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Visualizing Immune System Complexity
Michael L. Dustin (14 April 2009)
Science Signaling 2 (66), mr4. [DOI: 10.1126/scisignal.266mr4]
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MEETING REPORT
IMMUNOLOGY
Visualizing Immune System Complexity
Michael L. Dustin
A report on the EMBO workshop “Visualizing
Immune System Complexity,” Centre
d’Immunologie Marseille-Luminy, Marseille,
France, 15 to 17 January 2009.
Published 14 April 2009; Volume 2 Issue 66 mr4
New Tools for the Next Generation
of Researchers
The European Molecular Biology Organization (EMBO) workshop Visualizing Immune System Complexity presented many
exciting views of immunity, from single
molecules to whole organs, and occurred
just a week after a once-in-a-century blizzard had blanketed the dramatic limestone
spires of Les Calanques with snow. Lena
Alexopoulou, Hai-Tao He, and Didier Marguet, of Centre d’Immunologie MarseilleLuminy (CIML), and I organized the meeting. In his introductory remarks Eric Vivier, the director of CIML, pointed out that
advances in imaging technology and equally sophisticated analytical and modeling
methods were bringing us closer to addressing some enduring problems in immunology, from receptor function to patient care. An important aspect for the attendees, many of whom were students of
the Luminy Advanced Immunology
Course, is that many technologies that
would have been the purview of a small
biophysics elite a few years ago are available to a new generation of computersavvy young scientists who can access
powerful commercial imaging systems
through expanding core facilities in many
research centers. The meeting was divided
into sessions on the molecular, cellular, and
whole-organism level, and a keynote talk
by Diane Mathis (Harvard University,
Division of Molecular Pathogenesis, Skirball
Institute of Biomolecular Medicine, New York
University, New York, NY 10016, USA. E-mail,
michael.dustin@med.nyu.edu
Boston, USA) presented challenges for
bringing these advances to patient care.
Modeling Biology, a Two-Way Street
The meeting emphasized multidisciplinary modeling and simulation approaches with talks by Rob de Boer
(Utrecht University, the Netherlands),
Carsten Watzl (University of Heidelberg,
Germany), and Arup Chakraborty (Massachusetts Institute of Technology, Cambridge, USA). De Boer discussed his cellular
Potts model for simulating the natural
movement of naïve T lymphocytes (T
cells) and dendritic cells (DCs) within
lymphoid tissues, which was f irst described by Michael Cahalan’s laboratory
(University of California, Irvine, USA)
(1). This simulation incorporates experimental observations and describes cells as
occupying space and having defined surface energies that govern the cellular
movements of advancing and retracting,
as well as cell-cell interactions. The movements and interactions are then simulated
such that energy is minimized. With simple rules, he simulated the movement of T
cells and DCs and predicted behaviors
that had not yet been observed, like microstreams of similarly oriented cells,
which were then verified experimentally
(2). These simulations will be useful in
addressing important questions, such as
calculating how long it takes rare specific
T cells to find a pathogen and determining
the requirements for antigen-dependent
arrest of T cell movement—computational
results that will require validation through
experimental observations.
Watzl applied a mathematical modeling
approach to problems in signaling in natural
killer (NK) cells. NK cells integrated both
positive and negative signals (3). By including positive and negative feedback in his
model, Watzl accounted for the dominance
of inhibitory signaling over a large range of
inhibitory receptor densities, which is important to avoid self-injury. Continuing the
NK cell theme, Daniel Davis (Imperial College London, UK) reported that, in addition
to the conventional immunological synapses, signals initiated by the interaction of the
ligand MICA with the positive signaling receptor NKG2D can be maintained by long
nanotubes connecting cells that otherwise
appeared to have broken off interaction (4).
This is a twist that might require adjustments to signaling models with spatial
components because the nanotubes may allow signaling to persist even after cells appear to have separated.
Returning to T cells, Chakraborty described how the mechanism of Ras activation lends itself to bistability and switchlike
behavior. In his model, the key to bistability
is the priming of the Ras guanine nucleotide
exchange factor (GEF) SOS with active Ras
that is generated by the diacylglycerol
(DAG)–activated GEF RasGRP. This model
explains the switchlike behavior of Ras in
negative selection when stronger ligands
provide robust activation of phospholipase
C–γ (PLC-γ) (5). DAG may also be produced by PLD1 activation that is dependent
on the integrin LFA-1 (lymphocyte function-associated antigen–1), and this may also be important in activating RasGRP at the
plasma membrane, as described by Mark
Philips (New York University School of
Medicine, New York, USA) (6). Philips also
discussed the highly compartmentalized
process of the posttranslational modification
of Ras and of Ras signaling in T cells in
which RasGRP is activated on the Golgi by
T cell receptor (TCR) stimulation, but requires the LFA-1–dependent PLD1 signaling for activation at the plasma membrane.
Jacques Nunes (Centre de Recherche en
Cancérologie de Marseille, France) introduced additional negative regulation of Ras
by Dok4, a Ras GTPase-activating protein
(GAP)–associated protein, the recruitment
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The European Molecular Biology Organization (EMBO) meeting Visualizing
Immune System Complexity, held in January 2009, covered multiple scales,
from imaging single molecules to imaging whole animals. In addition to experimental details, there was an emphasis on modeling both for data analysis and
as a predictive tool to support experimental design. Imaging technologies discussed included total internal reflection fluorescence microscopy, fluorescence
correlation spectroscopy, two-photon laser scanning microscopy, and magnetic
resonance imaging. The biological systems included basic aspects of adaptive
and innate immunity. The type 1 diabetes model was used to illustrate how a
human disease was dissected at all the scales, from single-molecule analysis of
the interactions of T cell receptors with peptide-loaded major histocompatibility
complexes to dynamics of immune cell infiltrates by intravital microscopy, as
well as the application of imaging diagnostics in humans.
MEETING REPORT
of pMHC interacting with live T cells in
supported bilayers also containing the adhesion molecule ICAM-1 (intercellular cell
adhesion molecule–1) and the costimulatory ligand CD80. Because the fluorophores
on the single MHC molecules bleach rapidly, only short sequences of molecular positions are obtained. Computations from
many of these short runs allow a twodimensional (2D) affinity for TCR-pMHC
interactions to be calculated, but specific
assumptions need to be made about the
area in which TCR and MHC complexes
can interact. Mark Davis (Stanford University, Stanford, USA) reported results from
single-molecule fluorescence resonance energy transfer experiments, suggesting that
the duration of TCR-pMHC interactions in
the immunological synapse is about onetenth as long as in solution. This decrease
in half-life required F-actin. Data from our
laboratory and others show that these interactions take place in small microclusters,
which makes the modeling more complicated. The talks in this vein revealed a bidirectional impact of modeling on our understanding of immune complexity as an aid
in prediction and interpretation.
Lymphocytes in Action at
Multiple Scales
Lymphocyte function and kinesis are intertwined. These events are amenable to analysis by microscopic imaging because
functional modules from signaling microclusters to F-actin–rich filopodia are readily visualized and quantified. The challenge with immune cells is that they are
relatively small compared with other cellular models, and various strategies to overcome this and other challenges were presented. One way to parse submicrometer
lymphocyte str uctures was through
nanofabrication. Ruth Diez-Ahedo (GarciaParajo laboratory, Institute for Bioengineering of Catalonia, Barcelona) and Barbara Baird (Cornell University, Ithaca,
USA) used nanofabrication to observe and
manipulate patterns of ligands to study assembly of integrin and immunoglobulin E
(IgE) Fc receptor clusters (9). As described by Diez-Ahedo, integrin movement
into nanoclusters and ICAM-1–dependent
nanocluster assembly into microclusters
were diffusive, rather than directed, events.
Baird discussed an innovative use of
waveguides (holes in opaque substrates
that are smaller than the wavelength of
light) to determine if cells can generate
nanoscale protrusions, which could then
be subject to single-molecule measurements.
Determining the initial nanoscale organization of receptors was also an important
topic. Rachel Evans (Hogg laboratory, Cancer Research UK, London) showed that the
leading edge of migrating T cells is particularly rich in active conformations of LFA-1
that are in a complex with Lck and ZAP-70.
These active LFA-1 molecules are located
in microclusters. Diffusion and cytoskeletal
interactions are both important for cluster
formation and stability. The importance of
diffusion in receptor cluster formation was
also highlighted by Agniezka EssenglingOzdoba (Figdor laboratory, Nijmegen
Centre for Molecular Life Sciences, the
Netherlands) for ALCAM (activated leukocyte cell adhesion molecule), but the specific manner in which rebinding to actin in
the cluster stabilizes adhesion is not entirely
understood (10). Receptor clustering is not
limited to lymphocytes, but also occurs on
cells with which lymphocytes interact.
With dynamic fluorescence methods, Olga
Barreiro (Sanchez-Madrid laboratory,
National Center of Cardiovascular Research, Madrid, Spain) provided evidence
for domains organized by tetraspanins that
contain adhesion molecules VCAM (vascular cell adhesion molecule) and ICAM-1 on
endothelial cells that are involved in leukocyte attachment to endothelial cells (11).
M. Davis and Baird discussed the use of an
iterative single-molecule imaging process
called photoactivated localization microscopy to detect steady-state nanoclusters of
TCR and FcR. M. Davis proposed a model
in which all proteins exist in discrete islands
that allow basal segregation and liganddependent merging of specific classes of
transmembrane and lipid-modified proteins
to form signaling microclusters. In future, it
will be important to reconcile results from
high-resolution static imaging methods and
dynamic approaches that are not as clearly
supportive of static preclustering.
How cytoskeletal dynamics and vesicular
trafficking influence the formation of the
immunological synapse and regulate TCR
signaling was also an active area. Alcover
showed that knockdown of ezrin seemed to
prolong TCR signaling by preventing SLP-76
microcluster transport on microtubules. D.
Davis showed evidence that vesicles may
transport LAT (linker for activation of T
cells), which is critical for TCR signaling,
to various plasma membrane locations. The
importance of vesicle trafficking is also
emphasized by evidence reported by my
laboratory and Kumiko Sakata-Sogawa
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of which is mediated by lipids near the immunological synapse (7).
Ron Germain (National Institutes of
Health, Bethesda, USA) described Simmune, a software suite designed to democratize mathematical modeling by making
it available to any biologist who can schematize a putative signaling network
(http://www3.niaid.nih.gov/labs/aboutlabs/
psiim/computationalBiology/). Such modeling efforts require reliable data on the numbers of each molecule present in the cell.
For example, the kinase ZAP-70 is present
at more that 106 copies per T cell; perhaps
this many are needed in relation to the
~500,000 immunotyrosine-based activated
motifs (ITAMs) present in the ~50,000 TCR
complexes. With Simmune, Germaine’s
group identified an additional component
of the switchlike response of Ras signaling,
the positive feedback from the Ras pathway
effector extracellular signal–regulated
kinase (ERK) that protects the Src family
kinase Lck from inhibition by the tyrosine
phosphatase Shp-1. Germain’s efforts complement other efforts to make modeling
tools more accessible, like the Virtual Cell
project of the Center for Cell Analysis and
Modeling at the University of Connecticut
(http://www.ccam.uchc.edu/).
In addition to simulation methods,
mathematical modeling can be used to go
from stochastic single-molecule data to deterministic parameters like kinetic and
equilibrium rates. Cheng Zhu (Georgia Institute of Technology, Atlanta, USA) and
Marguet described two very different types
of single-molecule measurements—Zhu
the detection of single intermolecular interactions in the interface between two cells
after a defined period and area of interaction, and Marguet the detection of single
fluorescently tagged molecules in membrane nanodomains by fluorescence correlation spectroscopy. In both of these data
sets, the raw data consist of a series of 0 or
1 events that appear stochastic, but when
an appropriate mathematical model is applied, an enormous amount of information
can be extracted. For example, Zhu described the impact of CD8 interaction on
the interactions between the TCR and peptide-bound major histocompatibility complex (pMHC) (8). Marguet determined that
association of Fas with plasma membrane
nanodomains was dynamic and that domains were ~80 nm in size. Stephan Wieser
(Johannes Kepler University, Linz, Austria)
performed high-speed single-molecule total internal reflection fluorescence imaging
MEETING REPORT
member of the Toll-like receptor family
and recognizes bacterial DNA (15). Thus, it
was surprising that TLR9-deficient mice
succumb to autoimmunity (16), and this
led to further curiosity about crosstalk in
endosomal compartments. Alexopoulou reported that activation of TLR8 decreases
the activity of TLR7, leading to compensation in TLR8-deficient mice. In addition to
innate immune responses mediated by the
TLR family of receptors, there are TLRindependent responses that involve a multiprotein complex called the inflammasome,
which include proteins, such as NALP
family members, that recognize pathogenic
molecular patterns. Latz presented data on
the mechanisms by which inflammasomes
with the molecular pattern receptors
NALP3 or AIM2 are activated. He presented studies demonstrating that crystals of
monosodium uric acid or silica can damage
lysosomes in innate immune cells (17).
Lysosomal damage leads to activation of
NALP3, resulting in a state referred to as
pyroptosis in which interleukin-1β (IL-1β)
is activated and released. Latz furthermore
demonstrated how immune cells form an
inflammasome with the protein AIM2,
which recognizes double-stranded DNA
(dsDNA) introduced into the cytsosol by
virus infection or transfection (18).
The application of two-photon laser
scanning microscopy to questions in immunology has moved from its initial focus
on lymph nodes and the thymus into all
corners of the body where immune complexity is in evidence. Talks by Ellen
Robey (Berkeley), Philippe Bousso (Paris),
and Germain highlighted work in the brain,
tumors, and infectious foci. Robey described work on toxoplasma infection in
the brain and showed images of neutrophil
swarming (19) and T cell infiltration of the
central nervous system. Germain pointed
out that neutrophils also swarm while in
the process of uptake of Leishmania organisms after introduction by sand fly bites. I
presented collaborative work with Dorian
McGavern (National Institutes of Health,
Bethesda, USA) on meningitis caused by
lymphocytic choriomeningitis virus infection in which myelomonocytic cells were
recruited to the site of infection by CD8+ T
cells, leading to fatal immunopathology
(20). Bousso elaborated on the requirements for the antigen stop signal in lymph
nodes (introduced at the beginning of the
meeting by de Boer). He showed that for T
cells to pause or stop when they encounter
a DC required the continuous presence of
antigen, and competition for antigen reduced the stable interaction of a T cell with
the DC. Germain also emphasized the importance of chemokine deposition on the
reticular fiber network in driving migration
(21, 22). Intravital microscopy has offered
a unique window into issues of vaccination,
host response, and pathogenesis: It is likely
that we are only now scratching the surface
of the complexity in cellular responses involved in these events.
Investigations also extended to the genesis of these thymic and lymph tissues, and
efforts to reconstitute them in vitro in work
presented by Dimitris Kioussis (Medical
Research Council, London), Vivier, and
Stefanie Siegert (Luther laboratory, University of Lausanne, Epalinges, Switzerland). Kioussis has identified the receptor
RET, which is present in neural and lymphoid cells, and its ligands as key factors in
the ability of lymphoid-tissue inducer cells
to induce aggregates with CD11c + cells
and VCAM+ stromal cells that are the seed
for lymphoid tissues. These lymphoid aggregates were revealed to be highly dynamic. Vivier made an intriguing connection
between lymphoid aggregates in the gut
wall called cryptopatches and NKp46+ and
RORgt+ cells that suggested a connection
between cells with NK markers and ongoing lymphoid tissue generation. The study
of the immune system has been driven forward over the years by its in vitro systems.
Siegert has taken early steps to reconstitute
the stromal cell compartment of lymph
nodes in vitro by culturing VCAM+ gp38+
cells that secrete chemokines CCL19 and
CCL21 and cytokine IL-7. These cells were
cultured in a polyurethane sponge in which
3D fiber networks were observed. Incorporating stromal cells into in vitro cultures of
T cell with DCs and antigens may provide
advantages in studies of effector differentiation and memory in vitro.
Bringing It Together for
Type 1 Diabetes
I have saved the keynote talk for last because Mathis was the only speaker to present a story that spanned from a cellular
model to a mouse model to a human disease. The motivation for this work is type 1
diabetes, an autoimmune disease of the
pancreatic islets that strikes unpredictably,
often in childhood, and is usually not detected until it is too late to preserve the islet
mass with currently available procedures.
Mathis, collaborator Ralph Weissleder, and
colleagues (Harvard Medical School,
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(RCIA, Yokohama, Japan) that suggested
that TCR microclusters may be transported
to and from the immune synapse through
insertion and internalization. I showed that
signal termination through the formation of
the central supramolecular activation cluster (cSMAC) required Tsg101, a component of the ESCRT (endosomal sorting
complexes required for transport) complex,
functioning at the plasma membrane. Jan
Burkhardt (University of Pennsylvania,
Philadelphia, USA) reported that the lymphocyte homolog of the actin-binding protein
cortactin HS1 interacts with the kinases
ZAP-70, ITK, and c-Abl to regulate the
localized activation of Vav, a key GEF in
Rac activation (12). In addition, HS1 is required for receptor-mediated endocytosis,
providing another link between signaling
events and endocytic trafficking.
Anne Ridley (King’s College, London)
discussed the role of microtubules in cell
migration and presented data to suggest
that microtubules are not essential for migration of leukocytes, but regulate their directionality. This emphasizes the inherent
flexibility of biological systems to find alternative ways to carry out complex functions, but with increased potential for error.
An example of flexibility that is deleterious
to the animal is exhibited by animals with a
mutation in LAT. Signaling becomes
rerouted in T cells with the LAT Y136F
(Tyr136→Phe) mutation such that T cells
proliferate in the periphery after impaired
development in the thymus, leading to a T
helper 2 cell (TH2) lymphoproliferative disease as reported by Malissen (13).
Many of these experiments are done in
model systems in which the cell interacts
with a planar substrate to improve temporal
and spatial resolution compared to 3D
imaging of randomly oriented cell-cell interfaces. Stephane Oddos (D. Davis laboratory, Imperial College London, UK) reported that structures positive for TCR, SLP-76,
and LAT can be observed in cell-cell interfaces if the conjugate is optimally oriented
by means of laser tweezers (14). This
method has the potential to narrow the gap
in speed and signal-to-noise between the
planar bilayer systems and cell-cell systems, but it remains to be seen if this will be
as useful in complex T cell–DC interfaces.
Innate immunity was also illuminated
through application of visualization techniques based on talks from Alexopoulou
and Eicke Latz (University of Massachusetts Medical School, Worcester,
USA). TLR9 is an endosomally localized
MEETING REPORT
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10.1126/scisignal.266mr4
Citation: M. L. Dustin, Visualizing immune system complexity. Sci. Signal. 2, mr4 (2009).
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Joslin Diabetes Center,
Massachusetts General
Hospital, Boston) have exploited magnetic and fluorescent nanoparticles that can be used for
multimode, noninvasive imaging. With
these probes, they could detect the earliest
stages of leukocyte attack on the islets (insulitis) in a mouse model of diabetes, based
on the increase in vascular leakage and
macrophage engulfment of the probe that
accompany islet inflammation; they could
also predict months ahead of time which
mice would eventually develop clinical diabetes and which would not, as well as get an
early read on therapeutic reversal of disease. Mathis ended her talk by presenting
the first indications that this methodology
can potentially be applied to human type 1
diabetes patients, which opens important
possibilities for noninvasively predicting
the disease, aiding in difficult diagnoses,
and monitoring the effectiveness of drug
treatments. Type 1 diabetes can be attacked
at all scales. A multifaceted approach that
combines imaging from single molecules
and supramolecular networks in cells,
through cells in tissues to inflammation
of organs (Fig. 1), should prove worthwhile in other immune disorders. A model for the future is core facilities with
multiple advanced imaging technologies
under one roof.
Fig. 1. Visualizing immune cell activities in type 1 diabetes. Studies described
at the meeting spanned three scales, and current studies of type 1 diabetes
provide examples of all three. The immunological synapse modeled with a supported planar bilayer can be applied to study autoreactive T cells. Red, TCR;
green, phosphotyrosine; blue, ICAM-1. (Image provided by S. Vardhana, Skirball Institute, New York University School of Medicine, New York, USA) The
next level is intravital microscopy of the pancreatic islets with β cells in green
and vasculature in red. Monitoring the course of islet inflammation in individual
animals requires noninvasive imaging methods, which have been piloted in
mice (23). These preclinical studies have now led to the first efforts at human
translation. A 3D magnetic resonance image (MRI) with a pseudocolored T2* parametric map
overlaid on the pancreas is shown, indicating clearance of Ferumoxtran-10 magnetic 48
hours. [Intravital image provided by J. Hill, R. Kohler, and J. Figueirado (Harvard Medical
School, Boston); 3D MRI image provided by A. Guimaraes, J. Gaglia, and M. Harisinghani
(Harvard Medical School, Boston)]