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. 2007 Dec 18;104(51):20296-301.
doi: 10.1073/pnas.0710258105. Epub 2007 Dec 12.

Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells

Yoshihisa Kaizuka  1 , Adam D Douglass, Rajat Varma, Michael L Dustin, Ronald D Vale
Affiliations

Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells

Yoshihisa Kaizuka et al. Proc Natl Acad Sci U S A. .

Abstract

T cells interacting with antigen-presenting cells (APCs) form an "immunological synapse" (IS), a bull's-eye pattern composed of a central supramolecular activation cluster enriched with T cell receptors (TCRs) surrounded by a ring of adhesion molecules (a peripheral supramolecular activation cluster). The mechanism responsible for segregating TCR and adhesion molecules remains poorly understood. Here, we show that immortalized Jurkat T cells interacting with a planar lipid bilayer (mimicking an APC) will form an IS, thereby providing an accessible model system for studying the cell biological processes underlying IS formation. We found that an actin-dependent process caused TCR and adhesion proteins to cluster at the cell periphery, but these molecules appeared to segregate from one another at the earliest stages of microdomain formation. The TCR and adhesion microdomains attached to actin and were carried centripetally by retrograde flow. However, only the TCR microdomains penetrated into the actin-depleted cell center, whereas the adhesion microdomains appeared to be unstable without an underlying actin cytoskeleton. Our results reveal that TCR and adhesion molecules spatially partition from one another well before the formation of a mature IS and that differential actin interactions help to shape and maintain the final bull's-eye pattern of the IS.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Formation of an immunological synapse in Jurkat T cells interacting with planar lipid bilayers. (A) Schematic of the experimental system. Jurkat cells were adhered to planar lipid bilayers containing ICAM-1 (Alexa 488-conjugated) and anti-TCR antibodies (monobiotinylated anti-CD3ε antibodies bound to Texas red-conjugated streptavidin, which was attached to a biotinylated phospholipid) (see Materials and Methods). DOPC, dioleoylphosphocholine. (B) Confocal image of Jurkat cells bound to the lipid bilayer showing mature synapses at ≈30 min after initiation. Red indicates anti-TCR; green indicates ICAM-1. (C) Confocal image showing that ICAM-1 (green) and LFA-1 [labeled with cy3-conjugated anti-LFA-1 Fab (red)] colocalize at the pSMAC. (D) TIRF image of an actin–GFP expressing Jurkat cell at ≈30 min after initiation, showing that the pSMAC [labeled with cy3-anti LFA-1 Fab (red)] does not extend to the actin-poor cell center. (Scale bars, 5 μm.) Note that TIRF illumination tends to significantly amplify the intensity of surface proximal fluorophores in pSMAC and thus the pSMAC in D appears brighter than that in C.
Fig. 2.
Fig. 2.
Coupling of TCR and ICAM-1 microdomains to retrograde actin flow. Cells were applied to planar bilayers and imaged during the initial 10 min of synapse formation by spinning disk confocal microscopy. (A) (Upper) Maximum intensity projection of the TCR signal over a period of 4 min. Images were acquired at 1-s intervals. The majority of the bright fluorescent puncta are immobile aggregates of streptavidin in the bilayer. (Scale bar, 10 μm.) (Lower) Time course showing active transport of a single TCR microdomains (red arrow) in the ROI shown in the intensity projection. (B) (Upper) Maximum intensity projection of ICAM-1 over a period of 1 min. Images were acquired at 5-s intervals. (Scale bar, 10 μm.) (Lower) Time course showing retrograde movement of a single ICAM-1 microdomains over time. (C) (Upper) Maximum intensity projection of actin–GFP speckles over time. Images were acquired at 1.4-s intervals. (Scale bar, 10 μm.) (Lower) Time course showing retrograde movement of a single actin–GFP speckle (red arrow) in the boxed region shown in the intensity projection. (D) Histograms of transport rates of TCR (Top), actin–GFP (Middle), and ICAM-1 (Bottom) microdomains. (E) Trajectories of individual TCR (red) and ICAM-1 (yellow) microdomains, superimposed on an image of ICAM-1 in the mature synapse, reveal that TCR tends to have longer runs to the cell center. See SI Fig. 7 for more examples.
Fig. 3.
Fig. 3.
Segregation of TCR and ICAM-1 microdomains occurs before IS formation. Cells were applied to stimulatory bilayers and imaged by spinning-disk confocal microscopy during the early stages of synapse formation. (A) Time-lapse images of TCR (anti-CD3, red) and ICAM-1 (green) show that microcluster formation and centripetal movements precede the formation of both the cSMAC and pSMAC. (B) A close-up view of the boxed region shown in A demonstrates segregation of TCR and ICAM-1 into spatially segregated microclusters at the periphery. (Scale bar, 5 μm.)
Fig. 4.
Fig. 4.
The cSMAC creates a barrier to diffusion. A time series of single ICAM-1 molecules (imaged by TIRF) was superimposed on a single image of the cSMAC (fluorescent streptavidin linked to anti-TCR antibodies and imaged by epifluorescence), so that the behavior of single molecules could be analyzed relative to the boundaries of the cSMAC. (A) Trajectories of a single ICAM-1 particle (green) and its simulated counterpart (red) relative to a cSMAC, illustrating the simulation method. (Scale bar, 2 μm.) White dots indicate points illustrated in the bottom images, which show the single-molecule ICAM-1 images (green channel; red brackets) at three time points. The concentration of ICAM-1 molecules is comparable to other experiments, and the number of fluorescent ICAM-1 is adjusted for single-molecule imaging by photobleaching of most fluorophores. (B and C) Comparison of a population of real particles (B) and their simulated counterparts (C) in representative synapses (n = 252 trajectories). Particle trajectories were overlaid onto a population-level image of the cSMAC (grayscale). (Scale bar, 4 μm.)
Fig. 5.
Fig. 5.
The model for the segregation of TCR and adhesion molecules during the immunological synapse formation. Differential cluster nucleation, translocation, and diffusional exclusion of ICAM-1 and TCR drive immunological synapse formation. (A) At the initial contact of the Jurkat T cell with the planar lipid bilayer, interactions with the actin cytoskeleton form spatially separated TCR (red) and ICAM-1 (green) microdomains (note, ICAM–LFA-1 interactions likely form subsequent to TCR signaling). (B) After cells spread, separate TCR and ICAM-1 microdomains form primarily at the cell periphery, where new actin filaments are polymerizing. These microdomains are anchored to actin filaments and transported toward the cell center along with actin retrograde flow. (C) TCR microdomains populate the actin-sparse cell center and form the cSMAC, whereas ICAM-1 microdomains require anchoring to actin filaments for stability and do not extend beyond the actin boundary. (D) A diffusional barrier at the cSMAC periphery may hinder ICAM-1 from entering the mature cSMAC, thus helping to make the boundary.
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