Two‐Photon Imaging of the Immune System

Dzhagalov, Ivan; Melichar, Heather J.; Ross, Jenny O.; Herzmark, Paul; Robey, Ellen

doi:10.1002/0471142956.cy1226s60

Cited by 47 publications

(44 citation statements)

References 30 publications

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“…Mouse and human thymic slices were prepared essentially as described previously (55). For mouse tissue, individual thymic lobes were embedded in 4% low-melt agarose dissolved in HBSS.…”

Section: Discussionmentioning

confidence: 99%

Opposing chemokine gradients control human thymocyte migration in situ

Halkias¹,

Melichar²,

Taylor³

et al. 2013

J. Clin. Invest.

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The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4 + CD8 + double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.

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“…Mouse and human thymic slices were prepared essentially as described previously (55). For mouse tissue, individual thymic lobes were embedded in 4% low-melt agarose dissolved in HBSS.…”

Section: Discussionmentioning

confidence: 99%

Opposing chemokine gradients control human thymocyte migration in situ

Halkias¹,

Melichar²,

Taylor³

et al. 2013

J. Clin. Invest.

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“…Two-photon imaging was performed on 1-cm lengths of small intestine that were explanted, butterflied open, immobilized muscle side down on a glass coverslip, and perfused in warmed oxygenated medium (63). In some experiments, the intestinal epithelium was labeled by staining the explanted tissue with the styryl dye FM 4-64 (Molecular Probes) for 1 min on ice immediately before imaging.…”

Section: Methodsmentioning

confidence: 99%

Motile invaded neutrophils in the small intestine ofToxoplasma gondii-infected mice reveal a potential mechanism for parasite spread

Coombes

Charsar

Han

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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129

149

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Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laserscanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.neutrophil motility | dynamic imaging | gut | mucosal immunology T oxoplasma gondii infects around a third of humans worldwide and is widely dispersed in other warm-blooded hosts. Although clinical manifestations in the brain, eye, and developing fetus receive the most attention, T. gondii is an oral pathogen and first enters the body and establishes infection in the small intestine. Infection follows consumption of cyst-containing meat or oocyst-contaminated water and produce and is associated with the development of small intestinal pathology in a variety of nonhuman hosts (1). Most notably, experimental infection of C57BL/6 mice by the oral route results in an inflammation of the small intestine that shares immunological features with inflammatory bowel disease (2). This model is useful to further our understanding of host-pathogen interactions in the intestine and of common mechanisms underpinning the development of inflammatory bowel disease (3). Nevertheless, we have limited understanding of how and in which cells infection is established in the intestine, the extent to which the parasite replicates and spreads within the intestine, and how these factors contribute to the development of pathology (2, 4-9). The ability to label living parasites fluorescently and track them in the tissues of infected hosts provides an important tool for investigating these questions (10)(11)(12)(13)(14).Starting in the small intestine, T. gondii must travel long distances and surmount a variety of biological barriers to establish chronic infection in the brain. These barriers include the mucus, the intestinal epithelium, and the blood-brain barrier (7,15). Cells of the immune system are often highly motile and represent attractive transport vessels for pathogens seeking to reach and enter tissues while being protected from the external environment. Consequently, recent studies have focused on the role of immune cells in transporting parasites between tissues (4, 16-23). For example, cluster of differentiation 11b-positive (CD11b + ) cells have been implicated in the dissemination of T. gondii through the blood and across the blood-brain barrier (4, 19). Following oral infection, i...

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“…Vibratome-cut thymic slices were generated as described (6,49). Briefly, thymic lobes were embedded in 4% (wt/vol) low-melting agarose in HBSS, and 500-μm slices were prepared for imaging studies or 400-μm slices were prepared for flow-cytometric analysis.…”

Section: Methodsmentioning

confidence: 99%

Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

Ross

Melichar

Au-Yeung

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3-4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca 2+ ] i ) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca 2+ ] i over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus.Zap70 | thymic slice | two photon microscopy D eveloping thymocytes test their newly formed T-cell antigen receptors (TCRs) for their ability to bind self-peptide:major histocompatibility complex (MHC) complexes on thymic support cells. This process encompasses both positive and negative selection and ultimately leads to the formation of a functional and self-tolerant mature T-cell repertoire. During positive selection, CD4 + CD8 + [double positive (DP)] thymocytes with TCRs weakly reactive to self-peptide:MHC complexes receive signals to survive, mature, and give rise to CD4 or CD8 single positive (SP) cells. Positive selection requires close contact with thymic stromal cells and occurs over a period of several days (1-4). However, much of our information about T-cell development is based on analyses of steady-state thymocyte populations, and the individual steps that occur during the prolonged process of positive selection remain obscure.Thymocytes are highly motile within three-dimensional thymic tissue environments, and positive selection is tightly linked to thymocyte migration. The initial encounters with positive-selecting ligands on cortical thymic epithelial cells do not induce strong migratory stop signals, but instead occur as brief migratory pauses associated with transient elevations in intracellular calcium concentration ([Ca 2+ ] i ) (5-7). These brief, serial TCR signals are consistent with indications that positive selection is driven by weak recognition of self-peptide:MHC complexes and with the unique ability of DP thymocytes to respond to low-potency ligands (8, 9). However, there are also indications that thymocytes that have already received TCR signals still require further signaling to complete positive selection (10, 11). Thus, although the encounters with positive-selecting ligands last for...

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Two‐Photon Imaging of the Immune System

Cited by 47 publications

References 30 publications

Opposing chemokine gradients control human thymocyte migration in situ

Opposing chemokine gradients control human thymocyte migration in situ

Motile invaded neutrophils in the small intestine ofToxoplasma gondii-infected mice reveal a potential mechanism for parasite spread

Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

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Two‐Photon Imaging of the Immune System

Cited by 47 publications

References 30 publications

Opposing chemokine gradients control human thymocyte migration in situ

Opposing chemokine gradients control human thymocyte migration in situ

Motile invaded neutrophils in the small intestine ofToxoplasma gondii-infected mice reveal a potential mechanism for parasite spread

Distinct phases in the positive selection of CD8+T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

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Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns