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The Journal of Immunology, 2006, 176: 3356-3365.
Copyright © 2006 by The American Association of Immunologists

Soluble MHC-Peptide Complexes Containing Long Rigid Linkers Abolish CTL-Mediated Cytotoxicity1

Georgi S. Angelov*, Philippe Guillaume*, Marek Cebecauer*, Giovanna Bosshard*, Danijel Dojcinovic*, Petra Baumgaertner{dagger} and Immanuel F. Luescher2,*

* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and {dagger} Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital, Lausanne, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and coengage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (≥80 Å) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long Db-gp33 complexes strongly and specifically inhibited the Db-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Soluble MHC-peptide (pMHC)3 complexes, namely, avidin/streptavidin-based tetrameric complexes (tetramers) and IgG-based dimeric complexes (dimers), are widely used to enumerate and isolate Ag-specific T cells (1, 2, 3). Soluble pMHC complexes that are at least dimeric and coengage the coreceptor induce intracellular Ca2+ mobilization and diverse phosphorylation events, but not CTL degranulation (4, 5, 6, 7). To trigger degranulation, adhesion of CTL is required, which physiologically occurs when CTL conjugate with target cells (6, 7, 8, 9). The beta2 integrin LFA-1 ({alpha}L/beta2, CD11/CD18a) as well as beta1 and beta3 integrins play crucial roles in CTL-target cell conjugate formation and CTL-mediated target cell killing (6, 7, 8, 9). LFA-1- as well as beta1 and beta3 integrin-mediated adhesion is up-regulated upon TCR engagement, which increases their avidity for their respective ligands on target cells (6, 7, 8, 9, 10). LFA-1, by interacting with ICAM on target cells, plays a key role in formation of the immunological synapse and directional exocytosis of cytolytic granules (6, 8, 11). Activation-dependent up-regulation of adhesion is in part accounted for by conversion of LFA-1 from low to high affinity binding to ICAM and in part by clustering of LFA-1 (12). Similarly, beta1 and beta3 integrins exhibit increased binding to cell-associated VLA-4 and extracellular matrix proteins upon TCR triggering (7, 10, 12).

Dimeric pMHC-IgG peptide fusion proteins have been shown to inhibit CD4+ and CD8+ T cells in vitro and in vivo (13, 14, 15, 16, 17, 18). Such pMHC class II dimers can inhibit autoreactive CD4+ T cells in diabetes type I (16, 19) or multiple sclerosis (17, 18). The pMHC-IgG class I complexes have been reported to inhibit alloreactive CD8+ CTL in vitro and in vivo (13, 14, 15). The mechanism of this inhibition is not clear. Because such complexes activate CTL (14), it is possible that this inhibition is accounted for, at least in part, by activation-dependent death of CTL (20). Although pMHC complexes containing short spacers, i.e., short-MHC-MHC distances, strongly bind to and activate CD8+ CTL and induce CTL death, complexes containing long (≥ 80 Å) rigid polyproline spacers elicit little or no calcium flux, tyrosine phosphorylation, and CTL death (20, 21, 22). The remarkable, biologically inert nature of these long pMHC complexes prompted us to investigate their ability to inhibit CTL responses elicited by activating pMHC ligands.

A caveat for applications of soluble pMHC complexes, especially in vivo applications, is transfer of their peptide to cell-associated MHC molecules (23, 24). Such peptide transfer obscures investigations of the biological activity of soluble pMHC complexes and can cause the death of CTLs (25). For short periods of time, peptide transfer can be inhibited by brefeldin A and high concentrations of competitor peptide (26). In a more definitive manner, peptide transfer can be blocked by using covalent pMHC complexes. Such complexes can be obtained by fusing the peptide onto the MHC molecule (16, 17, 27) or by photoaffinity labeling with suitable photoreactive peptide derivatives (26, 28, 29). Based on the three-dimensional structures of pMHC class I complexes, a favorable position to introduce a photoreactive group is in the side chain of the peptide’s N-terminal amino acid (30).

In this study, we investigated the ability of soluble pMHC complexes containing long rigid polyproline spacers to inhibit Ag recognition by CD8+ CTL. Well-defined long dimeric, tetrameric, and octameric MHC complexes were prepared using site-specific alkylation of pMHC monomers with linkers containing polyprolines (20, 21, 22). Polyprolines in aqueous solution assume a rigid proline II helix, in which one residue spans 3.1 Å (21, 31). As a model system, we used cloned S14 CTL that recognize the PbCS peptide 252–260 (SYIPSAEKI) containing photoreactive 4-azido-benzoic acid (ABA) on PbCS K259 (PbCS(ABA)) in the context of H-2Kd (Kd) (32). These CTL are amenable to TCR photoaffinity labeling, which also allows stringent binding studies of small pMHC complexes. In this system, replacement of Plasmodium berghei circumsporozoite (PbCS) P255 with A(PbCS(ABA)P255A) increases pMHC binding and Ag recognition by 2- to 5-fold, whereas replacement of P255 with L (PbCS(ABA)P255L) yielded an antagonist-altered peptide ligand (31).

We report that dimeric, tetrameric, and octameric Kd-PbCS(ABA) complexes containing long rigid linkers inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. These findings were validated on lymphocytic choriomeningitis virus (LCMV)-specific CTL in vitro and in vivo. Infection of C57BL/6 (H-2b) mice with LCMV induces a strong CTL response, mainly directed against three immunodominant epitopes, two of which are derived from the viral glycoprotein (gp33 and gp276), and the third from the viral nucleoprotein (NP396) (30, 33, 34). More than 50% of the total LCMV-specific CTL activity is directed toward gp33 (KAVYNFATC), the only known immunodominant epitope that is restricted by H-2Db and H-2Kb (34). We show that long octameric Db-gp33 complexes efficiently inhibit Db-restricted, but not Kb-restricted, anti-gp33 LCMV CTL responses in vitro and in vivo. Long pMHC complexes interfere with the TCR-mediated signals that up-regulate the adherence of integrins, namely, LFA-1, and thus inhibit CTL target cell conjugate formation and killing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cells, and Abs

Six-week-old C57BL/6 (H-2b) mice were purchased from Harlan Sprague Dawley and maintained under pathogen-free conditions in accordance with Swiss federal regulations for work with experimental animals. S14 CTL and P815 cells were cultured and used as previously described (31, 32). Unless mentioned otherwise, in all experiments CTL were preincubated for 2 h with 30 µM brefeldin A (Sigma-Aldrich) and 10 µM PbCS 252–260 peptide to prevent peptide transfer from soluble to cell-associated MHC molecules (26). The following Abs were used: anti-LFA-1 (clone M17/4, {alpha}-CD11a) blocking Ab, anti-CD8beta mAb (clone H35), and anti-CD3{epsilon} mAb (clone 145.2C11), as described previously (4, 5).

Synthesis of peptide and linkers

Chemicals were purchased from Calbiochem-Novabiochem, Bachem, Sigma-Aldrich, Pierce, and Amersham Biosciences. Peptides were synthesized on solid phase, using F-moc for transient N-terminal protection. The photoreactive gp33 peptide was synthesized using F-moc-4-azido-phenylalanine (F-moc-F(N3)-OH). For orthogonal protection, very acid-labile groups were used; for polyproline linkers, sequential coupling with F-moc-P5-OH or F-moc-P2-OH, followed by N-acetylation, were used. Maleimides and Cy5 were introduced via hydroxysuccinimide esters of maleimide-acetic acid and Cy5, respectively. Biotin was introduced via F-moc-K(biotin)-OH or N-biotinyl-NH-(CH2-CH2-O)2-COOH. For biotin-(CH2-CH2-O)12-Dap(maleido-acetic acid)-P26-Dap(Mal)-P, the -(CH2-CH2-O)10 spacer was introduced via O-(N-F-moc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol. Peptides were deprotected with trifluoroacetic acid (5%) in dichloromethane and tri-isopropylsilane (5%) and purified by gel filtration chromatography on a Superdex Peptide HR 10/30 column (Amersham Biosciences) and on a semipreparative C-4 column (Vydac), which was eluted with a linear gradient of acetonitrile rising in 60 min from 0 to 75%. The purified peptides were characterized by MALDI-TOF mass spectrometry. The linkers under study are shown in Fig. 1; additional details on their synthesis, purification, and characterization were reported previously (20, 21, 22).


Figure 1
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  		FIGURE 1. Linkers and soluble pMHC complexes under study. A, The linkers used to produce the dimeric, tetrameric, and octameric pMHC complexes under study. Single amino acid letter code is used. Mal, beta-maleimidoacetyl; Dap, diaminopropionic acid; EO, ethyloxide. The indicated distances of the spacers refer to the extended conformation. B, Cartoons of the different dimeric, tetrameric, and octameric pMHC complexes. For the branched linkers, the spacer distance from the biotin to the branching point (Dap) is indicated as "x" and that from the branching point to the maleimide is indicated as "y." The CH-CH2-S- thioether linkages formed upon alkylation of the cysteine 275 of Kd spans ~6.5 Å.

 
Preparation of soluble Kd-PbCS(ABA) complexes

Monomeric Kd-PbCS(ABA) complexes containing a free cysteine in position 275 were prepared by refolding of H chain and human beta2-microglubin in the presence of PbCS(ABA) (SYIASAEK(ABA)I), or Cw3 170–179 peptide (RYLKNGKETL) as previously described (22, 23). The complexes were reduced with 15 mM glutathione and reacted with a 10-fold molar excess of the respective bis-maleimide linkers or with biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine for 2 h at room temperature under argon. The alkylated Kd-PbCS(ABA) complexes were purified by gel filtration on a Superdex S75 column. Dimeric Kd-PbCS(ABA) complexes were obtained by reacting the monomeric complexes with a 2-fold molar excess of reduced Kd-PbCS(ABA)-C and were purified by gel filtration on a Superdex S200 column (22, 23). Tetrameric and octameric pMHC complexes were obtained by reacting the corresponding biotinylated monomeric and dimeric pMHC complexes with Cy5-labeled streptavidin (Amersham Biosciences) and were purified by gel filtration on a Superdex 200 column. Db-gp33 peptide complexes were obtained by standard refolding of the Db H chain containing a free cysteine in position 277, beta2-microglobulin, and the gp33 peptide KAVYNFATA or its photoreactive derivative F(N3)-AVYNFATA. Covalent Db-F(N3)AVYNFATA complexes were obtained by UV irradiation of the latter complexes at 4°C for 30 s with a 90-W fluorescence UV lamp emitting at 312 ± 40 nm. Non-cross-linked complexes were removed by incubating the irradiated complexes for 4 h at 37°C and gel filtration chromatography on a Superdex S75 column. The P10/P10 octameric complexes used for in vivo experiments were prepared using covalent Db-F(N3)AVYNFATA complexes.

Ca2+ mobilization and binding studies

Calcium mobilization experiments were performed using the calcium-sensitive dye indo 1 and flow cytometry as previously described (20, 21). For binding studies, S14 CTL (1 x 107 cells/ml) in HBSS (Invitrogen Life Technologies) supplemented with 0.5% FCS, 10 mM HEPES, 2 µg/ml human beta2-microglobulin, 10 µM PbCS peptide, 5 mM EDTA, and 0.02% sodium azide (FACS buffer) were incubated with the indicated concentrations of fluorescent Kd-PbCS(ABA) complexes for 5 min at 37°C. After UV irradiation (35 s, 90 W, 312 ± 40 nm), the cells were washed twice in cold FACS buffer and analyzed by flow cytometry on a FACSCalibur (BD Biosciences). Data were analyzed with CellQuest software (BD Biosciences). For competition experiments, CTL were similarly incubated with 10 nM Cy5-labeled DapS/P10 octamer, 50 nM (EO)2 tetramer, 120 nM DapS dimer, or 120 nM P30 dimer together with graded amounts of unlabeled DapS dimer, P30 dimer, or monomer. Nonspecific binding was assessed using the corresponding Kd-Cw3 170–179 complexes and was subtracted.

Cytotoxic assays

For in vitro cytotoxicity assay, 51Cr-labeled P815 cells (5 x 103cells/well) transfected, or not, with Db or Kb (35) were incubated in DMEM supplemented with 5% FCS in 96-well microtiter plates for 30 min at 37°C with the indicated concentrations of IASA-YIPSAEK(ABA)I, F(N3)-AVYNFATA, or KAVYNFATA peptide. In the case of the IASA-YIPSAEK(ABA)I peptide, the cells were irradiated at ≥350 nm (34); in the case of the F(N3)-AVYNFATA peptide, they were irradiated at 312 ± 40 nM. After washing the target cells were incubated with cloned S14 CTL (1.5 x 104 cells/well; E:T cell ratio, 3:1). Alternatively, spleens were removed 7 days after LCMV WE infection (200 PFU/mouse i.v.), and bulk splenocytes (E:T cell ratio, 50:1) were used after RBC lysis in Tris-ammonium chloride for 2 min. After 4 h of incubation at 37°C, released 51Cr was determined, and specific lysis was calculated as: 100 x ((experimental – spontaneous release)/(total – spontaneous release)). For bystander killing, the same experiment was performed using unsensitized P815 cells, and CTL were pretreated with 30 µM brefeldin A for 2 h at 37°C.

An in vivo cytotoxicity assay was performed as described previously (36). Briefly, targets were prepared from Kb- or Db-expressing P815 cells. Each target cell type was divided into two populations. One population was loaded with 1 µM F(N3)-AVYNFATA peptide at 37°C for 90 min, followed by UV irradiation at 4°C for 40 s with a 90-W fluorescence UV lamp emitting at 312 ± 40 nm. Irradiated cells were washed extensively and labeled with a high CFSE (Molecular Probes) concentration (10 µM). The control population was incubated without peptide for 90 min at 37°C, irradiated, washed, and labeled with a low CFSE concentration (1 µM). Cells were resuspended at 5 x 107/ml, and equal volumes of peptide-pulsed CFSEhigh and CFSElow cells were mixed together. A total of 1 x 107 CFSE-labeled cells were injected i.v. into day 7 LCMV-infected C57BL/6 mice. To assess the effect of pMHC complexes on CTL-mediated killing, the mice were injected i.v. with covalent Db-F(N3)AVYNFATA P10/P10 octamers (10 µg/mouse) 30 min before the transfer of CFSE-labeled target cells. After 4 h, mice were killed, and their splenocytes were analyzed for the proportion of CFSE-labeled target cells. The two target populations were distinguished by their different CFSE fluorescence levels. A total of 5 x 103 CFSE+ cells were acquired for each condition. Two parameters were used to evaluate the in vivo cytotoxic activity of CD8 T cells. The ratio between peptide-pulsed targets and control targets (ratio = percentage CFSEhigh/percentage CFSElow) and the percentage of specific lysis that normalized the cytolytic activity between immunized and nonimmunized mice were determined. Specific lysis was calculated as: percentage specific lysis = (1 – ratio immunized/ratio nonimmunized) x 100.

CTL conjugation and adhesion assays

To measure stable conjugate formation, indo 1-labeled S14 CTL (6 x 106 cells/ml) in OptiMEM (Invitrogen Life Technologies) containing 2 µg/ml beta2-microglobulin and 10 µM PbCS peptide were incubated in the absence or the presence of the indicated concentrations of Kd-PbCS(ABA) complexes for 1 min at 37°C, with CFSE-labeled P815 previously sensitized with 10–8 M IASA-YIPSAEK(ABA)I peptide (E:T cell ratio, 1:1) and centrifuged for 1 min at 1000 x g. After another 1-min incubation at 37°C CFSE- and indo 1-associated fluorescence were analyzed by flow cytometry on an LSR flow cytometer (BD Biosciences). Data were processed using CellQuest software (BD Biosciences), and the percentage of conjugates was calculated as: conjugates (%) = 2 x CFSE+ indo 1+ counts/(2 x CFSE+ indo 1+ counts + CFSE+ indo 1– counts + CFSE– indo 1+ counts).

For LFA-1 adhesion experiments, Maxi-Sorb 96-well plates (Nalgen Nunc) were coated by adding under agitation 10 µl of affinity-purified ICAM1/GPI (0.5 µg/ml) in 16 mM CHAPS into wells containing 100 µl of PBS. After overnight incubation at 4°C, the wells were washed and saturated with OVA (1 mg/ml; Sigma-Aldrich). S14 CTL (2 x 105 cells/well) were added in 100 µl of HBSS supplemented with 1% OVA, followed by the indicated reagents and anti-CD3 mAb 145-2C11 (3 µg/ml), centrifuged for 1 min at 1000 x g, and incubated at 37°C for 30 min. Nonadherent cells were removed by vigorous washing with warm HBSS. The number of adherent cells was determined by staining with MTT as described previously (37). All incubations were performed in quadruplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ability of soluble pMHC complexes to induce calcium flux decreases with increasing spacer length

We prepared tetrameric Kd-PbCS(ABA) complexes containing linkers of different lengths connecting the pMHC entities with Cy5-labeled streptavidin (Fig. 1) and examined their ability to induce Ca2+ flux in cloned S14 CTL. The tetramer containing the 18-Å long flexible diethyl oxide spacer (EO)2 efficiently induced calcium mobilization even at low concentrations (Fig. 2A). For the two longer tetramers, we used linkers containing 14 and 24 prolines, spanning distances of 62 and 88 Å, respectively. Although the polyproline spacers were rigid (32), full freedom of mobility of the pMHC entities was warranted by the flexible lysine N{epsilon} biotin linkage to streptavidin as well as by the thioether linkage connecting the spacer to the pMHC complex. The P14 tetramer elicited calcium flux in S14 CTL ~60% lower than that with the (EO)2 tetramer, and the P24 tetramer induced only scant calcium mobilization (Fig. 2, B and C). Thus, the ability of tetramers to induce calcium flux decreased with increasing spacer length, as was the case for dimeric DapS Kd-PbCS(ABA) complexes (Fig. 2F) (21).


Figure 2
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  		FIGURE 2. Short, but not long, pMHC complexes induce strong calcium mobilization in S14 CTL. A–E, Indo 1-labeled S14 CTL were incubated at 37°C with 20 nM ({blacksquare}), 5 nM ({blacktriangleup}), 2 nM (•), and 0.5 nM ({diamondsuit} ) of the Kd-PbCS(ABA) (EO)2 tetramer (A), P14 tetramer (B), P24 tetramer (C), DapS/P10 octamer (D), or P10/P10 octamer (E), and calcium-dependent indo 1 fluorescence was measured by flow cytometry over the indicated period of time. The arrows indicate addition of the soluble pMHC complexes, and the empty squares indicate untreated cells. F, The maximal calcium responses were assessed upon incubation of indo 1-labeled S14 CTL with the indicated Kd-PbCS(ABA) complexes. The control (–) refers to untreated S14 cells. Mean values and SDs were calculated from three experiments.

 
The DapS/P10 octamer containing a short DapS dimer, followed by a P10 spacer after the branching point (Fig. 1), induced a slightly higher calcium mobilization than the (EO)2 tetramer even at the lowest concentration tested (Fig. 2D). Conversely, the P10/P10 octamer, containing P10 spacers before and after the branching point (Dap; Fig. 1), induced a maximal calcium response that was ~50% lower (Fig. 2E). The initial rise in the calcium response was more rapid for the octameric than the tetrameric complexes.

P30 dimer inhibits calcium mobilization more efficiently than pMHC binding

The DapS dimer, (EO)2 tetramer, and the DapS/P10 octamer induced not only strong calcium mobilization, but also extensive tyrosine phosphorylation of diverse signaling molecules and activation-dependent death of CTL (Fig. 2 and data not shown) (20, 22). Because this was not the case for the long complexes, we examined their ability to inhibit CTL activation. To this end, we tested whether the long P30 Kd-PbCS(ABA) dimer can inhibit the calcium mobilization induced by the short Kd-PbCS(ABA) DapS dimer. Half-maximal inhibition was observed between 1 and 3 nM P30 dimer, and nearly complete inhibition occurred at ~30 nM, i.e., at the same concentration as that used for the DapS dimer (Fig. 3A). Unlabeled P30 dimer inhibited the binding of Cy5-labeled DapS dimer less efficiently than the calcium response (Fig. 3A). For half-maximal inhibition of the binding, an ~10 times higher concentration of P30 dimer was needed. Monomeric Kd-PbCS(ABA) complexes exhibited weak, but significant, inhibition of the calcium response and DapS dimer binding at the highest concentrations tested (Fig. 3).


Figure 3
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  		FIGURE 3. Long pMHC complexes inhibit calcium mobilization induced by short pMHC complexes. A, Indo 1-labeled S14 CTL were incubated at 37°C with 30 nM Kd-PbCS(ABA) DapS dimer ({blacksquare}) or with 1 nM Kd-PbCS(ABA) DapS/P10 octamer ({square}) in the absence or the presence of the indicated concentrations of P30 dimer. As a specificity control, S14 CTL were incubated with DapS dimer and the indicated concentrations of Kd-PbCS(ABA) monomer ({cjs2089}). Calcium-dependent indo 1 fluorescence was measured by flow cytometry over a period of 3 min. The mean and SD of the maximal calcium responses were calculated from two or three experiments and are expressed as a percentage, with 100% being the value in the absence of a competitor. B, S14 CTL were incubated at 37°C for 5 min in the presence of EDTA (10 mM) and sodium azide (0.02%) with Cy5-labeled Kd-PbCS(ABA) DapS dimer ({blacksquare}) or Kd-PbCS(ABA) DapS/P10 octamer ({square}) and the indicated concentrations of unlabeled P30 dimers. As control, S14 CTL were incubated with DapS dimer and the indicated concentrations of Kd-PbCS(ABA) monomer ({cjs2089}). After UV irradiation, the cells were washed, and cell-associated Cy5 fluorescence was measured by flow cytometry. The nonspecific binding, as measured for the noncognate Kd-Cw3 DapS dimer, was subtracted. 100%, binding in the absence of a competitor.

 
A more striking difference was observed when the DapS/P10 octamer was used. Half-maximal inhibition of the calcium response was observed in the presence of an ~9-fold molar excess of P30 dimer (Fig. 3A). Conversely, the binding of the DapS/P10 octamer was inhibited by <40% in the presence of a 90-fold molar excess P30 dimer (Fig. 3B). These findings argue that the inhibition of the calcium response by P30 dimer cannot be solely explained by competition of an activating by a nonactivating ligand on the level of TCR binding.

Long Kd-PbCS(ABA) complexes ablate target cell killing by S14 CTL

We next tested the capacity of P30 dimer to inhibit S14 CTL-mediated cytotoxicity. The lysis of PbCS(ABA)-sensitized P815 cells was inhibited by P30 dimer in a concentration-dependent manner, requiring 10–20 nM for half-maximal inhibition (Fig. 4A). This inhibition was specific, because the noncognate Kd-Cw3 170–179 P30 dimer was inactive. The ability of long pMHC complexes to inhibit target cell lysis increased with their valence. Half-maximal inhibition was observed in the presence of 1.2–2.5 nM Kd-PbCS(ABA) P24 tetramer and only 0.3–0.6 nM P10/P10 octamer (Fig. 4, B and C). Conversely, monomeric Kd-PbCS(ABA) complexes caused barely significant inhibition (Fig. 4D).


Figure 4
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  		FIGURE 4. Long pMHC complexes inhibit target cell killing by cloned S14 CTL. S14 CTL were incubated at 37°C for 4 h with 51Cr-labeled P815 cells (E:T cell ratio, 3:1), previously sensitized with 10 nM of IASA-YIPSAEK(ABA)I in the absence or the presence of the indicated concentrations of Kd-PbCS(ABA)P255A P30 dimer (A), P24 tetramer (B), P10/P10 octamer (C), or monomer (D). A, Kd-Cw3 170–179 P30 dimer was included as a specificity control ({cjs2089}). E and F, P815 cells were sensitized with 100 nM IASA-YIPSAEK(ABA)I and incubated with the indicated concentrations of Kd-PbCS(ABA) P10/P10 octamers (E; {blacksquare}), P26/PEO10 octamers (E; {cjs2108}), or the antagonist peptide SYILSAEK(ABA)I (F). The mean values were calculated from three experiments.

 
The P10/P10 octamer also efficiently inhibited the strong lysis (~88%) of P815 cells sensitized with a 10 times higher concentration of PbCS(ABA) peptide (Fig. 4E). Half-maximal inhibition was observed in the presence of 0.7 and 1.5 nM P10/P10 octamer (Fig. 4E). The (EO)12-P26 octamer inhibited this strong lysis nearly twice as efficiently. In this octamer, the Kd-PbCS(ABA) entities in the subunit dimers were kept far apart by the rigid P26 spacer (81 Å; Fig. 1), which is the likely explanation for why this octamer was more inhibitory. In striking contrast, the antagonist peptide variant PbCS(ABA)P255L barely inhibited this strong lysis, even at high concentrations, although it was able to inhibit weak lysis (20–30%; Fig. 4F) (31). Taken collectively, these results demonstrate that soluble Kd-PbCS(ABA) complexes, in particular, octameric ones, unlike antagonist peptide ligands, efficiently inhibit target cell killing by S14 CTL at nanomolar concentrations.

Long Db-gp33 complexes inhibit LCMV-specific CTL in vitro and in vivo

To validate these results, we tested whether long pMHC complexes were able to inhibit the strong Db-restricted anti-gp33 CTL response in LCMV-infected C57BL/6 mice (33, 34). Seven days after infection, splenocytes from LCMV-infected mice lysed ~57% of Db-transfected P815 target cells (P815 Db) previously pulsed with 100 nM gp33 (KAVYNFATA) peptide (Fig. 5A). The lysis of unsensitized P815-Db cells was 5%. Remarkably, in the presence of Db-KAVYNFATA or non-cross-linked Db-F(N3)AVYNFATA monomers, even at low concentrations, the lysis of unsensitized P815 Db cells was comparable to that of gp33-sensitized target cells (Fig. 5A and data not shown). Conversely, the low background lysis of normal nontransfected P815 cells (H-2d) was not altered in the presence of Db-KAVYNFATA monomers (data not shown), indicating that the killing of P815 Db cells in the presence of Db-KAVYNFATA monomer was caused by transfer of gp33 peptide from soluble to cell associated Db.


Figure 5
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  		FIGURE 5. Long pMHC complexes inhibit target cell killing by LCMV-specific CTL in vitro. A, Splenocytes from LCMV-infected C57/BL6 mice (day 7) were incubated at 37°C for 4 h with 51Cr-labeled P815 Db-transfected cells (E:T cell ratio, 9:1) in the presence of 100 nM of the gp33 peptide KAVYNFATA ({cjs2108}) or the indicated concentrations of monomeric non-cross-linked Db-F(N3)AVYNFATA complexes ({blacksquare}) or covalent Db-F(N3)AVYNFATA monomers ({square}), and specific lysis was evaluated by the released 51Cr. Alternatively, the splenocytes were incubated with P815 Db- (B) or P815 Kb-transfected cells (C) in the presence of the indicated concentrations of the gp33 peptide KAVYNFATA ({diamondsuit}) or its photoreactive derivative F(N3)AVYNFATA ({blacksquare}), and specific lysis was assessed. D, Splenocytes from LCMV-infected C57/BL6 mice were incubated with P815 Db cells sensitized with 100 nM F(N3)-AVYNFATA peptide in the presence of the indicated concentrations of covalent DbF(N3)AVYNFATA monomer ({square}), P24 tetramer ({blacksquare}), or P10/P10 octamer ({cjs2089}), and specific lysis was assessed. Mean values and SD were calculated from three experiments.

 
To prevent peptide transfer, we prepared covalent Db-gp33 complexes by replacing the peptide’s N-terminal amino acid with photoreactive F(N3). This peptide derivative was recognized only slightly less efficiently than the gp33 wild-type peptide on both P815 Db (Fig. 5B) and P815 Kb (Fig. 5C) target cells, indicating that this substitution did not substantially affect gp33 peptide binding to Db and Kb and recognition by the gp33-reactive CTL from LCMV-infected mice. In the presence of covalent, i.e., photo-cross-linked Db-F(N3)AVYNFATA monomer, the background lysis of P815 Db cells remained near background levels, except for a slight increase at the highest concentrations tested (Fig. 5A). These results demonstrate that gp33 peptide is transferred from soluble to cell-associated Db and that this can be prevented by using covalent Db-gp33 complexes.

Covalent Db-gp33 P24 tetramers and P10/P10 octamers effectively inhibited the lysis of P815 Db cells sensitized with 100 nM F(N3)AVYNFATA (Fig. 5D). Half-maximal inhibition was observed in the presence of 6–12 nM P24 tetramer and 1.5–3 nM P10/P10 octamer. Monomeric Db-F(N3)AVYNFATA complexes were ineffective. As on S14 CTL, the long pMHC complexes failed to induce mobilization of intracellular calcium (Fig. 2 and data not shown). Importantly, covalent P10/10 octameric Db-F(N3)AVYNFATA complexes also inhibited at a very low dose, the killing of Db-P815 cells previously photo-cross-linked with F(N3)AVYNFATA peptide in LCMV-infected mice (Fig. 6, A and B). This inhibition was specific, because the Kb-restricted anti-gp33 CTL response was not affected. Moreover, Db-F(N3)AVYNFATA P10/P10 octamer selectively stained ~20% of CD8+ splenocytes from LCMV-infected C57BL/6 mice, but no splenocytes from normal mice (Fig. 6C). This rules out the idea that nonspecific octamer binding to endogenous cells could create unlabeled targets that could compete with the labeled targets. Taken together, these results demonstrate that long pMHC complexes can specifically inhibit the strong anti-LCMV CTL response in vitro and in vivo and corroborate the findings obtained on cloned S14 CTL.


Figure 6
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  		FIGURE 6. Long Db-gp33 P10/P10 octamer inhibits the anti-LCMV gp33 CTL response in vivo. A, Day 7 LCMV-infected or noninfected C57BL/6 mice were injected i.v. with covalent Db-F(N3)AVYNFATA P10/P10 octamer (10 µg/mouse) or PBS. Thirty minutes later, CFSE-labeled P815-Db or P815-Kb target cells previously photo-cross-linked with 1 µM F(N3)AVYNFATA peptide were injected i.v., and after 4 h, the CFSE-positive events were enumerated by flow cytometry. Specific lysis was established as explained in Materials and Methods. B, Mean values and SD were calculated from two independent experiments. Open bars refer to the Kb response, and filled bars refer to the Db response. C, Splenocytes from normal (LCMV –) or LCM-infected C57BL/6 mice (LCMV +) were stained with Cy5-labeled Db-F(N3)AVYNFATA P10/P10 octamer and FITC-labeled anti-CD8{alpha} mAb 53.6.72 and analyzed by flow cytometry.

 
Long Kd-PbCS(ABA) complexes inhibit CTL-target cell conjugate formation and LFA-1-mediated adhesion of S14 CTL

To learn more about the mechanism by which long pMHC complexes inhibit CTL-mediated cytotoxicity, we examined what effect they have on stable CTL-target cell conjugate formation. The long Kd-PbCS(ABA) complexes inhibited the conjugate formation of S14 CTL with PbCS(ABA)-sensitized P815 cells in a dose- and valence-dependent manner (Fig. 7A). Half-maximal inhibition was observed in the presence of 9–27 nM P30 dimer, 3–9 nM P24 tetramer, and 0.3–1 nM P10/P10 octamer. Compared with inhibition of lysis, these pMHC concentrations were 2- to 3-fold higher, arguing that it involves factors additional to conjugate formation (Figs. 4, A–C, and 7A).


Figure 7
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  		FIGURE 7. Long pMHC complexes impede CTL conjugate formation with target cells and activation of LFA 1. A, Indo 1-labeled S14 CTL were incubated at 37°C for 2 min in the absence or the presence of the indicated concentrations of Kd-PbCS(ABA) P30 dimer ({cjs2108}), P24 tetramer ({blacksquare}), P10/P10 octamer ({square}), or monomer. After addition of CFSE-labeled P815 cells (E:T cell ratio, 1:1) previously sensitized with 10 nM IASA-YIPSAEK(ABA)I peptide, the cells were cosedimented and after 2 min of incubation at 37°C were resuspended and fixed, and the percentage of conjugates formed was analyzed by flow cytometry. Mean values and SD were calculated from three experiments. B, S14 CTL were incubated for 30 min at 37°C in the absence or the presence of anti-CD3{epsilon} mAb 145.2C11 (3 µg/ml) and the indicated concentrations of Kd-PbCS(ABA) P30 dimer ({cjs2108}), P24 tetramer ({blacksquare}), P10/P10 octamer ({square}) or monomer in 96-well plates coated with GPI-linked ICAM-1. After washing off nonadherent cells, the percentage of adherent cells was determined. Mean values and SD were calculated from three experiments, each performed in triplicate.

 
Because CTL-target cell conjugate formation is mainly mediated by intercellular interactions of integrins, namely, by LFA-1, on the CTL with their respective ligands on the target cells (6, 7, 8, 9, 10), we examined what effect long Kd-PbCS(ABA) complexes have on LFA-1-mediated adhesion. The anti-CD3 Ab-induced adhesion of S14 CTL to immobilized ICAM-1 was strongly inhibited by all long pMHC complexes studied (Fig. 7B). Half-maximal inhibition was observed in the presence of ~3 nM P30 dimer, 0.3–1 nM P24 tetramer, and ~0.1 nM P10/P10 octamer. The pMHC complexes did not significantly compete with the binding of the anti-CD3 mAb (data not shown). Similar results were obtained when using plate-bound fibronectin, which binds to beta1 and beta3 integrins (7, 12), although higher pMHC concentrations were needed (data not shown). Collectively, these results indicate that long pMHC complexes inhibit CTL target cell conjugate formation and anti-CD3-induced, integrin-mediated adhesion of CTL.

Binding of Kd PbCS(ABA) complexes to S14 CTL

We next examined the binding of the long pMHC complexes to S14 CTL. The 37°C binding isotherms exhibited remarkably different maximal binding of the different Kd-PbCS(ABA) complexes (Fig. 8A). The highest binding was observed for DapS/P10 octamer, which was ~20% higher than that with the P10/P10 octamer. Similarly, the maximal binding for (EO)2, P14, and P24 tetramers decreased with increasing length of their spacers. As shown previously, the maximal binding of the short DapS dimer was ~2-fold higher than that with the long P30 dimer (Fig. 8A) (22). In all cases, the differences in binding could not be overcome by increasing the pMHC concentration (Fig. 8A and data not shown) (20, 21).


Figure 8
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  		FIGURE 8. Binding of Kd-PbCS(ABA) complexes to S14 CTL. A, S14 CTL were incubated at 37°C for 5 min in the presence of EDTA (10 mM) and sodium azide (0.02%) with the indicated concentrations of Cy5-labeled PbCS(ABA) DapS/P10 octamer ({diamondsuit}), P10/P10 octamer ({blacktriangleup}), (EO)2 tetramer ({blacksquare}), P14 tetramer (•), P24 tetramer ({diamond}), P30 dimer ({triangleup}), or monomer ({square}). After UV irradiation, the cells were washed, and cell-associated fluorescence was measured by flow cytometry. Nonspecific binding assessed on clones A1 Cw3 CTL was subtracted. Alternatively, S14 CTL were incubated with 3 nM Cy5-labeled DapS/P10 octamer (B), 10 nM PEO tetramer (C), 120 nM DapS dimer (D), or 120 nM P30 dimer (E) and the indicated concentrations of unlabeled PbCS(ABA) monomer ({diamondsuit}), DapS dimer ({blacksquare}), or P30 dimer ({blacktriangleup}), and cell-associated Cy5 fluorescence was determined as described in A. The numbers in the insets indicate the concentrations at which half-maximal competition occurred.

 
We next performed competition experiments in which S14 CTL were incubated with a constant concentration of Cy5-labeled DapS/P10 octamer and graded concentrations of unlabeled Kd-PbCS(ABA) monomer, DapS dimer, or P30 dimer. The P30 dimer competed more efficiently than the short DapS dimer, which, in turn, competed more effectively than monomeric complexes (Fig. 8B). The same was true when Cy5-labeled (EO)2 tetramer (Fig. 8C), DapS dimer (Fig. 8D), or P30 dimer (Fig. 8E) was used as the reference compound. Additionally, the competition curves of the DapS dimer, but not those of the P30 dimer, were biphasic. These findings argue that short pMHC complexes induce avidity, enhancing TCR aggregation, whereas long complexes interfere with it.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A key finding of the present study is that soluble pMHC complexes containing long rigid linkers abolished CTL-mediated cytotoxicity, even very strong lysis, at nanomolar concentrations in vitro and in vivo (Figs. 4–6). By contrast, peptide antagonists, at least in our system, significantly inhibited only weak lysis and only when used at high concentrations (Fig. 4F) (31). This is explained, on the one hand, by the fact that peptide antagonists compete with agonist peptides at the level of binding the restricting MHC molecule. In contrast, peptide antagonists often, but not always, exhibit weak TCR-pMHC interactions and fast dissociation kinetics (31, 38, 39, 40). In contrast, the binding of long pMHC complexes to Ag-specific CTL was of high avidity; it increased with the valence and was further strengthened when using strong agonist peptide variants (Fig. 8) (Ref.31 and our unpublished observations). This explains why these complexes, contrary to antagonist peptides, were effective at very low concentrations (Figs. 4 and 5). It is important that the distance between the individual pMHC entities in the complex is large, i.e., enforced by polyproline spacers, which in aqueous medium assume a rigid proline II helix of defined length (20, 41). For dimeric pMHC class II complexes, the ability to trigger CD4+ T cells decreased also with increasing length of the connecting spacer (up to 90 Å) (42); however, this decrease was much less dramatic than that with pMHC class I dimers (20). Because the spacers used in the former study were flexible and rigid in the latter, the importance of enforced large pMHC-pMHC distances for efficient inhibitory activity is strongly suggested.

Activation of T cells by soluble pMHC complexes requires that these be at least dimeric and coengagement of the coreceptor (4, 5, 20). Cross-linking of TCR/CD3 and CD8 activates Src kinases, namely, CD8-associated Lck, which is activated by cross-linking induced trans autophosphorylation of tyrosine 394 on the regulatory A loop (4, 20, 43). pMHC complexes containing long rigid polyproline spacers are unable to induce such cross-linking and, hence, fail to activate CTL, as evidenced by the scant calcium mobilization and tyrosine phosphorylations (Fig. 2) (20, 21). In addition, long pMHC complexes, unlike short ones, fail to induce the death of CD8+ CTL, which suggests their usefulness for the isolation of bona fide Ag-specific CTL.

Although this explains why long pMHC complexes fail to significantly activate CTL, it leaves open how they inhibit target cell killing by CTL. It is unlikely that this inhibition is accounted for solely by competition with cell-associated pMHC complexes at the level of TCR binding. The binding of cell-associated pMHC complexes to TCR (and CD8) on CTL is highly multivalent and is stabilized by diverse intercellular interactions, including integrins, as well as CD2, CD8, and noncognate pMHC complexes (6, 7, 8, 9, 10, 11, 12, 37, 44).

During the initial encounter of CTL with target cells, an Ag-specific, i.e., TCR-mediated signal, up-regulates the avidity of adhesion molecules, which, in turn, leads to tight CTL-target cell conjugate formation (6, 8, 9, 10, 11, 12, 44, 45). In particular, activation of LFA-1 is essential for the formation of stable conjugates, i.e., the immunological synapse (6, 42, 45); it relies on up-regulation of the affinity of LFA-1 for ICAM as well as on the avidity enhancing clustering of LFA-1 (44). The finding that the long pMHC complexes inhibit stable CTL-target cell conjugate formation as well as anti-CD3 Ab-induced LFA-1-mediated adhesion of CTL argues that they interfere with the TCR signaling that activates integrins (Fig. 7). Consistent with this is the observation that P30 dimer inhibited the calcium response triggered by short pMHC complexes more efficiently than pMHC binding (Fig. 3). Because the calcium response and the binding of DapS dimer exhibit linear concentration dependence in this concentration range and because P30 dimer does not elicit calcium mobilization (21), its inhibition of the DapS dimer-induced calcium response clearly seems to involve a mechanism(s) other than mere competition of activation by a nonactivating pMHC ligand at the level of TCR binding.

Our binding data support the view that long pMHC complexes interfere with pMHC-driven aggregation of TCR (and CD8) and, thus, with the Ag-specific activation of CTL. For several hormone, cytokine, and chemokine receptors, it has been shown that receptor dimerization is a means to strengthen receptor ligand binding and to promote receptor signaling (46, 47). Several reports suggest that TCR also undergo dimerization and aggregation (21, 48, 49). We have shown previously on S14 CTL that Kd-PbCS(ABA) complexes engage TCR in a dimeric mode (21). TCR aggregation correlates with translocation of TCR/CD3 into lipid rafts, activation of tyrosine kinases, and initiation of TCR signaling (4, 7, 50, 51). pMHC complexes containing long rigid polyproline spacers are incompatible with tight TCR aggregation and apparently interfere with aggregation induced by activating pMHC complexes. The more avidly long pMHC complexes bind to CTL, the more effectively they impede pMHC driven TCR aggregation and, hence, TCR signal induction (Figs. 4 and 5).

This view is supported by the following findings. First, the observation that the short activating soluble pMHC complexes bind more extensively to S14 CTL than the corresponding long nonactivating ones argues that the former, but not the latter, induced avidity enhancing TCR aggregation (Fig. 8A) (21). Second, although the long P30 dimer bound less efficiently to S14 CTL than the short DapS dimer, it competed more efficiently with the binding of soluble pMHC (Fig. 8) (21), arguing that the short dimer promoted and the long dimer inhibited TCR aggregation. Third, the competition curves of the DapS dimer, but not those of the P30 dimer, were biphasic, indicating that the DapS dimer at low concentrations promoted TCR aggregation, thus enhancing the binding of the fluorescence-labeled reference compound (Fig. 8). We also observed that blue (Cy5) and red (Cy3)-labeled DapS dimers on S14 CTL exhibited high colocalization, which was inhibited in the presence of unlabeled P30 dimer, but not monomer.

Dimeric pMHC-IgG dimers have been reported to inhibit CD4+ as well as CD8+ T cells in vivo and in vitro (13, 14, 15, 16, 17, 18, 19). However, contrary to our long pMHC complexes, these dimers activate CD8+ and CD4+ T cells, e.g., they trigger TCR-proximal phosphorylation events, cytokine release, up-regulation of activation markers (e.g., CD25 and CD69), and T cell proliferation (14, 16, 18, 19, 52). In fact, for CD4+ T cells, the inhibition of autoimmune responses by IgG-based pMHC dimers relies on their ability to induce production of certain cytokines, e.g., IL-10 (16, 19). Although the average MHC-MHC distance in these dimers is in the range of our long pMHC complexes, it is not fixed due to the flexibility of the IgG hinge region and the linkers joining the pMHC complexes to the IgG. In addition, these pMHC-IgG dimers contain an IgG Fc portion that binds to FcRs on cells and, hence, may favor intercellular interactions and contribute directly or indirectly to T cell activation.

How IgG-based pMHC class I dimers inhibit CD8+ CTL-mediated cytotoxicity is not clear. Because they activate CD8+ CTL, it is possible that they down-modulate TCR or induce activation-dependent death of CTL (14, 20). In addition, peptide can be transferred from soluble to CTL-associated MHC class I molecules (23, 24), and cognate peptides can cause fratricide of CTL (25). Our observation that Db-transfected P815 cells were killed by LCMV-reactive CTL in the presence of Db-gp33 monomer (Fig. 5) indicates that peptide transfer also occurs on cell types other than CTL. Because Kd-PbCS(ABA) complexes induce only scant peptide transfer that could be blocked by brefeldin A and competitor peptide, its extent apparently can vary considerably for different pMHC complexes.

Peptide exchange and related complications can be prevented by covalently linking the peptide to the MHC molecule. For MHC class II molecules, this can be accomplished by fusing the peptide via a linker to the N terminus of the beta-chain (16, 19, 27). For MHC class I molecules, analogous strategies have been reported; however, because the peptide binding groove is closed and both peptide termini are anchored (30), steric constraints and distortion of the MHC-peptide surface are a serious concern. Alternatively, the peptide can be photo-cross-linked to the MHC molecule by means of a suitable photoreactive peptide derivative (29, 32). The side chain of the first amino acid can usually be replaced with a photoreactive one without substantially altering the peptide’s MHC binding and recognition (Fig. 5) (26, 29, 35). In this position, the photoreactive group is in close proximity to the conserved tryptophan 167 on the MHC class I {alpha}2 helix (30). Because tryptophan reacts well with phenylazide-derived radicals, such photoreactive peptides efficiently photo-cross-link to MHC class I molecules (29, 30, 32).

In conclusion, the present study demonstrates that soluble pMHC complexes containing long rigid polyproline spacers can efficiently inhibit Ag-specific CTL responses in vitro and in vivo. Their inhibitory efficacy depends on their binding avidity, which increased with their valence and for octameric complexes was in the lower nanomolar range. Long pMHC complexes interfere with initial pMHC-driven TCR aggregation, TCR signal induction, and, hence, activation of integrins, namely LFA-1. The lack of adequate activation of LFA-1 impedes not only stable CTL target cell conjugate formation, but also targeted CTL degranulation, which requires LFA-1 adhesion and LFA-1 signaling (10, 53). Especially long covalent octameric complexes have a significant potential to inhibit Ag-specific CTL-mediated cytotoxicity in vivo and, hence, should be useful to blunt undesired CTL responses in autoimmune disorders such as diabetes type I or multiple sclerosis.


   Acknowledgments
 
We are most thankful to Dr. M. Dustin for the GPI-linked ICAM-1, to Drs. N. Boucheron and S. Mark for preliminary investigations related to this study, and to Rolf Cornaz for technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Swiss National Foundation (31-1946.00), the Swiss Cancer League (OCS 01421-08-2003), and the Stanley Thomas Foundation. Back

2 Address correspondence and reprint requests to Dr. Immanuel F. Luescher, Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland. E-mail address: immanuel.luescher{at}isrec.unil.ch Back

3 Abbreviations used in this paper: pMHC, peptide-MHC class I; ABA, 4-azidobenzoic acid; Dap, diaminopropionic acid; EO, ethylene oxide; IASA, iodo-azido-salicylic acid; F(N3), 4-azido-phenylalanine; LCMV, lymphocytic choriomeningitis virus; PbCS, Plasmodium berghei circumsporozoite. Back

Received for publication October 26, 2005. Accepted for publication January 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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