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Department of Chemistry, Stanford University School of Medicine, Stanford, California 94305-5080; and the Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, California 94305-5428
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The binding of T cell receptor to a specific MHC–peptide complex is the critical step in T cell activation as it initiates the intracellular signaling cascade in the T cell (reviewed in reference 1). The first known step in this cascade is the tyrosine phosphorylation of CD3 chains (2, 3). Among other downstream events, these phosphorylations lead to an increase in the level of intracellular calcium (reviewed in reference 4). Initially, calcium is released from intracellular stores, followed by an influx of exogenous calcium through calcium-specific channels in the cell membrane (5, 6). The direct coupling of CD3 phosphorylation to calcium mobilization has been demonstrated by reconstitution of this signaling pathway in COS-1 cells (7).
One major effect of the elevated calcium levels is the activation of calcineurin. This leads to the translocation of the cytoplasmic component of the T cell–specific transcription factor NF-AT1 to the nucleus and the initiation of transcription (8). The shape and mobility of T cells are also regulated by calcium (9, 10). Changes in intracellular calcium levels can be observed directly at the single cell level using ratiometric fluorescent dyes (9–14). While there is general agreement that the calcium signal is a specific consequence of T cell activation, the form and dose response of the signal remain controversial.
Apart from the calcium signal, other signaling pathways are activated following the engagement of the TCR. These include the pathway leading to the activation of the ERK kinases of the MAP kinase family via ras and raf (reviewed in reference 1). In addition, other T cell surface receptors such as B7 and cytokine receptors have been demonstrated to contribute to T cell activation (15, 16).
In addition to the TCR, the T cell coreceptor CD4 also binds to MHC class II, although its binding is not antigen specific. The cytoplasmic tail of CD4 can associate with the tyrosine kinase lck and recruit lck to the TCR complex (17). But even when the lck kinase domain is inactive (18) or in the absence of the CD4–lck association (19, 20) CD4 can contribute to the activation of T cells.
Altering antigenic peptides without decreasing their ability to bind to the MHC can have dramatic effects on T cell responses (reviewed in references 21–24). Some altered peptides elicit only a subset of the responses induced by the original peptide and thus have been labeled partial agonists (25, 26). Some inhibit an otherwise productive response to the original peptide and can thus be considered antagonists (27). Such antagonist peptides alone trigger some biochemical events in T cells, resulting in partial phosphorylation of the CD3-
In a previous study (36), we correlated different biological responses elicited by a set of altered peptides. This has been done in the presence or absence of blocking antibodies against CD4. We find that the different biological responses can be organized into a hierarchy of signaling, from ones elicited by most altered peptides to ones elicited only by the wild-type peptide and variants of it with minimal alterations. In this study, we characterize the form, frequency, and time course of the calcium response to different peptides at the single cell level. We show that the calcium signal reflects the postulated hierarchy of signaling and is predictive of subsequent T cell proliferation. In addition, we measure a delay between T cell–APC contact and the onset of the calcium signal, which is dependent on the strength of the activating stimulus. We suggest a model to explain this delay in the onset of the calcium signal in which the rate-limiting step in triggering the calcium response involves the accumulation of an intracellular signaling molecule with a half-life of a few minutes.
Cell Line.
Calcium Imaging.
chain (28–32). It has been found that the biological effect of a series of altered peptides correlates, with very few exceptions, with the half-life of the corresponding MHC–peptide–TCR complexes in vitro (28–30). Such a correlation has also been suggested based on work with agonists (33) and on theoretical grounds (34, 35).
Materials and Methods
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Abstract
Materials and Methods
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Peptides and Antibodies.
Peptides were synthesized with standard FMOC chemistry and purified by reverse phase HPLC. Anti-CD4 antibody GK1.5 was purchased from Becton-Dickinson (Mountain View, CA).
The moth cytochrome c (MCC)-specific 5C.C7 T cells were derived from the 5C.C7 TCR transgenic mouse (37, 38). Spleen cells were primed with 10 µM MCC peptide and expanded for 8 d in IL-2 containing medium. The T cells (1 x 106 cells/ml) were restimulated with irradiated (3,000 rads) B10.BR spleen cells and 10 µM MCC peptide. After 10 d of culture and expansion in the presence of IL-2, CD4+ cells were sorted by flow cytometry and restimulated in 10-d intervals.
For calcium imaging, 5C.C7 T cells were loaded for 30 min at room temperature with 2 µM Fura-2 in 10% FCS in PBS with 1 mM CaCl2 and 0.5 mM MgCl2. Chinese hamster ovary (CHO) cells transfected with I-Ek were grown in 8-well coverslips (Nunc, Naperville, IL) and peptide loaded with 25 µM peptide for 4–16 h. Peptide loading efficiency throughout this time range was almost constant. After washing unbound peptide away with 5% FCS in PBS with 1 mM CaCl2 and 0.5 mM MgCl2, Fura-2–loaded T cells were added onto a patch of confluent CHO cells on the microscope stage to start the experiment. Intracellular calcium in T cells was determined using C-Imaging1280 System hardware and the Simca Quantitative Fluorescence Analysis software package; both from Compix, Inc., Imaging Systems (Mars, PA). The imaging system was coupled to a Nikon Diaphot 300 inverted microscope, which was equipped with the epifluorescence attachment and a 75W Xenon arc lamp. Alternate excitation of Fura-2–loaded T cells at 340 and 380 nm was achieved using a Ludl high speed dual filter wheel (Ludl Electronic Products, Hawthorne, NY) controlled by the Simca software. Images were collected with a CCD camera (Dage-MTI CCD72) in combination with a SuperGenII intensifier, (DageMTI, Michigan City, IN) to amplify fluorescence. Analysis of intracellular calcium and the generation of figures was achieved with the Simca software package. Per run, 30 to 40 cells were picked at random and analyzed. Data of different runs were pooled. For the figures, data of different experiments in which the topic of the figure was studied in direct comparison of the conditions under question was pooled. Only the 102S titration series (part of Figs. 2 and 3) shows a single representative experiment. The fluorescence of the Fura-2–loaded T cell is only visible when the T cell is right on top of the CHO layer. Because the T cells are added to the chamber from the top, their appearance in the fluorescence image is taken as the time of first contact.
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The calcium response to well-characterized peptides in the MCC family was studied (Fig. 1). For all of them the anchor residues to the MHC are unchanged, and thus binding to I-Ek is very similar (39). Two of the three major TCR contact residues, positions 99 and 102 (39, 43), were changed more or less drastically in different peptides.
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Under different activation conditions, we find four types of calcium signals. Each starts with a rapid rise in intracellular calcium. However, the calcium levels after this initial rise vary. Using the MCC peptide, more than 95% of the cells respond with full signal, as shown in Fig. 2 and described in Table 1. With the altered peptides, we find three qualitatively different variants of the full signal, a reduced, partial, and transient signal (Fig. 2; Table 1). These describe the increasingly weaker calcium responses that we see and have predictive value.
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To determine the effect of low concentrations of stimulating peptide in a defined manner, the MCC and 102S peptides were each serially diluted into the neutral peptide 93E99T102A, such that the total peptide concentration during loading of the APCs is kept constant at 25 µM. As shown in Fig. 3, diluting the MCC peptide 1:1,000, resulting in a 25 nM MCC concentration, has no effect on the frequency or the type of the calcium signal. At a 1:10,000 dilution of the MCC (2.5 nM), more than 85% of the cells still respond with a full signal (see Fig. 5 A). When 102S is diluted into MCC93E99T102A in a dilution series ranging from 25 µM to 25 nM, the signaling frequency throughout the series is still almost 100% (Fig. 3). However, at a 25 nM (1:1,000 dilution), 70% of the cells respond with a reduced signal, as opposed to 30% at 25 µM.
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1 min for the activation of the first T cells. Using weaker stimuli, we find that the onset delay increases as the stimulus gets weaker (Fig. 4). This delay is seen both in the time it takes to activate the first cell, as well as in the average onset time of the T cells in a given experiment.
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Blocking CD4 and Antagonism.
We also studied the effect of blocking CD4 coreceptor engagement on the 102S and MCC calcium response. In the presence of 100 ng/ml of the anti-CD4 antibody GK1.5, the calcium signal induced by 25 nM MCC or 102S is reduced with respect to the frequency and type of signal (Fig. 5 A). The onset is not significantly delayed (Fig. 5 B). This suggests that CD4 plays a role predominantly after the onset of the calcium signal. The inhibition of the calcium signal by CD4 correlates with the data of Rabinowitz et al. (36), who have shown that GK1.5 blocks proliferation in response to MCC and 102S.
The peptides 99R and 102G have been shown to act as antagonists in cytokine secretion and proliferation assays (Reay et al., in preparation; 36). Furthermore, the partial agonist 102S can be changed into an antagonist in the presence of 100 ng/ml of the anti-CD4 antibody (36). To assess the effect of antagonists on the calcium response, the MCC peptide was serially diluted into the three different antagonist conditions. For a 1:10,000 dilution of MCC into 102S/GK1.5, resulting in 2.5 nM MCC concentration, 10% of the cells do not respond at all, and 30% respond with a reduced calcium signal (Fig. 6 A). This is essentially the same signal seen with the 102S peptide and GK1.5 alone without any MCC (see Fig. 5). A 1:10,000 dilution of the MCC into 99R and 102G leads to a reduction in the frequency of responding cells to less than 40%, and almost all calcium signals are partial (Fig. 6 B). Thus, the normal MCC calcium response can be efficiently antagonized under limiting activation conditions. In all three cases, the antagonist effect depends strongly on the concentration of the agonist, as MCC diluted to 1:1,000 shows only a small antagonistic effect, as judged by the slight reduction in type and frequency of the calcium signal (Fig. 6 A). Interestingly, however, all three antagonist conditions delay the onset of the calcium signal, ranging from slightly (about 1 min larger) to more than twice as long (Fig. 6 B).
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In the system used here, we find that the elevation of intracellular calcium varies from the full response to various reduced or oscillatory patterns and that this correlates with other bioassays as summarized in Table 2. A positive result in the acidification assay is always accompanied by some calcium signaling, whereas a proliferative response correlates with a full calcium signal in most of the cells. Even a small disturbance of the full signal, such as a delayed onset or a response from less than 100% of the T cells, correlates with an inability of the stimulus to induce proliferation of the whole population. This suggests that the need for paracrine help may require the proliferation of the large majority of the individual cells in a population. Furthermore, other intracellular signaling pathways that are necessary for full T cell activation may be activated independent of the calcium signal to some extent. Nevertheless, the importance of a sustained calcium signal has been shown by Timmerman et al. (48), who have demonstrated that only a sustained calcium elevation signal is able to keep NF-ATc in the nucleus. Similarly, Negulescu et al. (46) have shown that a sustained elevation of the intracellular calcium concentration is necessary for the activation of an IL-2 promoter reporter construct. From the data of Karttunen and Shastri (49) and Valitutti et al. (50), it also appears that a full calcium stimulus must be maintained for at least 30–60 min to induce an irreversible commitment to T cell activation, particularly at lower peptide concentrations. Given the importance of the sustained phase of the calcium signal, a small reduction might contribute significantly to a severe reduction in proliferation.
During activation, the T cell receives a wide variety of signals through a large number of individual receptors that have to be integrated within the signaling pathway to allow the cell to decide whether to proliferate or not. Our data allows us to estimate to what extent the calcium signal is already integrated. The pattern of elevation of the intracellular calcium elicited by different peptides is not only quantitatively different, but qualitatively as well. Antagonism is more effective at a higher dilution of the agonist peptides into the antagonist. A blocking anti-CD4 antibody reduces the calcium signal. These three observations suggest that a T cell has counted and qualified the different signals through TCR and CD4 before the elevation of the intracellular calcium concentration. In the process of signal integration, the calcium signal thus seems to be much closer to the final proliferation decision than to the events elicited directly by the single engaged receptors.
Altered Ligands.
With respect to TCR antagonist ligands, recently Sloan-Lancaster et al. (47) have shown that several antagonist ligands can induce weak calcium fluxes and suggest that this might be part of an antagonist signal. Our own data shows that whereas two different antagonist stimuli (102G alone or 102S + 
CD4 antibody) generate detectable, but reduced calcium responses, one (99R) does not. This indicates that the mobilization of intracellular calcium is not necessary for antagonist peptide–MHC ligands to block a response. Thus, their primary effect may lie upstream in the T cell signaling process. Similarly, Windhagen et al. (26) have described an antagonistic peptide that does not induce a calcium signal. Also consistent with a more TCR proximal effect of antagonism are the data of Sloan-Lancaster et al. (32) and Madrenas et al. (31) showing alterations in CD3-
phosphorylation and subsequent ZAP-70 association as a consequence of antagonist ligand engagement.
Kinetics of the Onset Delay: Evidence for a Rate-limiting Step in Triggering a T Cell Calcium Signal with a Half-life of a Few Minutes.
In this report, we determined the time between initial T cell–APC contact and onset of the calcium response. Comparing all activation conditions, we find that a minimal delay of 45 s is seen for the MCC peptide at high as well as low concentrations. Therefore, this initial delay must reflect events that are independent of the stimulus strength. Likely factors in this time frame are the time required for the establishment of cell–cell adhesion and for the transmission of intracellular signals from the membrane to the calcium stores.
Weak stimuli, such as partial agonist or antagonist peptides, result in an additional onset delay of up to 10 min. Recent studies have demonstrated that, in most cases, partial agonists and antagonists dissociate more rapidly from a given TCR than agonist ligands (28–30). Therefore, most altered peptide ligands should reach steady-state with respect to TCR binding more rapidly than agonist ligands. Furthermore, one characteristic of the onset delay is that large changes in the peptide dose result in only small changes in the activation time (Fig. 4). For example, going from 250 nM to 25 nM of the peptide 102S causes only a 40% increase in the onset time. If the formation of individual TCR–peptide–MHC pairs was the rate-limiting step in the induction of the calcium signal there would be, for concentrations of free MHC–peptide smaller than the concentration of free TCR, a linear increase in the onset time as the peptide concentration decreases. If the formation of TCR–peptide–MHC oligomers was critical there would be an even more dramatic increase in the onset time as a function of peptide concentration. Even more sophisticated models in which TCR–peptide–MHC mono- or oligomers need to interact for some minimal time in order for signaling to occur do not fit the observed peptide dependency of the onset delay (Kantor, R., C. Wülfing, and M.M. Davis, unpublished results). Thus, the increased onset time observed for weak stimuli does not appear to depend on some threshold level of TCR ligation or oligomerization. This is not to say that oligomerization does not occur or that the half-life of the TCR–peptide–MHC complex is not important, as other studies have shown that it is (30, 33), but that these factors may not be rate-limiting at the cellular level.
Instead, we suggest that the rate-limiting factor in this delay reflects intracellular reactions leading to the accumulation of signaling molecules. These reactions could involve a continuous competition between phosphorylation and dephosphorylation, or between production or breakdown of other parts of the signaling machinery. We hypothesize that T cell activation shifts the balance of this equilibrium towards the accumulation of signaling molecules. A simple kinetic model based on a reaction scheme of this type agrees well with the onset delay that we observe (Fig. 7). The crucial feature of this scheme is that the turnover of the rate-limiting signaling intermediate is determined by an activation strength-dependent first-order forward and an activation strength independent first-order reverse reaction:
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The concentration over time of this signaling intermediate B is given by B(t) = A0 x (1 + k–1 / k1)–1 x (1 – e(k1 + k–1)t) (see Materials and Methods for details).
The half-life of these reactions can be derived from our kinetic data. Under the very weak activation conditions of the antagonist 102G most calcium responses occur between 8–12 min. Because only a small percentage of the cells respond, the reaction leading to signaling must be nearly complete (i.e., at the new steady-state) before any responses occur. This is depicted in Fig. 7 C. Because the time to reach 95% of a new steady-state level of signaling intermediates is four times the half-time, 2 min is a good approximation for the half-time of the reaction towards the steady-state. At the same time, this is the lower limit for the half-times of the individual forward and reverse reactions of this steadystate. The half-times of this stimulus-independent reverse reaction and this forward reaction for 102G are a few minutes under the additional assumption that to trigger the calcium response the accumulation of the signaling intermediate has to be substantial but not complete (Fig. 7 C). The half-times of the forward reaction for stronger activation conditions would be shorter. As these measurements were done at 25°C, one must also allow for a two to four times acceleration in this value at 37°C. In a broader biological picture, we thus suggest that the accumulation of a signaling intermediate triggers the calcium signal. This intermediate is continuously degraded with a half-time of a few minutes. Therefore, every T cell stimulus that results in calcium signaling has to produce this intermediate with a halftime of less than a few minutes.
Other early processes in T cell activation occur within a similar time frame. One example is chain phosphorylation (31, 32), which reaches steady-state within a few minutes for agonist signaling and within 2–10 min for antagonist signaling (32). Such a reaction could produce the postulated critical signaling intermediate.
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Acknowledgments |
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Submitted: 16 January 1997
Revised: 11 March 1997
1Abbreviations used in this paper: CHO, Chinese hamster ovary; MCC, moth cytochrome C; NF-AT, nuclear factor of activated T cell.
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References |
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