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. 2010 Feb 23;107(8):3675-80.
doi: 10.1073/pnas.0905901107. Epub 2010 Feb 4.

Subcellular dynamics of T cell immunological synapses and kinapses in lymph nodes

Georges A Azar  1 , Fabrice Lemaître, Ellen A Robey, Philippe Bousso
Affiliations

Subcellular dynamics of T cell immunological synapses and kinapses in lymph nodes

Georges A Azar et al. Proc Natl Acad Sci U S A. .

Abstract

In vitro studies have revealed that T cell activation occurs during the formation of either dynamic or stable interactions with antigen-presenting cells (APC), and the respective cell junctions have been referred to as immunological kinapses and synapses. However, the relevance and molecular dynamics of kinapses and synapses remain to be established in vivo. Using two-photon imaging, we tracked the distribution of LAT-EGFP molecules during antigen recognition by activated CD4(+) T cells in lymph nodes. At steady state, LAT-EGFP molecules were preferentially found at the uropod of rapidly migrating T cells. In contrast to naïve T cells that fully stopped upon systemic antigen delivery, recently activated T cells decelerated and formed kinapses, characterized by continuous extension of membrane protrusions and by the absence of persistent LAT-EGFP clustering. On the other hand, activated CD4(+) T cells formed stable immunological synapses with antigen-loaded B cells and displayed sustained accumulation of LAT-EGFP fluorescence at the contact zone. Our results show that the state of T cell activation and the type of APC largely influence T cell-APC contact dynamics in lymph nodes. Furthermore, we provide a dynamic look at immunological kinapses and synapses in lymph nodes and suggest the existence of distinct patterns of LAT redistribution during antigen recognition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Naïve and recently activated T cells display different behaviors during Ag recognition in the lymph node. Naive or in vitro activated CD4+ T cells from WT15 TCR Tg mice were labeled with CFSE and adoptively transferred into BALB/c recipients. After 24 h, mice were injected with 50 μg LPS and 6 h later with 100 μg of LACK peptide. Control mice were injected with LPS only. Two-photon imaging of intact lymph nodes was performed 30 min after peptide injection. (A) Trajectories corresponding to 12 min of imaging are shown for naïve (Top) or activated T cells (Bottom) in response to LPS or LPS plus peptide. Naïve but not activated T cells completely arrested upon Ag recognition. (B) Quantitation of T cell velocities (Left) or straightness indexes (Right) in the different experimental conditions. Each dot represents an individual T cell. Low straightness indexes correspond to constrained behavior. *P < 0.05; **P < 0.01.
Fig. 2.
Fig. 2.
Generation and in vivo response of LAT-EGFP-expressing T cells. CD4+ T cells from WT15 TCR Tg mice were stimulated in vitro using anti-CD3/anti-CD28 coated beads and retrovirally transduced to express EGFP or LAT-EGFP. (A) LAT-EGFP expression by CD4+ T cells was assessed by flow cytometry 24 h after transduction. (B) Imaging of T cells in suspension after infection with retroviral particles encoding EGFP or LAT-EGFP. (C) LAT-EGFP-expressing T cells divide in response to in vivo immunization. WT15 CD4+ T cells were transduced to express LAT-EGFP, labeled with the SNARF dye, and adoptively transferred into BALB/c mice. Mice were injected with LPS and peptide or LPS alone. After 3 days, lymph nodes were harvested, and cells were stained with an anti-CD3 mAb. Flow cytometry shows that LAT-EGFP-expressing WT15 CD4+ T cells increased in size and proliferated (as reflected by the dilution of SNARF intracellular content) in the presence but not in the absence of peptide.
Fig. 3.
Fig. 3.
Two-photon imaging of T cells expressing LAT-EGFP in intact lymph nodes. WT15 CD4+T cells were transduced to express LAT-EGFP, labeled with SNARF dye, and transferred into BALB/c mice. Twenty-four hours after transfer, intact lymph nodes were subjected to two-photon imaging. (A–C) Three-dimensional reconstruction of an imaging volume within the T cell zone of the lymph node. Note that only a fraction of the T cells have been infected. GFP and SNARF signals are shown in B and C, respectively. Square side length = 5 μm. (D and E) Enlarged views of GFP and SNARF fluorescent signals for individual T cells. Note that GFP fluorescence allows for the detection of subcellular structures, including uropod and membrane protrusions that were not discernable with SNARF dye (white arrowheads). (Scale bar, 10 μm.)
Fig. 4.
Fig. 4.
Visualizing LAT-EGFP distribution in T cells during kinapses formation in lymph node. WT15 CD4+ T cells were transduced to express LAT-EGFP, labeled with SNARF dye, and transferred into BALB/c mice. After 24 h, mice were injected with 50 μg LPS and 6 h later with 100 μg of LACK peptide. Control mice were injected with LPS only. Intact lymph nodes were subjected to two-photon imaging 1 h after peptide injection. (A–C) WT15 CD4+ T cells decelerate but do not arrest in response to Ag. (Scale bar, 30 μm.) (A) Tracks of T cells (corresponding to 5 min of imaging) are shown for uninfected (GFP−SNARF+, red) and infected (GFP+SNARF+, green) WT15 CD4+ T cells in mice injected with LPS or LPS and peptide. (B) Mean velocities of individual GFP+ and GFP− WT15 CD4+ T cells in response to LPS or LPS and peptide. (C) Zoomed-in time-lapse imaging of a representative LAT-EGFP-expressing WT15 CD4+ T cell migrating vigorously in control mice (LPS only). Note that the T cell shape displays a typical elongated shape and that LAT-EGFP preferentially accumulates at the uropod. The last frame is a colored overlay of the T cell outlines at various time points. (Scale bar, 10 μm.) (D) Zoomed-in time-lapse imaging of two representative low-motile LAT-EGFP-expressing WT15 CD4+ T cells in mice that received both LPS and LACK peptide. WT15 CD4+ T cells sent out numerous and dynamic membranes protrusions, presumably while contacting host APCs. No stable accumulation of LAT-EGFP was evident during this process. Results are representative of three independent experiments. (Scale bar, 10 μm.)
Fig. 5.
Fig. 5.
Dynamics of LAT-EGFP in T cells forming stable immunological synapses with B cells in lymph nodes. WT15 CD4+ T cells were transduced to express LAT-EGFP and adoptively transferred into BALB/c mice. After 24 h, SNARF-labeled peptide-pulsed B cells were transferred, and two-photon imaging of lymph nodes was performed 6 h later. (A) 3D reconstruction of a representative T cell–B cell conjugate showing LAT-EGFP fluorescence accumulation at the cell interface. Scale bar = 5 μm. (B) LAT-EGFP fluorescence accumulate at the T cell–B cell interface in distinct clusters (arrows). (C) The intensity of GFP fluorescence was graphed along the outline shown in B (a.u, arbitrary units). (D and E) Time-lapse imaging of two representative T cell–B cell immunological synapses. Scale bar = 5 μm. (F) Example of a T cell establishing two immunological synapses simultaneously. LAT-EGFP accumulated at both sites of interaction until one of the B cells detaches. (G and H) Quantitation of the mean LAT-EGFP fluorescence at immunological synapse or in the rest of the T cell surface showed a sustained enrichment at the contact zone. Results are shown for a representative T cell–B cell pair. Representative of at least 10 time-lapse movies obtained in two independent experiments. (Scale bars, 5 μm.)
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