CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo

Abstract

In contrast to lymphocyte homing, little is known about molecular cues controlling the motility of lymphocytes within lymphoid organs. Applying intravital two-photon microscopy, we demonstrate that chemokine receptor CCR7 signaling enhances the intranodal motility of CD4(+) T cells. Compared to wild-type (WT) cells, the average velocity and mean motility coefficient of adoptively transferred CCR7-deficient CD4(+) T lymphocytes in T cell areas of WT recipients were reduced by 33 and 55%, respectively. Both parameters were comparably reduced for WT T lymphocytes migrating in T cell areas of plt/plt mice lacking CCR7 ligands. Importantly, systemic application of the CCR7 ligand CCL21 was sufficient to rescue the motility of WT T lymphocytes inside T cell areas of plt/plt recipients. Comparing the movement behavior of T cells in subcapsular areas that are devoid of detectable amounts of CCR7 ligands even in WT mice, we failed to reveal any differences between WT and plt/plt recipients. Furthermore, in both WT and plt/plt recipients, highly motile T cells rapidly accumulated in the subcapsular region after subcutaneous injection of the CCR7 ligand CCL19. Collectively, these data identify CCR7 and its ligands as important chemokinetic factors stimulating the basal motility of CD4(+) T cells inside lymph nodes in vivo.

Figure 1.

Analysis of CD4⁺ T cell motility in the subcapsular region of the pLNs before and after injection of CCL19 into the footpad. (A) Cryosection of a WT pLN stained for CCL19 plus CCL21 (red). FITC channel tissue autofluorescence (green) illustrates the outline of the LN capsule. Note the absence of CCR7 ligands in the subcapsular region (white arrow head). White frames indicate the positions of typical imaging volumes (270 × 270 × 40 μm) for the SCS region (frame 1; 20–60 μm below capsule) and T cell area (frame 2; 140–180 μm below capsule). Bar, 100 μm. (B) TAMRA-labeled WT or CCR7-deficient (CCR7^−/−) CD4⁺ T cells were adoptively transferred into WT recipients. The subcapsular region of the pLN was imaged by intravital microscopy after injection of 1 μg CCL19 into the footpad together with FITC-labeled dextran as a tracer to highlight the SCS (green area). Screenshots of two representative movies are shown (see also Videos S1 and S3). Bar, 50 μm. Within 20 min after injection of CCL19, large numbers of highly motile TAMRA-labeled WT CD4⁺ T cells accumulate in the SCS (white arrow heads; Video S1). In contrast, TAMRA-labeled CCR7^−/− CD4⁺ T cells do not migrate toward high concentrations of exogenous CCL19 (Video S3). Note that in both cases numerous nonlabeled WT host cells, visible as black “shadows” in the surrounding FITC-labeled dextran, migrate into the SCS (white arrows in 4× magnification; Video S1 and S3). (C) TAMRA-labeled WT CD4⁺ T cells were adoptively transferred into WT or plt/plt (plt) recipients and the subcapsular region of the pLN was imaged by intravital microscopy before (−) or after (+) injection of 1 μg CCL19 into the footpad. Circles represent average cell velocities of individual cells, and white bars indicate median values. Graphs summarize collective data of all experiments performed (at least three independent experiments for each setup). After injection of CCL19, the median of average cell velocities of WT CD4⁺ T cells is equally increased by a factor of 2.6 in WT and plt recipients.

Figure 2.

Quantitative analysis of CD4⁺ T lymphocyte movement behavior in the T cell zone. TAMRA-labeled WT or CCR7-deficient (CCR7^−/−) CD4⁺ T cells were adoptively transferred into WT or plt/plt (plt) recipients, and the T cell area of the pLN was imaged by intravital microscopy. (A) Automated tracking of CD4⁺ T lymphocyte migration in the T cell zone. 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 for each experimental setting (see also Videos S4–S6). In the bottom panel, images are rotated 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. (B) Average cell velocity. Circles represent average cell velocities of individual cells, and white bars indicate median values. (C) MC (mean ± SEM). (D) Mean displacement plot. The approximately linear curves indicate random walk movement in all experimental settings analyzed. (E) Meandering index (mean ± SEM). Motility parameters of WT B cells imaged in B cell follicles of WT recipients (WT B cells in WT) are shown for comparison. Graphs summarize collective data of all experiments (at least three independent experiments for each setup). *, P ≤ 0.1; **, P ≤ 0.05; ***, P ≤ 0.01. n.s., not significant.

Figure 3.

Motility analysis of naive CD4⁺ T lymphocytes in the T cell zone of WT recipients. (A) FACS analysis of lymphocyte subsets before cell sorting. Comparable proportions of naive (CD62L⁺CD44^low) CD4⁺ T cells are present in pooled peripheral LNs and spleens from WT (WT) and CCR7^−/− (CCR7^−/−) donor mice. After FACS sorting for CD4⁺CD44^low, TAMRA-labeled WT or CCR7-deficient (CCR7^−/−) naive T cells were adoptively transferred into WT recipients, and the T cell area of the pLN was imaged by intravital microscopy. Graphs summarize collective data of all experiments (two independent experiments for each setup). (B) Average cell velocity. Circles represent average cell velocities of individual cells, and white bars indicate median values. (C) MC (mean ± SEM). (D) Meandering index (mean ± SEM). *, P ≤ 0.1; **, P ≤ 0.05. (E and F) FACS analysis of adoptively transferred TAMRA⁺ WT (E) and CCR7^−/− (F) lymphocytes isolated from peripheral LNs of WT recipient mice after completion of intravital imaging. In both cases, the TAMRA⁺ population contains mostly naive (CD62L⁺CD44^low) CD4⁺ T cells. Numbers indicate percentage of gated cells. Data shown are representative for two experiments.

Figure 4.

Systemic CCL21 application restores the motility of WT CD4⁺ T lymphocytes in the T cell area of plt recipients. TAMRA-labeled WT CD4⁺ T cells were adoptively transferred into plt recipients, and the T cell area of the pLN was imaged by intravital microscopy before (−) and 2.5 h after (+) i.v. injection of 100 μg CCL21. Graphs summarize collective data of all experiments (two independent experiments). (A) Average cell velocity. Circles represent average cell velocities of individual cells, and white bars indicate median values. (B) MC (mean ± SEM). (C) Meandering index (mean ± SEM). *, P ≤ 0.1; **, P ≤ 0.05.