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Real-time imaging of lymphocytes in vivo
Michael D Cahalan
y
, Ian Parker
z
, Sindy H Wei
and Mark J Miller
New preparations, fluorescent probes and imaging techniques
are providing the means to observe the behavior of cells in the
tissue environment of lymphoid organs. In particular, when
combined with two-photon laser microscopy, intravital imaging
of surgically exposed lymph nodes provides a unique view of
lymphocyte migration and antigen presentation as it occurs
within the living animal. The view is emerging that lymphocytes
migrate randomly within lymphoid organs, and that lymphocyte
contact with antigen-presenting cells may be a stochastic
process rather than one guided by chemokine gradients.
Addresses Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA y e-mail: [email protected] zDepartment of Neurobiology and Behavior, University of California,
Irvine, California 92697, USA
Current Opinion in Immunology 2003, 15 :372–
This review comes from a themed issue on Immunological techniques Edited by Ronald N Germain
0952-7915/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0952-7915(03)00079-
Abbreviations APC antigen-presenting cell
DC dendritic cell
Introduction
The immune system consists of a distributed network of
trillions of cells that must operate independently to
provide antigen specificity and yet function in a coordi-
nated manner to defend us from a wide variety of patho-
gens. Communication between cells can be initiated by
direct cell contact, or can take place at some distance
within the tissue environment via chemokines.
Over the past 20 years, remarkable progress in molecular
immunology has defined the mechanism of antigen recog-
nition and identified a growing cast of molecules and
signaling pathways that link the T-cell receptor to the
nucleus. However, we still understand very little about
the basics of motility, compartmentalization and antigen
recognition in vivo, because these events occur within
densely packed lymphoid organs [1–3]. How do T cells, B
cells and dendritic cells (DCs) move within the native
tissue environment? How are T and B cell compartmental
boundaries established and maintained? How does a
T cell locate antigen within the lymph node — by
following chemokine gradients or by random collisions
with antigen-presenting cells (APCs)?
There is increasing recognition that events defined in vitro
may not correspond to the physiological situation in vivo
[4]. For example, contact between a T cell and an APC
leads to a redistribution of surface molecules and formation
of the ‘immunological synapse’ [5–7]. This type of mole-
cular redistribution has also been studied in vitro in planar
lipid bilayers with defined molecular constituents. Yet it is
still unclear whether stable synapses occur in the environ-
ment of the lymph node, or whether antigen recognition
naturally involves short-lived serial encounters.
Lymphoid organs have remained a black box into which
defined cell populations can be induced to home, but
from which we have been able to obtain only ‘snapshot’
views, by extracting cells or analyzing fixed tissue.
Clearly, there exists a strong need for imaging approaches
to visualize living cells within intact lymphoid tissue.
Seeing T cells in their native environment:
new preparations for imaging
To illuminate the black box of the tissue environment,
new in vitro preparations have been developed that more
faithfully represent the native tissue environment of
intact lymphoid organs. These include 3D collagen gel
matrices [8], monolayers of endothelial cells (ECs) bathed
with flowing solution to mimic the forces experienced by
cells [9], cultures of clusters or reaggregated tissue frag-
ments [
,11], engineered tissue surrogates [12], and
whole lymph node explants (Figure 1; [13,
]).
All of these preparations lack intact vascular and lymphatic
vessels, and therefore cannot be applied to investigate
processes such as lymphocyte trafficking. Furthermore,
the lack of blood and lymphatic vessels may disrupt the
distribution of important soluble factors or alter the phy-
siological levels of tissue oxygenation.
To overcome this limitation, several promising methodol-
ogies have been developed to visualize cells within the
in vivo tissue environment. Non-invasive methods include
bioluminescence imaging of cells engineered to express
luciferase [16], magnetic resonance imaging microscopy
to track cells labeled with superparamagnetic particles
[17], and positron-emission tomography (PET; [18]).
Although these methods can be applied to intact animals,
all three require cell engineering to derive populations
that can be detected and presently lack single-cell resolu-
tion. Instead, optical techniques offer cellular, and even
sub-cellular, levels of resolution. Intravital preparations of
Current Opinion in Immunology 2003, 15 :372–377 www.current-opinion.com
exposed lymphoid organs permit light microscopic imag-
ing in the native tissue environment with intact circula-
tory elements, but require anesthesia and surgery to bring
objective lenses close enough to the tissue.
To image cells at depths of more than about 50 mm, two-
photon microscopy is the technique of choice. When
combined with fluorescent probes, confocal microscopy or two-photon microscopy can reveal single cells at the plane of focus, either by imaging through a pinhole in the case of confocal microscopy, or by selectively exciting the fluorophore only at the plane of focus in the case of two- photon microscopy. Recently, we provided a detailed comparison of confocal and two-photon microscopy as
Figure 1
Z
Y
X
Model system Strengths Weaknesses
2D, important
native environmental
factors are missing,
non-native morphology.
High spatial and
temporal resolution,
control over molecular
interactions.
Planar lipid bilayer
Solution exchange is
difficult, may not mimic
secondary lymphoid
tissues.
Realistic morphology
and 3D behavior,
mimics natural peripheral
tissue substrate.
3D collagen gels
Missing physiological
blood flow and tissue
signals.
Permits adhesive and
transmigration behaviors
to be analyzed on a
physiologic substrate
under conditions of flow.
EC monolayer under flow
Lacking physiological
extracellular substrates
and long-range
environmental factors.
Preserves the immediate
microenvironment, for
example, cell to cell
contacts and short
range factors.
Reaggregates and clusters
Lacking lymphatic
inputs and normal
blood flow.
3D tissue environment,
physiological substrates,
natural cell to cell
interactions, long- and short-
range environmental effects.
Lymphoid tissue explants
True physiological Technically difficult.
environment.
Intravital preparation
Current Opinion in Immunology
The strengths and weaknesses of model systems in current use for the real-time imaging of lymphocytes. EC, endothelial cell.
In vivo imaging of lymphocytes Cahalan et al. 373
www.current-opinion.com Current Opinion in Immunology 2003, 15 :372– 377
genes, secretion of cytokines and cell proliferation. Two
different approaches have been used to visualize changes
in T-cell dynamics evoked by antigen. When antigen-
specific T cells were transferred into animals that had
been injected subcutaneously with specific antigen, clus- ters and swarms of enlarged T cells were observed one day following adoptive transfer [
]. At later times, cells divided and resumed a vigorous pattern of motility. Using
Figure 3
(a)
(b)
Current Opinion in Immunology
Intravital two-photon images of T cells in the inguinal lymph node of an anesthetized mouse. (a) Trajectories of four separate cells at varying times. The
colors represent cells at different depths, ranging from 100 to 150 mm below the surface of the lymph node, with blue representing the bottom and red the top of the imaging volume. Scale bar: 25 mm. (b) T cells (green), vessels and fibers (both red) labeled via tail vein injection with
tetramethylrhodamine dextran. Scale bar: 50 mm.
In vivo imaging of lymphocytes Cahalan et al. 375
www.current-opinion.com Current Opinion in Immunology 2003, 15 :372– 377
an alternative method of antigen priming, in which APCs
were differentiated in vitro from bone marrow cells,
pulsed with antigen and then injected subcutaneously,
Stoll et al. [
] observed contact between T cells and
APCs that lasted >15 hours in a one-to-one pattern of
association.
Recently, we have pursued an in vivo labeling method to
visualize antigen-primed DCs interacting with CD þ T
cells (MJ Miller, SH Wei, I Parker, MD Cahalan, unpub-
lished data). If the T cells can be likened to swimming fish,
DCs behave effectively as nets; they make contact with T
cells by throwing out long membrane tethers and rapidly
reeling them back in, constantly changing their shape and
greatly expanding their capture radius. It appears from
these early studies that the initial encounter between a T
cell and a DC relies upon dynamic cell behaviors that are
finely tuned to optimize the chance of random collisions.
Conclusions
Two-photon microscopy represents an optimal technique
for tracking the behavior of living cells deep within the
tissue environment. It is already feasible to image T cells
and other cells of the immune system within the circula-
tion, or in the tissue environment of lymph node, spleen,
Peyer’s patch, thymus and peripheral tissues. Video pre-
sentations of the data demonstrate the dynamic behavior
of T cells and B cells as they migrate within the lymph
node, and of DCs as they interact with T cells during
antigen presentation. Two-photon imaging will be adap-
table to a wide variety of new probes for second messen-
gers and gene expression, and to a broad range of
processes both physiological and pathological. Combined
with intravital imaging of surgically exposed lymphoid
organs, two-photon imaging is providing a unique view of
lymphocyte dynamics in vivo.
Update
A recent study used two-photon microscopy to examine the
interaction of dendritic cells labeled in vitro with motile
CD þ T cells in an explanted lymph node preparation
[
]. T cells made stable, long-lasting contacts with
antigen-pulsed DCs, rather than a series of short contacts.
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(^) of special interest (^) of outstanding interest
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