Interstitial motility

Lymphocytes entering the T cell zone in peripheral lymph nodes are exposed to high levels of CCR7 and CXCR4 binding chemokines. Our recent work suggests that these extravascular chemokines preferentially operate to trigger T cell motility and encounter of antigen presenting dendritic cells (DCs) in stromal-presented (two-dimensional) rather than soluble states. We have developed an in vitro live imaging set-up to follow how human T cells locomoting on immobilized chemokines encounter DCs and how specific antigenic signals stop lymphocyte subsets on differently stimulated DCs (Figure 1 and Movies 4, 5 and 6).

Figure 1 In vitro imaging of T cells (labeled with the inert dye BCECF) locomoting over immobilized CCL21, the key chemokine found in lymph node T zones. T cells are rapidly locomoting around monocyte-differentiated dendritic cells (grey), derived from a matching donor, but fail to generate any stable contacts in the absence of an antigen. The image is taken from Movie 6

The predominant T zone chemokine is CCL21. Most lymphocytes rapidly locomote on networks of fibroblastic reticular stromal cells (FRCs) presenting this chemokine in an immobilized state as they touch and scan FRC associated dendritic cells for antigenic signals. Assessing the mechanisms by which this chemokine and ICAM-1 can support random motility, we found that a lawn of immobilized chemokines (CCL21, CCL19 or CXCL12) is necessary and sufficient for rapid T cell motility, whereas soluble counterparts of these chemokines fail to promote motility even in the presence of integrin ligands. Notably, the ability of weak GPCR-ligand bonds to support contractile motility did not involve homologous GPCR desensitization, allowing the lymphocyte to retain persistent motility for hours without noticeable decay in responsiveness to these surface-presented chemokines. Most surprisingly, when the lymph node chemokines were encountered by motile T cells together with high levels of FRC or DC expressed integrin ligands, like ICAM-1 and VCAM-1, the integrin receptors to these ligands remained largely silent unless lymphocytes were exposed to external shear forces. Furthermore, neither the extent of motility nor the ability of motile cells to occasionally stop on chemokine-CAM coated surfaces was affected by integrin blockage. In vivo analysis of murine T cells lacking functional LFA-1 confirmed these results, although the speed of motile T cells was slightly reduced. Thus, LFA-1 does not contribute to any detectable T cell-T cell or T cell-DC sticking in vivo or in vitro, consistent with effective silencing of chemokine clustered LFA-1 in the absence of antigenic signals. In conclusion, while LFA-1 and VLA-4 adhesiveness is robustly activated when lymphocytes encounter chemokines and juxtaposed integrin ligands on endothelial cells in the presence of shear forces, these integrins are generally silenced in shear-free extravascular environments.

Figure 2 Surface-presented CCL21 triggers robust motility and polarized redistribution of LFA-1 and VLA-4 in migrating T cells. (a) Live images of LFA-1 (top) or of the α4 integrins VLA-4 and α4b7 (bottom) in T cells migrating in vitro. Representative images were taken at the indicated time intervals. Cells were labeled with either Alexa568-tagged TS2/4 (non-blocking anti-αL) or Alexa488-tagged B5G10 (non-blocking anti-α4). Upper rows depict consecutive merged DIC and fluorescence images. Lower rows depict spectrum analysis of the relative fluorescence intensity.