Visualization of T Cell–Dendritic Cell Encounter by Two-Photon Microscopy
Yoshiki Miyachi
Thursday, May 08, 2014
Introduction
The mechanism behind allergic contact
dermatitis (ACD) has been well investigated and described in
dermatology textbooks.1 When the causative agents invade
the skin, Langerhans cells (LCs) and dendritic cells recognize the
invaders and capture them moving to the regional lymph nodes, which
results in antigen presentation. Here, T cells become
sensitized to the causative agents and proliferate in preparation
for reinvasion in the future. Upon reinvasion of the agent, these
sensitized T cells become activated and produce various cytokines,
leading to epidermal spongiosis to wash out the invaders. This
pathogenesis of ACD is well known. However, have you ever visually
observed these reactions in the skin? Well, it is now possible to
appreciate the visualization of ACD using two-photon microscopy
(TPM; for a description of this tool, please review the DermQuest
Expert Opinion article '
Live Imaging of the Skin by Two-photon Microscopy').
LC mobility
By using Langerin-enhanced green
fluorescent protein mice in which LCs are stained green, we can
observe the distribution and movement of LCs by TPM as shown in
Figure 1a. In this image, keratinocytes are labeled red by a lectin
in order to identify the location of LCs accurately. When skin
inflammation is induced by topical application of an irritant, LCs
start moving in the intercellular space of keratinocytes as if
looking for the invaders (Figure 1b). If you look closely at these
LCs in 3D (Figure 2), you will notice that they elongate their
dendrites upward only, presumably in order to capture the invaders
entering from outside of the skin; not from the inside.

Figure 1a (see Movie 1a. LCs stained
green)

Figure 1b (see Movie 1b. LCs moving in the intercellular space
of keratinocytes as if looking for the invaders)

Figure 2 (see Movie 2. LCs in 3D elongating
dendrites)
LCs rendezvous with T cells in
lymph nodes
As shown in Figure 3, LCs (green)
demonstrate close contact with T cells (red) for a while in lymph
nodes, suggesting some information may be exchanged here. It is
unclear whether antigen presentations occur; however, one may infer
that this meaningful rendezvous is of great importance for contact
sensitization. Further studies are required to uncover this
mysterious encounter.

Figure 3 (see Movie 3. LCs (green) demonstrate
close contact with T cells (red) for a while in lymph
nodes)
Live imaging of T cells in
both irritant and allergic contact dermatitis
Figure 4 shows live-image movies of T
cells in both steady state (a) and inflammation (b). Under no
stimulation, we hardly observe T cell movement in the mouse ear
skin; though T cells occasionally appear like shooting stars. Upon
phorbol myristate acetate application to induce irritant cutaneous
inflammation, both CD4 T cells (green) and CD8 T cells (red) begin
moving around smoothly in the skin.

Figure 4a (see Movie 4a. T cells in steady
state)

Figure 4b (see Movie 4b. T cells in inflammation)
Figure 5 demonstrates T cell behavior
in dinitrofluorobenzene (DNFB)-painted ear skin in DNFB-sensitized
mice. DNFB-sensitized T cells were labeled green and
trinitrochlorobenzene (TNCB)-sensitized T cells were labeled red.
In these movies, it is possible to observe some DNFB-sensitized T
cells (green) stop migration for more than an hour (denoted by
white circles in the right-hand panel). Conversely, most
TNCB-sensitized T cells (red) moved actively. If the migration
velocity of T cells is measured (Figure 6), DNCB-sensitized T cells
demonstrate reduced velocity in DNFB-painted skin, but not in
TNCB-painted skin, and vice versa. This suggests that T cells
decrease their migration velocity when T cell receptor-specific
antigen exists.2

Figure 5 (see Movie 5. T cell behavior in DNFB-painted
ear skin in DNFB-sensitized mice)

Figure 6. Migration velocity
of T cells.T cells decrease their migration velocity when T cell
receptor-specific antigen exists (image from Egawa G,
et al., 20112)
Dendritic cells' encounter
with sensitized T cells in the skin in the elicitation phase of
contact dermatitis
During the elicitation phase of ACD,
by painting DNFB in DNFB-sensitized mouse skin, it seems likely
that T cells stop moving in the vicinity of dendritic cells for a
while (Figure 7), suggesting that the encounter of these two cell
populations occurs in the elicited skin. Although it still remains
unclear what kind of communications are exchanged here, we
speculate that there is a very good chance of antigen presentation
in the skin.

Figure 7 (see Movie 7. T cells stop moving in the
vicinity of dendritic cells for a while)
Conclusion
Thanks to the new technology of TPM,
the immunological response of contact dermatitis can now be
visualized as real-time movies, which demonstrate the behaviors of
both T cells and dendritic cells. By following the footsteps of
contact sensitization as well as elicitation visually, we can
appreciate the skin as an important immunological organ. More
precise visual analysis of the process will reveal how
immunological musicians play in the orchestra on the stage of the
skin.
References
- Honda T, Egawa G, Grabbe S, Kabashima K. Update of immune
events in the murine contact hypersensitivity model: toward the
understanding of allergic contact dermatitis. J Invest
Dermatol 2013;133:303-15.
- Egawa G, Honda T, Tanizaki H, Doi H, Miyachi Y, Kabashima K.
In vivo imaging of T-cell motility in the elicitation
phase of contact hypersensitivity using two-photon microscopy.
J Invest Dermatol 2011;131:977-9.
Back