Making friends in out-of-the-way places: how cells of the immune system get together and how they conduct their business as revealed by intravital imaging.

A central characteristic of the immune system is the constantly changing location of most of its constituent cells. Lymphoid and myeloid cells circulate in the blood, and subsets of these cells enter, move, and interact within, then leave organized lymphoid tissues. When inflammation is present, various hematopoietic cells also exit the vasculature and migrate within non-lymphoid tissues, where they carry out effector functions that support host defense or result in autoimmune pathology. Effective innate and adaptive immune responses involve not only the action of these individual cells but also productive communication among them, often requiring direct membrane contact between rare antigen-specific or antigen-bearing cells. Here, we describe our ongoing studies using two-photon intravital microscopy to probe the in situ behavior of the cells of the immune system and their interactions with non-hematopoietic stromal elements. We emphasize the importance of non-random cell migration within lymphoid tissues and detail newly established mechanisms of traffic control that operate at multiple organizational scales to facilitate critical cell contacts. We also describe how the methods we have developed for imaging within lymphoid sites are being applied to other tissues and organs, revealing dynamic details of host-pathogen interactions previously inaccessible to direct observation.

Highways, byways and breadcrumbs: directing lymphocyte traffic in the lymph node.

The lymph node (LN) is charged with a crucial function in the mammalian immune system: to facilitate physical interactions between extremely rare cells arriving from different tissue compartments. Paramount to carrying out this function is its unique placement at the interface between the blood and lymphatic systems, thus enabling tissue-derived antigen and antigen-presenting cells, especially dendritic cells (DCs) to gather in close proximity to blood-derived antigen-specific motile lymphocytes. A generally held view is that this accumulation of cells, coupled with stochastic migration, is itself sufficient to facilitate a physiologically adequate frequency of cell-cell contacts due to random migration within the confined space of the LN. Based on recent data, we propose an expanded model of LN function in which unique architectural features and chemical signals together provide a means of enhancing otherwise unlikely encounters between sparse DCs and rare antigen-specific lymphocytes.

Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement.

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.

Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells.

In contrast to naïve T cells that recognize short antigen-derived peptides displayed by specialized antigen-presenting cells, immunoglobulin receptors of B lymphocytes primarily recognize intact proteins. How and where within a lymph node such unprocessed antigens become available for naïve B cell recognition is not clear. We used two-photon intravital imaging to show that, after exiting high-endothelial venules and before entry into lymph node follicles, B cells survey locally concentrated dendritic cells. Engagement of the B cell receptor by the dendritic cell (DC)-associated antigen leads to lymphocyte calcium signaling, migration arrest, antigen acquisition, and extrafollicular accumulation. These findings suggest a possible role for antigen-specific B-DC interactions in promoting T cell-dependent antibody responses in vivo.

Natural killer cell behavior in lymph nodes revealed by static and real-time imaging.

Natural killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo during the early phases of an adaptive immune response, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and shortly after infection with Leishmania major. In the LNs of naive mice, NK cells reside in the medulla and the paracortex, where they closely associate with DCs. In contrast to T cells, intravital microscopy revealed that NK cells in the superficial regions of LNs were slowly motile and maintained their interactions with DCs over extended times in the presence or absence of immune-activating signals. L. major induced NK cells to secrete interferon-gamma and to be recruited to the paracortex, where concomitant CD4 T cell activation occurred. Therefore, NK cells form a reactive but low mobile network in a strategic area of the LN where they can receive inflammatory signals, interact with DCs, and regulate colocalized T cell responses.

An extended vision for dynamic high-resolution intravital immune imaging.

The past few years have seen the application of confocal and especially two-photon microscopy to the dynamic high-resolution imaging of lymphocytes and antigen presenting cells within organs such as lymph nodes and thymus. After summarizing some of the published results obtained to date using these methods, we describe our view of how this technology will develop and be applied in the near future. This includes its extension to a wide variety of non-lymphoid tissues, to the tracking of functional responses in addition to migratory behavior, to the analysis of molecular events previously studied only in vitro, to dissection of the interplay between hematopoietic and stromal elements, to visualization of a wider array of cell types including neutrophils, macrophages, NK cells, NKT cells and others, and to the interaction of the host with infectious agents. Reaching these goals will depend on a combination of new tools for genetic manipulations, novel fluorescent reporters, enhanced instrumentation, and better surgical techniques for the extended imaging of live animals. The end result will be a new level of understanding of how orchestrated cell movement and interaction contribute to the physiological and pathological activities of the immune system.

Illuminating the landscape of in vivo immunity: insights from dynamic in situ imaging of secondary lymphoid tissues.

A central feature of the immune system is the migratory behavior of its cellular components. Thus, fully understanding the generation and maintenance of immune responses must include consideration of how hematopoietic cells home to, interact within, and exit from secondary lymphoid organs as well as peripheral tissues. Recent advances in in situ imaging techniques now permit direct observation of these events in their physiologic settings with high spatiotemporal resolution. This review summarizes progress in this area of investigation from a lymphocentric perspective. We highlight controversies, point out key unanswered questions, and briefly outline what we believe are some of the near-term directions that in situ microscopic analysis of the immune system will take.