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).
Jurkat T cells formed much larger contacts with PLL-coated glass than with OKT3-coated glass or uncoated glass (Supplementary Movies 1–3 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.
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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Nature Immunology (2018)