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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  • 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.


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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).

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Author notes

  1. These authors contributed equally: Miroslav Hons, Jens V. Stein, Michael Sixt.


  1. Institute of Science and Technology Austria, Klosterneuburg, Austria

    • Miroslav Hons
    • , Aglaja Kopf
    • , Robert Hauschild
    • , Alexander Leithner
    • , Florian Gaertner
    • , Jörg Renkawitz
    •  & Michael Sixt
  2. Theodor Kocher Institute, University of Bern, Bern, Switzerland

    • Miroslav Hons
    • , Jun Abe
    •  & Jens V. Stein


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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.

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

Integrated Supplementary Information

  1. 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.

  2. 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.

  3. 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.

  4. 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).

  5. 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

  1. Supplementary Figures

    Supplementary Figures 1–5

  2. Reporting Summary

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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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