Watching Immune Cells in Action

In contrast to most adult tissues, a unique feature f the vertebrate immune system is the motility of the mmune cells. Both innate and adaptive immune reponses are critically dependent on the migratory caacity of myeloid and lymphoid cells. For example, ffector cells of the innate immune system rapidly nter infected tissues, whereas sentinel dendritic cells DCs) in these sites mobilize and transit to lymph odes [1-5]. Lymphoid and myeloid cells also enter eripheral tissues in response to local signals such as hemokines and adhesion molecules present on the urface of endothelial cells [3]. In all of these sites, ells of the innate and adaptive immune systems comunicate extensively [6,7], each guiding the differeniation of the other, leading to changes in their paterns of gene expression, alterations in their repertoire f surface proteins, and production of effector moleules. These orchestrated series of events ensure the roper quality and magnitude of adaptive humoral and ell-mediated immune responses to infectious agents 8,9]. Equally important, the immune cells also interct with cells of the neighboring stroma, which has an mportant contribution to the eventual outcome of the mmune response. Until recently, support for this description of the mmune system comes from more than a century of xperimental analyses, with conclusions about in vivo ell dynamics inferred largely from serial static analsis of tissue sections (at relatively long intervals comared with the rate at which the underlying events ctually transpired) and dynamic studies of dissociated ells from in vitro cultures (in the absence of intact tromal elements normally present in situ) [10]. Even ith improvements in staining reagents, flow cytoetric equipment [11], and microscopy tools [12-14], hese early dynamic studies of immune cell behavior ave been limited to in vitro imaging of cultured cells, hich was used to clarify the molecular reorganization hat occurs during cell-cell contacts [15-17] and to ingle-color analysis of leukocyte behavior in small essels [18], which helped to confirm the rolling, stopd ing, and transmigration model of cell extravasations rom the blood to the tissues in situ [19]. However, ecent advances with in vivo imaging modalities have egun to afford direct visualization of these events in iving animals and tissue explants, allowing a renew ppreciation of the dynamics of immune cell behavior n situ. Positron emission tomography, magnetic resnance imaging, and luminescence imaging [20,21] ave been used to track immune cells in vivo. Alhough useful in tracking bulk population behavior, hese methods failed to offer the spatial resolution and ime scale required for single-cell analysis during an mmune response. Fortunately, advances in confocal nd, most recently, 2-photon laser scanning microsopy tools, combined with new intravital surgical aproaches that preserve the structural integrity, nervous nnervations, and blood and lymphatic circulation of the issues studied, have made high spatialand temporalesolution imaging of immune cell interaction in any tissues possible and have revolutionized our bility to probe more directly how immune cells oprate in vivo [22]. Two-photon microscopy was espeially attractive as a tool for intravital imaging because f the increased depth of tissue penetration with high patial resolution, high signal-to-noise ratio, and dereased photobleaching of out-of-focus planes [22,23], hus allowing prolonged periods of high-resolution ata collection on events that occur deep in intact tissues f living animals or intact tissue explants (Table 1). To date, data generated by 2-photon microscopy ethods have provided new perspectives and insights nto our understanding of how, where, and when cells nteract during the initiation of an immune response nd during the delivery of effector function at periphral sites [24-31]. Some initial studies have tracked the ovements of naive B and T cells after adoptive transer in the absence of antigenic stimulation, revealing he unexpectedly rapid pace of lymphocyte migration n secondary lymphoid tissues [25,26,31-38], a finding hat had important implications for our current modls of how rare antigen-specific lymphocytes and anigen-presenting cells (APCs) find one another to prouce an effective response. When a migrating T cell