Lymphocyte motility is vital for trafficking within lymphoid organs and for initiating contact with antigen-presenting cells. Visualization of these processes has previously been limited to in vitro systems. We describe the use of two-photon laser microscopy to image the dynamic behavior of individual living lymphocytes deep within intact lymph nodes. In their native environment, T cells achieved peak velocities of more than 25 micrometers per minute, displaying a motility coefficient that is five to six times that of B cells. Antigenic challenge changed T cell trajectories from random walks to "swarms" and stable clusters. Real-time two-photon imaging reveals lymphocyte behaviors that are fundamental to the initiation of the immune response.

The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naïve CD4 ؉ T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of Ϸ11 m⅐min ؊1 . T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 m⅐min ؊1 . An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naïve T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells. C D4 ϩ T cells play a pivotal role in the initiation and subsequent coordination of the immune response. Recirculation of naïve T cells between the blood and secondary lymphoid organs is critical for the detection of foreign antigens in tissues throughout the body (1-4), and the organized structure of lymphoid tissues (5, 6) makes it possible for T cells to sample a local landscape of antigen, discriminating foreign from selfdeterminants. Evidence has been reviewed recently (7-11) that chemokine gradients orchestrate the cellular organization of the lymphoid organs and direct the trafficking of lymphocytes within these tissues. However, these dynamic phenomena have never been observed in vivo. Despite the enormous progress in understanding the molecular and cellular details that shape the immune response, we still know little about the motility and migratory behavior of individual T cells as they pass through the secondary lymphoid organs (12).Lymphocyte function is contingent on numerous environmental factors that cannot readily be replicated by in vitro studies. For example, motility has been studied in vitro by using activated T cells and T cell lines on a variety of substrates, but with little consensus on velocities or control by intracellular signaling pathways (13-17). Moreover, striking differences in T cell͞ antigen-presenting cell interaction dynamics and activation requirements have been observed depending on the culture system (16,(18)(19)(20). Clearly, in vivo studies are essential for understanding lymphocyte function as it occurs in the natural tissue environment. Intravital microscopy has been suc...

Dendritic cells (DCs) ingest antigens in peripheral tissues and migrate to lymph nodes where they present MHC class II-bound antigen to CD4 ؉ T cells. We used two-photon microscopy to image the single-cell dynamics of interactions between DCs and T cells within intact lymph nodes in the absence of relevant antigen. DCs were fluorescently labeled in vivo by cutaneous injection of alum adjuvant including carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE-positive DCs (CD11c ؉ , CD11b ؉ , and low-to-intermediate CD8 ؉ ) were observed in draining lymph nodes 24 -72 h later. Labeled DCs meandered slowly (2-3 m⅐min ؊1 ) in the T cell zone near B cell follicles but vigorously extended long agile dendrites. Encounters between T cells and DCs arose as T cells moved autonomously along random paths. Moreover, T cells did not accumulate around DCs, and their relative velocities approaching and departing DCs were equivalent, implying that T cells are not attracted toward DCs by chemotactic gradients but rather encounter them by chance. T cell͞DC contacts occurred primarily on dendrites at arm's length from the DC soma and typically lasted Ϸ3 min, enabling an individual DC to interact with up to 5,000 T cells per hour. We conclude that dynamic DC gesticulation and random T cell motility together enhance the stochastic scanning of the T cell repertoire, thereby enabling rapid initiation of the immune response.two-photon microscopy ͉ T lymphocyte

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

Many lymphocyte functions, such as antigen recognition, take place deep in densely populated lymphoid organs. Because direct in vivo observation was not possible, the dynamics of immune-cell interactions have been inferred or extrapolated from in vitro studies. Two-photon fluorescence excitation uses extremely brief (<1 picosecond) and intense pulses of light to 'see' directly into living tissues, to a greater depth and with less phototoxicity than conventional imaging methods. Twophoton microscopy, in combination with newly developed indicator molecules, promises to extend single-cell approaches to the in vivo setting and to reveal in detail the cellular collaborations that underlie the immune response.Scientific questions drive the development of technology, and new technologies, in turn, influence the type of questions that we pose in an iterative process of discovery. Applied to immunology, this has prompted the innovation and application of powerful techniques for analysing the microscopic world of the immune system. Two methodological lines of investigation have dominated the field: in vivo experiments that examine the behaviour of populations of cells in living animals during an immune response; and in vitro experiments that use individual cells in artificial environments. An immune response is the sum of many complex and dynamic individual cellular behaviours that are shaped by many environmental factors. In vivo experiments maintain this natural environment, but they cannot resolve the behaviours of individual cells. By contrast, in vitro experiments provide information at the subcellular and molecular levels, but they cannot replicate adequately the full repertoire of environmental factors. There is a pressing need for techniques that allow real-time observation of single cells and molecules in intact tissues. Recent developments in imaging technology now make this possible. In this review, we provide a guide to the application of biophotonic techniques to the field of immunology and, in particular, to the use of two-photon laser microscopy. We discuss recent results obtained using this approach and pinpoint some key questions that might be resolved by tissue imaging. The biophotonics tool kitImmunologists have long been adept at raiding other disciplines to acquire new research tools. Fluorescence techniques -for example, flow cytometry, video imaging and more exotic imaging modalities, such as FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) and two-photon microscopy -combined with new probes to track Ca 2+ signalling, Correspondence to M.D.C. mcahalan@uci.edu. NIH Public Access Imaging T-cell dynamics in vitroThe single-cell approach has intrinsic value, because asynchronous behaviours are not indicated by population measurements. The need for live-tissue imagingIn vivo approaches have elucidated the basic properties of immune cells and lymphoid tissues, whereas in vitro cell-culture systems have provided a high-definition analysis of the cellsurface receptors and intracellular signalling pat...