Our primary objective was to describe cell behavior and interactions in native tissues during T cell priming, from initial contact with antigen-bearing DCs to cell division. To accomplish this, we used two-photon microscopy to image OVA-specific CD4+
T cells with endogenous DCs in intact lymph nodes. Because it is not currently possible to monitor continuously a given T cell from initial contact with an antigen-bearing DC to cell division, we used an experimental protocol ( A) designed to synchronize responses within the adoptively transferred T cell population. The key features of this approach are as follows: (a) the use of abundant antigen (~100 μg of OVA, bound to alum) to activate a majority of antigen-specific T cells in the system, and (b) the adoptive transfer of T cells only after allowing sufficient time (18–24 h) for antigen-bearing DCs to traffic to the lymph node (19
). This procedure also avoided the presence of soluble antigen within the lymph node (23
) and ensured that the DC pool arriving from the periphery contained CFSE-labeled DCs that were competent to present antigen (19
Figure 1. Two-photon imaging of T cell activation in the lymph node. (A) Time line of our experimental protocol. (B–E) Time lapse images of T cells (red) and DCs (green), displayed as maximum intensity projections along the z axis (top view) through image (more ...)
Within 1 h of adoptive transfer, most T cells homed to lymph nodes and few remained in the circulation (Video S1, available at http://www.jem.org/cgi/content/full/jem.20041236/DC1
). Thus, the great majority of labeled T cells in the draining node encounter antigen-bearing DCs at approximately the same time, thereby synchronizing their responses. Moreover, this period of synchronization would persist during the first day because T cells that had initially homed to nondraining nodes remain sequestered there for ~24 h (25
). We imaged T cells and DCs in lymph nodes during continuous periods of 30 min–2.5 h at different times after T cell transfer (time zero). As illustrated in , and Videos S2–S7 (available at http://www.jem.org/cgi/content/full/jem.20041236/DC1
), T cells exposed to cognate antigen progress through distinct stages defined by their behavior. In the following sections, we describe these stages in sequential order, provide a quantitative comparison of T cell behavior at each stage, and conclude with an assessment of T cell activation induced by this immunization protocol.
Default T Cell Trafficking and Antigen-independent DC Interactions in the Lymph Node.
As a prelude to examining antigen-induced T cell behaviors, we first established whether T cells exhibited time-dependent changes after adoptive transfer and homing in the absence of cognate antigen.
shows examples of noncognate interactions between T cells and DCs, and illustrates our procedure for cell tracking and contact analysis. Individual T cell tracks are superimposed on true-color images ( A, dotted lines), and contacts between T cells and DCs (arrowheads) were confirmed if both cells were at the same depth in depth-encoded images ( B). As reported previously (19
), T cells encountered DCs randomly, decelerating only slightly while in contact with DCs, and quickly migrated away after ~3 min ( C and Video S2). T cells from sham-immunized mice showed no significant change in T cell velocity (averaging 9.6 μm/min−1
; D), motility coefficient ( E), or contact duration with DCs (averaging 3.2 min) when imaged at 2, 8, 12, 18, or 24 h after adoptive transfer. This description of baseline naive T cell behavior serves as a basis of comparison with cognate interactions in OVA-primed mice.
Figure 2. Noncognate interactions at various times after T cell homing. (A–C) Representative interactions between T cells and DCs 12 h after adoptive transfer in a control mouse without antigen. (A) True-color image of a single time point showing a T cell (more ...)
Cognate Interactions at <2 h: Dynamic and Serial T Cell–DC Contacts.
In contrast with the default, noncognate interactions described before, distinct differences in T cell behavior were apparent within <2 h in the lymph nodes of OVA-challenged mice. Most T cells moved in characteristic looping patterns, making serial contacts with the same or with neighboring DCs (, and Videos S3 and S4). As a result, the overall T cell motility decreased sharply (mean velocity = 5.4 μm/min−1 and motility coefficient = 9.7 μm2/min−1: ). Contacts occurred preferentially on DC dendrites, fluctuated rapidly in size over tens of seconds, and involved an average membrane area of ~10 μm2. Interactions were more prolonged in the presence of antigen (mean: 11.4 min vs. 3.2 min in sham-immunized mice), but usually remained intermittent (), with the exception of a few T cells that showed associations lasting >1 h (e.g., ). Perhaps the most striking observation is that interactions between T cells and dendritic cells are for the most part unstable early in the immune response.
Figure 3. Early cognate interactions, 1–3 h. (A–C) Three examples of T cell–DC interactions imaged 1–3 h after adoptive transfer into an OVA-challenged mouse. Each row shows a true-color snapshot with overlain T cell track. Bars, (more ...)
Interactions at 2–14 h: T Cell Clusters.
The next stage was characterized by the formation of dense T cell clusters around DCs (). Contact areas at this stage were roughly 16 μm2, and contact durations increased such that many T cells remained associated with DCs for longer than the typical duration (~60 min) of our imaging sequence. Nevertheless, the T cell clusters were dynamic entities because cells continuously changed their relative positions and individual T cells were sometimes added or lost (, and Video S5). T cell–DC interactions were terminated either by the T cell moving away or by the DC withdrawing its dendrite. In some instances (), an entire cluster of T cells transferred from one DC to another (Video S5). T cells in clusters showed spherical morphology, and their movement resulted primarily from cells being carried along on migrating DCs. During this period, the average T cell velocity was only 2.6 μm/min ( G), and the motility coefficient was 2.3 μm2/min ( H).
Figure 4. T cell clusters after T cell transfer. (A–E) Cognate T cell–DC interactions imaged 8–10 h after adoptive transfer into an OVA-challenged mouse. Images and measurements from 39 T cells were obtained as in . Numbers within (more ...)
The aforementioned results were obtained with our standard immunization protocol (~100 μg OVA), which produced clustering behavior in >80% of T cells. With lesser amounts of OVA (<10 μg), far fewer T cells were observed in clusters, and many were freely motile (Video S6).
Interactions at 16–24 h: T Cell Swarms.
By this time, the clusters had largely dissociated, and T cells were visibly enlarged. These T cell blasts moved slowly in a looping pattern within a local area ( and Video S7), a behavior we termed “swarming” (15
). Although some cells remained stably associated with DCs (), most swarmed around DCs, making intermittent, sweeping contacts, often involving successive contacts with several DCs (). These contacts lasted on average 20 min and involved roughly 24 μm2
of membrane surface area. In comparison with the preceding cluster stage, T cell velocities increased to 4.1 μm/min, and the motility coefficient to 6.3 μm2
Late T cell swarms. (A–E) Cognate T cell–DC interactions imaged 16–18 h after adoptive transfer into an OVA-challenged mouse. Images and measurements from 23 cells were obtained as in –.
T Cell Behaviors at >24 h: Proliferation and Resumption of Autonomous Motility.
After 24 h, T cell swarming behavior diminished and many T cell blasts migrated autonomously, making only infrequent, brief (mean ~12 min) contacts with DCs ( and Video S8, available at http://www.jem.org/cgi/content/full/jem.20041236/DC1
). The overall T cell velocity averaged 4.6 μm/min, but individual blasts often showed appreciably higher mean velocities (8–9 μm/min). At this time, we observed many instances of cell division ( E and Video 9, available at http://www.jem.org/cgi/content/full/jem.20041236/DC1
). T cell blasts stopped abruptly, rounded up, paused for ~15 min, and cleaved into daughter cells within ~5 min. The daughter cells rapidly regained motility, sometimes trailing long membrane tethers as they moved away from each other. Finally, by 40 h, most T cells had undergone one or more rounds of division (assessed by dilution of CFSE fluorescence) and were migrating randomly with a mean velocity of 9.5 μm/min, similar to that of naive T cells (15
Figure 6. T cell proliferation and resumption of motility. (A–D) Time lapse sequence showing T cell motility and cell division 26 h after T cell transfer. (left) Full-frame images with superimposed tracks of four T cells (different colors). Bars, 25 μm. (more ...)
Progressive Changes in T Cell Motility and DC Contact Durations.
summarizes the progressive changes in T cell velocity, motility coefficient, and DC contact duration throughout T cell priming. Changes in T cell motility are most readily apparent in the superimposed cell tracks shown in A, and in the plot of motility coefficient ( C), which provides a measure of how far a cell would have moved on average from its starting point after any given time. In the absence of cognate antigen, T cells roamed widely along random paths with high motility coefficient, a strategy that enhances efficient scanning of the T cell repertoire (19
). After transfer into OVA-challenged mice, T cell displacement decreased rapidly ( A), illustrated by the dramatic compression of cell tracks at 2 and 8 h. Motility gradually recovered as shown by cell tracks that covered a broader territory at 16, 24, and 40 h. By 40 h, most T cells had resumed autonomous migration, dispersing throughout the T cell region. The transient reduction in T cell velocity and motility coefficient () retained T cells near antigen-bearing DCs, and was associated with increased T cell–DC contact durations ( D). In OVA-challenged mice, contact durations increased by 2 h and peaked at 8 h, by which time the majority of contacts lasted longer than the imaging record. After this clustering period, contact durations decreased to ~20 min at 16 h, and further to ~10 min at 24 h. Without OVA, T cell–DC interactions lasted only minutes, seen as a rapid decay in the contact persistence plot.
Figure 7. Analysis of T cell motility at different stages of activation. (A) Tracks of individual T cells (different colors, normalized to their starting coordinates) showing representative motility of cells in control experiments (without OVA) and at various times (more ...)
Population Measures of T Cell Activation, Proliferation, and Effector Function.
To correlate our single cell imaging data with functional measures of T cell activation, we assayed CD69 expression, proliferation, and DTH. By 2 h, ~40% of antigen-specific T cells had up-regulated CD69 ( A, B), and by 24 h this increased to ~80% ( C). There was no evidence of proliferation in draining lymph nodes at this time ( D), but after 3 d T cells had undergone as many as six rounds of division ( E). By day 5, the number of divisions increased to >10 ( F). Thus, beginning at ~24 h when we first observed instances of cell proliferation directly by two-photon imaging, T cells proliferated with a minimum doubling time of ~8 h. On day 5, distal nondraining nodes in OVA-challenged mice contained T cells that had divided more than four times, in addition to a population of undivided T cells ( G), indicating that many OVA-specific T cells were trafficking throughout the body as central memory cells (26
). Finally, the capacity of expanded OVA-specific T cells to mount an effector response was confirmed by the presence of a DTH reaction after injection of soluble OVA into the ear on day 9 ( H). Moreover, OVA-specific T cells that had divided more than six times were present in ears exhibiting DTH, but not in the ears of sham-immunized animals that were similarly challenged ( I). Together, these results provide evidence that the single cell behavior we have described here reflects productive T cell–DC interactions.
Figure 8. Kinetics of T cell activation, proliferation, and effector response. (A–C) CD69 expression by DO11.10 T cells in control and OVA-challenged mice at the times indicated. (D–F) T cell proliferation assessed by CFSE dilution measured 1, 3, (more ...)