This is a postprint of an article published in
Reichardt, P., Gunzer, M.
The biophysics of T lymphocyte activation in vitro and in vivo
(2006) Results and Problems in Cell Differentiation, 43, pp. 199-218
"Cell Communication in Nervous and Immune System"
Chapter: "The biophysics of T lymphocyte activation in vitro and in vivo"
Running title: Biophysics of T cell activation
Peter Reichardt; Matthias Gunzer
Junior Research Group of Immunodynamics, German Research Centre for Biotechnology
(GBF), Mascheroder Weg 1, D-38124 Braunschweig,
Address correspondence to Dr. Matthias Gunzer, Immunodynamics, German Research Centre
for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany
Office: 49 (0)531-6181-977
Fax : 49 (0)531-6181-312
Keywords: T cell activation, DC, cell-cell interaction, Biophysics
Document Word Count: 5.164
Document Character Count (with spaces): 31.716
N° of References: 85
T cell activation is crucial for the development of specific immune reactions. It requires the
physical contact between T cells and antigen-presenting cells (APC). Since these cells are
initially located at distinct positions in the body, they have to migrate and find each other
within secondary lymphoid organs. After encountering each other both cells have to maintain
a close membrane contact sufficiently long to ensure successful signaling. Thus, there is the
necessity to temporarily synchronize the motile behavior of these cells. Initially, it had been
proposed, that during antigen recognition T cells receive a stop signal and maintain a stable
contact with APCs for several hours when an appropriate APC has been encountered.
However, direct cell observation via time-lapse microscopy in vitro and in vivo has revealed a
different picture. While long contacts can be observed, many interactions appear to be very
short and sequential despite efficient signaling. Thus, two concepts addressing the biophysics
of T cell activation have emerged. The single encounter model proposes that after a period of
dynamic searching a T cell stops to interact with one appropriately presenting APC until
signaling is completed. The serial encounter model suggests that T cells are able to collect a
series of short signals by different APC until a critical activation threshold is achieved. Future
research needs to clarify the relative importance of short and dynamic versus long-lived T-
APC encounters for the outcome of T cell activation. Furthermore, a thorough understanding
of the molecular events underlying the observed complex motility patterns will make these
phenomena amenable for intervention, which might result in the identification of new types of
immune modulating drugs.
T lymphocytes are positioned at the crossroads of the human immune system. They are
central for efficiently mounting an adaptive immune response. To fulfill their tasks, T cells
undergo a complex maturation process and display a characteristic migratory pattern where
they physically and functionally interact with many cell types of the body. Thus, for proper
functioning of the immune system, the ability to migrate in an autonomous fashion is an
indispensable prerequisite . In case of the de novo induction of a specific immune response,
two diverse cell types, a T cell and an antigen-presenting cell (APC: mainly dendritic cells
(DC), B cells or macrophages (Mφ)) have to physically engage and need to exchange specific
signals. T cells recognize a complex of a peptide antigen in combination with an MHC
molecule on the surface of APC via their antigen specific T cell receptor (TCR). During
thymic development, the clonotypic TCRs are generated by a chance recombination of a huge
number of genetic building blocks, also including mechanisms to add variety. This is done by
each developing T cell individually, until a functioning TCR is obtained. Thus, although each
T cell expresses only one type of TCR, the diversity of the TCRs in the whole T cell
compartment is immense [2,3]. However, although there are millions of different TCR, each
one is able to recognize a spectrum of peptide-MHC complexes with different affinity, thus,
each antigen will be recognized by a number of individual T cells, albeit with distinct
efficiency. This system may help finding a balance between the ability to recognize a broad
set of antigens and to mount a specific immune response in a short enough time .
Only contacts with peptide-MHC complexes of sufficiently high TCR affinity will ultimately
lead to an activation associated change in one or both partners, e.g. blast formation and
proliferation in T and B cells or antibody production and antibody class switch in B cells,
only. However, all cell types, that need to interact initially reside in very diverse areas of the
body. Moreover, only very few of a body’s antigen-presenting cells will carry the specific
antigen looked for by a specific T cell. In contrast to hormonal signaling, which allows
communication between far apart cells by means of small, transportable molecules, from all
we know, it is not possible to communicate between an APC and a T cell, which antigen has
entered the body, other than by direct physical cell-cell contact. We will see later how, despite
these obstacles, the body ensures efficient T cell activation leading to a successful immune
response by creating unique immunological “marketplaces” of intense cell communication
and cell-cell contact, the lymph nodes. To better understand the functional importance of the
migration and cell-cell interactions of T lymphocytes, we will first follow a developing T cell
along its way from the bone marrow to the thymus, where precursor T cells get prepared for
their central task, the encounter of a foreign antigen and the appropriate response to it. The
activation of T cells by antigen occurs in the lymph nodes, the anatomy of which we will
therefore briefly discuss. Mapping the T cell’s workplace will then open the ground for
having a closer look at experimental data obtained from direct observations of cell-cell
interactions and at the mechanistic models generated from these data trying to explain how
cellular contacts will enable a T cell to get activated in vivo.
Like all hematopoetic cells T lymphocytes originate in the bone marrow. As T cell precursors,
they leave the bone marrow via the bloodstream to begin a rigorous two-step test for
functionality and self-destructive potential in the thymus termed positive and negative
selection. Details of the thymic selection processes have been described in a number of
excellent reviews [3-5]. Very briefly, thymocytes must try to establish contact via their then
only half-completed TCR with MHC molecules on specialized cortical thymic epithelial cells
and DCs, respectively, which both present a large variety of self peptides. During these
interactions the T cell precursors are sorted by the strength of signals resulting from binding
of their TCR to the peptide-MHC complexes presented in the thymus [6, 7]. T cells are
positively selected as a result of a weak TCR signal, whereas no signal results in death by
neglect and a too strong signal results in negative selection . For effective interactions with
stromal cells thymocytes decrease their motility, and establish sustained calcium oscillations
that may enhance the expression of genes enabling positive selection . The signaling
strength mediated by the TCR is believed to be the key to both positive and negative
selection. Thus, T cells are selected for survival, if the signals received from antigen-
presenting DCs residing in the medulla of the thymus do not exceed a certain threshold. If
The role of T cells within the immune system - a T cell biography
T cell Selection in the Thymus
they do, suspicion is raised that this high affinity towards the selecting peptide might pose a
risk for this T cell to later develop into an auto-reactive T-cell thereby priming the organism
for the development of auto-immune disease. Consequently, the potentially dangerous T cell
is driven into apoptosis. The duration and strength of signal also participates in the decision to
which of the two main T cell lineages a particular T cell will belong to in the future: to the
CD8+ (killer) T cells bearing direct cytotoxic potential or to the CD4+ (helper) T cells
providing support to CD8 cells for killing, to B cells for producing antibodies and to
macrophages/monocytes to kill intracellular pathogens such as listeria . While there is
evidence that positive and negative selection occur spatially distinct from each other and
consecutively in time [6, 11], both processes are aimed at achieving a single goal, namely to
provide the highest possible number of T cell specificities available for fighting invaders
while minimizing the risk of attacking self tissue. This process is rigorous, more than 90
percent of all T lymphocytes entering the thymus fall prey to it and die without ever leaving
the thymus .
2.2 T cell Circulation and Homeostasis
At the end of the thymic selection process, the now mature naïve (i.e. antigen-inexperienced)
T cells are released into the blood and circulate through the periphery . This process
comprises the migration of T cells through secondary lymphoid organs and peripheral
nonlymphoid tissues. The persistent recirculation is fundamental to the maintenance of
immune surveillance. A variety of proteins on the T cell surface facilitates their interaction
with resident endothelial, epithelial, stromal and immune cells and with matrix components of
secondary lymphatic organs such as spleen, lymph nodes, and the lymphoid tissue of the
respiratory tract and intestine [13-15]. Initially, naïve T cells will randomly enter secondary
lymphoid organs (e.g. lymph nodes) [16, 17]. Upon scanning the environment for a cognate
antigen, T cells will predominantly encounter self peptides with which they undergo serial
antigen-unspecific, short-lived contacts providing sub-threshold survival signals and being
implicated in T cell homeostasis [17a]. It could be shown that recirculating naïve T cells
constitutively contact high numbers of DC as they migrate through the perifollicular regions
of the lymph node . The lymph node is also the place where the most important cell-cell
interactions occur with respect to pro-inflammatory T cell activation towards antigens of
invading organisms. Here, antigen drained from the site of infection/inflammation arrives
within afferent lymphatic vessels, in soluble form or bound to antigen presenting cells, mostly
DC . The close proximity in the lymph node of foreign antigens, APC and T cells is
essential for the transformation of a resting naïve T cell into an activated, armed effector T
2.3 T cell activation in the lymph node – a marketplace analogy
In order to get activated T cells needs to find APC presenting the correct peptide-MHC
complexes on their surface. However, there exists an intrinsic information problem. Neither
does a T cell have any knowledge, whether the antigen for which its TCR is specific has been
taken up somewhere in the body. Nor does a DC know, which T cell is specific for the
pathogen that it just had phagocytosed and whose processed peptide antigens are now
presented on its MHC molecules. Likewise, a B cell cannot tell, which T cell can provide help
for the antigen, that fits into its B cell receptor (BCR), a membrane bound version of the
antibody, that is being produced by this particular B cell.
The way nature has solved this problem is probably best explained by a town’s marketplace
analogy. A marketplace is a defined region where all town’s inhabitants go to check, what is
being offered by the merchants. Merchants go to this place and set up their booths because
they know, that they will have a very high chance to get in contact with the town´s
inhabitants. Inhabitants entering have a preference for a specific good, but they don’t know
who is selling this particular product. Therefore, they have to migrate over the entire market
place and interact with many merchants to check the goods. As a result, two individuals, who
did not know of each other’s existence before and were initially far apart meet and interact
Transferred to the immune system, the marketplace is a lymph node, the merchants would be
APC and the customers would be T cells. The “goods” that are being presented are self and
foreign peptide antigens, which are loaded onto MHC molecules of the APC. Thereby
powerful APCs such as DCs, which carry 1-8*106 MHC complexes on their surface ,
most likely present several hundred if not thousands of different peptides and thus can serve
more than one distinct T cell clone (Fig. 1, [19, 20]).
However, it is not only the T cell that moves towards the lymph node, DCs may travel long
ways, too. DC usually reside in peripheral tissues of the body and form a tight network of
gatekeepers for invading foreign antigen (Fig. 2, ).
Fig. 1: The lymph node as busy marketplace for T cell activation and B cell help.
Fig. 2: Langerhans cells, the dendritic cells of the skin, form a tight meshwork of cells in
the epidermis of a mouse ear.
They leave their position once they have taken up a sample of this antigen by phagocytosis
and start a migration process, that ends when the DC reaches the lymph node draining the area
of infection, usually 1-2 days later . This process has been particularly well documented
for Langerhans cells (LC), the DC of the epidermis . Activating tissue resident LC, e.g. by
painting contact sensitizers on the skin or simply by explanting skin and putting it in culture,
induces the rapid emigration of skin resident LC into the lymph vessels of the dermis  and
from there into draining lymph nodes .
The lymph node architecture comprises an inner medullary area rich in plasma cells and
macrophages and an outer cortical region. The node is surrounded by a sinus and then a
capsule. Afferent lymphatics drain into this sinus, thereby bringing new antigens to the node
for presentation to T-cells [18, 25-27]. An unstimulated node's cortex contains primary
follicles which are mainly composed of B cells and some DC surrounded by a DC rich T cell
zone. Lymphocytes entering the lymph node from the blood stream exit the blood vessels via
crossing the walls of specialized post-capillary or high endothelial venules (HEV), around
which DCs may line up in strategic positions to immediately interact with newly arriving T
cells [28, 29]. In fact, DCs will be the major antigen presenters as they are also the most
effective professional antigen-presenting cells (APC) . However, B cells may also
actively participate in antigen presentation, if they were able to internalize soluble antigen via
their B cell receptor, process the antigen and load the generated peptides onto MHC
molecules an then transport these MHC/peptide complexes to the cell surface . B cells
will then migrate with directional preference toward the B-zone-T-zone boundary by
upregulating the chemokine receptor CCR7, which enables the B cells to sense a CCR7-
ligand gradient, which extends from the T zone into the border of the B cell follicle [32, 33].
Immigrant T cells will start scanning the surrounding APCs for a peptide/MHC complex
capable of binding with high affinity to their TCR. By doing this, they display an interesting
and diverse spectrum of biophysical interactions with each of these types of APC [28, 34],
which will be looked at in detail below. If the search is finally successful, the T cell will
respond to the new situation with proliferation and differentiation into a primed effector cell.
For CD4+ T cells, this would be a T helper cell whose main function is to support B cells,
whose presented peptides are recognized, to produce antibodies corresponding to the protein,
from which these peptides were derived. Another important function of helper cells is to
support the generation of cytotoxic effector cells. Consequently, a part of the pre-activated
CD4+ T cell will march towards the lymph node’s B cell zone , while others stay in the T
cell zone or leave the lymph node to function in the periphery . A CD8+ cell, in contrast,
would turn into a cytotoxic T cell (CTL) possessing various direct and indirect possibilities of
killing infected cells. In the much more frequent case that a T cell will fail to meet an
appropriate antigenic partner, the T cell will leave the lymph node and re-start circulating the
2.4 Homing of activated T cells
Following a successful activation, T cells differentiate into effector cells with a certain tissue-
specific phenotype guiding their homing preferences [13, 35-37]. Key factors that collectively
determine the homing of leukocytes are interactions between integrins or selectins with their
tissue adhesion molecules as well as interactions between chemokine receptors and their
specific ligands, the chemokines [13, 35-37]. In addition, the presence of inflammatory
stimuli modulates the homing behavior of immune cells . Activated T cells start homing
to peripheral tissues where they exert pro-inflammatory regulatory effector functions or
recirculate as memory cells . At the target location, T cells leave the vessels and enter the
local tissue ready to exert their effector function [13, 13a]. Alternatively, antigen primed T
cells might become memory cells, an option which may occur early during T cell
development as well as after differentiation into an effector cell . Memory cells either
reside centrally in lymphoid tissues or in the periphery with a very high likelihood to expand
rapidly upon re-encounter of the stimulating antigen .
2.5 T cell Regulation
A special case emerges when the T-cell/APC contact does not generate a full T-cell activation
status aimed at performing the optimal immune response, but rather results in T cells
suppressing the immune response. It is currently a matter of debate, whether a regulatory cell
is a differentiation status of any given T cell or rather a specific type of T cell, that is
preferentially expanded in situations where suboptimal activation is prevailing. Evidence for
naturally occurring regulatory cells has been obtained . But antigen presentation by
immature and semimature DC in vivo might lead to incomplete T cell activation and de novo
induction of regulation [42, 43]. Regulatory T cells have attracted great interest in recent
years and certainly play a very important role in maintaining an efficient and safe immune
system [44, 45]. The role of cell-cell interactions during this process has not yet been studied
in detail .
3. The biophysics of T cell activation
The direct microscopic observation of T cells revealed that upon migration T cells (which
appear round in liquid cell culture) show a very dynamic cell shape with clear polarity: a tail
is formed by a motile uropod, as well as a head consisting of the cytoskeletal leading edge by
which cell-cell contacts are initiated (Fig. 3, [28, 47-50]).
Fig. 3: A naïve T cell (green) contacting an antigen specific naïve B cell (orange). Note
the intense cell-cell contact as well as the membrane protrusions of the T cell engaging
the B cell. The fibrous structures are protein strands consisting of collagen type 1, which
was used to simulate a 3-D environment. A dynamic aspect of such cell pairs can be
obtained from the supplemental material of  on the webpage of the BLOOD journal.
When a firm contact is established, the T cell can give up its migratory morphology and round
up again or remain stationary [51, 52]. However, it may also maintain a dynamic state of
motility and crawl along the APC surface with substantial velocities of up to 10 µm per
minute [28, 34, 49, 53]. How long will such individual contacts persist in vivo and where are
they formed during an immune response? In the following we will now have a closer look at
the available experimental data and on the models of T cell activation derived from these
3.1 Classical in vitro studies of T-APC interaction
Initially, due to the absence of better technology, researchers were only able to take a static
picture of the T-APC interaction. Classical experiments from Kupfer and colleagues showed,
that antigen-specific T cells physically interacted with B-cells presenting the relevant peptide
antigen. Thereby the microtubule organizing center (MTOC) of both cells became reoriented
towards each other . It was the same group that 9 years later showed that upon interaction
the two cells formed a highly organized multi-molecular assembly of proteins at the
interaction plane that is now being referred to as the immunological synapse (IS) . At the
same time it was demonstrated in liquid culture, that naïve T cells need a certain period of
time in contact with APC to reach their full effector potential. This was ~ 6h for the best
possible APC, but could be as long as 30 h for weak APC. Thereby, powerful APC. Like DC
were able to induce T cell proliferation within a few hours of contact, even at low doses of
antigen, while weak APC such as B cells needed long contact times, even at high levels of
pMHC occupancy . In addition, the first approaches using live cell imaging of T cells
migrating on lipid bilayers found that in the absence of recognizable peptide-MHC complexes
T cells were highly mobile and migrated on ICAM-1 layers . However, when in addition
to ICAM-1, a specific peptide-MHC complex was available within the lipid bilayers, the T
cells stopped moving and remained at the MHC-peptide containing spot .
3.2 The single encounter model of T cell activation
The above data were used to establish a model of T cell activation in lymph nodes stating that
naïve T cells are actively motile in lymphoid tissues thereby scanning the surface of all
available APC for specific peptide-MHC complexes. However, as soon as a high affinity
(cognate) MHC-peptide complex is discovered, T cells stop and remain at this location,
forming a stable cell contact with the APC for several hours until full activation is reached.
During that time, an immunological synapse is formed which is essential for signaling and
which is possibly also involved in stopping T-cell motility [58, 59]. The concept of a cognate
cell pair stopping and thus stabilizing its contact was appealing as it provided a solution to the
apparent problem that in vivo a rather low number of motile peptide-loaded APC had to
activate an also very limited number of motile T cells  specific for an invading pathogen. It
appeared logical that in the rare event of a specific APC hitting a specific T cell this
interaction had to be stabilized in order to maximize the chance of a successful T cell
activation. Thus, the complete stopping of the two cellular partners was considered a general
rule for all situations of specific T cell activation.
3.3 Challenges introduced by live cell imaging experiments
This concept, however, was challenged by the first observations employing live cell imaging
of antigen presentation in liquid cultures with T cell blasts and with macrophages serving as
APC. Here it was found that T cells migrate over APC bodies stopping only for a limited
amount of time on one APC but then resume motility and wander on to the next APC. From
these observations it was concluded that a T cell sequentially could gain signals from more
than one APC . However, one point of critique regarding these experiments was that the
particular behavior of the T-cells used in this study could result from their pre-activated state
and thus might not reflect the behavior of a naïve, non-activated T-cell. Therefore, in a new
approach, naïve T cell receptor (TCR) transgenic T cells plus peptide-loaded highly activated
DC were used as antigen-specific activation pair and the two types of cells were embedded in
a 3D collagen matrix as approximation of the 3D environment present within lymphatic
tissue. Using this experimental system, it was shown that it was a general feature of naïve T
cells to form contacts with specific antigen-loaded DC only transiently, for a few minutes
rather than hours (Fig. 4, ).
Fig. 4: T cells (green) interacting with an antigen specific DC (orange) within 3-D
collagen. Not the huge size of the DC relative to the T cells as well as the ability of the
DC to “serve” three T cells at the same time. In vivo, DC can contact up to 200 cells at
any given time point .
In addition, T cells were observed to engage multiple DC in a serial fashion. It was assumed
that although each of these contacts was able to induce signals within the T cell, none of these
short-lived contacts was sufficient to activate the T cell but rather a sequence of several
contacts was needed . It could later be shown that T cells may indeed gradually increase
the level of signaling intermediates  and that the same level of activation can be reached
by a sequence of interrupted contacts or with one stable contact . The intracellular events
underlying these phenomena are not yet fully elucidated. Accumulation of transcription
factors such as cJun over time following suboptimal TCR ligation has been demonstrated
; it was postulated that a counting molecule exists allowing for a stepwise buildup of
activated signaling intermediates finally leading to T cell activation . The question,
whether such a system works like a capacitor, that “deloads” in one stroke, once it has
reached the loading capacity or whether a more analog system exists, that can generate a
smooth response reflecting different levels of activation is currently only a matter of
3.4 A serial encounter model of T cell activation
These findings were put together in the serial encounter model for T cell activation [64, 65].
Based on the findings in 3D collagen matrices the serial encounter model stated that there is
no difference in the biophysical dynamics of T cells contacting low affinity or cognate APC.
However, only cognate APC were able to deliver a signal to the T cell. Thereby, the number
of available pMHC complexes on one cell would not be essential, as it has been shown, that
even one pMHC molecule is sufficient to trigger T cell responses [64a]. Since one contact
was not enough, the T cell was forced to undergo additional contacts to other APC presenting
the same antigen. Such a mechanism would be helpful in “measuring” the amount of antigen
presenting APC in the migratory reach of a T cell and thereby inhibit unnecessary immune
activation in the case of minor infections with very low numbers of antigen specific APC
3.5 The road to intravital imaging – phase models of T cell activation
The results on T cell biophysics obtained in 3D collagen matrices were challenged mainly by
the fact that in true lymphatic tissue collagen fibers are not directly accessible to migrating
cells. Rather, they are ensheathed by follicular reticulum cells of the lymphatic environment
[26, 66]. Thus, the behavior of T cells in 3D collagen might not necessarily represent the in
vivo reality [67-69].
The goal to observe T cell activation in true lymphatic tissue was finally reached by two
groups working with naïve T cells and DC in explanted lymph nodes [70, 71] and by a group
examining thymocytes in reaggregate thymic organ cultures . All three studies were able to
demonstrate effective migration of T cells in lymphatic tissue. This was very remarkable
given the extreme crowding of cells in these organs . In fact, for T and B cells, the
measured motility parameters in intact lymphatic organs were identical to the values that were
obtained for cells migrating in artificial 3D systems, which are only sparsely populated such
that individual cells only rarely touch each other [34, 72-74]. Within T cell zones of lymph
nodes dendritic cells, in contrast, were found to be more static. Rather than migrating within
lymph nodes, they remained mainly stationary, but displayed highly motile dendrites which
were constantly reaching for T cells [49, 75]. Interestingly, the studies arrived at opposite
conclusions concerning the duration of antigen-specific contacts between T cells and DC.
While the studies by Bousso et al. , and Miller et al.  showed that contacts between T
cells and APC were an equal mixture of short-lived and more extended interactions, Stoll et
al. found exclusively long-lasting contacts between adoptively transferred mature DC and
naïve T cells upon antigen presentation . It was possible that the differential results were
due to differences in treatment of explanted tissues or that changes in the behavior of cells
after removal of the lymph nodes occurred. Therefore, it was clear that it was necessary to
approach imaging in lymph nodes in situ, i.e. in the living animal.
This goal was initially reached by Miller et al., albeit only by looking at T cell migration .
The first intravital study of naïve T cells interacting with DC in a lymph node was completed
by Mempel et al. . Monitoring CD8+ T cells this study showed that the T cell activation
followed a three step mechanism. T cells which had just entered the lymph node engaged
antigen-specific DC in short, transient encounters during the first 6-8 hours. After this time
point interactions became more stable (in the range of one or more hours) and the production
of effector cytokines was induced. This phase lasted for ~ 12 h. A third stage was again
characterized by profound motility and only transient encounters with DC. It was during this
phase that T cell divisions became detectable . These findings were essentially confirmed
for CD4+ T cell activation in vivo  and in an explanted lymph node model . In
addition, the group of Amigorena showed that CD8+ T cells contacting DC under a regime
inducing tolerance rather that activation displayed short-lived interactions at any time point,
while Mempel’s three phase model with long interactions in a middle phase  could be
confirmed for an activatory situation . Thus, current data suggest, that a complete absence
of static interactions to DC is associated with less efficient T cell activation or even T cell
tolerization for CD8 cells, while there seems to be no obvious difference of interaction
behavior of CD4 T cells, whether the outcome is anergy or activation . In any case, most
available studies clearly show the presence of dynamic interactions at different time points
during effective T cell stimulation.
The exact function and underlying molecular mechanisms of each respective phase are
unclear, however, the sequence may reflect expression changes of surface receptors and
cytoskeletal regulators during T cell activation. It is possible that the longevity of T-DC
interactions in a phase might indicate the formation of a mature synapse-like contact zone
. While both dynamic and stable contacts contribute to T cell activation and proliferation,
long-lasting interactions may be needed for maximum IL-2 production and gaining of full
effector functions . The phase model or kinetic transition model of T cell interaction
expands the serial encounter model by postulating an integration of several signals of different
duration and strengths into a common pathway of T cell activation.
3.6 The role of the APC
The results of the recent studies lead to another important question: What cell makes the
decision on maintaining the interaction and co-migration? The T cell or the APC? The studies
by Mempel  and Miller  concluded that it was the status of the T cell which decided
about the duration of APC interaction. However, even in the rather homogeneous population
of naïve TCR transgenic T cells, interactions with DC were shown to differ substantially in
vitro, ranging from short-lived to long-lived . The first study addressing the role of the
APC for deciding on the duration of interaction used two types of effective APC, DC and
activated B cells, as well as naïve B cells as a model for a weak, ineffective APC. The APC
were peptide-loaded and incubated separately with naïve TCR transgenic T cells. It was found
that the T cells contacted effective APC, DC and activated B cells, in a short-lived manner,
while they formed long-lasting contacts with inefficient APC, naive B cells. This pattern was
demonstrated both in 3D in vitro systems and in lymph nodes in vivo . The findings were
even more unexpected as the long-lasting contacts with naïve B cells were shown to be the
least efficient in terms of T cell activation, while the much more dynamic contacts to DC and
activated B cells rapidly induced T cell activation .
3.7 Summary: A multitude of T-APC interaction kinetics
For the original problem of how to synchronize the migration of cells initially separated in
space, live cell imaging experiments of the past five years have offered a wide range of
solutions. Synchronization can occur transiently by limiting the migration of T cells to the
surface of APC, either with an almost unchanged velocity compared to free migration or in a
more stable manner with strongly reduced motility down to complete stopping of T cells on
the surface of DC. In a most extreme form, hour-long coordinated migration of a cell pair
occurs, as in the example of naïve B cells as APC . During the co-migration, very motile
T cells were shown to push B cells in vivo through lymph node parenchyma, whereas B cells,
when activated, may take the lead themselves and pull T cells . The latter finding was
later confirmed for B cells which were activated by loading antigen onto their B cell receptor
. Another issue covered by the studies mentioned was directional migration. In the
marketplace analogy, this would mean that customers on the market would somehow be
preferentially attracted to or by specific booths. Currently available data do not support this
idea. In contrast, there is evidence that migration of T cells in lymph nodes is entirely random
and that encounters with APC are stochastic events [76, 80]. However, other researchers call
these conclusions into question . What is agreed upon is the sole fact that the lymph node
per se is attractive to T cells, B cells and APC by means of the chemokines CCL19 and
CCL21 binding to the chemokine receptor CCR7 .
Lastly, it was shown that the character of interaction is not only modulated by antigenic load
and type of APC, but the architecture of the surrounding tissue may influence the dynamics of
cell interactions, too. Tight dendritic cell networks within the lymph node facilitate in vivo T
cell contacts with DC and the transition between them, allowing the T cells to be in contact
with DC for most of the time [75, 83].
4. Conclusions and Outlook
Great advantages in imaging techniques in recent years have led to unique insights into the
complexity of cell-cell interactions in vivo. However, the resulting models of T cell activation
still vary in their interpretation of physical interaction modes of T cells and many aspects
remain yet descriptive. There is now evidence from several research groups for a multi-
step cell interaction process during T cell activation. However, the molecular basis for the
different interaction kinetics observed remains unclear. Other questions in need of
clarification are e.g.: What is the relative importance of long over short contacts for T cell
activation? Are both contact types necessary or is one dispensable in vivo? By what molecular
principle do short cell contacts differ from long ones? Do short contacts “prepare” cells for
long ones and if yes, how? How exactly is the surrounding tissue influencing the duration or
outcome of interaction? Apart from studying effector cell induction, the mechanism of APC
activation (especially of B cells) needs to be investigated. Experimental models need to be
expanded to include locations other than inguinal or polietal lymph nodes, e.g. lymphatic sites
in the gut and the lung. More physiologically relevant models together with pathogen-related
antigens are needed as well as ways to overcome one major obstacle for in vivo imaging
studies, the low number of precursor cells in wild type animals.
In conclusion, T cells display an intriguingly complex pattern of cellular interaction with APC
during their activation. A more complete understanding of the underlying mechanisms in vivo
is needed to modulate or mimic such processes. This would be one prerequisite to make them
amenable for therapeutic intervention. Based on the excellent advances intravital imaging
made in recent years we can be very positive about future insights gained from this powerful
We thank Priyanka Narang for critical reading of the manuscript.
1. Friedl, P. and E.B. Brocker, TCR triggering on the move: diversity of T-cell
interactions with antigen-presenting cells. Immunol Rev, 2002. 186: p. 83-9.
Nikolich-Zugich, J., M.K. Slifka, and I. Messaoudi, The many important facets of T-
cell repertoire diversity. Nat Rev Immunol, 2004. 4(2): p. 123-32.
Goldrath, A.W. and M.J. Bevan, Selecting and maintaining a diverse T-cell repertoire.
Nature, 1999. 402(6759): p. 255-262.
Sebzda, E., et al., SELECTION OF THE T CELL REPERTOIRE. Annu. Rev.
Immunol., 1999. 17(1): p. 829-874.
Correia-Neves, M., et al., The shaping of the T cell repertoire. Immunity, 2001. 14(1):
Bousso, P., et al., Dynamics of thymocyte-stromal cell interactions visualized by two-
photon microscopy. Science, 2002. 296(5574): p. 1876-80.
Richie, L.I., et al., Imaging synapse formation during thymocyte selection: inability of
CD3zeta to form a stable central accumulation during negative selection. Immunity,
2002. 16(4): p. 595-606.
Alberola-Ila, J., et al., Selective requirement for MAP kinase activation in thymocyte
differentiation. Nature, 1995. 373(6515): p. 620-3.
Bhakta, N.R., D.Y. Oh, and R.S. Lewis, Calcium oscillations regulate thymocyte
motility during positive selection in the three-dimensional thymic environment. Nat
Immunol, 2005. 6(2): p. 143-51.
Yasutomo, K., et al., The duration of antigen receptor signalling determines CD4+
versus CD8+ T-cell lineage fate. Nature, 2000. 404(6777): p. 506-10.
Witt, C.M., et al., Directed migration of positively selected thymocytes visualized in
real time. PLoS Biol, 2005. 3(6): p. e160.
Palmer, E., Negative selection--clearing out the bad apples from the T-cell repertoire.
Nat Rev Immunol, 2003. 3(5): p. 383-91.
Butcher, E.C. and L.J. Picker, Lymphocyte homing and homeostasis. Science, 1996.
272(5258): p. 60-6.
Gunzer, M., et al., Systemic administration of a TLR7 ligand leads to transient
immune incompetence due to peripheral blood leukocyte depletion. Blood, 2005.(in
Potsch, C., D. Vohringer, and H. Pircher, Distinct migration patterns of naive and
effector CD8 T cells in the spleen: correlation with CCR7 receptor expression and
chemokine reactivity. Eur J Immunol, 1999. 29(11): p. 3562-70.
Moser, B. and P. Loetscher, Lymphocyte traffic control by chemokines. Nat Immunol,
2001. 2(2): p. 123-8.
Mackay, C.R., Homing of naive, memory and effector lymphocytes. Curr Opin
Immunol, 1993. 5(3): p. 423-7.
Bradley, L.M. and S.R. Watson, Lymphocyte migration into tissue: the paradigm
derived from CD4 subsets. Curr Opin Immunol, 1996. 8(3): p. 312-20.
Brocker, T., Survival of mature CD4 T lymphocytes is dependent on major
histocompatibility complex class-II expressing dendritic cells. J.Exp.Med., 1997. 186
(8): p. 1223-1232.
Itano, A.A., et al., Distinct dendritic cell populations sequentially present antigen to
CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity,
2003. 19(1): p. 47-57.
Cella, M., et al., Inflammatory stimuli induce accumulation of MHC class II complexes
on dendritic cells. Nature, 1997. 388(6644): p. 782-7.
20. Kedl, R.M., et al., T cells compete for access to antigen-bearing antigen-presenting
cells. J Exp Med, 2000. 192(8): p. 1105-13.
Mellman, I. and R.M. Steinman, Dendritic cells: specialized and regulated antigen
processing machines. Cell, 2001. 106(3): p. 255-8.
Romani, N., et al., Migration of dendritic cells into lymphatics-the Langerhans cell
example: routes, regulation, and relevance. Int Rev Cytol, 2001. 207: p. 237-70.
Lukas, M., et al., Human cutaneous dendritic cells migrate through dermal lymphatic
vessels in a skin organ culture model. J Invest Dermatol, 1996. 106(6): p. 1293-9.
Mehling, A., et al., Overexpression of CD40 ligand in murine epidermis results in
chronic skin inflammation and systemic autoimmunity. J Exp Med, 2001. 194(5): p.
Gretz, J.E., A.O. Anderson, and S. Shaw, Cords, channels, corridors and conduits:
critical architectural elements facilitating cell interactions in the lymph node cortex.
Immunol Rev, 1997. 156: p. 11-24.
Gretz, J.E., et al., Lymph-borne chemokines and other low molecular weight molecules
reach high endothelial venules via specialized conduits while a functional barrier
limits access to the lymphocyte microenvironments in lymph node cortex. J Exp Med,
2000. 192(10): p. 1425-40.
Ushiki, T., O. Ohtani, and K. Abe, Scanning electron microscopic studies of reticular
framework in the rat mesenteric lymph node. Anat Rec, 1995. 241(1): p. 113-22.
Mempel, T.R., S.E. Henrickson, and U.H. Von Andrian, T-cell priming by dendritic
cells in lymph nodes occurs in three distinct phases. Nature, 2004. 427(6970): p. 154-
Bajenoff, M., S. Granjeaud, and S. Guerder, The strategy of T cell antigen-presenting
cell encounter in antigen-draining lymph nodes revealed by imaging of initial T cell
activation. J Exp Med, 2003. 198(5): p. 715-24.
Fuchs, E.J. and P. Matzinger, B cells turn off virgin but not memory T cells. Science,
1992. 258(5085): p. 1156-9.
Rodriguez-Pinto, D. and J. Moreno, B cells can prime naive CD4+ T cells in vivo in
the absence of other professional antigen-presenting cells in a CD154-CD40-
dependent manner. Eur J Immunol, 2005. 35(4): p. 1097-105.
Okada, T., et al., Antigen-Engaged B Cells Undergo Chemotaxis toward the T Zone
and Form Motile Conjugates with Helper T Cells. PLoS Biol, 2005. 3(6): p. e150.
Reif, K. et al. Balanced responsiveness to chemoattractants from adjacent zones
determines B cell position. Nature, 2002. 416: p. 94-9.
Gunzer, M., et al., A spectrum of biophysical interaction modes between T cells and
different antigen-presenting cells during priming in 3-D collagen and in vivo. Blood,
2004. 104(9): p. 2801-9.
Kannagi, R., Regulatory roles of carbohydrate ligands for selectins in the homing of
lymphocytes. Curr Opin Struct Biol, 2002. 12(5): p. 599-608.
Springer, T.A., Adhesion receptors of the immune system. Nature, 1990. 346(6283): p.
Springer, T.A., Traffic signals for lymphocyte recirculation and leukocyte emigration:
the multistep paradigm. Cell, 1994. 76(2): p. 301-14.
Hwang, J.M., et al., A critical temporal window for selectin-dependent CD4+
lymphocyte homing and initiation of late-phase inflammation in contact sensitivity. J
Exp Med, 2004. 199(9): p. 1223-34.
Sallusto, F., J. Geginat, and A. Lanzavecchia, Central Memory and Effector Memory T
Cell Subsets: Function, Generation, and Maintenance. Annu. Rev. Immunol., 2004.
22(1): p. 745-763.
40. Seder, R.A. and R. Ahmed, Similarities and differences in CD4+ and CD8+ effector
and memory T cell generation. Nat. Immunol., 2003. 4(9): p. 835-842.
Sallusto, F., et al., Two subsets of memory T lymphocytes with distinct homing
potentials and effector functions. Nature, 1999. 401(6754): p. 708-12.
Jonuleit, H., et al., Identification and functional characterization of human
CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. J
Exp Med, 2001. 193(11): p. 1285-94.
Probst, H.C., et al., Inducible transgenic mice reveal resting dendritic cells as potent
inducers of CD8+ T cell tolerance. Immunity, 2003. 18(5): p. 713-20.
O'Neill, E.J., et al., Natural and Induced Regulatory T Cells. Ann NY Acad Sci, 2004.
1029(1): p. 180-192.
Sakaguchi, S., Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in
immunological tolerance to self and non-self. Nat Immunol, 2005. 6(4): p. 345-52.
Fontenot, J.D., et al., Regulatory T cell lineage specification by the forkhead
transcription factor foxp3. Immunity, 2005. 22(3): p. 329-41.
Negulescu, P.A., et al., Polarity of T cell shape, motility, and sensitivity to antigen.
Immunity., 1996. 4(5): p. 421-430.
Valitutti, S., et al., Sustained signaling leading to T cell activation results from
prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton. J.Exp.Med.,
1995. 181: p. 577-584.
Miller, M.J., et al., T cell repertoire scanning is promoted by dynamic dendritic cell
behavior and random T cell motility in the lymph node. Proc Natl Acad Sci U S A,
2004. 101(4): p. 998-1003.
Miller, M.J., et al., Imaging the single cell dynamics of CD4+ T cell activation by
dendritic cells in lymph nodes. J Exp Med, 2004. 200(7): p. 847-56.
Lee, K.H., et al., The immunological synapse balances T cell receptor signaling and
degradation. Science, 2003. 302(5648): p. 1218-22.
Shakhar, G., et al., Stable T cell-dendritic cell interactions precede the development of
both tolerance and immunity in vivo. Nat Immunol, 2005, 6 (7): p. 707-714
Gunzer, M., et al., Antigen presentation in extracellular matrix: interactions of T cells
with dendritic cells are dynamic, short lived, and sequential. Immunity, 2000. 13(3):
Kupfer, A. and S.J. Singer, The specific interaction of helper T cells and antigen-
presenting B cells. IV. Membrane and cytoskeletal reorganizations in the bound T cell
as a function of antigen dose. J Exp Med, 1989. 170(5): p. 1697-713.
Monks, C.R., et al., Three-dimensional segregation of supramolecular activation
clusters in T cells. Nature, 1998. 395(6697): p. 82-6.
Iezzi, G., K. Karjalainen, and A. Lanzavecchia, The duration of antigenic stimulation
determines the fate of naive and effector T cells. Immunity, 1998. 8(1): p. 89-95.
Dustin, M.L., et al., Antigen receptor engagement delivers a stop signal to migrating T
lymphocytes. Proc Natl Acad Sci U S A, 1997. 94(8): p. 3909-13.
Dustin, M.L. and A.C. Chan, Signaling takes shape in the immune system. Cell, 2000.
103(2): p. 283-94.
Lanzavecchia, A., G. Lezzi, and A. Viola, From TCR engagement to T cell activation:
a kinetic view of T cell behavior. Cell, 1999. 96(1): p. 1-4.
Underhill, D.M., et al., Dynamic interactions of macrophages with T cells during
antigen presentation. J Exp Med, 1999. 190(12): p. 1909-14.
Borovsky, Z., et al., Serial triggering of T cell receptors results in incremental
accumulation of signaling intermediates. J Biol Chem, 2002. 277(24): p. 21529-36.
Faroudi, M., et al., Cutting edge: T lymphocyte activation by repeated immunological
synapse formation and intermittent signaling. J Immunol, 2003. 171(3): p. 1128-32.
63. Rosette, C., et al., The impact of duration versus extent of TCR occupancy on T cell
activation: a revision of the kinetic proofreading model. Immunity, 2001. 15(1): p. 59-
Friedl, P. and M. Gunzer, Interaction of T cells with APCs: the serial encounter
model. Trends Immunol, 2001. 22(4): p. 187-91.
D. J. Irvine, M. A. Purbhoo, M. Krogsgaard, and M. M. Davis. Direct observation of
ligand recognition by T cells. Nature 419 (6909):845-849, 2002.
Rachmilewitz, J. and A. Lanzavecchia, A temporal and spatial summation model for
T-cell activation: signal integration and antigen decoding. Trends Immunol, 2002.
23(12): p. 592-5.
Kaldjian, E.P., et al., Spatial and molecular organization of lymph node T cell cortex:
a labyrinthine cavity bounded by an epithelium-like monolayer of fibroblastic
reticular cells anchored to basement membrane-like extracellular matrix. Int
Immunol, 2001. 13(10): p. 1243-53.
Dustin, M.L. and A.R. de Fougerolles, Reprogramming T cells: the role of
extracellular matrix in coordination of T cell activation and migration. Curr Opin
Immunol, 2001. 13(3): p. 286-90.
Dustin, M.L., P.M. Allen, and A.S. Shaw, Environmental control of immunological
synapse formation and duration. Trends Immunol, 2001. 22(4): p. 192-4.
Lanzavecchia, A. and F. Sallusto, Antigen decoding by T lymphocytes: from synapses
to fate determination. Nat Immunol, 2001. 2(6): p. 487-92.
Miller, M.J., et al., Two-photon imaging of lymphocyte motility and antigen response
in intact lymph node. Science, 2002. 296(5574): p. 1869-73.
Stoll, S. Delon, J., Brotz, T. M. and R. N. Germain. Dynamic imaging of T cell-
dendritic cell interactions in lymph nodes. Science, 2002, 296(5574): p. 1873-1876.
Friedl, P., et al., CD4+ T lymphocytes migrating in three-dimensional collagen lattices
lack focal adhesions and utilize beta1 integrin-independent strategies for polarization,
interaction with collagen fibers and locomotion. Eur J Immunol, 1998. 28(8): p. 2331-
Friedl, P., P.B. Noble, and K.S. Zanker, T lymphocyte locomotion in a three-
dimensional collagen matrix. Expression and function of cell adhesion molecules. J
Immunol, 1995. 154(10): p. 4973-85.
Friedl, P., et al., Locomotor phenotypes of unstimulated CD45RAhigh and
CD45ROhigh CD4+ and CD8+ lymphocytes in three-dimensional collagen lattices.
Immunology, 1994. 82(4): p. 617-24.
Lindquist, R.L., et al., Visualizing dendritic cell networks in vivo. Nat Immunol, 2004.
5(12): p. 1243-50.
Miller, M.J., et al., Autonomous T cell trafficking examined in vivo with intravital two-
photon microscopy. Proc Natl Acad Sci U S A, 2003. 100(5): p. 2604-9.
Hugues, S., et al., Distinct T cell dynamics in lymph nodes during the induction of
tolerance and immunity. Nat Immunol, 2004. 5(12): p. 1235-42.
Lee, K.H., et al., T cell receptor signaling precedes immunological synapse formation.
Science, 2002. 295(5559): p. 1539-42.
Hurez, V., et al., Restricted clonal expression of IL-2 by naive T cells reflects
differential dynamic interactions with dendritic cells. J Exp Med, 2003. 198(1): p.
Wei, S.H., et al., A stochastic view of lymphocyte motility and trafficking within the
lymph node. Immunol Rev, 2003. 195: p. 136-59.
Germain, R.N. and M.K. Jenkins, In vivo antigen presentation. Curr Opin Immunol,
2004. 16(1): p. 120-5.
82. Forster, R., et al., CCR7 coordinates the primary immune response by establishing Download full-text
functional microenvironments in secondary lymphoid organs. Cell, 1999. 99(1): p. 23-
Westermann, J., et al., Naive, effector, and memory T lymphocytes efficiently scan
dendritic cells in vivo: contact frequency in T cell zones of secondary lymphoid organs
does not depend on LFA-1 expression and facilitates survival of effector T cells. J
Immunol, 2005. 174(5): p. 2517-24.