To the editor — Full understanding of lymphocyte activation will require thorough characterization of the ‘resting’ state and how it changes. Surfaces coated with the cationic homopolymer poly-l-lysine (PLL) are widely used for total internal reflection fluorescence (TIRF) imaging of the organization of surface proteins on resting lymphocytes1,2,3,4,5 because PLL is assumed to be inert. Here we found that PLL initiated T cell signaling and profoundly altered the activity of membrane proteins such as the T cell antigen receptor (TCR). Therefore, the emerging idea that receptors and signaling proteins cluster by default1,2,3,4,5, which has been based mostly on studies of lymphocytes interacting with PLL-coated surfaces, needs reconsideration.

We investigated whether PLL-coated surfaces are actually inert by testing their ability to induce calcium signaling in primary CD4+ T cells and Jurkat human T lymphocytes. Cells were labeled with the fluorescent calcium indicator Fluo-4AM and then were transferred to coverslips coated with PLL or the OKT3 monoclonal antibody to the TCR invariant chain CD3 (Supplementary Methods). Signaling occurred in ~80% of primary T cells and Jurkat T cells after 30 s on PLL-coated coverslips and in 90% of cells after 30 s on OKT3-coated coverslips (Fig. 1a). Similar results were obtained for Jurkat T cells with the ratiometric reporter Fura-2 (N = 270 cells in n = 3 experiments; Supplementary Fig. 1a). The dynamics of the responses of Jurkat T cells to each surface were also comparable (Fig. 1b). Signaling was largely insensitive to the concentration, coating time or polymer size of the PLL coating (Fig. 1c and Supplementary Fig. 1b). Lck-deficient Jurkat T cells (J.CaM1.6 cells) and TCRβ-deficient Jurkat T cells (J.RT3-T3.5 cells) exhibited diminished responses, relative to those of Jurkat T cells (Fig. 1a), that were restored by re-expression of Lck and TCRβ, respectively (Supplementary Fig. 1c); this indicated that PLL induced true TCR signaling. PLL induced TCR-proximal signaling, detected as phosphorylation of the tyrosine kinase ZAP70, albeit weakly (Supplementary Fig. 1d), and induced TCR-distal upregulation of the activation marker CD69 (Fig. 1d). Although these cells were somewhat more refractory, calcium signaling was induced by PLL in mouse (C57BL/6) primary CD4+ T cells and in two of three T cell hybridomas tested (for example, DO11.10; Supplementary Fig. 1e). PLL also induced signaling in a B cell line (A20) that was dependent on the B cell antigen receptor (Supplementary Fig. 1f).

Fig. 1: PLL induces strong T cell signaling and profoundly alters TCR diffusional activity and organization.
Fig. 1

a, Fraction of primary (CD4+) T cells and wild-type and mutant Jurkat T cells (horizontal axis) in which calcium responses were elicited (detected with Fluo-4AM) after contact with surfaces coated with OKT3 or PLL (key). b, Change in fluorescence intensity of Fluo-4AM, plotted against contact time, for randomly chosen Jurkat T cells (n = 100) on surfaces coated with OKT3 or PLL (key), presented in arbitrary units (AU). c, Fraction of Jurkat T cells responding on an uncoated glass surface (far left) or glass surfaces coated with various concentrations (horizontal axis; weight per volume (w/v)) of high-molecular-weight PLL (High MW; 150 kDa) or low-molecular-weight PLL (Low MW; 70 kDa) (key). d, Surface expression of CD69 on cultured Jurkat T cells (Resting) or on Jurkat T cells after overnight incubation on surfaces coated with PLL or OKT3 (top right corners). e, Single-molecule TIRF-based tracking of TCRs (labeled with fluorescent Fab fragments) at the basal surfaces of Jurkat T cells contacting glass surfaces coated with PLL or agarose (above images); track colors indicate diffusion coefficient (mean-square-displacement analysis) (key). Areas outlined in main images are enlarged in insets. f, TCR diffusion coefficients from single-molecule tracking for Jurkat T cells on PLL, agarose or agarose and PLL (horizontal axis), calculated from ensemble average mean-square-displacement curves. *P < 0.001 and **P < 1 × 10−10 (Student's t-test). g, Stimulated emission depletion (STED) imaging of TCRs (labeled with tetramethylrhodamine-based HaloTag fluorescent ligand) at the basal surfaces of Jurkat T cells contacting a PLL-coated glass surface or suspended in hydrogel (above images). Areas outlined in main images are enlarged in insets to show variation in fluorescence intensity (key); top right corners, experimental set-up. Scale bars (e,g), 2 μm (main images) or 500 nm (insets). Data are from three experiments with n = 195–479 cells per condition (a; mean ± s.d.), n = 100 cells per condition (b; mean (solid lines) and s.d. (shaded areas)), n = 215–513 cells per condition (c; mean ± s.d.) or n = 21–33 cells per condition (f; mean ± s.d. for cell-to-cell variation) or are representative of three experiments (d,e,g).

Jurkat T cells formed much larger contacts with PLL-coated glass than with OKT3-coated glass or uncoated glass (Supplementary Movies 13 and Supplementary Fig. 1g). To determine whether PLL perturbs receptor activity, we used single-molecule tracking of fluorescence-labeled TCRs. On PLL-coated coverslips, TCR mobility was immediately reduced (diffusion coefficient (D) = 0.018 ± 0.01 µm2/s (mean ± s.d.), versus D = 0.06 µm2/s measured at the apical surface of a mouse T cell hybridoma6; Fig. 1e,f and Supplementary Movie 4). To determine whether this required hard surfaces, we spin-coated agarose onto coverslips, which produces a softer surface still suitable for TIRF imaging. Under these conditions, the TCR exhibited substantial mobility (D = 0.18 ± 0.07 µm2/s (mean ± s.d.); Fig. 1e,f and Supplementary Movie 4) that was reduced to near-static levels when PLL was added to the agarose (D = 0.04 ± 0.03 µm2/s (mean ± s.d.); Fig. 1f and Supplementary Movie 4). Therefore, PLL directly perturbed TCR dynamics, presumably via electrostatic polymer–receptor interactions.

But how might PLL initiate signaling, and how could such effects be avoided? The receptor-type tyrosine phosphatase CD45 showed depletion of ~50% at PLL-mediated contacts, relative to the abundance of CD3 (n = 16 images; P < 0.001 (Student’s t-test); Supplementary Fig. 1h,i); this might favor signaling by decreasing dephosphorylation of the TCR. Similar levels of phosphatase segregation suffice to initiate TCR phosphorylation and downstream signaling on supported lipid bilayers7. The immobilization of CD45 and TCRs in PLL-mediated contacts might also potentiate signaling by constraining receptor dephosphorylation. Overall, the results indicated that for imaging of truly resting cells, surfaces might be best avoided altogether. Surfaces coated with the integrin ligand ICAM-1 allow T cells to be captured without triggering strong calcium signaling8, but integrin ‘out-to-in’ signaling would probably nevertheless shift such cells away from the resting state9. Although TCR diffusion was apparently unimpeded on agarose surfaces (Fig. 1e,f), for imaging it was necessary to hold the cells in place through the use of agarose pads, which induced signaling (in 79% ± 10% of responding cells (mean ± s.d.; N = 80 cells in n = 3 experiments); data not shown). In >70% of experiments, however, signaling was undetectable in Jurkat T cells taken from culture and immediately suspended in hydrogels10 (Supplementary Fig. 2a).

Super-resolution, stimulated emission depletion imaging revealed that the TCR organization on Jurkat T cells suspended in hydrogel was profoundly different from that of cells placed on PLL-coated surfaces (Fig. 1g). Intensity-based analysis revealed that the degree of TCR clustering was significantly higher for cells contacting PLL-coated glass than for cells in hydrogel (n = 5–14 images; P < 0.001 (Student’s t-test); Supplementary Fig. 2b). This suggested that the high levels of receptor and signal-protein clustering reported previously for lymphocytes interacting with PLL-coated surfaces1,2,3,4,5 is probably not reflective of the activity of resting cells. The extent to which the deformable surfaces that non-activated T cells encounter in vivo affect receptor and signal-protein organization, if at all, is a separate matter.


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Supported by a Royal Society University Research Fellowship (UF120277 to S.F.L.) and Research Professorship (RP150066 to D.K.); the EPSRC (EP/L027631/1 to A.P.,); the Wellcome Trust (098274/Z/12/Z to S.J.D., and WT101609MA to R.A.F.); PA Cephalosporin Fund (C.E.); the Wolfson Imaging Centre Oxford (funded by the Wolfson Foundation and Wellcome Trust; 104924/14/Z/14); the Micron Advanced BioImaging Unit (Wellcome Trust Strategic Award 091911); the Medical Research Council (MC_UU_12010/Unit Programmes G0902418 and MC_UU_12025); an MRC/BBSRC/EPSRC award (MR/K01577X/1); and a Marie Skłodowska-Curie Intra-European grant (707348 to I.U.).

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  1. These authors contributed equally: Ana Mafalda Santos, Aleks Ponjavic, Marco Fritzsche and Ricardo A. Fernandes.


  1. Radcliffe Department of Medicine, University of Oxford, Oxford, UK

    • Ana Mafalda Santos
    • , Marco Fritzsche
    • , Ricardo A. Fernandes
    • , Jorge Bernardino de la Serna
    • , Martin J. Wilcock
    • , Falk Schneider
    • , Iztok Urbančič
    • , Consuelo Anzilotti
    • , Meike Aßmann
    • , Simon J. Davis
    •  & Christian Eggeling
  2. MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK

    • Ana Mafalda Santos
    • , Marco Fritzsche
    • , Ricardo A. Fernandes
    • , Jorge Bernardino de la Serna
    • , Martin J. Wilcock
    • , Falk Schneider
    • , Iztok Urbančič
    • , Consuelo Anzilotti
    • , Meike Aßmann
    • , Richard J. Cornall
    • , Simon J. Davis
    •  & Christian Eggeling
  3. Department of Chemistry, University of Cambridge, Cambridge, UK

    • Aleks Ponjavic
    • , James McColl
    • , Kristina A. Ganzinger
    • , David Klenerman
    •  & Steven F. Lee
  4. Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK

    • Marco Fritzsche
    • , David Depoil
    •  & Michael L. Dustin
  5. Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK

    • Richard J. Cornall


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A.M.S., A.P., M.F., R.A.F., J.B.S., M.J.W, F.S., I.U., J.M., C.A., K.A.G., M.A. and D.D. performed experiments; A.M.S., A.P., M.F., R.A.F., R.J.C, M.L.D., D.K., S.J.D., C.E. and S.F.L. drafted and/or edited the manuscript; D.K., S.J.D., C.E. and S.F.L. conceived of the study; and M.L.D., S.J.D., C.E. and S.F.L. supervised the experiments.

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The authors declare no competing financial interests.

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Correspondence to Christian Eggeling or Steven F. Lee.

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