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Address correspondence to M.M. Davis, Dept. of Microbiology and Immunology, Stanford University School of Medicine, 279 Campus Dr., Beckman Center, B 221, Stanford, CA 94305. Tel.: (650) 725-4755. Fax: (650) 723-1399. email: mdavis{at}cmgm.stanford.edu
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Abstract |
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Key Words: lymphocyte activation; macromolecular systems; three-dimensional imaging; T cell receptor; cell communication
Abbreviations used in this paper: APC, antigen-presenting cell; APL, altered peptide ligand; cSMAC, central supramolecular activation complex; GPI, glycosyl-phosphatidyl inositol; IRM, interference reflection microscopy; MCC, moth cytochrome c; pMHC, peptidemajor histocompatibility complex; pSMAC; peripheral supramolecular activation complex; TCR, T cell receptor.
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Introduction |
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Altered peptide ligands (APLs) are variants of wild-type TCR-activating peptides that produce a range of effects from differential cytokine production to null peptides with no activity (Sloan-Lancaster and Allen, 1996). Antagonist peptides are a rare subset of APL that block T cell activation to otherwise stimulatory concentrations of agonist ligand (De Magistris et al., 1992; Sloan-Lancaster et al., 1994). In general, antagonist ligands have lower affinities and faster off-rates for a given TCR than agonists (Lyons et al., 1996; Wu et al., 2002), with some exceptions (Sykulev et al., 1998). Structural studies have indicated that in at least one case, the poor affinity of an antagonist ligand can be explained by a packing defect in its association with TCR when complexed to MHC (Baker et al., 2000). The filling of this space increases the affinity and is sufficient to revert the peptide into an agonist. Antagonist pMHC complexes usually need to be in excess of activating (agonist) pMHC complexes on the antigen-presenting cell (APC) surface to block T cell activation, although there are exceptions, notably in the class I MHC/CD8+ T cell system (Purbhoo et al., 1998; Sykulev et al., 1998). Antagonist peptides clearly have physiological relevance, as some pathogens may use antagonist peptide mutants of dominant MHC epitopes to hinder the T cell response during the course of infection. Such antagonist variant epitopes are thought to play a role in HIV and HBV viral modulation of T cell immunity (Bertoletti et al., 1994; Klenerman et al., 1994; Purbhoo et al., 1998).
Recently, it has been found that T cell activation occurs in the context of a highly organized membrane junction between the T cell and the APC termed the immunological synapse (Monks et al., 1998; Grakoui et al., 1999). The hallmarks of a mature synapse are binding and transport of pMHC complexes into the central supramolecular activation complex (cSMAC), surrounded by a more extensive integrin-rich region, the peripheral supramolecular activation complex (pSMAC; Bromley et al., 2001a). Integrins link cytoskeletal rearrangement and cell adhesion to engagement of extracellular matrix or cell surface ligands. As adhesion precedes mature synapse formation, integrins serve an indispensable function in the formation of the immunological synapse (Dustin and Chan, 2000). LFA-1 (Lß2) is arguably the most important integrin for leukocyte function, regulating T cell activation and contact formation via interactions with its cell surface ligands ICAM-1, -2, or -3. TCR signaling causes an increase in the affinity/avidity/valency of LFA-1 for its ligands, through an "inside-out" signaling process involving differential cytoskeletal association and ligand induced conformational changes (Kucik et al., 1996; Takagi et al., 2002; Kim et al., 2003). Signaling through LFA-1 also aids synapse formation by synergizing with CD28 in triggering the active transport of TCR/CD3 to the cSMAC in activated (Wulfing and Davis, 1998; Wulfing et al., 2002), but not naive, T cells (Bromley and Dustin, 2002). Lastly, integrin engagement ("outside-in") along with signals downstream of the TCR complex serve to modulate the cessation of T cell migration upon activation, collectively termed the "stop signal" (Dustin et al., 1997; Dustin, 2002). Correlating with a mature synapse pattern, the stop signal arises from Ca2+-dependent arrest of cell motility and down-modulation of myosin motor function as well as T cell cytoskeletal remodeling that stabilizes the cellcell interface, allowing a long-lived contact and continuous TCR-driven signaling (Dustin, 2003; Huppa et al., 2003; Jacobelli et al., 2004). Hence, as corroborated by in vivo intravital imaging in mouse lymph nodes, antigen recognition on activated dendritic cells by naive T cells leads to stable contact formation concurrent with cytokine production (Mempel et al., 2004).
Here, we characterize the effects of TCR antagonism on the formation of the immunological synapse, using both a supported lipid bilayer system and live B cells to present pMHC complexes. Simultaneous presentation of antagonist and agonist peptide-loaded MHC molecules in the presence of integrin ligands reduces T cell adhesion to membranes at limiting antigen concentration. Although a similar fraction of adherent T cells are able to cluster MHC, the density of MHC in the cSMAC is significantly reduced in the antagonized contacts compared with the null peptide control. This decrease correlates with a block in T cell proliferation that is not rescued by incorporation of the costimulatory ligands B7-1 and CD48 into the bilayer. For the majority of T cells unable to stop and form a stable contact, integrin engagement as monitored by ICAM-1 accumulation occurs in a unique crescent-shaped pattern at the leading edge. Surprisingly, the density of integrin ligand in these moving contacts is equivalent to that of pSMAC-localized integrins in a mature, stable synapse, suggesting that up-regulation of LFA-1 binding to ICAM-1 does occur but is not sufficient for T cell arrest in the bilayer system. We extend these findings to live three-dimensional imaging of the TB cell interface and demonstrate that copresentation of antagonist and agonist pMHCs invokes similar ICAM-1 accumulation patterns, although these T cells maintain contact with the B cell surface. Nonetheless, antagonist pMHCs compromise downstream effector functions such as elevated intracellular Ca2+ and IL-2 secretion. Hence, TCR antagonists hinder synapse formation by attenuating the stop signal but not integrin activation, manifested as a dense sickle-like pattern of integrin ligand engagement. In addition, antagonists decrease pMHC transport to the cSMAC in the contacts that can overcome this effect, which may be the result of direct competition for TCR binding while lacking sufficient interaction for complete signaling.
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Results |
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Readhesion blocked by antagonism
TCR antagonism is thought to create an anergic state that renders the T cell unable to respond to antigen for up to several days following exposure to antagonist peptides (Sloan-Lancaster and Allen, 1996). To investigate anergy in our system, we took advantage of our previous observation that formation of a tight interface between bilayer and T cell observable by interference reflection microscopy (IRM) is highly dependent on productive TCR signaling (Grakoui et al., 1999). After allowing T cells to adhere to bilayers with 1:10 dilutions of MCC into the APL in a flow chamber, we disrupted the contacts by pulsing with cold buffer, which causes gradual detachment of the cell from the bilayer membrane. The temperature was then returned to 37°C and the T cells were scored using IRM for the ability to reform tight contacts. When we compared T cell contact reinitiation on bilayers where either antagonist (99R) peptide or the null (99A) variant were combined with agonist, we saw a significant decrease in the percentage of T cells that were able to reestablish tight contact (Fig. 1 e, 6.4% vs. 20.2%, respectively). It is important to note that initial adhesion rates for the two conditions at this peptide dilution were very similar (Fig. 1 a, adhesion at 1:10 was 60% vs. 59%). This refractory state lasted for at least half an hour. Hence, T cells were inhibited from activation, at least with respect to adherence and synapse formation, by prior exposure to a mixture of agonist and antagonist ligands.
We have recently shown that the "null" peptide MCC99A, although having no biological activity on its own, can nevertheless participate in agonist-driven MHC clustering and augment T cell activation at low MHC densities (Wulfing et al., 2002). The bilayer experiments described here are at higher densities where this synergistic effect has not been observed. Nevertheless, to rule out that the deleterious effects of 99R compared with 99A on T cell activation is not simply a result of the loss of this property of 99A, we have also made comparisons with 99E, another MCC variant that forms a pMHC complex that does not contribute in any detectable manner to synapse formation or activation (Wulfing et al., 2002). We found that 99R still displayed antagonism when compared with 99E (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200404059/DC1), which is consistent with previous results (Rabinowitz et al., 1996).
Unique ICAM-1 engagement pattern during TCR antagonism
Studying the patterns of GPI-linked ICAM-1 accumulation in the presence of antagonist peptides, among the majority of T cells that were unable to form a bona fide synapse with MHC accumulation, we noticed a prominent crescent- or sickle-like accumulation of ICAM -1 (Fig. 2, a and b). This phenotype predominated at higher agonist pMHC densities while maintaining excess antagonist, and was clearly different than the patterns of ICAM-1 on bilayers without agonist or with antagonist pMHC alone (Grakoui et al., 1999), but was similar to ICAM-1 engagement in mobile contacts formed by ionomycin-anergized Th1 cells (Heissmeyer et al., 2004). Cells with this aberrant integrin accumulation did not stop and reorient toward the bilayer, but slowly crawled (3 µm/min ± 0.7 µm) in the direction of the polarized ICAM-1 sickle. Their migration paths resembled long alternating arcs (r = 15 µm) directed away from the site of initial contact (Fig. 2 c). LFA-1 on the T cell could apparently engage and aggregate its ligand ICAM-1, despite the fact that the stop signal was blocked by TCR antagonism. Similar patterns were also observed with another antagonist variant of MCC, T102G (unpublished data). The addition of PMA, a phorbol ester that activates PKC, was unable to provide the stop signal or alter the pattern of ICAM-1 accumulation (unpublished data). To further investigate this event at more optimal concentrations of agonist for T cell activation and LFA-1 up-regulation, we formed a two-component pMHC system featuring high-density mobile pMHC coupling through His-tagged MHC I-Ek and Ni-NTA lipid derivatives, as well as lipids (GPI) covalently coupled to MHC as used here and in previous experiments (Grakoui et al., 1999). Using this new method, we could achieve mobile MHC densities of several thousand molecules per square micron, which could activate T cells and cluster into synapses (unpublished data).
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Integrin engagement and TCR antagonism in live TB cell interface
To extend these results to live, costimulation-competent APCs, we used the B cell lymphoma CH27 expressing a GFP-linked ICAM-1 molecule. Consistent with previous results (Wulfing et al., 1998), B cells loaded with the MCC peptide caused T cells to form tight contacts leading to elevated intracellular calcium and the early accumulation of ICAM-1 into a small, dense cluster at the center of the synapse (Fig. 4 a). At this peptide concentration, 1.5% of the MHCs on the B cell surface are occupied by the peptide as detected with pMHC specific antibody staining (unpublished data). Given the MHC density of 220 per square micron for these cells, this resulted in an average plasma membrane agonist-MHC density of 3 pMHC molecules per square micron, or
2400 pMHCs per cell. When we diluted the agonist into 100-fold excess of antagonist peptide, resulting in 0.03 agonist pMHC per square micron (
20 per cell), we saw that the T cells were still able to adhere to the APCs, and form flat interfaces and flux calcium, albeit intermittently. Approximately half these contacts showed very little ICAM-1 accumulation over background and correlated with little or no calcium flux (Fig. 4 b, top right). However, most of the T cells that formed productive contacts with elevated calcium accumulated ICAM-1 in asymmetric patterns very similar to the sickle-like ones seen on planar bilayers (Fig. 4 b, bottom left), although the clusters often appeared as patches of higher intensity arrayed in an elongated shape with positive curvature around the perimeter of the T cell contact rather than the contiguous regions of more uniform intensity on bilayers. Consistent with these observations, we found that at these agonist/antagonist ratios significantly fewer T cells overall were fluxing calcium at any given time (Fig. 4 c).
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Discussion |
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The stop signal that is most evident using the two-dimensional system of supported lipid bilayers can be viewed as a model for antigen recognition in vivo, which is accompanied by T cell motility arrest in nearly every experimental approach to date. One notable exception is a recent study of naive T cells entering peripheral lymph nodes, which show an initial phase of rapid, "random-walk" migration even in the presence of potent activating signals in the form of antigen-loaded dendritic cells. This phase, lasting several hours, occurs before the T cells settle down to stable, long-lived interactions consistent with the stop signal and presumably involving the formation of a mature synapse (Mempel et al., 2004). The cellular mechanism(s) responsible for the initial refractive phase of high T cell motility in the face of antigen exposure is not known, and evidently not recapitulated in the model systems used in this work. This behavior may be a result of additional cues only present in intact lymph nodes, including pro-migratory factors such as chemokines that have been shown to overcome the stop signal under certain conditions (Bromley et al., 2000). Alternatively, the T cells themselves may be altered in some way due to their recent extravasation through the high endothelial venules, perhaps via metalloprotease-mediated proteolysis of adhesion receptors, and require this initial phase to reexpress surface proteins or signaling intermediates that are necessary for T cell motility arrest.
Current models for TCR antagonism invoke a range of phenomena from kinetic competition to conformational changes in the TCR subunits or negative signal spreading (Sloan-Lancaster and Allen, 1996; Dittel et al., 1999). Several studies using dual TCR expressing T cells to study antagonism (Daniels et al., 1999; Dittel et al., 1999; Stotz et al., 1999; Wang and Grey, 2003) have reported conflicting results. Antagonist peptides can alter tyrosine phosphorylation patterns on the CD3 chain (Sloan-Lancaster et al., 1994; Madrenas et al., 1995) although this has not been universally observed (Liu and Vignali, 1999) and its relevance to antagonism has been challenged (Pitcher et al., 2003). Nonetheless, these partially-phosphorylated molecules could inhibit T cell activation. As seen in Fig. 3 c, the ICAM-1 sickle pattern is lost over time with continued exposure to antagonist peptides. Likewise, in our readhesion assay (Fig. 1 e), T cells on agonist-antagonist bilayers were unable to reestablish a disrupted contact. These findings suggest that continuous engagement by antagonist pMHC creates a pool of inactive TCR that gradually disables the ability of the T cell to respond to agonist ligand.
A subset of peptides that are unable to activate T cells (null peptides) can nonetheless cocluster in the synapse in the presence of agonists and augment T cell activation within a lower range of MHC density (Wulfing et al., 2002). Null and antagonist versions of TCR ligands both possess weaker binding kinetics and lower half-lives of interaction with the TCR (Lyons et al., 1996; Wu et al., 2002). However, these two types of APL have different effects on TCR signaling and synapse formation when copresented with agonist ligands. In particular, null pMHC complexes have no measurable affinity for their cognate TCR (Lyons et al., 1996; Wu et al., 2002), but clearly do bind as they are recruited into the synapse in a TCR dependent manner (Wulfing et al., 2002). Furthermore, they clearly do not act as antagonists. We propose that this apparent discrepancy might be best addressed by a kinetic model (Fig. 6), where decreasing the half-life of interaction (t1/2 = ln2/koff) of an agonist ligand would produce effects ranging from reducing the activating potential of the peptide (weak agonist), to creating ligands with antagonist properties, and finally null ligands that may be compatible with TCR-MHC binding but do not engage long enough to signal. Mathematical modeling of receptor engagement in the immunological synapse has shown the importance of kinetic boundary conditions that affect molecular patterning (Lee et al., 2002b). Rapid, transient interactions whose kinetics fit the postulated optimal range for antagonism (Fig. 6) may disrupt the cytoskeletal-driven TCR transport to the center of the synapse that characterizes agonist engagement (Wulfing and Davis, 1998) hinder CD4-TCR association (Zal et al., 2002) or prevent TCR oligomerization as suggested by the results of Reich et al. (1997). This model also poses the testable prediction that for any given TCR there exists a kinetic half-life window where any ligand falling within this range will behave as a TCR antagonist.
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Similar to our previous work (Wulfing et al., 2002), another study has described the coclustering of null pMHC with agonist using effector T cells and dendritic cells as APCs (Chmielowski et al., 2002). In contrast, naive T cells "expel" null peptides from the agonist pMHC core, which is postulated to occur via an active mechanism specific to antigen inexperienced cells. Antagonist peptide, but not another null peptide, interferes with this expulsion and causes resdistribution across the whole T cell contact. These observations can now be put into context by the work presented here: the exclusion effect in naive T cells is likely due to less efficient null pMHC retention arising from less TCR accumulation at the cSMAC compared with effector T cells. TCR transport deficiency in noneffector T cells is well documented, evidenced by slower or only transient pMHC clustering in naive T cells (Lee et al., 2002a) and thymocytes (Richie et al., 2002). In addition, a recent paper shows that human naive (but not effector) CD8+ T cells are deficient in their ability, independent of antigen, to cluster integrins into a pSMAC ring pattern termed the "presynapse" (Somersalo et al., 2004). Hence the enhanced cytoskeletal activity of effector T cells would be better able to retain null pMHC corralled at the cSMAC. Antagonists may hinder this effect simply by reducing MHC cluster density, as we show here (Fig. 1 b). The fact that antagonism leads to null pMHC spreading out across the whole interface argues against an active "expulsion process" in naive T cells. If that were indeed the case, antagonists would block this putative process and a central cluster of null pMHC would rather be observed, as with effector T cells. Finally, we note that sickle-like ICAM-1 patterns have also been recently observed in a study of ionomycin-anergized Th1 cells (Heissmeyer et al., 2004). The stop signal is similarly compromised, despite some MHC clustering. Hence, that work and ours together support the hypothesis that an aberrant integrin engagement pattern and an abrogated stop signal may comprise novel hallmarks of T cell anergy. In summary, our work complements and extends these studies in quantifying the precise molecular densities of MHC and ICAM-1 and showing how antagonist peptide APLs perturb the density and geometry of molecules at the T cell synapse.
The value of TCR antagonism to a pathogen could be to blunt the immune response toward rare agonist epitopes to defuse T cell help during an infection, especially for small and highly mutagenic viral genomes with a limited number of MHC epitopes for the T cell repertoire. Likewise, HIV can generate Gag epitope variants that act as antagonists to block T cell activation (Klenerman et al., 1994). Our results demonstrate that antagonist peptides, in addition to their role in inhibiting MHC accumulation in the mature synapse, can also disrupt synapse formation by bypassing the stop signal that is normally elicited by antigen engagement, leading to an aberrant integrin pattern and slow but sustained T cell motility. We also show that an increase in integrin avidity alone through inside-out signaling is not sufficient to initiate the T cell stop signal.
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Materials and methods |
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T cell functional assays
Proliferation of T cells on bilayers was assayed in a 96-well format. Bilayers were formed from 1µl of vesicle droplets on the bottom of the well, sandwiched by a heat treated (450°C for 8 h) 5-mm coverslip, blocked with 0.2 µm filtered BSA solution (2% BSA [Sigma-Aldrich] in 50 mM citrate/phosphate, and 50 mM NaCl) and loaded with peptides overnight at 37°C. After peptide loading, the wells were washed with 10% FCS/RPMI and each glass coverslip flipped in solution to rest with the bilayer side facing up. T cells were added for 48h at 37°C, followed by 1216 h of [3H]thymidine (Amersham Biosciences) incorporation, transfer to filtermat, and quantification by liquid scintillation (1205 Betaplate; Wallac). IL-2 was quantified from a T cellB cell coculture assay (relevant peptides added to 1 x 105 T and 104 B cells/well in 10%FCS/RPMI and incubated at 37°C for 56 h) by a standard sandwich ELISA using epitope-matched antibodies (BD Biosciences) and Europium detection (Victor; Wallac).
Microscopy and image analysis
Two fluorescent videomicroscopy systems outlined in detail previously (Dustin et al., 1997; Wulfing et al., 2002) were used to collect and process images. The first is a custom inverted microscope (Yona Microscopes) using a 100x objective (model Neofluar; Carl Zeiss MicroImaging, Inc.), cooled CCD camera (Photometrics) and IP Lab control software (Signal Analytics). The second is an Axiovert S100TV (Carl Zeiss MicroImaging, Inc. or Universal Imaging Corp.) system using a 63x Neofluar objective, 175W Xenon lamp (Sutter Instrument Co.), and an interline cooled CCD-1330-V/HS (Princeton Instruments). Bilayers for imaging were formed in a parallel plate flow chamber (FCS2; Bioptechs) or in 8-well chambers (Labtek; Nunc) with 5-mm coverslips. GPI-protein density in contacts from flat-field and background corrected images was quantified as reported previously (Grakoui et al., 1999). ICAM-1 accumulation patterns at the TB cell interface were generated by three-dimensional reconstruction of background corrected 1 µm z-planes collected with a piezo PIFOC motor (Physik Instrumente) mounted underneath the objective, using Metamorph software (Universal Imaging Corp.). Additional image processing used Photoshop, Illustrator (Adobe), and Graphic Converter (Lemke Software). Activation state was monitored by a calcium sensitive dye (Fura-2; Molecular Probes). To judge readhesion efficiency, ice-cold imaging buffer (deficient RPMI [GIBCO BRL], 10% FCS, and 10 mM Hepes) was pulsed into the flow chamber, leading to gradual detachment of the T cells which remained in the field, enabling tracking. As the media warmed back to 37°C, the percentage of T cells previously in contact with the bilayer that could reform a tight contact was scored.
Online supplemental material
Fig. S1 shows that 99R displays TCR antagonism whether compared with 99A or 99E. The same experimental conditions as in Fig. 5 were used, representative of two experiments. Videos 13 correspond respectively to the experiments in Fig. 3, panels ac. Original image capture was at 30 seconds per frame (shown at 1 second per frame), and each video represents 10 min of imaging. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200404059/DC1.
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Acknowledgments |
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This work was supported by the National Institutes of Health and the Howard Hughes Medical Institute.
Submitted: 8 April 2004
Accepted: 6 July 2004
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