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4D Tracking with Imaris

Immunology

Naive T cells constantly recirculate through lymph nodes (LNs) in search of their cognate antigen. Several studies analyzing intranodal T cell migration have established a model of naive T cells migrating in a mode of random walk while scanning the T cell area. In contrast to lymphocyte homing, only little is known about molecular cues controlling the motility of lymphocytes within lymphoid organs. Dr. Tim Worbs, Prof. Reinhold Förster and colleagues from the Institute of Immunology at Hannover Medical School, are applying intravital two-photon microscopy to investigate the modulatory effects of different chemokines on the basal motility level of T cells.

Figure 1: Quantitative analysis of CD4+ T lymphocyte movement behavior in the T-cell zone Automated tracking of CD4+ T lymphocyte migration in the T cell zone using Imaris: TAMRA-labeled wild type (WT) or CCR7-deficient (CCR7−/−) CD4+ T cells were adoptively transferred into WT or plt recipients. 150 randomly chosen trajectories of individual cells are displayed as color-coded tracks to represent increasing time from blue (start of imaging) to yellow (end of imaging). A representative imaging session of 15 min is shown. In the bottom panel, images are rotated in 3D to display the z-dimension of the imaged volumes. Grid spacing (distance between major tick marks) is 10 μm for x, y, and z orientation in all images.

Figure 2: Average cell velocity. Circles represent average cell velocities of individual cells, white bars indicate median values.

The present study demonstrates that chemokine receptor CCR7 signaling enhances the motility of CD4+ T cells inside the popliteal LNs (pLNs). To address the role of CCR7 signaling for the intranodal motility of CD4+ T cells, the paracortex, the LN compartment in which CCR7 ligands are naturally present was analyzed using intravital two-photon laser-scanning microscopy. Adoptively transferred wild type (WT) or CCR7-deficient (CCR7-/- ) CD4+ T cells were imaged in the paracortical T cell areas of WT and plt (plt/plt mice lacking CCR7 ligands) recipients. Imaris was used for four-dimensional image analysis and automated tracking of cells. The accuracy of the automated tracking was manually controlled, and only tracks with durations of >60 s were included in the analysis. Average cell velocity and meandering index were calculated using Imaris. At first glance, the overall mode of movement seemed to be comparable in the presence or absence of CCR7 signaling (see Figure 1). However, the velocity was obviously reduced for CCR7-/- T cells in WT recipients and for WT T cells migrating in the T cell zone of plt recipient (see Video 1, Video2, and Video3, created with Imaris), and quantitative analysis by automated cell tracking confirmed a significant decrease of the median of the average cell velocities of CCR7 -/- T cells (see figure 2).

These data provide for the first time direct in vivo evidence for a chemokinetic function of CCR7 and its ligands stimulating the basal motility of CD4+ T cells inside lymph nodes. The enhanced motility allows for a faster, more widespread movement of T cells in the T cell area, thereby potentially enhancing the likelihood of encounters with resident dendritic cells (DCs) presenting rare cognate antigens.

Author: Dr. T. Worbs and Prof. R. Förster and colleagues, Hannover Medical School, Germany

Category: Application Note

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