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The Journal of Immunology, 2004, 172: 7466-7475.
Copyright © 2004 by The American Association of Immunologists

Distinct Footprints of TCR Engagement with Highly Homologous Ligands1

Fabio R. Santori*, Kaisa Holmberg{dagger}, David Ostrov{ddagger}, Nicholas R. J. Gascoigne{dagger} and Stanislav Vukmanovic2,*,§

* Michael Heidelberger Division of Immunology, Department of Pathology and New York University Cancer Center, New York University School of Medicine, New York, NY 10016; {dagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; {ddagger} Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610; and § Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, Washington, DC 20010


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell receptor engagement promotes proliferation, differentiation, survival, or death of T lymphocytes. The affinity/avidity of the TCR ligand and the maturational stage of the T cell are thought to be principal determinants of the outcome of TCR engagement. We demonstrate in this study that the same mouse TCR preferentially uses distinct residues of homologous peptides presented by the MHC molecules to promote specific cellular responses. The preference for distinct TCR contacts depends on neither the affinity/avidity of TCR engagement (except in the most extreme ranges), nor the maturity of engaged T cells. Thus, different portions of the TCR ligand appear capable of biasing T cells toward specific biological responses. These findings explain differences in functional versatility of TCR ligands, as well as anomalies in the relationship between affinity/avidity of the TCR for the peptide/MHC and cellular responses of T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The biological response elicited by TCR ligands is dependent on the developmental stage of the T cell and the affinity/avidity of interaction (1, 2). Relatively high affinity/avidity TCR ligands induce cell death of immature CD4+CD8+ thymocytes (negative selection), while proliferation, cytokine secretion, and/or cytolytic activity (agonist activity) are elicited in mature T cells. The low affinity/avidity TCR engagement promotes positive selection, i.e., survival and differentiation of immature thymocytes into mature CD4+CD8– or CD4–CD8+ T cells. In mature T cells, low affinity/avidity ligands may antagonize T cell responses induced by agonist ligands in vitro (3) and possibly in vivo (4), and can also promote survival and/or homeostatic expansion of naive T cells (1). Although the spectrum of biological activities elicited by peptide/MHC usually correlates with the affinity for the TCR, numerous exceptions have been identified (5 , www-ermm.cbcu.cam.ac.uk/01002502h.htm). For example, peptides with low affinity/avidity are capable of fully stimulating mature T cells (6, 7, 8, 9, 10). In addition, some of the same peptides that stimulate proliferation of mature T cells can also promote positive selection (11, 12, 13, 14, 15). The kinetics of the TCR engagement was proposed to be the principal determinant of stimulatory potential of TCR ligands (16). Although biological activity correlates better with the t1/2 of peptide/MHC-TCR interaction than with the affinity/avidity of association (7), exceptions to this rule have also been noted (10).

Stimulation by anti-TCR Abs with specificities for TCR{alpha} or TCR{beta} chain had distinct effects on thymocytes, suggesting that physical engagement of TCR{alpha} may induce signaling events distinct from engagement of TCR{beta} (17, 18). Indeed, disposal of TCR{alpha}-connecting peptide motif was detrimental for positive (and not negative) selection, for extracellular signal-regulated kinase activation by the ligands that promote positive selection, and for recruitment of CD3{delta} chain into TCR/CD3 complex (19). In contrast, deletion of a counter structure in TCR{beta} chain prevented negative, but not positive selection (20). Collectively, these findings may suggest that TCR{alpha}, as opposed to TCR{beta}, is uniquely equipped for generating signals leading to positive selection. This notion is further supported by selective usage of certain V{alpha} families in CD4+ or CD8+ T cells (21, 22). If this is true, then physiological ligands that induce positive vs negative selection should be expected to follow distinct rules of TCR engagement, focusing preferentially on contacting TCR{alpha} or TCR{beta}, respectively. Furthermore, similar differences could be expected in peripheral T cells because ligands that induce positive or negative selection can also initiate cellular responses in mature T cells. Different types of TCR engagements can be envisioned if ligands that promote distinct T cell responses are structurally different. However, in most experimental models, similarity to Ag is essential for biological activity of ligands that induce positive selection and antagonist activity (23, 24, 25). This similarity is especially important at the TCR contact residues in the C terminus of the peptide, which interacts with TCR{beta} (26, 27, 28). To assess whether different cellular responses of T cells require distinct TCR engagements, we performed comparative mapping and mutational analysis of three homologous peptides that promote distinct T cell responses in the H-Y-specific TCR transgenic model. Our results demonstrate that distinct rules of TCR engagement operate for different cellular responses of T cells, despite high degree of similarity between the specific ligands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide binding to MHC class I

Custom synthesized peptides were purchased from Research Genetics (Huntsville, AL). Peptide binding to and dissociation from H-2Db was verified by RMA-S stabilization assay. Briefly, 2 x 105 RMA-S cells were plated overnight at room temperature with different concentrations of peptide. Cells were then incubated at 37°C for 1 h, washed in PBS, and stained for 30 min with anti-H-2Db Ab KH95 (BD PharMingen, San Diego, CA). Cells were then washed in PBS and stained for 30 min with secondary anti-mouse FITC or PE. After secondary staining, cells were washed and analyzed by FACS. For dissociation assays, we pulsed 1 x 106 RMA-S cells overnight at room temperature with 30 µM test peptide. Cells were then washed 1x with PBS, and incubated for 0, 2, 4, or 6 h at 37°C. After each time point, cells were stained, as described above.

Proliferation assay

Spleen cells (1 x 105) from H-Y-specific TCR transgenic mice (purchased from Taconic Farms, Germantown, NY) were incubated with irradiated 5 x 106 spleen cells from C57BL/6 mice pulsed with a range of concentrations of the test peptide 100–0.001 µM agonist in round-bottom 96-well plates in RPMI 1640 medium supplemented with 5 x 10–5 M 2-ME, 1 mM sodium pyruvate (Life Technologies, Gaithersburg, MD), 0.1 mM nonessential amino acids solution (Life Technologies), and 10% FCS (PM-10 medium). After 48–72 h, as indicated, each microculture was pulsed with 0.5 µCi of [3H]thymidine (ICN Biomedicals, Costa Mesa, CA) overnight, and thymidine incorporation was subsequently measured on a beta scintillation counter 1450 MicroBeta (Wallac, Turku, Finland). To test the antagonist activity of peptides, 5 x 106 irradiated splenocytes of C57BL/6 female mice were incubated for 1 h at 37°C with a suboptimal dose of the original Smcy738–746 peptide (100 nM). The cells were washed once with PM-10 medium, and 5 x 105 cells were incubated with 1 x 105 H-Y-specific T responder cells in the continuous presence of indicated concentrations of peptides tested for antagonism (23).

Coreceptor down-modulation dulling assay

The assay was performed, as described (29). C57BL/6 mice were injected i.p. with 3 ml of thioglycolate solution. After 3–4 days, mice were sacrificed and the activated macrophages were collected with cold PBS. The cells were washed, resuspended in PM medium supplemented with 20% FCS, and plated in flat-bottom 96-well plates at the density of 1.5 x 105 cells/well. Macrophages were incubated for at least 1 h at 37°C when they received 4 x 105 thymocytes/well derived from either male or female H-Y, B10.D2, recombination-activating gene-2–/– mice (Taconic Farms). The cultures were then pulsed with the desired concentration of peptide and incubated overnight at 37°C. The next day, the thymocytes were collected and stained for FACS analysis with anti-mouse CD4 PE and CD8 CyChrome Abs (BD PharMingen). Dulling was quantified based on the shift from the gate of untreated double-positive (DP) 3 thymocytes from those of peptide-treated DP thymocytes. Peptide-treated thymocytes were also collected and tested for apoptosis using a FITC-based TUNEL assay kit (Boehringer Mannheim, Indianapolis, IN).

Fetal thymus organ cultures (FTOC)

The FTOCs were performed using gestation day 16 fetuses (23). Fetal thymus lobes were cultivated on sponge-supported filters (Millipore, Bedford, MA) in medium supplemented with peptide at 300 µM (unless otherwise stated). Cultures were arranged so that one lobe was treated as experimental, while the other lobe from the same fetus was treated as control. As negative control, we used peptides that bind H-2Db well, but do not induce positive selection of H-Y thymocytes. After 10 days, lobes were dissociated and cells and fetal thymuses were screened with anti-mouse V{beta}8 FITC (BD PharMingen) to ascertain expression of H-Y TCR and anti-H-2Kb PE (BD PharMingen) to ascertain for TAP1–/– status. Fetal lobes were further analyzed by triple FACS staining with monoclonal anti-mouse CD4 PE, CD8 CyChrome, and CD24 FITC Abs (BD PharMingen). Remaining cells were used in proliferation assay, as described above.

Molecular modeling

The atomic coordinates for the peptide/H-2Db complex most similar to Ube1x509–517, ARX54–62, and Smcy738–746 provided the basis for molecular models. Two crystal structures were used as the basis for molecular modeling and yielded indistinguishable results: 1INQ, which is the structure of SSVVGVWYL/H-2Db, and SVNL9 peptide, which is the structure of SSVVNVWYL/H-2Db and does not have a Brookhaven Protein Data Bank code (30). The latter structure contains two key anchor residues at positions 5 (N) and 9 (L). The coordinates for the peptide in the crystal structure of SVNL9 complexed to H-2Db were mutated to the corresponding residues in Ube1x509–517, ARX54–62, and Smcy738–746 using the program O, version 7 (31). The atomic coordinates were subjected to 200 steps of conjugate gradient energy minimization using the program CNS (32). The molecular models of Ube1x509–517, ARX54–62, and Smcy738–746 bound to Db were displayed using the program SETOR (33). The peptides and residues 1–135 of the H-2Db H chain are displayed. The residues corresponding to the {alpha}2-helix 136–176(136–176) were omitted for clarity.

Tetramer-binding and inhibition experiments

H-2Db containing a biotinylation sequence and human {beta}2-microglobulin were expressed in Escherichia coli and refolded, as described (34, 35, 36). Briefly, inclusion bodies dissolved in 8 M urea were mixed with peptide using 4.5 mg of H chain, 1.5 mg of L chain, and 1 mg of peptide in the presence of 10 mM DTT (34, 36). The mixture was dialyzed against 4 L of 20 mM Tris, pH 8.0, 150 mM NaCl at 4°C for 48–72 h. Refolded pMHC complex was separated with a Superdex HR200 column (Pharmacia Biotech, Uppsala, Sweden). Biotinylation was performed using the BirA enzyme and reagents from Avidity L.L.C. (Denver, CO) (35, 36). For tetramerization, pMHC was mixed with streptavidin-PE (Molecular Probes, Eugene, OR) in a molar ratio of 4:1. Tetramers were separated with the Superdex HR200 column. Concentration of tetramer was determined by Bradford assay. C57BL/6 splenocytes were irradiated and pulsed with 50–100 µM Smcy738–746 for 1 h. After washing, pulsed APC were mixed with H-Y TCR transgenic thymocytes and cultured in RPMI 1640 medium supplemented with IL-2. The cells were restimulated 10 days later, and thereafter, cells were restimulated once per week. After several stimulations, only CD8highT3.70high (Id-positive) cells were left in the culture. Cells were washed twice with FACS buffer (PBS, 2% FCS, 0.1% NaN3) before staining with peptide/MHC tetramers. Tetramer-staining experiments were performed, as described (36). Briefly, for equilibrium binding, cells were stained with tetramer at concentrations from 0.1 to 600 nM together with anti-TCR C{beta} mAb (H57-597). Crystallography studies have demonstrated that this Ab does not interfere with binding of peptide/MHC complexes (37). For inhibition, we stained with Ube1x509–517L4R tetramer (20 nM) in the presence of unlabeled pMHC monomer (5 x 10–5 to 10–8M). Staining was for a minimum of 2 h at 4°C. Cells were washed twice and fixed with 1% formaldehyde in PBS before flow cytometry. T cells were gated based on anti-TCR C{beta} staining.

Tetramer decay experiments

Tetramer decay was performed largely as described (10, 36, 38). Lymphocytes were stained with tetramer (20–100 nM), washed twice with FACS buffer, and kept on ice until they were mixed with excess anti-H-2Db mAb 28-14-8s supernatant and incubated at room temperature, to allow tetramer dissociation. Although mAb 28-14-8s binds to the {alpha}3 domain of H-2Db, it prevents both association and rebinding of H-2Db tetramers to the P14 TCR (39) (K. Holmberg and N. R. J. Gascoigne, unpublished observations). After 0–30 min, cells were washed and fixed for flow cytometry. The natural logarithm of percentage of geometric mean fluorescence (GMF) at each time point (compared with 0 min) was plotted against time. The tetramer t1/2 was derived from the slope by t1/2 = ln2/slope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Biological responses of T cells require distinct TCR contacts

The Smcy738–746 peptide (KCSRNRQYL) is an Ag for the H-Y TCR (40). Mature TCR transgenic CD8+ cells proliferate (Fig. 1a), while immature CD4+CD8+ thymocytes down-modulate CD4 and CD8 molecules (Fig. 1b) and undergo apoptosis (Fig. 1c) in response to this ligand. CD4/CD8 down-modulation can occur in response to ligands that induce positive selection (24, 41), and is therefore not a direct measure of cell death. Nevertheless, ligands that induce positive selection induce strong coreceptor down-modulation only if the signals are nonspecifically enhanced. Therefore, strong coreceptor down-modulation induced by a ligand alone is a good indication of negative selection. The self peptide Ube1x509–517 (KSNLNRQFL) antagonizes Ag-induced CD8+ cell proliferation (Fig. 1d) and promotes generation CD4–CD8+ thymocytes (Fig. 1e). These CD4–CD8+ thymocytes are mature both phenotypically (low levels of CD24; Fig. 1f) and functionally (capable of responding to Ag; see Fig. 5c). In contrast, putative self peptide ARX54–62 (VSNLNRQFL) that differs from Ube1x509–517 only at position 1 (p1) induces antagonist activity, but not positive selection (23). Ube1x509–517 and ARX54–62 were discovered using homology to the Ag (Smcy738–746)-based bioinformatics algorithm. All three peptides have classical anchors at p5 and p9, described as motif for peptide binding to H-2Db (42). To determine which of the remaining seven residues are potential TCR contacts for the H-Y TCR, 1 aa at a time of these three peptides was substituted by alanine, except for p5 and p9. Each peptide variant was tested for induction of following biological activities: agonist or antagonist activity in mature T cell proliferation assays; positive and negative selection of thymocytes.



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  		FIGURE 1. Induction of distinct biological responses in the H-Y-specific TCR transgenic model. a, Comparison of mature CD8+ T cell proliferation induced by Smcy738–746 and Smcy738–746K1A peptides. b, Deletion of CD4+CD8+ thymocytes by Smcy738–746 in vitro. The level of CD4/CD8 down-modulation (dulling) was calculated by setting an elliptic gate on DP thymocytes from the negative control (100% of DP). Dulling in the experimental samples was estimated from the loss of DP thymocytes in the elliptic gate in percentage. c, Apoptosis of CD4+CD8+ thymocytes stimulated in vitro in the absence (CTRL) or presence of Smcy738–746 (0.1 µM) was determined by TUNEL. d, Ube1x509–517 induces antagonism of CD8+ T cell proliferation. e and f, Thymocyte-positive selection by Ube1x509–517. TAP1–/–, H-Y TCR transgenic FTOC were treated with 300 µM Ube1x509–517 or control peptide (Ube1x509–517K1H). After 10 days of culture, thymocytes were stained with anti-CD4, anti-CD8, and anti-CD24 mAbs, as described in Materials and Methods. Shown are CD4/CD8 dot plots and histograms with expression levels of CD24 in the gated CD4–CD8+ subpopulation.

 


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  		FIGURE 5. The role of peptide p4 in positive selection. a, Intermediate contacts at p4 can be essential for thymocyte-positive selection. Shown are CD4 and CD8 dot plots and histograms with expression levels of CD24 in the gated CD4–CD8+ subpopulation of thymocytes from H-Y TCR transgenic, TAP1-deficient FTOC treated with indicated peptides (300 µM). b, Peptides Ube1x509–517L4R and ARX54–62L4R show differential capacity to induce positive selection of CD4–CD8+ thymocytes in FTOC. Peptides were added at concentrations of 10 or 20 µM, as indicated. Shown is a bar graph with percentage of selected CD4–CD8+ thymocytes (mean and SEs). c, Thymocytes selected by Ube1x509–517L4R and ARX54–62L4R in FTOC are functionally responsive to cognate Ag. After 10 days in FTOC with the indicated peptides, 1 x 105 H-Y TCR transgenic TAP-1-deficient thymocytes/well were stimulated for 3 days with Smcy738–746. Proliferation was determined by tritiated thymidine incorporation during last 16 h of culture. Shown are means and SDs.

 
Substitution at p4, p6, and p7 completely abolished the capacity of Smcy738–746 to induce proliferation of CD8+ T cells (Fig. 2a) or negative selection of CD4+CD8+ thymocytes (Fig. 2b). Substitution at p8 had the same effect (Table I), but is not shown in the figure because subsequent experiments indicated that Smcy738–746Y8A did not bind to H-2Db (Table I). To address the issue of p8 as a possible TCR contact, we synthesized another variant peptide (Y8L) that did bind to H-2Db (Table I). Inability of this variant to induce proliferation (Table I) suggests that p8, too, is a TCR contact residue. Two Smcy738–746 variants (R4A and Q7A) that lost the ability to induce T cell proliferation acquired the ability to induce TCR antagonist activity and promote positive selection (data not shown), a phenomenon observed in other experimental systems (11, 13, 43, 44). In contrast, Smcy738–746R6A had no biological activity (Table I), suggesting a crucial role for peptide p6 in engaging the H-Y-specific TCR. The most striking activity was that of Smcy738–746K1A, which was indistinguishable from the original peptide (Fig. 1a). Even though a subtle quantitative loss in activity could be ascribed to the mutation at p1, due to slightly better binding to the H-2Db of the mutant peptide (Table I), the lack of a deeper impact of p1 came as a surprise. p1 was anticipated to be relatively important TCR contact based on differences between biological activities of Ube1x509–517 and ARX54–62 that differ only at p1 (K vs V). To test whether V at p1 might have specific prohibitory effects, we made a K1V substitution in the Smcy738–746. Potent proliferation of H-Y-specific CD8+ T cells induced by this peptide, as well as positive selection induced by ARX54–62 with more reactive side chain at p8 (F to Y substitution; Table I), excluded negative effects of V at p1.



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  		FIGURE 2. Alanine scan mutagenesis of TCR ligands with distinct biological activities. Replacement of single amino acids by alanine at each peptide position was performed in Smcy738–746 (a and b) or Ube1x509–517 (c–e). Variant Smcy738–746 peptides were tested at concentration of 1 µM for capacity to induce proliferation of mature T cells (a), or CD4/CD8 coreceptor down-modulation in CD4+CD8+ thymocytes (b). Variant Ube1x509–517 peptides were tested at concentration of 300 µM for positive selection of CD4–CD8+ T cells in FTOC (c) and response of FTOC-derived T cells to the Ag Smcy738–746 (d), or at concentration of 10 µM for antagonism of mature T cell proliferation (e). Each experiment was performed several times with equivalent results, as indicated in Table I.

 

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  		Table I. Summary of biological activities of variants of Smcy738–746, Ube1x509–517, and ARX54–62 peptidesa

 
In Ube1x509–517, p1, p2, p6, p7, and p8 were essential for positive selection in FTOC (Fig. 2, c and d). However, the same amino acid replacements had a different impact on antagonist activity of this peptide. Although p6, p7, or p8 was essential, p1 and p2 were dispensable for antagonist activity of Ube1x509–517 (Fig. 2e). All Ube1x509–517 variant peptides bound to H-2Db (Table I). The same residues (p6, p7, and p8) were essential for antagonist activity of the peptide ARX54–62 (Table I). Collectively, these results demonstrate that the residues essential for the induction of distinct biological responses of T cells are not necessarily identical even if the same TCR ligand is used (antagonist activity vs positive selection induced by Ube1x509–517).

The impact of N-terminal peptide residues is not imposed by indirect structural effects

The difference in biological activity of peptides could result from conformational differences between peptides or structural modifications of H-2Db itself when bound to different peptides. Therefore, we generated molecular models of peptide/MHC complexes based on structural data of available peptide/H-2Db complexes. These models suggest that the side chains of K1 in Smcy738–746 and Ube1x509–517 are positioned in a very similar, if not identical manner, and that Smcy738–746, Ube1x509–517, and ARX54–62 have very similar overall conformation (Fig. 3). In addition, side chains of most of the peptide residues are exposed to solvent, suggesting that amino acid alterations in the peptide could be accommodated without significant perturbations of the overall structures. The predicted orientation of the side chains is in agreement with all of the structures of peptide/H-2Db complexes solved to date. These models therefore suggest that distinct identity of peptide N-terminal residues neither induce gross differences in the overall peptide conformation, nor cause alterations of the MHC class I structure that would prevent interactions with the H-Y TCR.



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  		FIGURE 3. Molecular modeling of H-2Db associated with Smcy738–746, Ube1x509–517, or ARX54–62 peptides. Side view of the {alpha}1 and {alpha}2 domains of H-2Db complexed with Ube1x509–517, ARX54–62, or Smcy738–746 peptides. For better visibility of peptides, the {alpha}2 helix projecting in front of the peptide was removed. Blue represents basic, red represents acidic, and gray represents neutral portions of the peptide. Green is the {alpha}1 helix; orange are {beta}-pleated sheets. Numbers designate amino acids of the peptide.

 
We have also tested possible structural alterations in peptide/H-2Db functionally. If R at p4 is a crucial TCR contact for induction of T cell proliferation (and negative selection), and p1 in ARX54–62 causes no structural alterations incompatible with activation of the H-Y TCR, then Ube1x509–517 and ARX54–62 with L to R substitution at p4 should induce the same level of T cell proliferation and negative selection. This was indeed the case (Table I). If anything, proliferation induced by ARX54–62L4R was minimally, but repeatedly superior to that induced by Ube1x509–517L4R (see Fig. 4b). These two peptides were less potent than Smcy738–746 due to presence of a less reactive F (compared with Y) at p8, because the activity of ARX54–62 with double mutation (L4R and F8Y) was comparable to that of Smcy738–746 (Table I). Collectively, these data argue against direct or indirect interference of peptide p1 on interactions with the H-Y TCR.



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  		FIGURE 4. The role of peptide p1 in biological responses of T cells. a, The ability of side chain at p1 to form noncovalent bonds is essential for positive selection by Ube1x509–517. Variants of Ube1x509–517 in which K at p1 was substituted by R or M, or control peptide (ARX54–62), were added at concentration of 300 µM to H-Y-specific, TAP-1-deficient FTOC. Shown are CD4 and CD8 dot plots and histograms with expression levels of CD24 in the gated CD4–CD8+ subpopulation. b and c, Peptide p1 contribution to proliferation requires suboptimal contacts at p4. Spleen cells from H-Y TCR transgenic mice (H-2b) were stimulated with peptides, as indicated, and cell proliferation was determined by tritiated thymidine incorporation. Shown are means and SDs of triplicate cultures.

 
Qualities of side chains at p1 that promote positive selection

To understand the role of side chains at the peptide N terminus during positive selection, we focused on p1 because both the Ag and the ligand involved in positive selection have an identical residue at p1 with a side chain predicted to protrude out of the peptide-binding cleft further away than side chains of residues at p2 and p3. We replaced K1 in Ube1x509–517 with 7 aa in a decreasing order of similarity to the original residue (R, H, N, M, A, G, and Y), as defined by the PAM250 matrix (45). Only one of these replacements (R) did not alter the ability of Ube1x509–517 to induce positive selection. Twenty-six percent of CD4–CD8+ thymocytes were found in the Ube1x509–517K1R-treated, vs 13% found in control peptide-treated FTOCs (Fig. 4a), while the original peptide generated an average net gain of ~20% CD4–CD8+ thymocytes (see Figs. 1e and 2c). Of the remaining replacements, the most illustrative is that of M, whose side chain has similar volumes and length as K. Even though Ube1x509–517K1M bound to H-2Db as well as the original peptide and exhibited antagonist activity in T cell proliferation assays (Table I), it was unable to induce positive selection (net increase of only 3% of CD4–CD8+ over control peptide-treated lobes; Fig. 4a). These results suggest that the ability of side chain at p1 to form salt bridges is crucial for induction of positive selection. The fact that other amino acids were also unable to promote positive selection when placed at p1 suggests that length of the side chain at p1 is also important factor for induction of positive selection of H-Y-specific T cells by Ube1x509–517.

Compensatory contacts of p1 on T cell proliferation and negative selection are context dependent

Taken together, the results presented so far suggest differential impact of central or N-terminal peptide residues on cellular responses of T cells. The requirement for distinct TCR contacts appears independent of the maturational stage of T cells, but specific for biological responses. To address the latter possibility directly, we first evaluated potential effects of p1 on mature T cell proliferation and negative selection of thymocytes. Comparison of biological activities of Smcy738–746 and the variant with K1A replacement at a wide range of concentrations showed only minor quantitative effects of p1 in the context of optimal TCR contacts at p4, p6, p7, and p8 (Fig. 1a). Biological activities of Ube1x509–517L4R and ARX54–62L4R (Fig. 4b) further suggest that the presence of contacts at p1 does not compensate for suboptimal contacts near the peptide C terminus (F at p8). To assess the role of p1 and p4 in a highly controlled manner, we designed a series of multiple position variants of Smcy738–746 with A fixed at p2 and p3 and interchanged contacts at p1 and p4. SmcyKAAR induced mildly stronger proliferation of CD8+ T cells than SmcyAAAR (Fig. 4c). The residue at p1 could also contribute when suboptimal contact was provided at p4: SmcyKAAK and SmcyAAAK peptides showed a weak agonist and an antagonist activity, respectively (Table I). However, p1 had no influence in the absence of contacts at p4 (Table I; compare SmcyRAAA with SmcyAAAA). Similar findings were obtained by studying negative selection of CD4+CD8+ thymocytes (Table I). These findings suggest that the contribution of p1 to T cell proliferation or negative selection is dependent on the presence of contacts at p4.

Suboptimal contacts at peptide p4 can be used to induce positive selection

We next tested whether contacts at p4 can replace the requirement for contacts at p1 and/or p2 for positive selection. SmcyAAAK, but not SmcyAAAA, induced efficient positive selection (15.6 vs 6% CD4–CD8+ thymocytes, respectively; Fig. 5a), suggesting that K at p4 was essential for positive selection. However, optimal TCR contacts at p4, p6, p7, and p8, as in peptides SmcyAAAR and SmcyKAAR, induced strong negative selection (Table I). R at p4 was partially tolerated when suboptimal TCR contacts were provided at the C terminus of the peptide as in ARX54–62L4R (F at p8), which was able to induce positive selection at low peptide concentration (Fig. 5, b and c). Ube1x509–517L4R was even more efficient at the induction of positive selection due to the presence of K at p1. Therefore, p4 can replace N-terminal contacts to induce positive selection only if the TCR contacts at the C terminus of the peptide or at p4 itself are weakened.

Biological activity of peptide/MHC complexes does not correlate with the affinity/avidity of interaction with the TCR

Preferential use of p1/p4 residues could be ascribed to the effect they might exert on the affinity/avidity of peptide/MHC-TCR interaction. According to the affinity/avidity paradigm, biological activities of peptides would predict following rank: Smcy738–746 ≥ Smcy738–746K1A > ARX54–62L4R ≥ Ube1x509–517L4R > Ube1x509–517 ≥ ARX54–62. To test this prediction experimentally, we generated soluble MHC tetramers and assessed their binding to living cells (10, 36, 38, 46). Smcy738–746 had the strongest binding of all peptides (Fig. 6a). However, we were unable to reach the saturation point of tetramer binding with this peptide, consistent with previous reports (47). Smcy738–746 also displayed longest t1/2 and the most potent competition as a monomer (Fig. 6, b–d). The next strongest peptide in all aspects was Ube1x509–517L4R, followed by Smcy738–746K1A and ARX54–62L4R. The weakest binding, t1/2, and inhibition values were obtained with Ube1x509–517 and ARX54–62L4R.



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  		FIGURE 6. Binding of H-2Db peptide/MHC ligands to H-Y T cells. Tetramers were prepared with H-2Db-peptide complexes and assayed for binding to restimulated HY transgenic T cells. a, Binding of pMHC tetramers. Binding is plotted as GMF against peptide/MHC concentration. Binding of Smcy-R4A was intermediate between Smcy-K1A and Ube1x (data not shown). The rank order of binding (Smcy738–746 > Ube1x509–517L4R > Smcy738–746K1A ≥ ARX54–62L4R > Smcy738–746R4A > Ube1x509–517 ≥ARX54–62) was reproducible from experiment to experiment, although the quantity of tetramer bound was variable. b, Dissociation kinetics for peptide/MHC tetramers bound to H-Y T cells in the presence of blocking anti-Db mAb. Binding is presented as the natural logarithm of percentage of GMF, in which maximal binding at time = 0 is 100% (36 38 ). The t1/2 obtained from this experiment are shown as insert. c, Inhibition of tetramer binding by monomeric peptide/MHC. Monomers of various H-2Db/peptide complexes were titrated into a binding experiment with 20 nM Ube1x509–517LR4 fluorescent tetramer. The amount of monomer required to inhibit tetramer binding by 20% was determined and used to calculate the relative inhibitory efficacy for the different peptide/MHCs. d, Summary of normalized data from several experiments on dissociation kinetics and monomer inhibition. All data are normalized to the values obtained with Smcy738–746. Normalization was performed before determination of mean and SD to compensate for day-to-day variability between experiments. Actual values for Smcy738–746 were t1/2 = 6.07 ± 2.10 min and monomer inhibition of 20% tetramer binding = 7.58 x 10–6 ± 3.34 x 10–6 M.

 
Comparison of these measurements with functional activities of peptides shows that affinity/avidity can only predict the biological activities of peptides with the most extreme values of the affinity/avidity scale. Thus, Smcy738–746 with the strongest binding promotes agonist activity and negative selection. Conversely, peptides with low affinity/avidity binding promote antagonist activity (ARX54–62) and positive selection (Ube1x509–517). However, biological activity of peptides in the intermediate range of affinity/avidity was independent of their affinity/avidity rank. Thus, Smcy738–746K1A readily induced negative selection at wide range of concentrations, while Ube1x509–517L4R or ARX54–62L4R at low concentrations promoted positive selection. At the same low concentration, these three peptides clearly had strikingly different potential to delete CD4+CD8+ thymocytes in FTOC (Fig. 7, a and b). Likewise, Smcy738–746K1A more efficiently induced mature T cell proliferation than did Ube1x509–517L4R or ARX54–62L4R (Figs. 1a and 4b). Smcy738–746K1A engaged the H-Y TCR with an affinity/avidity lower than Ube1x509–517L4R or ARX54–62L4R. The affinity/avidity theory would require the latter two peptides to induce stronger negative selection than the Smcy738–746K1A. Therefore, the preferential use of p1 for positive selection and of p4 for mature T cell responses and negative selection is not determined by the impact of p1 and p4 on the affinity/avidity of TCR engagement.



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  		FIGURE 7. Potency to induce negative selection does not correlate with the affinity/avidity of peptide/MHC-TCR interaction. H-Y TCR transgenic, TAP-1-deficient FTOCs were treated for 8–10 days with 10 µM Smcy738–746K1A, Ube1x509–517L4R, or ARX54–62L4R. Shown are representative CD4 and CD8 dot plots (a) and a bar graph with percentages of CD4+CD8+ and CD4–CD8– cells summarizing data from three experiments (b). Schematic representation of correlation between affinity/avidity of peptide/MHC-TCR interaction and biological activities for peptides tested in this study (c).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Taken together, careful mapping of TCR contact residues in the H-Y TCR model shows that biological activity is distributed over p1, p2, p4, p6, p7, and p8. A core group of residues (p6–8) is required for all biological activities of MHC-bound peptide and is sufficient for TCR antagonist activity. One of the core TCR contacts (p6 in this case) represents the major contact because peptides with nonconserved mutations at this position are unable to elicit any biological response (Table I; Figs. 1d and 2). Besides the core, additional contacts are required to induce T cell responses other than antagonist activity. The central portion of the peptide (p4) is critical for T cell proliferation and negative selection, while the effects on positive selection are context dependent. Reciprocally, the N-terminal region (p1 and/or p2) is required for positive selection, but the induction of T cell proliferation and negative selection is context dependent. The functional importance of additional TCR contacts is asymmetric and cannot be explained by compensatory effects of secondary TCR contacts in the absence of primary ones (48). Hence, we call these contacts response preference TCR contacts.

A question of theoretical and practical importance is the general applicability of the present findings. All self-restricted peptide/MHC-TCR interactions resolved structurally to date follow the common docking mode, differing only by up to 35 degrees in orientation (26, 27, 28). This means that interactions between specific portions of the TCR with corresponding portions of the peptide/MHC complex are more or less comparable between different TCRs. In that respect, TCR{alpha} chain interacts with N terminus, whereas TCR{beta} chain interacts with C-terminal peptide portion. So, are core and response preference TCR contacts likely to operate in TCRs other than the one used in this study? Data on the vital importance of central and/or C-terminal peptide residues for agonist activity are readily available (11, 13, 48, 49, 50, 51). However, the use of the same TCR contacts for other T cell responses is known only in few of these examples. Homology of C-terminal TCR contacts to Ag was shown to be the key quality of self peptides that promote positive selection of the OT-I TCR transgenic thymocytes. Nonconservative replacements at p6 and/or p7 in this model abrogated the activation of both mature T cells by the cognate Ag (50) and of immature thymocytes by two physiological self peptide/MHC complexes (24, 41). Thus, the major contact for the OT-I TCR is located close to the C terminus, as is the case for the H-Y TCR. However, loss of all biological activity in another model occurred by mutations at p4 (13). Thus, the major TCR contact is typically located in the central and/or C-terminal portion of the peptide. In the vicinity of the major contact are secondary TCR contacts (48), which could belong to the core set of residues, or could be agonist preference contacts.

Although the importance of contacts between the TCR and central peptide residues is well established, the functional role of N-terminal residues of peptide is less clear. Direct or indirect (through water molecules) contacts between the complementarity-determining region 1{alpha} (CDR1{alpha}) peptide p1 were seen in most of the structures of peptide/MHC-TCR complexes (26). Mutational analysis has shown that interactions of the CDR1{alpha} are important for the overall binding of the TCR to the peptide/MHC complex (52). For most TCRs, contacting p1 was not important to induce agonist activity, although some exceptions have been noted (11, 13). Our data presented in this work support possible use of p1 for agonist activity, but only when contact at p4 was suboptimal. Perhaps this is also the case with natural epitopes in the above experimental models. Mutations at p1 have enabled agonist ligands to induce positive selection (43, 44). However, the role of p1 in these cases was most likely different from the one observed in this study. Altering p1 in epitopes reduced the affinity/avidity of peptide/MHC-TCR interaction (2), whereas alterations in our case led to an increase. Thus, the true test for our observations would include alanine-scan analysis of positively selecting peptides. This has only been performed in the OT-I TCR transgenic model, in which two such ligands are known. For one ligand, p1 did not appear essential for activation of immature thymocytes (24), while in the other, a peptide with a replacement at p1 did not bind MHC well, and was therefore not tested functionally (41). C-terminal contacts in the former self peptide were identical with the Ag (24), and therefore did not create suboptimal conditions required for the use of p1. The same occurred in the H-Y model when C-terminal contacts identical with the Ag allowed selection without the role for p1 (peptide SmcyAAAK). Similar to the role of p2 in selection of the H-Y thymocytes, p2 was also partially involved in OT-I thymocyte activation by the latter self peptide, which has less conserved core TCR contacts (41). Even though the role of p1 in this ligand could not be tested, these data suggest that peptide N terminus can be involved in selection of the OT-I TCR too.

Thus, there may be at least two ways to induce positive selection by a single peptide: with or without using interactions with the N terminus of the peptide. Under physiological conditions, several peptide species collaboratively promote selection of a given TCR, and each peptide is essential for selection (53). Thus, even though some self peptides may not select in an N terminus-dependent manner, selection of any given TCR may likely depend on contacts with N terminus of one or more peptides involved in collective selection. Consistent with this notion are data suggesting the crucial role of CDR1{alpha} in positive selection (21, 22), as well as the positioning of MHC class I-binding anchor motifs that allow selective exposure of the N-terminal peptide to the TCR. Although the C-terminal anchor is usually the very last amino acid whose side chain is buried deep into the peptide-binding cleft, the N-terminal anchors are as a rule never at p1, but instead at p2 or p5 (42). As a result, the p1 side chain is usually exposed to solvent and therefore available for contact by the TCR. Our data and the existing literature, therefore, suggest that contacting the N terminus of the peptide is a requirement for selection of most TCRs, albeit not necessarily with all of the self peptides involved in selection.

The amino acid composition of the CDR regions may explain the unequal effect of individual response preference TCR contacts (p4 > p1) on the affinity/avidity of peptide/MHC-TCR interaction. Although ~50% of CDR1{alpha} residues represent those capable of forming alcohol-based hydrogen bonds such as Y, S, or T (54), CDR3 regions are richer in amino acids that form salt bridges (55). However, biological activity of peptides did not correlate with the affinity/avidity of peptide/MHC-TCR interaction, especially in the intermediate range (Fig. 7c). This was even the case with dissociation rates, which in general tend to correlate with biological activities of peptides better than other parameters of TCR binding to peptide/MHC (7). Thus, Smcy738–746K1A that has a faster dissociation rate from the H-Y TCR than Ube1x509–517L4R or ARX54–62L4R is a stronger promoter of mature T cell proliferation and negative selection. Functional potentials of Ube1x509–517L4R and ARX54–62L4R themselves are also inconsistent with the affinity/avidity paradigm. Despite slightly better binding to the H-Y TCR, the former peptide was mildly more potent in inducing positive selection. Lack of strict correlation between the affinity/avidity of peptide/MHC-TCR interaction and the functional potential of TCR ligands has been described before on several occasions (5, 6, 7, 8, 9, 10), but has not been adequately explained. In our model, the presence or the absence of the response preference TCR contacts in the context of stronger or weaker core TCR contacts explains the functional activity of peptides with intermediate affinity/avidity. The effect of response preference TCR contacts cannot, we believe, override an unfavorable extreme affinity/avidity (either low or high) between the TCR and peptide/MHC. Instead, it can act within the intermediate affinity/avidity range to provide specific functional information to the engaged T cell. This view agrees with a model in which each peptide/MHC complex has intrinsic efficacy (3), i.e., inherent bias to induce specific biological responses independent of the affinity/avidity of the TCR engagement. Intrinsic efficacy was used to explain why strong agonist peptides induce thymocyte negative selection in all dose ranges (56).

T cells during thymic development learn to blunt the impact of the most N-terminal portion of peptides that engage the TCR with low affinity/avidity. This finding can partly explain higher sensitivity of CD4+CD8+ thymocytes than of mature T cells observed in other experimental models (57, 58), and most likely is a consequence of a new threshold set by the selecting ligand (59). One interesting aspect of developmentally regulated TCR plasticity is the underlying molecular mechanism(s). An attractive speculation may be inferred from the ability of Abs to adopt different binding site configurations (60). These different configurations were not dependent on induced-fit mechanisms following binding to the ligand. Instead, different structural isomers were found to pre-exist in equilibrium. Similar pre-existing isomers of TCR have been proposed for TCR as well, of which only one would be competent for ligand binding (28). We suggest that at least two of the many structural TCR isomers are capable of ligand binding, one form more sensitive than the other to the N-terminal end of the peptide. Both forms may be represented in different ratios by mature and immature T cells, resulting in dissimilar sensitivities for TCR preference contact residues.


   Acknowledgments
 
We thank John Hirst for FACS analysis, and John Altman (Emory University, Atlanta, GA) for the H-2Db cDNA with BirA sequence.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI48837 and AI41573 to S.V., GM39476 and GM48002 to N.R.J.G., and National Cancer Institute Core Support Grant 5P30 CA16087. K.H. was a recipient of a postdoctoral fellowship from Cancer Research Institute. Back

2 Address correspondence and reprint requests to Dr. Stanislav Vukmanovic, Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010. E-mail address: svukmano{at}cnmc.org Back

3 Abbreviations used in this paper: DP, double positive; CDR, complementarity-determining region; FTOC, fetal thymus organ culture; GMF, geometric mean fluorescence; p, position. Back

Received for publication December 3, 2003. Accepted for publication April 7, 2004.


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 Results
 Discussion
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F. R. Santori and S. Vukmanovic
Delineation of Signals Required for Thymocyte Positive Selection
J. Immunol., November 1, 2004; 173(9): 5517 - 5523.
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