatial association of dendritic cells with the fibroblastic reticular cell network within lymph nodes and to microscopy data about T-B lymphocyte synapse formation. Such analyses that yield important information about the molecular mechanisms determining cellular interaction behavior would be very difficult to perform with approaches that rely on manual/semi-automated analyses. This protocol integrates adaptive threshold segmentation, object detection, adaptive color channel merging and neighborhood analysis and permits rapid, standardized, quantitative analysis and comparison of the relevant features in large data sets.

Treg are key players in maintaining immunhomeostasis but have also been shown to regulate immune responses against infectious pathogens. Therefore Treg are a promising target for modulating immune responses to vaccines in order to improve their efficacy. Using a viral vector system, we found that Treg act on the developing immune response early after infection by reducing the extent of dendritic cell costimulatory molecule expression. Due to this change and the lower IL-2 production that results, a substantial fraction of CD8+ effector T cells lose CD25 expression several days after activation. Surprisingly, such Treg-dependent limitations in IL-2 signaling by antigen-activated CD8+ T cells prevent effector differentiation without interfering with memory cell formation. In this way Treg fine-tune the numbers of effector T cells generated, while preserving the capacity for a rapid recall response upon pathogen re-exposure. This selective effect of Treg on a subpopulation of CD8+ T cells indicates that while manipulation of the Treg compartment might not be optimal for prophylactic vaccinations, it can be potentially exploited to optimize vaccine efficacy for therapeutic interventions.

Summary

Granulomas play a key role in host protection against mycobacterial pathogens, with their breakdown contributing to exacerbated disease. To better understand the initiation and maintenance of these structures, we employed both high-resolution multiplex static imaging and intravital multiphoton microscopy of Mycobacterium bovis BCG-induced liver granulomas. We found that Kupffer cells directly capture blood-borne bacteria and subsequently nucleate formation of a nascent granuloma by recruiting both uninfected liver-resident macrophages and blood-derived monocytes. Within the mature granuloma, these myeloid cell populations form a relatively immobile cellular matrix that interacts with a highly dynamic effector T cell population. The efficient recruitment of these T cells is highly dependent on TNFα-derived signals, which also maintain the granuloma structure through preferential effects on uninfected macrophage populations. By characterizing the migration of both innate and adaptive immune cells throughout the process of granuloma development, these studies provide a new perspective on the cellular events involved in mycobacterial containment and escape.

Cells lining the gastrointestinal tract serve as both a barrier to and a pathway for infectious agent entry. Dendritic cells (DCs) present in the lamina propria under the columnar villus epithelium of the small bowel extend processes across this epithelium and capture bacteria, but previous studies provided limited information on the nature of the stimuli, receptors, and signaling events involved in promoting this phenomenon. Here, we use immunohistochemical as well as dynamic explant and intravital two-photon imaging to investigate this issue. Analysis of CD11c–enhanced green fluorescent protein (EGFP) or major histocompatibility complex CII-EGFP mice revealed that the number of trans-epithelial DC extensions, many with an unusual “balloon” shape, varies along the length of the small bowel. High numbers of such extensions were found in the proximal jejunum, but only a few were present in the terminal ileum. The extensions in the terminal ileum markedly increased upon the introduction of invasive or noninvasive Salmonella organisms, and chimeric mouse studies revealed the key role of MyD88-dependent Toll-like receptor (TLR) signaling by nonhematopoietic (epithelial) elements in the DC extension response. Collectively, these findings support a model in which epithelial cell TLR signaling upon exposure to microbial stimuli induces active DC sampling of the gut lumen at sites distant from organized lymphoid tissues.

The major histocompatibility complex (MHC)-dependent presentation of processed tissue-specific self-antigens can contribute to either peripheral (extrathymic) tolerance or the differentiation of autoreactive T cells. Here, we have studied the MHC class II molecule presentation of gastric parietal cell (PC)-specific H+/K+-ATPase, which induces a destructive autoimmune gastritis in BALB/c mice lacking CD4+ CD25+ regulatory T cells. Immunofluorescence microscopy showed physical association of CD11c+ dendritic cells (DCs) with PCs in the gastric mucosa. H+/K+-ATPase protein was found within vesicular compartments of a few CD11c+ DCs only in the draining gastric lymph node (LN) and these antigen-containing DCs increased markedly in number with the onset of tissue destruction in autoimmune animals. Both CD8αhi and CD8αlo gastric DCs, but not peripheral or mesenteric DCs, showed evidence of constitutive in vivo processing and presentation of H+/K+-ATPase. These data provide direct support for a widely held model of local tissue antigen uptake and trafficking by DCs in normal animals and demonstrate that DCs in the draining LN can present a tissue-specific self-antigen under noninflammatory conditions without fully deleting autoreactive T cells or inducing active autoimmunity.

Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50–100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000–30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.

Intravenous (i.v.) injection of high amounts of soluble proteins often results in the induction of antigen-specific tolerance or deviation to helper rather than inflammatory T cell immunity. It has been proposed that this outcome may be due to antigen presentation to T cells by a large cohort of poorly costimulatory or IL-12–deficient resting B cells lacking specific immunoglobulin receptors for the protein. However, previous studies using T cell activation in vitro to assess antigen display have failed to support this idea, showing evidence of specific peptide–major histocompatibility complex (MHC) class II ligand only on purified dendritic cells (DC) or antigen-specific B cells isolated from protein injected mice. Here we reexamine this question using a recently derived monoclonal antibody specific for the T cell receptor (TCR) ligand formed by the association of the 46-61 determinant of hen egg lysozyme (HEL) and the mouse MHC class II molecule I-Ak. In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61–Ak complexes on their surface. DC readily isolated from spleen also display this TCR ligand on their surface. Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells. These results demonstrate that in the absence of activating stimuli, both lymphoid DC and antigen-unspecific B cells present to a similar extent class II–associated peptides derived from soluble proteins in extracellular fluid. The numerical advantage of the TCR ligand–bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.

T cell receptor (TCR) recognition of peptide–major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4+ cell population. Low concentrations of TCR ligand elicit only interferon-γ (IFN-γ) production. Increasing ligand recruits more cells into the IFN-γ+ pool, increases IFN-γ produced per cell, and also elicits IL-2, but only from cells already making IFN-γ. Most cells producing only IFN-γ show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-γ synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-γ/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-γ and not IL-2, and the amount of IFN-γ exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.

Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II β chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for Ii-dependent determinants. This demonstrates that related leucine-based trafficking signals in Ii and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation of ligand from an engaged TCR and (b) recruitment of lck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide–MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-ζ, reduced tyrosine phosphorylation of CD3ε, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR–ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide–MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment of immunological tolerance.

Signaling through the T cell receptor for antigen (TCR) has been studied for years by conventional biochemical means. More recently, attempts have been made to develop computational models of signaling through this receptor, with a specific focus on understanding how this recognition system discriminates between closely related (self and non-self) ligands. Here we discuss recent advances centered on the role of feedback regulation, especially the key finding that a combination of digital and analog control circuits is fundamental to the discrimination properties of the TCR. We end by pointing to future, more biologically accurate models that incorporate spatial aspects of molecular organization in antigen-engaged T lymphocytes with this underlying biochemistry.

Summary

Cell-mediated adaptive immunity is critical for host defense, but little is known about T cell behavior during delivery of effector function. Here we investigate relationships among antigen presentation, T cell motility, and local production of effector cytokines by CD4+ T cells within hepatic granulomas triggered by Bacille Calmette-Guérin or Mycobacterium tuberculosis. At steady-state, only small fractions of mycobacteria-specific T cells showed antigen-induced migration arrest within granulomas, resulting in low-level, polarized secretion of cytokines. However, exogenous antigen elicited rapid arrest and robust cytokine production by the vast majority of effector T cells. These findings suggest that limited antigen presentation and/or recognition within granulomas evoke a muted T cell response drawing on only a fraction of the host’s potential effector capacity. Our results provide new insights into the regulation of host protective functions, especially how antigen availability influences T cell dynamics and in turn, effector T cell function during chronic infection.

Chemotaxis and chemorepulsion of osteoclast precursors depends on S1P concentrations and expression of the receptors S1PR1 and S1PR2, which act to regulate osteoclast precursor localization.

Sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, controls the dynamic migration of osteoclast (OC) precursors (OPs) between the blood and bone, in part via the S1P receptor 1 (S1PR1) which directs positive chemotaxis toward S1P. We show that OPs also express S1PR2, an S1P receptor which mediates negative chemotaxis (or chemorepulsion). OP-positive chemotaxis is prominent in gradients with low maximal concentrations of S1P, whereas such behavior is minimal in fields with high maximal S1P concentrations. This reverse-directional behavior is caused by S1PR2-mediated chemorepulsion acting to override S1PR1 upgradient motion. S1PR2-deficient mice exhibit moderate osteopetrosis as a result of a decrease in osteoclastic bone resorption, suggesting that S1PR2 contributes to OP localization on the bones mediated by chemorepulsion away from the blood where S1P levels are high. Inhibition of S1PR2 function by the antagonist JTE013 changed the migratory behavior of monocytoid cells, including OPs, and relieved osteoporosis in a mouse model by limiting OP localization and reducing the number of mature OCs attached to the bone surface. Thus, reciprocal regulation of S1P-dependent chemotaxis controls bone remodeling by finely regulating OP localization. This regulatory axis may be promising as a therapeutic target in diseases affecting OC-dependent bone remodeling.

The success of a non-live vaccine requires improved formulation and adjuvant selection to generate robust T cell immunity following immunization. Here, using protein linked to a TLR7/8 agonist (conjugate vaccine), we investigated the functional properties of vaccine formulation, the cytokines, and the DC subsets required to induce protective multifunctional T cell immunity in vivo. The conjugate vaccine required aggregation of the protein to elicit potent Th1 CD4+ and CD8+ T cell responses. Remarkably, the conjugate vaccine, through aggregation of the protein and activation of TLR7 in vivo, led to an influx of migratory DCs to the LN and increased antigen uptake by several resident and migratory DC subsets, with the latter effect strongly influenced by vaccine-induced type I IFN. Ex vivo migratory CD8–DEC205+CD103–CD326– langerin-negative dermal DCs were as potent in cross-presenting antigen to naive CD8+ T cells as CD11c+CD8+ DCs. Moreover, these cells also influenced Th1 CD4+ T cell priming. In summary, we propose a model in which broad-based T cell–mediated responses upon vaccination can be maximized by codelivery of aggregated protein and TLR7/8 agonist, which together promote optimal antigen acquisition and presentation by multiple DC subsets in the context of critical proinflammatory cytokines.