Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy - PubMed (original) (raw)

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

Mark J Miller et al. Proc Natl Acad Sci U S A. 2003.

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 naive 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 approximately 11 microm x min(-1). T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 microm x min(-1). An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naive 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.

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Figures

Figure 1

Figure 1

Intravital 2P imaging system. (Top and Middle) Schematic diagram of the 2P microscope and intravital imaging chamber. PMT, photomultiplier tube. (Bottom) The structure of a lymph node, showing the relative positions of B cell follicles, T cell areas in the diffuse cortex, HEVs, overlying capsule, and lymphatic and circulatory connections.

Figure 2

Figure 2

Intravital imaging of vessels and cells in a living lymph node. (A) 3D reconstruction representing a 85 × 120 × 75-μm volume of the T cell area, centered 120 μm below the surface of the lymph node. (Scale bars, 30 μm in all axes.) CFSE-labeled naïve T cells (green) are observed in the vicinity of a presumptive HEV (red), identified by i.v. injection of tetramethylrhodamine dextran. (B) Video-rate imaging of a T cell flowing in a small vessel within a T cell region of the node. Image is a superposition of nine consecutive video frames acquired at 34-ms intervals and shows the progression of a single CFSE-labeled T cell traveling at ≈20 mm⋅min−1 (0.03 cm⋅s−1) within a blood vessel. (Scale bar, 25 μm.)

Figure 3

Figure 3

In vivo motility of T cells. (A) Sequence of images at ≈1-min intervals illustrating T cell migration. Each panel shows a compression along the z axis (top view) derived from a 51-μm-deep _z_-stack. Four individual cells and their corresponding tracks (dots tracked at intervals of 10 s) are pseudocolored for illustration. Times are in min/s. (Scale bar, 20 μm.) (B) Velocity fluctuations of five individual T cells. Velocities were computed point by point from positions during consecutive 10-s intervals. (C) Fourier analysis of velocity fluctuations derived from the velocity traces in B, illustrating cyclical fluctuations with a characteristic period of ≈2 min. (D) Distribution of instantaneous T cell velocities. Lateral (x_–_y) velocities were measured from point-to-point tracks at 10-s intervals (n = 2,930 measurements in three experiments). Velocities <3 μm⋅min−1 correspond to pauses of motile cells rather than to a population of nonmotile cells.

Figure 4

Figure 4

T cell migration proceeds autonomously through the T cell areas. (A) Overlay of 39 individual T cell tracks plotted after aligning their starting positions. Cells were tracked over a 12-min period. (Inset) Shown are the mean coordinates of entire populations of T cells. Units are in μm. (B) Plot shows the mean absolute displacement of individual T cells (n = 3 experiments) away from their starting points as a function of square root of time. A random walk process is expected to yield a straight line on this transformed scale, and the red line shows a linear regression to the data. Error bars are ±SEM.

Figure 5

Figure 5

Axial motion of T cells. (Scale bars, 25 μm.) (A) Color encoding of axial position of T cells. Representative images derived from a single _z_-stack, showing compressions along the z axis (top view) and y axis (side view). The axial (z) depth of cells within the stack is encoded by a pseudocolor scheme as illustrated in the side view [red, top 6 μm of stack (closer to the capsule); yellow, 6–15 μm; green, middle 30 μm; cyan, 36–45 μm; blue, 45–51 μm]. (B) Time-lapse image sequence, using color-encoding to allow tracking of T cells in 3D. Boxes frame a cell that began at the bottom of the imaging volume and moved upward toward the capsule. The complete image sequence can be viewed in Movie 1. (C) Distribution of T cell speed in the z axis (up or down). (D) Bar graph showing numbers of cells tracked as moving upward (toward the capsule) or downward. Data are from three experiments. Error bars indicate 95% confidence interval for a binomial distribution consistent with random cell trafficking in the z axis.

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