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The Journal of Immunology, 2006, 176: 1498-1505.
Copyright © 2006 by The American Association of Immunologists

CD8 T Cells, Like CD4 T Cells, Are Triggered by Multivalent Engagement of TCRs by MHC-Peptide Ligands but Not by Monovalent Engagement1

Jennifer D. Stone*,{dagger} and Lawrence J. Stern2,{dagger},{ddagger}

* Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; and {dagger} Department of Pathology and {ddagger} Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell activation is initiated by recognition of antigenic peptide presented in complex with MHC molecules on the surface of APCs. The mechanism by which this recognition occurs is still unclear, and many models exist in the literature. CD4 T cells have been shown to respond to soluble oligomers of activating class II MHC-peptide complexes, but not to soluble monomers. In determining the reactivity of CD8 T cells to soluble activating class I MHC-peptide complexes, a complicating phenomenon had been observed whereby peptide from soluble complexes was loaded onto cell surface MHCs on the T cells and re-presented to other T cells, clouding the true valency requirement for activation. This study uses soluble allogeneic class I MHC-peptide monomers and oligomers to stimulate murine CD8 T cells without the possible complication of peptide re-presentation. The results show that MHC class I monomers bind to, but do not activate, CD8 T cells whether the cells are in solution or adhered to a surface. Monomeric MHC class I binding can antagonize the stimulation triggered by soluble oligomers, a phenomenon also observed for CD4 T cells. Dimeric engagement is necessary and sufficient to stimulate downstream activation processes including TCR down-regulation, Zap70 phosphorylation, and CD25 and CD69 up-regulation, even in T cells that do not express the MHC coreceptor CD8. Thus, the valency dependence of the response of CD8 T cells to soluble MHC-peptide reagents is the same as previously observed for CD4 T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In vivo T cells become activated when they recognize specific MHC-peptide complexes on the surface of APCs. However, despite much work in this area, the specific molecular event that is detected by the T cell upon Ag-specific TCR engagement has been difficult to identify. In other immune cells such as B cells or mast cells in which the Ag recognized is a soluble particle, multivalent receptor engagement has been observed to be crucial for stimulation (1, 2, 3). Conversely, T cells are triggered by antigenic stimuli presented on the surface of another cell, and the specific interaction is accompanied by a multitude of non-Ag-specific interactions including: binding of adhesion molecules, engagement of costimulatory receptors, possible contributions of nonstimulatory MHC-peptide complexes, and MHC coreceptor binding. One way that the molecular trigger of T cell activation has been addressed has been to develop recombinant MHC-peptide complexes of defined valency and use them to treat T cells. This type of study in CD4 T cells showed that dimers and higher oligomers of cognate class II MHC-peptide complexes were able to stimulate many T cell markers such as TCR down-regulation (4), CD69 up-regulation (5), and CD25 up-regulation (6); conversely, monomeric MHC-peptide complexes were not able to induce such markers (7, 8, 9, 10). When similar studies have been undertaken treating CD8 T cells with soluble, recombinant class I MHC-peptide complexes, conflicting studies reported activation in response to oligomeric complexes (10, 11, 12, 13, 14, 15), to monomeric complexes (13, 14, 15), and even to free peptide (14, 15). A partial explanation for this discrepancy may be "cross-presentation" of peptide by T cells, wherein peptide adventitiously released from soluble MHC monomers can be loaded onto T cell endogenous class I MHC molecules with resultant T-T presentation and activation (14, 15). When T cells that do not express endogenous MHC are used, neither peptide nor class I MHC monomers caused down-regulation of TCR or up-regulation of activation markers (14, 15).

There remains a question as to the molecular trigger for CD8 T cells reacting to specific MHC-peptide complex. Cytolysis by CD8 cells seems to be an ultrasensitive response, not requiring the formation of a stable immunological synapse associated with full activation in CD4 cells (16). Some evidence suggests that class I MHC monomers are sufficient to induce signaling in T cells that have been "adhesion primed" by Ag nonspecific interactions on an APC or a surface coated with Abs to T cell surface molecules (17). Indeed, T cell triggering has been observed for a single activating peptide on an APC for both CD8 and CD4 T cells (18, 19), and nonactivating MHC-peptide complexes have been shown to aid in robust signaling both in membranes and as soluble heterodimers with agonist complexes (20, 21).

In this study, we took advantage of the murine 2C TCR, which reacts specifically to both the syngeneic complex KbSIY and the allogeneic complex LdQL9 (22, 23). Soluble monomers and oligomers of allogeneic class I MHC-peptide complexes were used to treat CD8 T cells to observe the reaction when peptide re-presentation is not a complicating factor. The experiments conducted showed that CD8 T cells, like CD4 T cells, are stimulated by dimers and higher-order oligomers of soluble MHC-peptide complexes, but are not activated by soluble monomers. In fact, at stimulatory concentrations of oligomeric MHC-peptide reagents, increasing concentrations of soluble monomers can compete off the activation observed. However, dimeric MHC-peptide complexes can stimulate activation regardless of the involvement of the class I MHC coreceptor CD8.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials and peptides

Fluorescent mAbs to cell surface molecules (human CD3 and CD69 and murine CD3, TCR, CD8, CD69, and CD25) labeled with either PE, fluorescein, or allophycocyanin were obtained from BD Pharmingen. Calibration beads for PE and fluorescein for use in flow cytometry were obtained from Spherotech. Unlabeled Abs to cell surface molecules (murine CD11a) were obtained from Leinco Technologies. Size exclusion chromatography was performed using Superdex 200 gel filtration columns (Amersham Biosciences); often two columns were used in series to achieve higher resolution for purification of oligomeric MHC species. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences).

The following peptides were all synthesized using 9-fluorenylmethoxycarbonyl chemistry and verified using mass spectrometry: SIY (SIYRYYGL) found from a combinatorial library to activate the 2C TCR in complex with H2-Kb (22); OVA (SIINFEKL) derived from OVA; QL9 (QLSPFPFDL) modified from an endogenous murine peptide presented on H2-Ld; influenza hemagglutinin (HA)3 (PKYVKQNTLKLAT); and A2 (VGSDWRFLRGYHQYA) derived from the human class I MHC allele HLA-A2. For use in direct treatment of T cells or in making soluble class II MHCs, the peptides were purified using reverse-phase HPLC; crude peptide was used for refolding into soluble class I MHCs.

Production, labeling, and cross-linking of soluble class I MHC-peptide complexes

Soluble murine class I MHC H chains H2-Kb and H2-Ld and the L chain beta2-microglobulin were expressed as separate inclusion bodies in Escherichia coli and folded in vitro by rapid dilution in the presence of excess peptide as previously described for human class I MHCs (24). MHC class I H chains Kb and Ld were produced in two ways: carrying a C-terminal biotinylation signal peptide (25, 26) and a naturally occurring cysteine at position 121. Cys121 does not form intermolecular disulfide bonds between folded complexes. These complexes were fluorescently labeled at Cys121 at roughly 1 label per protein by incubating with 20-fold molar excess of fluorescein maleimide (Molecular Probes) at room temperature for 1 h in the dark, and then quenching with 1 mM DTT and purifying by size exclusion chromatography. The level of label incorporation was determined using UV-Vis absorbance measurements. These constructs also could be C-terminal biotinylated using the birA enzyme (Avidity). Alternately, class I MHC H chains Kb and Ld were produced carrying a C121R mutation and a newly introduced, uniquely reactive C-terminal cysteine at position 282. Freshly reduced and purified C-terminal cysteine-containing complexes were chemically biotinylated by incubating with 20-fold molar excess of biotin-polyethylene oxide (PEO)-maleimide (Pierce) for 1 h at room temperature, and then purified by size-exclusion chromatography. Biotinylated class I MHCs were incorporated into streptavidin-linked tetramers by stepwise addition of streptavidin to purified biotinylated MHC to a final molar ratio of 1:4 (27). Tetrameric complex was then isolated by size-exclusion chromatography using a Superdex 200 column (Amersham Biosciences).

The class I MHC constructs carrying the uniquely reactive C-terminal cysteine at position 282 were also formed into disulfide-linked dimers by incubating with no DTT, and in the presence of 1.3 mM 1,10-phenanthroline and 0.25 mM copper sulfate (Sigma-Aldrich) for 1 h at room temperature. The MHC dimers were purified by size exclusion chromatography using two Superdex 200 columns in series for additional resolution (Amersham Biosciences).

Production, labeling, and cross-linking of soluble class II MHC-peptide complexes

The soluble human class II MHC HLA-DR1 was produced in E. coli as separate inclusion bodies for the extracellular domains of the {alpha}- and beta-chains and folded in vitro by rapid dilution in the presence of excess peptide as previously described (28). In some proteins, a uniquely reactive cysteine was introduced at the C terminus of either the {alpha} or beta extracellular domain for the ability to cross-link into oligomers. For homodimers of DR1-peptide complexes, a disulfide bond between the C-terminal reactive cysteines on folded purified monomers could be created by incubating with 1.3 mM 1,10-phenanthroline and 0.25 mM copper sulfate (Sigma-Aldrich) as previously discussed for class I MHCs.

T cell clones

The murine CD8+ T cell clone L3.100 (29), expressing the 2C TCR specific for KbSIY and LdQL9 complexes (22, 23), was stimulated weekly using irradiated P815 cells and recombinant murine IL-2. The human CD4+ T cell clone HA1.7 specific to HLA-DR1 in complex with the HA peptide (30) was stimulated weekly using peptide-pulsed irradiated APCs and recombinant human IL-2. T cells were maintained in RPMI medium containing 10% FCS at 37°C, 5% CO2, and were used for assays after resting 7–10 days poststimulation.

Ex vivo T cell purification

2C TCR transgenic (31) or OT-1 TCR transgenic (32) mice (H-2b) were used as a source for CD8 T cells. Lymph nodes were extracted from mice and processed into a single-cell suspension. RBCs were lysed with a brief wash in ammonium sulfate buffer, and the resulting lymphocytes were depleted of APCs and CD4+ T cells using magnetic beads (Dynal Biotech). Lymphocytes were at least 99% TCR+ after purification. For 2C T cells, 75–80% of T cells were CD8+ and 20–25% were CD8–. There may be incomplete exclusion of native TCR {alpha}-chains by the transgenic TCR-{alpha} in the 2C transgenic mice in which the transgene is not expressed on a Rag–/– background, allowing some small fraction of the T cells to express a non-2C TCR-{alpha}.

T cell activation and binding assays

T cell assays were conducted in 96-well plates with 50,000–100,000 cells/well. For assays in which immobilized protein was used, the plates were incubated with the protein in PBS overnight at 4°C. All wells were blocked before incubation with cells using 1% BSA in PBS at 4°C overnight to prevent spurious immobilization of soluble MHCs during the assay. Activation responses in T cells were assayed by incubation with dilutions of peptide or MHC in medium for 3–6 h for CD8+ T cells or 12–24 h for CD4+ T cells at 37°C. After the incubation, the samples were chilled on ice, and then stained on ice for 30–45 min using fluorescent Abs to cell surface markers (BD Pharmingen). MHC binding to T cells was assayed by incubation with dilutions of fluorescent-labeled MHC and Abs to cell surface markers in FACS buffer (0.02% azide and 1% FCS in PBS) for 45 min at 4°C to prevent the internalization of complexes. The cells were washed, fixed with 1% paraformaldehyde (Sigma-Aldrich), and analyzed by flow cytometry. Calibration of fluorescent-labeled MHC-peptide complexes associated with lymph node-derived 2C T cells was conducted by comparing with SPHERO Rainbow Calibration Particles (Spherotech).

For intracellular staining of phosphorylated Zap70, 2C transgenic splenocytes were incubated with soluble stimulus for 10 min at 37°C, and then fixed and permeabilized on ice for 20 min, using ice-cold Cytofix/Cytoperm solution (BD Pharmingen) containing 20 mM NaF and 1 mM Na3VO4 (Sigma-Aldrich) to prevent alterations in the phosphorylation state. Next, the cells were extensively washed in Perm/Wash solution (BD Pharmingen), and then stained in Perm/Wash solution for phospho-Tyr493 on Zap70 (Cell Signaling Technology) for 30 min on ice. The cells were washed three times with Perm/Wash solution, and then the anti-phospho-Zap70 Ab was detected with a FITC-labeled goat anti-rabbit polyclonal Ab (Chemicon International) while costaining for surface markers in Perm/Wash solution for 30 min on ice. The cells were washed three times again in Perm/Wash solution, resuspended in 1x PBS containing 1% paraformaldehyde, and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Soluble H-2Ld monomers and oligomers

To investigate the valency dependence of CD8 T cell activation, it was necessary to construct soluble, allogeneic MHC-peptide complexes as monomers and oligomers of defined valency. To make soluble H-2Ld oligomers, a Ld H chain construct was designed in which the cysteine at position 121 was mutated to an arginine. This design is as found in the native sequence of several other murine class I MHC proteins, including H-2Kd and H-2Db. In addition, a new, uniquely reactive cysteine was introduced at position 282, which is the C terminus of the soluble construct. This protein was produced in E. coli as an insoluble inclusion body, solubilized in urea, and folded in vitro in the presence of beta2-microglobulin and excess QL9 peptide. The yield of refolded protein was similar to the yield obtained with the unaltered soluble H-2Ld construct, with the naturally found cysteine at position 121 and a biotinylation signal peptide at the C terminus.

The freshly reduced, purified LdCys-QL9 was oxidized to form a disulfide bond, creating a covalent dimer (Fig. 1A). This peak was cleanly separated from remaining monovalent MHC by size-exclusion chromatography before performing an experiment. The dimerization gives near quantitative yield, and the purified dimer is stable for over 6 mo at 4°C (data not shown).


Figure 1
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  		FIGURE 1. Characterization of soluble allogeneic class I MHC-peptide complex monomers and oligomers. A, Gel filtration traces show refolded LdQL9 complexes using two Superdex 200 columns in series in 1x PBS (pH 7.4). Traces show absorbance at 280 nm. LdQL9 complexes dimerized (top trace) by oxidizing the uniquely reactive cysteine at the C terminus of the H chain into a disulfide bond. LdQL9 monomeric complex (second trace) that does not contain the reactive C-terminal cysteine. Molecular weight standards are shown in bottom trace. B, Gel filtration traces of H2-Ld soluble extracellular domain with a uniquely reactive cysteine engineered at the C terminus, refolded with human beta2-microglobulin and QL9 peptide. A single Superdex 200 column was used in 1x PBS (pH 7.4). Cysteine modified with biotin-PEO-maleimide (top trace). FITC-labeled streptavidin (second trace). A mixture of biotinylated LdQL9 and streptavidin-FITC (third trace) at a 4:1 molar ratio, where streptavidin has been added stepwise in small aliquots and allowed to bind biotinylated complexes. For experiments with LdQL9 tetramers, the mixture (molecular mass ~240 kDa) was freshly isolated (shaded peak) and used to treat T cells. Molecular mass standards are represented in bottom trace.

 
Alternatively, the purified LdCys-QL9 was biotinylated by addition of 20-fold excess biotin-PEO-maleimide, and the excess biotin removed by gel filtration. The biotinylated monomer was cross-linked by stepwise addition of streptavidin to a 1:4 molar ratio with the monomer. This resulted in a mixture of oligomeric states, the predominant species of which contains tetravalent Ld according to estimated molecular mass (~240 kDa) (Fig. 1B). This peak represented by the shaded area was freshly isolated when performing stimulations with LdQL9 tetramers.

Allogeneic MHC-peptide complexes activate CD8 T cells as oligomers, but not monomers

T cells that carry the 2C TCR have a well-characterized response to a specific allogeneic complex: H2-Ld in complex with the QL9 peptide (23). We have previously shown that this MHC-peptide does not activate T cells carrying the 2C TCR as a soluble monomer (15), but we wanted to investigate whether this same complex could trigger T cell activation as a soluble oligomer, as has been seen for dimeric class II MHCs in CD4+ T cells (8). We tested the ability of the allogeneic MHC-peptide monomers and oligomers to induce activation in naive CD8+ T cells isolated from the lymph nodes of 2C TCR transgenic mice (Fig. 2, A and B). Dimers and higher-order oligomers were able to induce activation responses such as TCR down-regulation (Fig. 2A) and CD69 up-regulation (Fig. 2B). Soluble monomers of LdQL9 were unable to induce activation responses even at high concentrations (Fig. 2, A and B). The valency requirement for activation observed in these experiments is the same as in previous experiments with CD4+ T cells (8).


Figure 2
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  		FIGURE 2. CD8 T cells are activated by allogeneic MHC oligomers, but not by monomers, regardless of surface adhesion. A and B, CD8+ T cells isolated from lymph nodes of 2C TCR transgenic mice were stimulated for 3 h at 37°C with soluble LdQL9 monomer (circles), dimer (diamonds), or tetramer (squares) in wells that were either precoated with 5 mg/ml anti-CD11a Ab (open symbols, dashed line) or were not coated (closed symbols). Activation in terms of A, TCR down-regulation and B, CD69 up-regulation was seen for a dimer or higher order oligomer of LdQL9, but not for a monomer, with or without anti-CD11a included in the assay. C, Murine H2-Kb+ CD8+ T cells isolated from 2C TCR transgenic (not Rag–/–) lymph nodes express the adhesion molecule CD11a on their surface. Live 2C T cells purified from lymph nodes, both CD8+ and CD8–, were stained with anti-CD11a (black histogram) and an isotype control (gray histogram). D, Dot plot of 2C transgenic T cells purified from lymph nodes by depletion of CD4+ and B220+ cells using magnetic beads. The relative percentages of CD8+ and CD8– T cells is roughly constant from sample to sample. E and F, CD8– T cells isolated from lymph nodes of 2C TCR transgenic mice were stimulated for 3 h at 37°C with soluble LdQL9 monomer (circles), dimer (diamonds), or tetramer (squares) in wells which were precoated with 5 mg/ml anti-CD11a Ab (open symbols, dashed line), compared with not coated (closed symbols). Activation was measured as TCR down-regulation (E) and CD69 up-regulation (F).

 
It has been proposed that in the context of an adherent cell, a soluble monomeric MHC-peptide complex would be sufficient to trigger T cell activation (17). In that study, when CD8+ T cells were allowed to adhere to a surface coated with Abs to cell surface molecules not involved in the Ag-specific response, the adherent T cells showed calcium flux in response to soluble syngeneic monomeric class I peptide-MHC molecules. The study used a syngeneic class I MHC-peptide as a stimulus that may be susceptible to the complication that peptide could be recognized after loading onto T cell surface class I MHCs, as observed in other studies (14, 15), although it was argued that the responses were measured in a time frame too rapid to allow for peptide re-presentation. To investigate the possibility that CD8+ T cells could be activated by MHC monomers after adhesion priming, but without the potential for re-presentation of peptide, we studied the responses of the 2C lymph node T cells to soluble allogeneic monomeric and oligomeric MHCs after adhesion of the T cells to plastic coated with anti-CD11a. Assay wells were either blocked as normal or coated with 5 mg/ml Ab against CD11a, which is expressed highly on the surface of 2C TCR transgenic T cells (Fig. 2C). The amount of immobilized anti-CD11a used has been shown to enhance cytokine responses to coimmobilized agonist MHC-peptide complexes for both CD8+ and CD4+ T cells (33). The 2C T cells in both sets of wells were treated identically with soluble LdQL9 monomers, dimers, or tetramers (Fig. 2, A and B). The activation responses seen from this treatment were indistinguishable in magnitude and concentration dependence whether or not the adhesion molecule was included on the surface for both TCR down-regulation (Fig. 2A) and CD69 up-regulation (Fig. 2B).

Engagement of the MHC coreceptor CD8 has been proposed to play an important role in activation of CD8 T cells (12, 21, 34). In the current experiments, we had an opportunity to study the activation responses of CD8+ 2C T cells (~75–80% of lymph node T cells) vs a naturally occurring population of CD8– T cells also found in the 2C transgenic mice (~20–25% of lymph node T cells) (Fig. 2D). CD8– 2C T cells responded to LdQL9 dimers or higher-order oligomers in a concentration range similar to CD8+ 2C T cells. Soluble LdQL9 monomers did not stimulate CD8– T cells (Fig. 2, E and F). Again, as with the CD8+ T cells (Fig. 2, A and B), no change in responsiveness from the CD8– T cells was seen with vs without the inclusion of immobilized Abs against CD11a (Fig. 2, E and F).

The concentrations of soluble LdQL9 monomers used in these experiments were expected to be sufficient to bind to a large portion of 2C T cell receptors based on previous binding studies. The Kd of KbSIY for 2C T cells has been reported to be 0.33 µM, with an LdQL9 Ig-fusion binding even more strongly and showing less dependence on CD8 expression (35). To confirm the anticipated binding behavior of soluble MHC monomers, we tested their ability to bind specifically to the 2C T cells. Soluble, fluorescently labeled class I MHC-peptide monomers were used to stain 2C T cells. Clear, specific binding is seen for soluble, fluorescent LdQL9 (allogeneic) and KbSIY (syngeneic) MHC monomers, but not with KbOVA (nonspecific complex) (Fig. 3A). The number of MHCs bound to the cell was calculated for CD8+ (Fig. 3B) and CD8– (Fig. 3C) 2C T cells, using the CD3–CD8– cells (non-T cells) found in lymph nodes to measure the level of nonspecific binding. The number of specifically bound complexes indicates that both of the stimulatory MHCs, LdQL9 and KbSIY, bind at levels that correspond to a large fraction of the total cell surface T cell receptors (~8000–24,000 CD3 complexes per cell, data not shown). Thus, under conditions in which no activation was observed, LdQL9 monomers were engaging many TCR on the surface of the 2C T cells.


Figure 3
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  		FIGURE 3. Allogeneic MHC binding and activation measured on 2C transgenic T cells. A, Raw flow cytometry data from cells stained with fluorescent class I MHC monomers. Histogram traces are shown for LdQL9- and KbSIY-specific MHC-peptide complexes and for the KbOVA nonspecific MHC-peptide complex on CD8+ T cells, CD8– T cells, and non-T cells. B and C, Quantitative binding of fluorescent-labeled, soluble LdQL9 (•), KbSIY (x), and KbOVA ({circ}) class I MHC monomers on T cells isolated from lymph nodes of 2C TCR transgenic mice. Fluorescent signals were normalized by subtracting out staining levels in the non-T cell population (CD3–CD8–), and then calibrating the number of specifically bound fluorescent molecules using SPHERO Rainbow calibration particles (Spherotech). Binding is shown for CD8+ T cells (B) and CD8– T cells (C). D and E, Activation levels correlated with MHC binding levels for allogeneic MHC monomers and tetramers. TCR down-regulation as a percentage of the maximum (D) and CD69 up-regulation (E).

 
To further investigate the relationship between binding and activation for the soluble allogeneic class I MHC, we plotted activation levels induced by LdQL9 monomers and oligomers against measured fluorescent MHC-peptide complex binding. The number of fluorescein molecules per MHC was calibrated so the total numbers could be compared between monomer and tetramer. Maximal amounts of activation signal were observed at relatively low numbers of MHC bound for the streptavidin-linked tetramer (≤5000 MHCs associated) for both TCR down-regulation (Fig. 3D) and CD69 up-regulation (Fig. 3E). Similar binding levels were seen for LdQL9 soluble monomer (≥6000 MHCs associated) with essentially baseline levels of activation markers.

These data indicate that TCR engagement by a soluble MHC monomer does not lead to sustained signaling and modulation of cell surface molecules typical of an activation response. Thus, a binding valency of dimer or higher is required to activate CD8 T cells with soluble MHC, and an MHC dimer is sufficient to trigger T cells that do not express the coreceptor CD8. Adhesion priming through CD11a does not allow soluble MHC monomers to lead to productive sustained signaling. Although some possibility exists that the disulfide-linked MHC dimers cause activation by actually cross-linking one T cell to another, the lack of any apparent dimeric T cell population in the analyses (data not shown) combined with the weak binding seen for soluble LdQL9 monomers, particularly for CD8– T cells, suggests that the activation response is caused by both MHC molecules from the Ld dimer binding to the same T cell.

Early activation markers are induced by soluble allogeneic MHC-peptide dimers

Soluble LdQL9 dimers or oligomers were able to induce TCR down-regulation and CD69 up-regulation, whereas monomers were not. However, some possibility remains that LdQL9 monomer binding could trigger some intracellular signaling that did not result in changes in the surface expression of these markers. To further investigate the functional consequences of soluble MHC binding, we studied the effect of these treatments on the very early activation marker of phosphorylation of intracellular Zap70 (36). After a brief, 10 min stimulation with MHC-peptide complexes, 2C T cells were fixed, permeabilized in the presence of phosphatase inhibitors, and stained intracellularly for phospho-Tyr493 of Zap70, which is an indicator of induced TCR signaling. This experiment showed that for both CD8+ (Fig. 4A) and CD8– (Fig. 4B) T cells, addition of an allogeneic LdQL9 dimer (Fig. 4, A and B, top histogram) is sufficient to induce increased phosphorylation of Zap70, whereas addition of a high concentration of LdQL9 monomer (Fig. 4, A and B, middle histogram) is essentially indistinguishable from an unstimulated sample (Fig. 4, A and B, bottom histogram). The increase in Zap70 phosphorylation is clearly observed with LdQL9 dimer addition, but at 10 min, the increase is small and does not encompass the entire population (Fig. 4, A and B). Similar behavior is seen for later activation markers such as TCR down-regulation and CD69 up-regulation if they are assayed early in the response.


Figure 4
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  		FIGURE 4. Soluble allogeneic MHC dimers, but not monomers, induce an increase in phosphorylated Zap70. Splenocytes from 2C transgenic mice (not on a Rag–/– background) were stimulated for 10 min at 37°C with soluble LdQL9 monomer or dimer, free SIY peptide (stimulatory), or soluble Kb OVA monomer (nonstimulatory). The cells were fixed and permeabilized in the presence of phosphatase inhibitors, and then stained for phosphorylated Zap70 levels. A and B, Offset histograms of phospho-Zap70 staining for unstimulated (light gray histogram), 2 µM LdQL9 monomer (medium gray histogram), and 1 µM LdQL9 disulfide-linked dimer (black histogram) in A, CD8+ 2C T cells and B, CD8– 2C T cells. C and D, Percentage of phospho-Zap70 high staining cells (n = 4) for a relevant concentration range of soluble LdQL9 monomer and dimer, free SIY peptide, and soluble KbOVA monomer in CD8+ 2C T cells (C) and CD8– 2C T cells (D). Results are representative of multiple experiments.

 
When looking at a wider concentration range, it is apparent that LdQL9 dimer treatment (Fig. 4, C and D) gives a concentration-dependent increase in phospho-Zap70, whereas treatment with LdQL9 monomer (Fig. 4, C and D) does not result in increased Zap70 phosphorylation. The fraction of the population responding in the CD8– T cells (Fig. 4D) was much smaller than in the CD8+ T cells (Fig. 4C), but the difference between LdQL9 monomer and dimer was consistent in four experiments. A control nonactivating MHC monomer, KbOVA (Fig. 4, C and D), resulted in a small increase in phospho-Zap70; however, that signal does not lead to productive downstream activation markers (TCR down-regulation and CD69 up-regulation) as seen with LdQL9 dimers. Some phospho-Zap70 increase was seen when SIY peptide alone was added to provoke T-T cross-presentation (Fig. 4, C and D), although the 10 min timescale of the experiment may be somewhat short to get robust, population-wide stimulation by this mechanism.

A 2C+ T cell clone is similarly activated by soluble allogeneic MHC oligomers

Naive T cells are less sensitive to Ag stimulation than effector or memory T cells, and previous work determining the MHC valency dependence for T cell stimulation was performed using long-term CD4+ T cell clones (8, 9). To address the potential that effector CD8+ T cells may require a different TCR engagement valency than naive CD8+ T cells, we tested the ability of soluble allospecific MHC monomers and oligomers to stimulate a CD8+ T cell clone, L3.100, carrying the 2C TCR (Fig. 5). The soluble MHC-peptide complex LdQL9 was able to induce TCR down-regulation (Fig. 5A) and CD69 up-regulation (Fig. 5B) when it was presented as a covalently linked dimer or tetramer, but soluble monomers generally did not activate the L3.100 clone (Fig. 5, A and B). A small amount of TCR down-regulation and CD69 up-regulation was observed for this highly sensitive clone at the highest concentrations of LdQL9 monomer. To investigate this activation, we looked at the relationship between the number of MHCs associated with the cells and the amount of activation seen, as we had in Fig. 3 for the naive 2C T cells. L3.100 cells consistently show much more activation response in terms of TCR down-regulation (Fig. 5C) and CD69 up-regulation (Fig. 5D) for a tetramer of LdQL9 as compared with a monomer of LdQL9 at the same amount of MHC binding. The apparent activation response of the LdQL9 monomer in fact is similar to the nonspecific complex KbOVA. Thus, we find no evidence for induction of specific activation responses by the LdQL9 monomers in the L3.100 T cell clone. It is possible that the small amount of stimulation seen at high concentrations of LdQL9 monomer could be due to small amounts of aggregation of the MHC-peptide complex during the assay, although no evidence of such behavior was observed in the soluble complex stored at 4°C by gel filtration (see Fig. 1).


Figure 5
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  		FIGURE 5. A CD8+ T cell clone is activated by allogeneic MHC oligomers, but not by monomers. A and B, L3.100, a murine H2-Kb+ CD8+ T cell clone carrying the 2C TCR, which reacts specifically with KbSIY and has a characterized allospecific response to the LdQL9 complex, was stimulated for 6 h at 37°C with soluble LdQL9 monomers (•), disulfide-linked dimers ({diamondsuit}), streptavidin-linked tetramers ({blacksquare}), or KbOVA immobilized on the surface of the wells ({boxtimes}, dashed line). Robust activation responses were induced for treatment with LdQL9 dimers and tetramers, but little or none for soluble LdQL9 monomers or immobilized KbOVA complex when detected by mean fluorescence intensity of TCR down-regulation (A) or mean fluorescence intensity of CD69 up-regulation (B). C and D, The relationship between activation responses and MHC binding for allogeneic stimulus. L3.100 CD8+ T cells were stimulated for 6 h at 37°C with soluble syngeneic KbSIY monomer (x), nonstimulatory KbOVA monomer ({circ}), allogeneic LdQL9 monomer (•), or LdQL9 tetramer ({blacksquare}). Complexes were fluorescently labeled, and binding was measured simultaneously with activation markers. C, TCR down-regulation vs MHC-peptide complexes bound to the cell, both in mean fluorescence intensity. D, CD69 up-regulation vs MHC-peptide complexes bound to the cell, both in mean fluorescence intensity.

 
Two other aspects of this system are illustrated by this analysis. Cross-link-dependent activation by soluble MHC oligomers is predicted to decrease at high concentrations because of increased monovalent binding of oligomers as previously described (37). The reduction of CD69 level at high LdQL9 tetramer concentration seen in this study (Fig. 5B) may reflect this behavior, although it is unclear because of the noise level of the assay. Also, the syngeneic stimulatory monomer KbSIY (Fig. 5, C and D) shows near maximal activation even at undetectable binding levels, as its main mechanism of activation is through peptide re-presentation on cell surface H2-Kb molecules, as seen previously (15).

Monomeric MHC-peptide binding antagonizes activation responses induced by MHC-peptide oligomers

Because productive activation responses can be induced by allogeneic MHC-peptide complexes when presented as a multivalent oligomer, we tested whether the binding seen with addition of monomeric MHC-peptide complexes would inhibit activation induced by an oligomer, or whether monomer binding could contribute to signaling in the context of a productive activation signal initiated by multivalent MHC-peptide binding. The highly sensitive CD8+ T cell clone L3.100 carrying the 2C TCR was activated by a constant 20 nM concentration of LdQL9 streptavidin-linked tetramer. Increasing concentrations of either LdQL9 or nonspecific KbOVA monomer were added to the cells. The activation response in terms of TCR down-regulation was dramatically reduced when monomeric LdQL9 complex, but not KbOVA, was added to the T cells in the same concentration range that monomer LdQL9 binding is observed (Fig. 6, A and B). This inhibition of T cell activation by MHC monomer binding seems to indicate that small amounts of activation seen in this clone with high concentrations of allogeneic MHC monomers (Fig. 5, A and B) are likely to be artifactual.


Figure 6
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  		FIGURE 6. Monovalent MHC binding antagonizes T cell responses to MHC oligomers. A, L3.100 T cells, a murine CD8+ 2C TCR+ clone, was tested for binding of soluble monomeric LdQL9 (•) and KbOVA ({circ}). The number of MHCs bound, calibrated by using SPHERO Rainbow calibration particles (Spherotech), may be read on the y-axis. B, L3.100 T cells were also tested in a separate experiment for the ability of monomeric MHCs to reduce the activation response to 20 nM LdQL9 tetramer. The amount of TCR down-regulation can be read on the y-axis when increasing amounts of LdQL9 ({blacksquare}) or KbOVA ({square}) were added to the cells with the stimulatory LdQL9 tetramer. The binding of soluble monomeric LdQL9 complex to murine CD8+ 2C TCR transgenic T cells isolated from lymph nodes was measured (C) (•). The ability of soluble monomeric LdQL9 complex to reduce activation responses of the cells to 10 nM LdQL9 tetramer with regard to TCR down-regulation shown in mean fluorescence intensity (MFI) (D) and CD69 up-regulation shown in mean fluorescence intensity (E). F, HA1.7 T cells, a human CD4+ T cell clone specific to HLA-DR1 in complex with the HA peptide, was incubated with 20 nM DR1-HA disulfide-linked dimer with increasing concentrations of either soluble monomeric DR1-HA complex ({blacksquare}, dashed line) or the nonspecific soluble monomeric DR1-A2 complex ({square}, dashed line) (38 ).

 
Monomeric MHC-peptides can antagonize activation responses to MHC-peptide oligomers in ex vivo 2C transgenic CD8+ T cells as well as in T cell clones. The direct binding of LdQL9 monomer to 2C T cells is shown (Fig. 6C). Addition of LdQL9 monomer decreases TCR down-regulation (Fig. 6D) and CD69 up-regulation (Fig. 6E) induced from a constant concentration of LdQL9 streptavidin tetramer of 10 nM, similar to the behavior seen in the L3.100 T cell clone.

This monomer antagonism of T cell activation is also seen in the human CD4+ T cell clone HA1.7, which is specific to the human class II MHC protein HLA-DR1 in complex with the HA peptide (30). The TCR down-regulation induced by treating the HA1.7 T cells with a DR1-HA dimer is reduced when DR1-HA is added to the cells in the form of a monomer (38) (Fig. 6F). The nonspecific complex DR1-A2 does not compete with the TCR down-regulation response from the specific dimer (Fig. 6F). These experiments confirm that soluble MHC-peptide monomers bind to the TCR, but do not activate T cells in situations in which peptide re-presentation is not a complicating factor.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have shown that CD8+ T cell triggering by soluble MHC-peptide complexes follows the same patterns as CD4+ T cell triggering (8) when not complicated by peptide re-presentation on endogenous cell surface MHC molecules. A dimer of MHC-peptide complexes is necessary and sufficient to induce T cell activation markers such as TCR down-regulation and CD69 up-regulation, with or without expression of the MHC coreceptor CD8+. Adhesion priming by immobilized Abs to T cell adhesion molecules does not lower this threshold to allow allogeneic MHC monomers to activate these processes. In fact, activation by an oligomer of MHC-peptide complexes is antagonized by high concentrations of soluble MHC-peptide monomer in both CD8+ and CD4+ T cells. These results taken together suggest that the molecular triggers for Ag recognition in CD8+ T cells and CD4+ T cells are likely to be the same.

Although the valency requirements for T cell activation seem to be clear, the nature of the Ag-specific signal as provided by an APC to the TCR is still not completely understood. The fact that a single soluble MHC-peptide complex binding to the TCR does not result in sustained signaling argues for a model in which TCR clustering is a critical event, similar to other immune cells. In the environment of T cell stimulation by an APC, a single activating peptide could create a signal through TCR clustering by promoting binding to endogenous MHC-peptide complexes, using the activating peptide as an anchor around which weaker TCR interactions can play a larger role (20, 21). The threshold for these interactions may be lowered by the involvement of MHC coreceptors CD4 and CD8, either by simply increasing the affinity of the TCR for nonstimulatory MHC-peptide complexes or by a complex bridging model in which a coreceptor bound to one MHC is associated with the transmembrane domain of a separate MHC-bound TCR, causing phosphorylation (19). However, it is clear from the results presented in this study that high affinity TCR interactions with dimeric MHC-peptide complexes do not require a bridging CD8 coreceptor to result in signaling.

There has been some discussion of a potential conformational change in the TCR or possibly an allosteric rearrangement of the receptor subunits, upon MHC-peptide binding, which could initiate signaling (39). In a previous study, productive TCR engagement correlated with exposure of an intracellular binding epitope for Nck; this epitope is revealed extremely early in the activation of T cells (40). Whether the exposure of this epitope is a result of interaction with a single stimulatory MHC-peptide complex propagated through some conformational change, or a result of appropriate TCR clustering, is not determined. It is known that the cytoplasmic domains of several components of the TCR complex tend to homo-oligomerize at high concentrations (40); perhaps ligand-induced clustering of the TCR drives the cytoplasmic domains of proximal receptors to rearrange, exposing the Nck binding epitope and propelling other signaling cascade processes. These results are intriguing for further study.


   Acknowledgments
 
We thank Jennifer Cochran (Stanford School of Medicine, Stanford, CA) for the HA1.7 monomer inhibition experiment, Jonathan Lamb (University of Edinburgh Medical School, Edinburgh, U.K.) for the HA1.7 T cell clone, Herman Eisen and Carol McKinley (Massachusetts Institute of Technology, Cambridge, MA) for the L3.100 T cell clone, Cynthia Chambers and Airiel Davis (University of Massachusetts Medical School, Worcester, MA) for transgenic mice and for lymph node isolation, and John Altman and John Lippolis (National Institutes of Health Tetramer Facility, Emory University, Atlanta, GA) for soluble class I MHC constructs.


   Disclosures
Top
 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 National Institutes of Health U19-AI-057319-03. Back

2 Address correspondence and reprint requests to Dr. Lawrence J. Stern, Departments of Pathology and Biochemistry and Molecular Biology, University of Massachusetts Medical School, Room S2-127, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: lawrence.stern{at}umassmed.edu Back

3 Abbreviations used in this paper: HA, influenza hemagglutinin; PEO, polyethylene oxide. Back

Received for publication July 14, 2005. Accepted for publication October 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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