Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy

Abstract

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...

Characterizing T Cell Movement within Lymph Nodes in the Absence of Antigen

“…From these experiments, the locations of individual T cells moving in lymphoid organs are obtained as a function of time (5)(6)(7)(8). The instantaneous and mean velocities of T cells, their mean free paths, and their trajectories in the presence and the absence of Ag-presenting dendritic cells can thus be determined.…”

Video-rate two-photon imaging of mouse footpad – a promising model for studying leukocyte recruitment dynamics during inflammation

“…Moreover, since the mice are unharmed during the imaging process, it might be possible to perform longitudinal studies on individual research subjects. The first report of intravital 2P being used to study lymphoid tissues appeared in 2003 [11] and since then several variations of this approach have been widely adopted to study lymphocytes and antigen presenting cells in vivo [12]. This technique has changed our static view of the immune system and provided a tantalizing glimpse of the complex cellular dynamics that underlie the immune response [13].…”

T cell–antigen-presenting cell interactions visualized in vivo in a model of antigen-specific inflammation

“…Since the uveitis model employed for this study involves the introduction of a foreign antigen and the use of a TLR 4 agonist, LPS, the APCs should be stimulated and are presumably elaborating chemokines that attract T cells. Mean displacement plots (MDPs) are commonly employed as a method to determine if cell movement is random, directed, or confined as by a structure such as a vessel or a collagen bundle [26]. The MDPs in Figure 3 include data from 42 DO11.10 T cells in the irises of seven mice that had been injected with ovalbumin and in 24 DO11.10 T cells in the irises of ten control mice injected with BSA.…”

“…Lymphocytes with the T cell zone of a lymph node also follow paths that have been classified as analogous to random walks by mean displacement plots [26]. More recently it has been shown that the vast majority of lymph node T cells actively follow along fibers of the fibroblastic reticular cell network.…”