By
From the Basel Institute for Immunology, CH 4005 Basel, Switzerland
ADynamic View of T Cell Behavior. For many years it has
been known that T lymphocytes engage APCs in a
long-lasting interaction that results in the generation of a
rapid and sustained signal in T cells. Lasting almost 1 h, this
sustained signal is required by T cells to maintain the transcription factors translocated in the nucleus and, ultimately,
to become activated (1). However, in view of the low
number of ligands recognized and of the low affinity of
TCR, the question of how T cells maintain the sustained signal and become activated remained a puzzle (2).
Unlike the antigen-antibody interaction that can be easily studied by measuring the proportions of the reactants at
equilibrium, the interaction of the TCR with its ligand has
proved to be much more difficult to analyze. This interaction occurs in the limited space between the opposing membranes of two motile cells, the T cell carrying ~30,000
TCRs and the APC displaying few ligands, typically ~100
complexes for recognition by T helper cells (3, 4) and perhaps as few as one complex for recognition by cytotoxic T
cells (5).
Because of the small number of ligands recognized, it
was originally thought that only few TCRs would be engaged and would be sufficient to initiate and sustain the signal transduction cascade for the time necessary to lead to T
cell activation. In other words, it was thought that, once
the appropriate molecular interactions had been set in place,
the signal would be self sustained. In the last three years a
kinetic and stoichiometric analysis of the TCR-peptide-
MHC interaction has revealed a very dynamic type of interaction.
Two experimental approaches have proved particularly
useful for studying the dynamics of TCR-peptide-MHC
interaction in living cells. The first is the continuous monitoring of the TCR-mediated signaling that can be achieved
by measuring intracellular calcium elevations or medium
acidification. These methods have been used to demonstrate that the sustained signal in T cell-APC conjugates requires a continuous engagement of TCRs with peptide-
MHC complexes on APC. Indeed, treatments that prevent
these continuous engagements such as addition of anti-MHC
antibodies, blocking of the actin cytoskeleton, or removal
of peptide antigen, can terminate the signaling process within
2-4 min, demonstrating that the signal emanating from
triggered TCR is short lived (6, 7).
The second approach is the measurement of the stoichiometry of the TCR-ligand interaction. Valitutti et al. have
shown that TCR downregulation is a reliable readout of
the number of TCRs triggered. Using this method it was
shown that, over time, as many as 20,000 TCRs are triggered by as few as 100 peptide-MHC complexes (8). Thus
the fast kinetics of TCR-ligand interaction appears to be a
key feature allowing multiple TCR engagements by a single agonist (for review see reference 9). In addition, T cells
appear to be able to take a "count" of the number of TCRs triggered and respond by proliferation and cytokine production when an activation threshold is reached (10).
Three papers in The Journal of Experimental Medicine have
used these methods to reveal new aspects of TCR signaling
induced by peptide-MHC complexes with agonistic or antagonistic properties and by APC with different costimulatory capacities.
Sustained Signaling Inhibited by TCR Antagonists.
The paper by
Wülfing et al. (11) analyzes the calcium signal at single cell
level in T cells activated by a variety of ligands alone or in
combination. The authors distinguish four patterns of intracellular calcium elevation. Although agonist induced a
robust response that correlated with T cell proliferation, three
antagonist complexes gave very different release patterns, or none at all. In addition, when added together with the
agonist, the antagonists markedly reduced the calcium response. These results demonstrate that antagonism does not
require calcium release, but can inhibit the calcium signal
triggered by agonists. Interestingly, the authors report an
inverse correlation between the strength of the stimulus
and the time required for the onset of the calcium signal after T-APC contact. The dose dependence of this delay has
been interpreted to indicate that the rate-limiting step in the calcium response involves accumulation of some form
of intracellular complex or molecule with a half-life of a
few minutes.
TCR Occupancy and T Cell Activation. A third paper by Cai et al. (17) investigates the requirements for TCR downregulation and T cell activation in naive CD8+ T cells using peptide-MHC complexes of different affinities for the TCR. The APCs used by the authors are Drosophila cells, engineered to express extremely high levels of a single peptide- MHC complex in the total absence or in the presence of adhesion and costimulatory molecules. Using these extreme experimental conditions, TCR downregulation can be dissociated from T cell activation. The authors show that when costimulatory APCs are used, T cell activation can occur in the absence of measurable TCR downregulation, whereas with APCs that display the agonist in the absence of adhesion and costimulatory molecules, no activation is observed in spite of an almost complete TCR downregulation. The fact that the authors do not find a correlation between the extent of TCR downregulation and T cell activation is not entirely surprising. Indeed, it is well known that the final outcome of TCR triggering (signal 1 as measured by TCR downregulation) is modulated by additional stimuli provided by costimulation (signal 2) (18). In a previous study done on human T cells, it was shown that in the absence of costimulation, a relatively large number of TCR have to be triggered (~8,000) to activate a T cell, whereas this threshold is decreased to ~1,500 if costimulation is provided (10).
The findings by Cai et al. are also important for our understanding of the nature of the ligands that induce TCR downregulation, as well as for the mechanism of downregulation itself. As to the first point, all the data reported so far indicate a good correlation between the capacity of a ligand to induce TCR downregulation, and its strength as an agonist (8, 10, 12, 17). Indeed, weak agonists have a lower capacity to downregulate TCR that exactly reflect their reduced capacity to induce calcium signal and to activate T cells to produce cytokines, while antagonists fail to do so. As to the mechanism, Cai et al. show that TCR downregulation is not inhibited by genistein or azide. Further evidence indicates that TCR downregulation is also resistant to inhibitors or depletion of protein tyrosine kinases and results from targeting and degradation of TCRs in the lysosomes (19).From Serial Engagements to T Cell Activation.
The sustained signaling in T-APC conjugates appears to be maintained by a
very dynamic mechanism (Fig. 1). The fast kinetics of interaction allows a single peptide-MHC complex to engage
many TCRs over time. The fate of these engagements can
vary depending on the stability of the interaction which
can be influenced by the presence of a coreceptor. Engagements that are stable enough result in complete assembly of
the signal transduction machinery and triggering of the
TCR. This leads to the production of second messengers
that accumulate to the point where a calcium signal is released. However, these messengers are short lived and, in the absence of a continuous TCR triggering, the signal will
stop. This happens experimentally when we interfere with
TCR-agonist interaction or spontaneously as a consequence
of TCR downregulation (20). By serially engaging and
triggering TCRs, few agonists can maintain sufficient levels
of second messengers to sustain the signal.
How can antagonists interfere with serial triggering by agonists? Because of their fast kinetics (16), antagonists can engage TCRs very efficiently. However, although we cannot exclude the possibility of a transient inactivation of some substrates of the signal transduction cascade, it seems that challenge with antagonists per se does not lead to any form of signal transduced, neither is there any long- or short-term consequence on T cells. The serial spoiling of TCRs by antagonists could be the basis for their capacity to effectively reduce the rate of triggering by agonists. How do we go from serial engagement to T cell activation? The possibility of measuring signal 1 using TCR downregulation has provided a quantitative framework to the notion that the effect of the signals emanating from TCRs can be influenced by signals delivered by costimulatory molecules (18). Clearly, the level of TCR engagement required to trigger a T cell is very high in the absence, and much lower in the presence, of costimulation. Understanding the nature of the costimulatory signals and how they are integrated those from TCR will be a major task for future research.
Address correspondence to Antonio Lanzavecchia, Basel Institute for Immunology, Grenzacherstrasse 487, CH 4005 Basel, Switzerland.
Received for publication 2 March 1997.
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