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Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces

Nature Immunology volume 8, pages 1076–1085 (2007)Cite this article

Abstract

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that 'decorate' lymph node stroma. In a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma. The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail to stimulate stable integrin adhesiveness in extravascular shear-free environments.

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Figure 1: Immobilized but not soluble CCL21 promotes sustained locomotion of resting peripheral blood T lymphocytes without apparent desensitization.
Figure 2: Immobilized CCL21 triggers polarized redistribution of LFA-1 and α4 integrins.
Figure 3: LFA-1 on T cells migrating on CCL21 does not bind ICAM-1.
Figure 4: LFA-1 mediates stable adhesions in lymphocytes moving on immobilized CCL21 and ICAM-1 under shear stress but not in shear-free conditions.
Figure 5: VLA-4 is not adhesive in T cells migrating on CCL21 and juxtaposed integrin ligands in shear-free conditions.
Figure 6: Microtubules contribute to firm CCL21-induced LFA-1 adhesiveness under shear stress but are not required for lymphocyte motility on CCL21 and ICAM-1.
Figure 7: LFA-1 is not required for lymphocyte motility toward and over DCs and stroma expressing ICAM-1.
Figure 8: Slight increase in T cell velocity in lymph nodes by CD18.

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Acknowledgements

We thank D. Zipori and A. Nagler for providing human stromal cells; M. Krummel for help with the two-photon microscope; G. Shakhar for discussions; and S. Schwarzbaum for editorial assistance. CD18-deficient mice were provided by E. Brown (University of California, San Francisco); antibodies TS1/18 and TS2/4 were from T. Springer (Harvard University); monoclonal antibody 13 was from K. Yamada (National Institutes of Health); antibody B5G10 was from M. Hemler (Harvard University); and 240Q and A308926 (control compound, A270412) were from D.E. Staunton (ICOS). Supported by the Cancer Research Institute (I.G.), the German Research Foundation (M.S.), the Max Planck Society (M.S.), the National Institutes of Health (AI45073 to J.G.C.), the Linda Jacobs Chair in Immune and Stem Cell Research (R.A.), the Israel Science Foundation (R.A.), the Minerva Foundation (R.A.) and MAIN, the EU6 Program for Migration and Inflammation (R.A.).

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Authors and Affiliations

  1. Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100, Israel

    Eilon Woolf, Adi Sagiv, Valentin Grabovsky, Sara W Feigelson, Ziv Shulman, Tanja Hartmann & Ronen Alon

  2. Department of Microbiology and Immunology and Howard Hughes Medical Institute, University of California, San Francisco, 94143, California, USA

    Irina Grigorova & Jason G Cyster

  3. Max Planck Institute of Biochemistry, Munich, 82152, Germany

    Michael Sixt

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Contributions

E.W., most in vitro assays; I.G., multiphoton imaging; A.S., assistance with lymphocyte motility studies; V.G., flow chamber studies; S.W.F., motility analysis and integrin staining; Z.S., cytoskeleton staining; T.J., immunoblots; M.S., immunohistochemistry; J.G.C., in vivo experimental design and contributions to manuscript preparation; R.A., conceptual design and manuscript preparation.

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Correspondence to Ronen Alon.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 795 kb)

Supplementary Video 1

Surface bound CCL21 triggers robust and random motility of CD3+ PBL. Representative time lapse movie corresponding to Fig. 1a,b, depicting human T lymphocytes migrating between 10 and 20 min on immobilized CCL21 (600 sites/ μm2) co-immobilized with collagen and fibronectin. 1 sec movie = 1 min. (MOV 3075 kb)

Supplementary Video 2

Soluble CCL21 fails to promote T cell locomotion. Representative time lapse movie corresponding to Fig. 1a. Human CD3+ PBL were treated with 30nM CCL21 and plated on a collagen and fibronectin matrix devoid of chemokines. The movie was taken 10 min after cell plating. 1 sec movie = 1 min. (MOV 2494 kb)

Supplementary Video 3a

(a) T cells migrate on isolated CCL21. Human CD3+ PBL were pre-labeled with the lipophilic tracer 'DiD' [DiIC18(5)]. 1 sec movie = 2 min. (MOV 651 kb)

Supplementary Video 3b

(b) T cells migrate on isolated CXCL12. T cells were pre-labeled with the lipophilic tracer 'DiD' [DiIC18(5)]. 1 sec movie = 2 min. (MOV 431 kb)

Supplementary Video 4

α4-integrins are confined to the uropod of T lymphocytes locomoting on immobilized CCL21. Human CD3+ PBL were pre-labeled with Alexa488-tagged B5G10 (a non-blocking 4-specific antibody) and allowed to migrate on CCL21 co-immobilized with collagen and fibronectin. The video corresponds to Fig. 2a, bottom panel. 1 sec movie = 0.5 min. (MOV 2312 kb)

Supplementary Video 5

LFA-1 is enriched at the leading edge of T lymphocytes locomoting on immobilized CCL21. Human CD3+ PBL were pre-labeled with Alexa568-tagged TS2/4 (a non-blocking αL-specific antibody) and allowed to migrate on CCL21 co-immobilized with collagen and fibronectin, as in Supplementary Video 4. The video corresponds to Fig. 2a, top panel. 1 sec movie = 0.5 min. (MOV 2225 kb)

Supplementary Video 6

A T cell colliding into an ICAM-1 coated bead. Represeantative T lymphocyte locomoting on immobilized CCL21 and colliding into an ICAM-1 coated bead. The video corresponds to Fig. 3a. 1 sec movie = 1 min. (MOV 1300 kb)

Supplementary Video 7

A T cell colliding into the uropod of a passing T cell. CD3+ PBL were pre-labeled with Alexa568-tagged TS2/4 (non-blocking αL-specific antibody) and allowed to migrate on CCL21 co-immobilized with collagen and fibronectin. The movie shows a T cell moving toward a neighboring cell, touching it, and moving away. The video corresponds to Fig. 3b. 1 sec movie = 1 min. (MOV 1952 kb)

Supplementary Video 8

Artificial activation of LFA-1 on T lymphocytes migrating on immobilized CCL21 results in long lived contact with the uropod of neighboring T cells. Lymphocytes were pretreated with a β2-activating F(ab) 240q and settled on immobilized CCL21 co-immobilized with collagen and fibronectin. Note the two lymphocytes that form a stable contact and a long bridge tether following their collision. The video corresponds to Fig. 3c. 1 sec movie = 1 min. (MOV 1894 kb)

Supplementary Video 9

T cells migrate towards and over autologous DCs in the presence of immobilized CCL21. Monocyte-differentiated DCs were plated and spread for 10 min on a matrix containing immobilized CCL21 and collagen and fibronectin. The DCs were overlaid with 30 nM CCL21 for an additional 5 min and washed. Autologous BCECF-labeled CD3+ PBL were added and cells were tracked 5 min later. The video corresponds to Fig. 7a–c. 1 sec movie = 1 min. (MOV 2763 kb)

Supplementary Video 10

T cells migrate towards and over human LN-derived stromal cells in the presence of immobilized CCL21. Human stromal cells were plated as described in the Methods. BCECF-labeled CD3+ PBL were added and cells were tracked 5 min later. The video corresponds to Fig. 7d–f. 1 sec movie = 1 min. (MOV 2618 kb)

Supplementary Video 11

Motility of wildtype and CD18−/− T cells in the lymph node T zone. Two-photon imaging was performed on an explanted inguinal lymph node from a mouse that had received wildtype GFP+ T cells (green) and CMTMR labeled CD18−/− T cells (red) one day before. The imaging volume is 240 by 288 μm in xy plane with 60 μm projection in the z axis. Imaging frames were collected every 20 sec for 30 minutes. A representative movie from more than three experiments. (MOV 2469 kb)

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Woolf, E., Grigorova, I., Sagiv, A. et al. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces. Nat Immunol 8, 1076–1085 (2007). https://doi.org/10.1038/ni1499

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