motility ͉ two-photon microscopy N atural killer (NK) cells are a specialized subset of lymphocytes capable of mediating effector functions without the need for prior sensitization. To avoid aberrant autoimmune attack, NK cells express a diverse repertoire of inhibitory receptors that suppress lytic activity. Polymorphic Ly49 molecules specific for self-MHC class I constitute one of the best characterized family of inhibitory receptors (1). Under normal conditions, persistent engagement of Ly49 by cognate selfligands results in recruitment of SHP-1 to the receptor's cytoplasmic immunoreceptor tyrosine-based inhibitory motif domain and subsequent dephosphorylation of activating signaling pathways (2). This inhibition of NK cell effector function is alleviated in the absence of self-molecules and is often the first step in acute rejection of allogeneic grafts. Although NK cellmediated clearance of circulating MHC-mismatched targets is well established (3), the ability of NK cells to migrate to and eliminate allogeneic target cells from peripheral lymph nodes (LN) under homeostatic conditions remains poorly defined.Recent evidence suggests that NK cells may exert important functional responses within LN. Specifically, enhanced recruitment of NK cells to antigen-stimulated LN facilitated the induction of a productive immune response (4). Moreover, Chen et al. (5) showed that LN NK cells efficiently lysed tumor cells and suppressed B16 melanoma metastasis to draining LN. These findings suggest that NK cells actively patrol the LN in search of cognate target and transformed cells, perhaps exhibiting robust motility similar to other lymphocyte subsets, such as T and B cells (6, 7). Therefore, it was surprising that NK cells isolated by DX5-positive selection were reported to move with slow velocity (2.75 Ϯ 0.17 m/min) when imaged in LN by two-photon microscopy (8).DX5 is a monoclonal antibody commonly used to identify and isolate murine NK cells. DX5 binds the ␣ 2 -integrin CD49b (9). Integrins are heterodimers comprised of ␣-and ␤-subunits and are of key importance in several complex biological processes, including cell adhesion and migration (10). Experimental evidence indicates that integrin engagement can result in the activation of key signaling events (11). Specifically, cross-linking of ␤ 1 -integrins expressed on human NK cells transduces intracellular signals, leading to the activation of the Ras-MAPK cascade, subsequent IFN-␥ production, and enhanced cytotoxicity (12). Therefore, CD49b cross-linking could induce signaling events that render positively selected NK cells immotile. To investigate this possibility, we used two-photon microscopy to visualize the in situ basal motility of unmanipulated NK cells within intact LN and examine their interaction dynamics with syngeneic and allogeneic B cells. Our results demonstrate that CD49b cross-linking strongly modulates motility, adhesion to collagen, and effector function, whereas unmanipulated NK cells are highly motile and preferentially form stable conjugate...

T cell activation by APC requires cytosolic Ca2+ ([Ca2+]i) elevation. Using two-photon microscopy, we visualized Ca2+ signaling and motility of murine CD4+ T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca2+ spikes associated with sustained slowing. Inflammation reduced velocities and Ca2+ spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca2+ spike frequency and elevated basal [Ca2+]i. These Ca2+ signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca2+]i and spiking frequency constitute a Ca2+ signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.

Direct presentation of foreign MHC molecules expressed by donor-derived dendritic cells (DCs) has generally been considered the dominant pathway of allorecognition in acute transplant rejection. However, recent studies implicate preferential activation of the indirect pathway by host DCs. The respective importance of each pathway and the mechanisms that determine their relative contributions remain to be clearly established. In this study, using two-photon microscopy, we visualized host NK cell interactions with syngeneic and allogeneic DCs within intact lymph nodes of mice. Upon contact with allogeneic DCs, NK cells formed prolonged interactions that led directly to target cell lysis. This rapid elimination limited the ability of allogeneic DCs to stimulate primary and recall T cell responses. To discriminate whether donor or host DCs are principally involved in presenting Ag derived from allografts, we used CD11c-diphtheria toxoid receptor mice to conditionally ablate CD11c+ DCs and to show that direct presentation by donor DCs is alone insufficient to elicit acute allograft rejection. We thus propose that rapid elimination of allogeneic DCs limits direct Ag presentation and thereby favors the indirect pathway of alloreactivity.

We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests. Analysis of in vitro data showed that these periods frequently coincided with contacts with target cells, sometimes leading to target cell lysis. However, NK cells were also commonly observed to stop independently of contact with other cells. In order to objectively characterize the migration of NK cells, we implemented a simple method to discriminate when NK cells stop or have low motilities, have periods of directed migration or undergo random movement. This was achieved using a sliding window approach and evaluating the mean squared displacement (MSD) to assess the migration coefficient and MSD curvature along trajectories from individual NK cells over time. The method presented here can be used to quickly and quantitatively assess the dynamics of individual cells as well as heterogeneity within ensembles. Furthermore, it may also be used as a tool to automatically detect transient stops due to the formation of immune synapses, cell division or cell death. We show that this could be particularly useful for analysis of in situ time-lapse fluorescence imaging data where most cells, as well as the extracellular matrix, are usually unlabelled and thus invisible.

International audienceT lymphocytes are highly motile cells that decelerate upon antigen recognition. These cells can either completely stop or maintain a low level of motility, forming contacts referred to as synapses or kinapses, respectively. Whether similar or distinct molecular mechanisms regulate T-cell deceleration during synapses or kinapses is unclear. Here, we used microfabricated channels and intravital imaging to observe and manipulate T-cell kinapses and synapses. We report that high-affinity antigen induced a pronounced deceleration selectively dependent on Ca 2+ signals and actin-related protein 2/3 complex (Arp2/3) activity. In contrast, low-affinity antigens induced a switch of migration mode that promotes T-cell exploratory behavior, characterized by partial deceleration and frequent direction changes. This switch depended on T-cell receptor binding but was largely independent of downstream signaling. We propose that distinct mechanisms of T-cell deceleration can be triggered during antigenic recognition to favor local exploration and signal integration upon suboptimal stimulus and complete arrest on the best antigen-presenting cells