Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs

Abstract

The immune system is the most diffuse cellular system in the body. Accordingly, long-range migration of cells and short-range communication by local chemical signaling and by cell-cell contacts are vital to the control of an immune response. Cellular homing and migration within lymphoid organs, antigen recognition, and cell signaling and activation are clearly vital during an immune response, but these events had not been directly observed in vivo until recently. Introduced to the field of immunology in 2002, two-photon microscopy is the method of choice for visualizing living cells deep within native tissue environments, and it is now revealing an elegant cellular choreography that underlies the adaptive immune response to antigen challenge. We review cellular dynamics and molecular factors that contribute to basal motility of lymphocytes in the lymph node and cellular interactions leading to antigen capture and recognition, T cell activation, B cell activation, cytolytic effector function, and antibody production.

Figure 1

Basics of motility analysis. (a) Snapshot visualization of the 3-D locations of fluorescently labeled cells throughout the imaging volume at a single time point. Tracks of three cells are superimposed. The cells are CFSE-labeled allogeneic CD8⁺ T cells (green), CMTMR-labeled allogeneic CD4⁺ T cells (red), and CMF2HC-labeled syngeneic CD4⁺ T cells (blue) in the lymph node of B6D2F1 (H-2b/d) mice (Video 1a provided by Y. Yu). The total volume is (86 μm × 57 μm × 50 μm). (b) Flower plot representation of 2-D tracks of several cells, superimposed after normalizing their starting coordinates to the origin (adapted from Reference 26). (c) Schematic showing the track of a cell at five successive time points, t₁, t₂, etc. Instantaneous velocities are calculated from the net distance d traveled during each time interval Δt, and the turn angle θ is the angle through which the cell turns between time steps. The displacement D is the straight-line distance of the cell from its origin at any given time. Path length is given by the sum of d₁, d₂, d₃, etc. Persistence time (or length) is the time for which a cell continues to move without turning appreciably, as is illustrated for time points t₂–t₄. (d) The instantaneous velocity of T cells fluctuates in a characteristic manner over time, accompanied by changes in shape index (long axis/short axis) as the cell elongates while moving faster (adapted from Reference 2). (e) A plot of mean displacement from origin of multiple cells is expected to follow a straight line when plotted as a function of square root of time, if cell motility follows a random walk (adapted from Reference 26). The slope of this line can be used to derive a motility coefficient M, analogous to the diffusion coefficient for Brownian motion. Deviations from a straight-line relationship may be indicative of directed migration (upward curvature) or migration constrained by physical or biological barriers (plateau). (f) Chemotactic index. This measure, defined as path length/displacement from origin, remains constant with a value of unity for linear-directed motion but decreases progressively with time for cells following a random walk. Video may be viewed separately by following the Supplemental Materials link on the Annual Reviews website at http://www.annualreviews.org.

Figure 2

Structural and functional aspects of lymph node organization. (a) Macroscopic organization of lymph node regions, illustrating B cell follicles, T cell localization within the paracortex, and high endothelial venules (HEVs). (b) Schematic illustrating the motility of labeled T cells (orange) along and across the fibroblastic reticular scaffolding, in the presence of DC and an excess of unlabeled T cells (yellow), only a small fraction of which are shown in one region. (c) Hypothesis for regulation of lymphocyte egress by sphingosine 1-phosphate (S1P), illustrated under conditions of steady-state egress (left) and lymphopenia (right). T cells are shown with S1P₁ receptors either exposed or internalized in the presence of S1P. Junctions between the lymphatic endothelial cells close in the presence of an S1P₁ agonist.

Figure 3

Cell motility and interactions in the absence of antigen. T and B cells are depicted migrating randomly in diffuse cortex and follicles, respectively. A recently homed B cell is on its way toward the follicle. Macrophages and FDCs within the follicle are devoid of specific antigen. A T cell is shown interacting transiently with a resident DC, while another T cell crosses the lymphatic endothelium to gain access to the efferent lymphatic vessel. Shown on right are frames from Videos 3a, 3b, and 3c, respectively: (a) Montage of T cell and B cell dynamics, imaged separately in their respective regions (2); (b) T cells interacting transiently with a DC (96) (panel on the left is a “true” color maximum intensity projection along the z-axis and that on the right shows the same sequence after color depth encoding); (c) T cells migrating across the lymphatic endothelium (endothelial cells are stained with red fluorescent lectin) (84). Videos may be viewed separately by following the Supplemental Materials link on the Annual Reviews website at http://www.annualreviews.org.

Figure 4

Cellular choreography with antigen. B cells move by chemotaxis to the follicle edge, after picking up antigen from macrophages and from FDCs. A T cell/B cell conjugate pair is shown at the edge of a follicle, together with T cells clustering around a dendritic cell bearing cognate antigen. T cells proliferate at this time, and egress is blocked. Shown on right are frames from the following videos: (a) B cell chemotaxis (146); (b) T cell/B cell motile pairs (146); (c) T cell/DC clustering (84, 117); (d) stereo movie of T cell proliferation (26); (e) egress block and subsequent recovery following removal of S1P₁ agonist at lymphatic barrier. Video Links 4a–4e may be viewed separately by following the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org.