Chemokines and integrins independently tune actin flow and substrate friction during intranodal migration of T cells


Although much is known about the physiological framework of T cell motility, and numerous rate-limiting molecules have been identified through loss-of-function approaches, an integrated functional concept of T cell motility is lacking. Here, we used in vivo precision morphometry together with analysis of cytoskeletal dynamics in vitro to deconstruct the basic mechanisms of T cell migration within lymphatic organs. We show that the contributions of the integrin LFA-1 and the chemokine receptor CCR7 are complementary rather than positioned in a linear pathway, as they are during leukocyte extravasation from the blood vasculature. Our data demonstrate that CCR7 controls cortical actin flows, whereas integrins mediate substrate friction that is sufficient to drive locomotion in the absence of considerable surface adhesions and plasma membrane flux.

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Fig. 1: Cell shape and speed are tightly coupled in naïve T cells migrating in vivo.
Fig. 2: LFA-1 and CCR7 contribute distinctly to naïve T cell migration in vivo.
Fig. 3: LN stroma guides T cell migration in the absence of LFA-1 or CCR7.
Fig. 4: CCR7 drives rapid cortical actin flows.
Fig. 5: Polarized T cells show no membrane flows.
Fig. 6: Substrate adhesiveness and force transmission efficiency controls T cells migration under confinement.
Fig. 7: Decreasing ICAM-1 concentration leads to LFA-1–ICAM-1 clutch slippage.

Change history

  • 06 July 2018

    In the version of this article initially published, the links for Supplementary Videos 1 and 2 incorrectly showed Supplementary Videos 9 and 10, respectively. The correct video files are now provided.


  1. 1.

    Gérard, A. et al. Detection of rare antigen-presenting cells through T cell-intrinsic meandering motility, mediated by Myo1g. Cell 158, 492–505 (2014).

  2. 2.

    Krummel, M. F., Bartumeus, F. & Gérard, A. T cell migration, search strategies and mechanisms. Nat. Rev. Immunol. 16, 193–201 (2016).

  3. 3.

    Dobbs, K. et al. Inherited DOCK2 deficiency in patients with early-onset invasive infections. N. Engl. J. Med. 372, 2409–2422 (2015).

  4. 4.

    Worbs, T. & Förster, R. A key role for CCR7 in establishing central and peripheral tolerance. Trends Immunol. 28, 274–280 (2007).

  5. 5.

    Kishimoto, T. K., Hollander, N., Roberts, T. M., Anderson, D. C. & Springer, T. A. Heterogeneous mutations in the β subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell 50, 193–202 (1987).

  6. 6.

    Ley, K., Laudanna, C., Cybulsky, M. I. & Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678–689 (2007).

  7. 7.

    Hogg, N., Patzak, I. & Willenbrock, F. The insider’s guide to leukocyte integrin signalling and function. Nat. Rev. Immunol. 11, 416–426 (2011).

  8. 8.

    Campbell, J. J. et al. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279, 381–384 (1998).

  9. 9.

    Warnock, R. A., Askari, S., Butcher, E. C. & von Andrian, U. H. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187, 205–216 (1998).

  10. 10.

    Stein, J. V. et al. The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J. Exp. Med. 191, 61–76 (2000).

  11. 11.

    Boscacci, R. T. et al. Comprehensive analysis of lymph node stroma-expressed Ig superfamily members reveals redundant and nonredundant roles for ICAM-1, ICAM-2, and VCAM-1 in lymphocyte homing. Blood 116, 915–925 (2010).

  12. 12.

    Lehmann, J. C. U. et al. Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking. J. Immunol. 171, 2588–2593 (2003).

  13. 13.

    Shulman, Z. et al. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin. Immunity 30, 384–396 (2009).

  14. 14.

    Legate, K. R., Wickström, S. A. & Fässler, R. Genetic and cell biological analysis of integrin outside-in signaling. Genes Dev. 23, 397–418 (2009).

  15. 15.

    Miller, M. J., Wei, S. H., Parker, I. & Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

  16. 16.

    Bajénoff, M. et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25, 989–1001 (2006).

  17. 17.

    Katakai, T., Habiro, K. & Kinashi, T. Dendritic cells regulate high-speed interstitial T cell migration in the lymph node via LFA-1/ICAM-1. J. Immunol. 191, 1188–1199 (2013).

  18. 18.

    Woolf, E. 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).

  19. 19.

    Worbs, T., Mempel, T. R., Bölter, J., von Andrian, U. H. & Förster, R. CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo. J. Exp. Med. 204, 489–495 (2007).

  20. 20.

    Lämmermann, T. et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453, 51–55 (2008).

  21. 21.

    Friedl, P., Entschladen, F., Conrad, C., Niggemann, B. & Zänker, K. S. CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize β1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion. Eur. J. Immunol. 28, 2331–2343 (1998).

  22. 22.

    Fukui, Y. et al. Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412, 826–831 (2001).

  23. 23.

    Nombela-Arrieta, C. et al. A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress. J. Exp. Med. 204, 497–510 (2007).

  24. 24.

    Faroudi, M. et al. Critical roles for Rac GTPases in T-cell migration to and within lymph nodes. Blood 116, 5536–5547 (2010).

  25. 25.

    Mitchison, T. J. & Cramer, L. P. Actin-based cell motility and cell locomotion. Cell 84, 371–379 (1996).

  26. 26.

    Bretscher, M. S. Getting membrane flow and the cytoskeleton to cooperate in moving cells. Cell 87, 601–606 (1996).

  27. 27.

    Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

  28. 28.

    Mueller, J. et al. Load adaptation of lamellipodial actin networks. Cell 171, 188–200.e16 (2017).

  29. 29.

    Barnhart, E. L., Lee, K.-C., Keren, K., Mogilner, A. & Theriot, J. A. An adhesion-dependent switch between mechanisms that determine motile cell shape. PLoS Biol. 9, e1001059 (2011).

  30. 30.

    Renkawitz, J. et al. Adaptive force transmission in amoeboid cell migration. Nat. Cell Biol. 11, 1438–1443 (2009).

  31. 31.

    Jacobelli, J., Bennett, F. C., Pandurangi, P., Tooley, A. J. & Krummel, M. F. Myosin-IIA and ICAM-1 regulate the interchange between two distinct modes of T cell migration. J. Immunol. 182, 2041–2050 (2009).

  32. 32.

    Bray, D. & White, J. G. Cortical flow in animal cells. Science 239, 883–888 (1988).

  33. 33.

    Ruprecht, V. et al. Cortical contractility triggers a stochastic switch to fast amoeboid cell motility. Cell 160, 673–685 (2015).

  34. 34.

    Callan-Jones, A. C., Ruprecht, V., Wieser, S., Heisenberg, C.-P. & Voituriez, R. Cortical flow-driven shapes of nonadherent cells. Phys. Rev. Lett. 116, 028102 (2016).

  35. 35.

    Ofer, N., Mogilner, A. & Keren, K. Actin disassembly clock determines shape and speed of lamellipodial fragments. Proc. Natl Acad. Sci. USA 108, 20394–20399 (2011).

  36. 36.

    Bieling, P. et al. Force feedback controls motor activity and mechanical properties of self-assembling branched actin networks. Cell 164, 115–127 (2016).

  37. 37.

    Kucik, D. F., Elson, E. L. & Sheetz, M. P. Cell migration does not produce membrane flow. J. Cell Biol. 111, 1617–1622 (1990).

  38. 38.

    Keren, K. et al. Mechanism of shape determination in motile cells. Nature 453, 475–480 (2008).

  39. 39.

    Lee, J., Gustafsson, M., Magnusson, K. E. & Jacobson, K. The direction of membrane lipid flow in locomoting polymorphonuclear leukocytes. Science 247, 1229–1233 (1990).

  40. 40.

    Comrie, W. A., Babich, A. & Burkhardt, J. K. F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse. J. Cell Biol. 208, 475–491 (2015).

  41. 41.

    Nordenfelt, P., Elliott, H. L. & Springer, T. A. Coordinated integrin activation by actin-dependent force during T-cell migration. Nat. Commun. 7, 13119 (2016).

  42. 42.

    Nordenfelt, P. et al. Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration. Nat. Commun. 8, 2047 (2017).

  43. 43.

    Liu, Y.-J. et al. Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell 160, 659–672 (2015).

  44. 44.

    Schweitzer, Y. & Kozlov, M. M. Cell motion mediated by friction forces: understanding the major principles. Soft Matter 9, 5186–5195 (2013).

  45. 45.

    Bergert, M. et al. Force transmission during adhesion-independent migration. Nat. Cell Biol. 17, 524–529 (2015).

  46. 46.

    Mayya, V. et al. Durable Interactions of T cells with t cell receptor stimuli in the absence of a stable immunological synapse. Cell Rep. 22, 340–349 (2018).

  47. 47.

    Stein, J. V. T cell motility as modulator of interactions with dendritic cells. Front. Immunol. 6, 559 (2015).

  48. 48.

    Takeda, A. et al. Fibroblastic reticular cell-derived lysophosphatidic acid regulates confined intranodal T-cell motility. eLife 5, e10561 (2016).

  49. 49.

    Campbell, E. J. & Bagchi, P. A computational model of amoeboid cell swimming. Phys. Fluids 29, 101902 (2017).

  50. 50.

    Driscoll, M. K. et al. Cell shape dynamics: from waves to migration. PLOS Comput. Biol. 8, e1002392 (2012).

  51. 51.

    Schaefer, B. C., Schaefer, M. L., Kappler, J. W., Marrack, P. & Kedl, R. M. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell. Immunol. 214, 110–122 (2001).

  52. 52.

    Förster, R. et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99, 23–33 (1999).

  53. 53.

    Ding, Z.-M. et al. Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration. J. Immunol. 163, 5029–5038 (1999).

  54. 54.

    Riedl, J. et al. Lifeact mice for studying F-actin dynamics. Nat. Methods 7, 168–169 (2010).

  55. 55.

    Wilson, R. W. et al. Gene targeting yields a CD18-mutant mouse for study of inflammation. J. Immunol. 151, 1571–1578 (1993).

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We thank N. Darwish-Miranda, S. Caballero Mancebo, J. Schwarz, J. Alanko and F. P. Assen for advice and help with experiments. Further, we thank the Microscopy Imaging Center of the University of Bern for contribution to optical setups and the scientific support facilities of IST Austria for technical support. This work was funded by grants from the European Research Council (ERC StG 281556 and CoG 724373) and the Austrian Science Foundation (FWF) to M.S. and by Swiss National Foundation (SNF) project grants 31003A_135649, 31003A_153457 and CR23I3_156234 to J.V.S. F.G. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 747687, and J.R. was funded by an EMBO long-term fellowship (ALTF 1396-2014).

Author information

M.H. designed research, performed experiments, analyzed the data and wrote the manuscript. A.K. A.L., F.G. and J.R. performed experiments and analyzed the data. R.H. performed formal analysis. J.A. performed experiments and contributed to generating mouse lines and bone marrow chimeras. J.V.S. and M.S. designed and supervised the research and wrote the manuscript.

Correspondence to Miroslav Hons or Jens V. Stein or Michael Sixt.

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Integrated Supplementary Information

Supplementary Figure 1 Two-photon intravital microscopy and analysis showing morphodynamical parameters of T cells migrating in LN parenchyma after transfer into wild-type mice.

(a) Examples of actual cell shapes and idealized ellipses of a given shape factor. (b) Comparison of cell shape (left) and skeletal length (right) of WT T cells on an example dataset. (c) 2D morphometrical parameters of WT T cells from sample dataset as in b, Spearman correlation coefficient (r) as indicated, **** P < 0.0001 (two-tailed), n = 226 shapes. (d) Example 3D reconstruction, 2D projection and morphometrical parameters from a track as in Fig. 1b. (e) Speed of primary naïve T cells migrating in LN parenchyma as determined by 2D versus 3D tracking. Left, T cells tracked from 2D videos as in Fig. 1 and Fig. 2, 6 s intervals, minimum track span 2 min. Dots represent individual cell tracks. WT n = 69 cells, Ccr7-/- n = 66 cells, Itgal-/- n = 61 cells, Ccr7-/- Itgal-/- n = 55 cells, Dock2-/- n = 15 cells. Right, tracks from independent experiments analyzed in 3D, tracked at 20 s intervals, minimum track span 7 min. Dots show individual cell tracks. WT n = 227 cells, CCR7-/- n = 217 cells, LFA-1-/- n = 245 cells, CCR7-/- LFA-1-/- n = 155 cells. Box plot center line, median; box limits, upper and lower quartiles; whiskers represent Tukey fences.

Supplementary Figure 2 Two-photon intravital microscopy showing morphodynamical parameters of wild-type, Ccr7–/–, Itgal–/– and Ccr7–/–; Itgal–/– T cells migrating in LN parenchyma after transfer into wild-type mice.

(a) Cell speed – shape correlation as in Fig. 2a, e, f. Lines show linear correlation fit, stairs show median values in respective bins. Spearman correlation coefficients (r) as indicated. **** P < 0.0001 (two-tailed). (b) Cell shape frequency distribution of Ccr7-/- and Itgal-/- T cells as in Fig. 2b. (c) Relationship between periodicity of cell shape, track mean speed and track median cell shape of WT and all (WT, Ccr7-/-, Itgal-/- and Ccr7-/- Itgal-/-) T cells analyzed. Dots show individual tracks. WT n = 69 tracks, all n = 251 tracks. Spearman correlation coefficient (r) as indicated, NS = not significant, **** P < 0.0001. (d) Shape frequency distribution of WT and Ccr7-/- T cells co-transferred in WT mice and analyzed from movies where both cell phenotypes were present. WT n = 553 shapes, Ccr7-/- n = 726 shapes. Box plots correspond to distribution plots. Box plot center line, median; box limits, upper and lower quartiles; whiskers represent Tukey fences. Mann-Whitney test, **** P < 0.0001 (two-tailed). (e) Temporal shift of speed - shape correlation and cross-covariance of speed and shape factor of WT, Ccr7-/-, Itgal-/- and Ccr7-/- Itgal-/- T cells. Dots indicate correlation coefficient or cross-covariance values. Curves show spline interpolation. (f) Evolution of cell shape in time illustrated on two example cell tracks. Dots and circles show actual measurements, curves show Fourier smoothing. (g) Spectral analysis of respective cell tracks from f.

Supplementary Figure 3 Two-photon intravital microscopy showing interactions of T cells with LN stroma after transfer of fluorescently labeled T cells into bone marrow chimeric mice expressing GFP in the LN stroma.

(a) Example Ccr7-/- T cell interacting with local areas of LN stroma. Top view and upper left view (below) on a time-lapse sequence of a 3D reconstitution. (b) Itgal-/- T cell interacting with local areas of LN stroma. Top view and bottom up view (below) on a time-lapse sequence of a 3D reconstitution. (c) Ccr7-/- Itgal-/- T cell interacting with local areas of LN stroma (green). Top view and upper right view (below) on a time-lapse sequence of a 3D reconstitution. a-c represent examples of data as described in Fig. 3. In a-c, side of one square is equivalent to 6.3 µm.

Supplementary Figure 4 Characterization of the under-agarose assay.

(a) FRAP recovery of CCL19-AF555 in the agarose block. Grey areas indicate SD, n = 11 bleaches. Data show one experiment. (b) CCL19 immobilization on surfaces. Fluorescent signal from UV-ablated areas on PEG-coated slides incubated with 100 nM CCL19-AF555 (left). Subsequently, slides were incubated with 5 µg/mL fibrinogen-AF488 (Right). Image on top shows UV-ablated pattern highlighted with fibrinogen-AF488, bar indicates 10 µm. Data show one experiment. (c) Agarose stiffness interpolated with cell speed as a function of agarose concentration. Agarose Young’s modulus was determined by atomic force microscopy, 25 measurements per condition were taken in a 5 × 5 array. Primary WT T cells were migrating on dishes coated with 2 µg/mL ICAM-1 under agarose supplemented with 10 nM CCL19. 0.5% n = 126 cells, 1% n = 159 cells, 1.5% n = 111 cells. Right, tracks speeds of cells migrating under 4 µm high PDMS confiner, n = 112 cells. Dots indicate individual cell tracks or individual AFM measurement. Box plot center line, median; box limits, upper and lower quartiles; whiskers represent Tukey fences. Data show one experiment. (d) Representative 3D reconstructions of cells under 0.5% agarose or 4 µm high PDMS confiner. Cells were placed on PEG coated surfaces in the presence of 10 nM CCL19. Side of one square is equivalent to 5.2 µm. (e) Speeds of WT and integrin β2-deficient T cells on 10% BSA or ICAM-1 (2 µg/mL) blocked with 10% BSA. Cells were placed under agarose supplemented with 5% BSA and 10 nM CCL19. WT on ICAM-1 n = 210 cells, Itgb2-/- on ICAM-1 n = 108 cells, WT on BSA n = 95 cells. (f) Speed of WT and integrin β2-deficient T cells on ICAM-1 immobilized on biotinylated PEG or on biotinylated PEG only. Cells were placed under agarose supplemented with 5% BSA and 10 nM CCL19. WT on ICAM-1 n = 229 cells, Itgb2-/- on ICAM-1 n = 25 cells, WT on PEG n = 88 cells. In e and f dots indicate individual cell tracks. Box plot center line, median; box limits, upper and lower quartiles; whiskers represent Tukey fences. Kruskal-Wallis test, NS = not significant, * P < 0.05, **** P < 0.0001 (two-tailed). Data represent pool of two experiments. (g) Correlation of retrograde actin flow and cell protrusion speed of T cells migrating on 10 ng/mL ICAM-1 blocked with BSA as in Fig. 7c. Cells were confined under agarose supplemented with 5% BSA with 10 nM CCL19. Data represent pool of three experiments. n = 26 cells, r = Spearman correlation coefficient, **** P < 0.0001 (two-tailed).

Supplementary Figure 5 Scheme of T cell shape and speed determination.

(a) T cell elongation is given by speed of cortical actin flow. High actin flow speed correlates with increase in cell elongation (below). (b) In absence of integrin engagement T cells can not migrate and actin flow speed and cell elongation are at maximum. Force transmission of actin flow through LFA-1 engagement with ICAM-1 leads to actin flow deceleration, cell speed increase and cell rounding (below).

Supplementary information

Supplementary Figures

Supplementary Figures 1–5

Reporting Summary

Supplementary Video 1

Workflow used to determine T cell morphodynamics from intravital 2PM of T cells transferred in WT mice and migrating in intact LNs.

Supplementary Video 2

Intravital 2PM showing WT T cells migrating in WT LN parenchyma. 3D reconstructions, one square unit side length is equivalent to 10 µm (left). Cell shape outlines correspond to a track highlighted in blue (right). Time in min:s.

Supplementary Video 3

Intravital 2PM showing migration of WT, Ccr7-/-, Itgal-/- and Ccr7-/- Itgal-/- T cells in context of LN stroma. T cells in magenta, stroma in green. 2D projection, time in min:s. Bar indicates 20 µm.

Supplementary Video 4

Intravital 2PM showing WT, Ccr7-/-, Itgal-/- and Ccr7-/- Itgal-/- T cells interacting with local areas of LN stroma. T cells in magenta, stroma in green. Top view (left) and tilted view (right) on a 3D reconstitution. Side of one square unit is equivalent to 6.3 µm, time in min:s.

Supplementary Video 5

Phase contrast microscopy of primary naïve T cells on a PEG-coated dish confined under an agarose block. Agarose supplemented with 1 or 100 nM CCL19. Time in min:s.

Supplementary Video 6

TIRFM of a Lifeact-GFP-expressing primary naïve T cell confined under an agarose block on a PEG-coated dish. Agarose supplemented with 100 nM CCL19. Time in min:s.

Supplementary Video 7

TIRF-FRAP of Lifeact-GFP (left) and CellMask membrane dye (right) recovery after photobleaching. Primary naïve T cells were confined under an agarose block on a PEG-coated dish. Agarose was supplemented with 100 nM CCL19. Time in s:ms.

Supplementary Video 8

Phase contrast microscopy of primary naïve T cells migrating on 2 µg/mL ICAM-1-coated dish under an agarose block. Agarose supplemented with 100 nM CCL19. Time in min:s.

Supplementary Video 9

Interference reflection microscopy of primary naïve T cells confined under an agarose block on PEG or ICAM-1-coated glass slide. Agarose was supplemented with 10 nM CCL19. Time in min:s:ms.

Supplementary Video 10

TIRFM of Lifeact-GFP-expressing primary naïve T cells confined under an agarose block on glass slides coated with BSA or indicated concentration of ICAM-1. Agarose was supplemented with 10 nM CCL19. Time in min:s:ms.

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