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Fine Specificity and MHC Restriction of Trinitrophenyl- Specific CTL¹

Alessandra Franco^*, Takashi Yokoyama, Dung Huynh^*, Cole Thomson, Stanley G. Nathenson and Howard M. Grey^2,*

^* La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; Pharmaceutical Research Laboratory, Kirin Brewery, Takasaki, Gunma, Japan; and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461

	Abstract

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In this study, the fine specificity and MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was analyzed. Based on the structure of peptide/K^b complexes and ternary TCR/Ag/MHC complexes, four TNP peptides, two octamers, and two nonamers were chosen for eliciting anti-TNP CTL responses. Hapten was conjugated at position 4 in the octamers and at position 5 in the nonamers, positions which should allow engagement of the hapten by TCRs. Potent CTL activity for each of the TNP peptides was obtained that was highly hapten-specific; however, there were considerable differences in the extent of cross-reactivity with other TNP peptides, with the octamers generating more cross-reactive CTL than the nonamers. MHC restriction analysis suggested that anti-hapten responses were less dependent on MHC recognition than anti-peptide responses. This was evidenced by the relative ease of detecting cross-reactivity to haptenated peptides presented by allo-MHC and by the relative insensitivity of anti-hapten vs anti-peptide CTL to mutations in the K^b molecule at potential TCR interaction sites. One potential explanation for this insensitivity to MHC mutation was the finding that the anti-hapten response appeared to be of higher avidity, since a >100-fold difference in the amount of Ag required to sensitize target cells was found between these two types of Ags.

	Introduction

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It is widely accepted that hapten-specific T lymphocytes can play an important role in immunopathology. In several allergic diseases, not only hapten-specific B cells but also T cells are pivotal. Nitrobenzene derivatives such as trinitrochlorobenzene are well known for their capacity to induce a contact hypersensitivity that is mediated by hapten-specific CD4⁺ and CD8⁺ T cells 1, 2, 3 . CD4⁺ T cells specific for the ß-lactamic ring of penicillin have been generated from peripheral blood of allergic patients 4 , and CD4⁺ and CD8⁺ T cells specific for the hapten urushiol play an important role in the hypersensitivity caused by contact with poison ivy or oak 5 . Autoreactive hapten-specific T cells may also play a role in certain autoimmune diseases. For instance, in a murine collagen arthritis model, the immunodominant epitope is a glycopeptide in which an oligosaccharide O linked to hydroxylysine is critical for T cell recognition 6 .

Because of the unique structure of haptenated peptides that results in longer, bulkier side chains extending from the peptide backbone than are normally found, and because many haptens represent unique chemical structures that are distinctively different from the natural endogenous peptides involved in thymic selection, we have, in this study, investigated several parameters of the T cell response to the extensively studied TNP³ hapten, including the role of the peptide backbone in defining the specificity, MHC restriction of the response, and relative avidity of the CTL for Ag/MHC complexes.

	Materials and Methods

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Mice

Female C57BL/6 mice (8–12 wk old) purchased from The Jackson Laboratory (Bar Harbor, ME) were used in all the experiments.

Peptide synthesis

The peptides used in this study were synthesized by Fmoc chemistry using a multiple peptide synthesizer (Symphony/Multiplex; Protein Technologies, Tucson, AZ). Peptides were cleaved automatically on the synthesizer using trifluoracetic acid as a cleavage reagent. Peptides were >90% pure as assessed by C18 reverse phase HPLC, and the identity of the peptides was verified by mass spectroscopy. TNP modifications were introduced by using -N-TNP lysine derivatives for peptide synthesis (Bachem Bioscience, King of Prussia, PA).

MHC purification and MHC peptide binding assay

EL-4 cells were used as a source of K^b and D^b molecules, and RDM4 as a source of K^k molecules. Nonidet P-40 cell lysates from large-scale (10¹⁰ to 10¹¹) cell cultures were filtered through 0.45-µm filters and purified by affinity chromatography as described 7 . To measure peptide binding to MHC molecules, previously identified MHC binding peptides were ¹²⁵I-radiolabeled (sequences vesicular stomatitis virus (VSV) NP 52–59 for K^b, Y240 substituted adenovirus, E1a 234–243 for D^b, and Y240 substituted Flu-HA 240–248 for K^k) and incubated with 5–10 nM of purified MHC molecules for 48 h in PBS containing 5% DMSO, 0.05% Nonidet P-40, and protease inhibitors. The K^b, D^b, and K^k complexes were subsequently separated from free peptide by gel filtration TSK columns 7 . The binding capacity of peptides to these MHC molecules was measured by their capacity to inhibit binding of the radiolabeled ligand. The affinity of the binding was estimated by determining the quantity of peptide required to inhibit by 50% (IC₅₀) the binding of the radiolabeled peptide.

Generation and characterization of T cell lines and clones

To generate hapten-specific CTL, C57BL/6 mice were immunized s.c. at the base of the tail with 50 µg of TNP-conjugated peptide in IFA, together with 140 µg of a known Th epitope (hepatitis B virus surface Ag 128–140) 8 . Two weeks after priming, mice were sacrificed, and splenocytes were stimulated in vitro with the same haptenated peptide in the presence of irradiated syngeneic LPS/Dextran SO₄ (LPS from Salmonella Typhosa: Sigma, St. Louis, MO; and Dextran Sulfate: Pharmacia Biotech AB, Uppsala, Sweden)-activated B cell blasts as an APC source. Culture medium consisted of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 20 mM glutamine, 100 µg streptomycin, 100 U/ml penicillin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (Life Technologies), 50 µM 2-ME, and 10% heat-inactivated FCS (Life Technologies). Six to seven days later, T cells were tested for CTL activity, and, in some cases, they were cloned by limiting dilution 9 . Hapten specificity of T cell lines and clones was studied in a standard ⁵¹Cr release assay by comparing the ability of effector T cells to lyse targets pulsed with haptenated peptides with their ability to lyse targets pulsed with nonhaptenated peptides.

The peptides utilized in this study were: 1) two K^b binding poly(A) octamer and nonamer sequences: AIIKFAAL and AIIAKFAAL; and 2) three lysine (K)-modified sequences derived from known immunodominant CTL epitopes: VSV nucleoprotein 52–59 (RGYKYQGL), Sendai virus (SEV) nucleoprotein 324–332 (FAPGKYPAL), and OVA 257–264 (SIIKFEKL). The K in position 4 or 5 of these peptides was the residue to which TNP was conjugated. For MHC-restriction studies, a panel of transfected cells that contain mutant K^b molecules was used as targets for killing by TNP-specific CTL. The K^b mutants, along with a control wild-type K^b, were expressed in the Abelson virus-transformed pre-B cell line R8 as previously described 10, 11 . Cytotoxicity was quantified by expressing the data as LU. For determining cross-reactivity with other haptenated peptides and reactivity on mutant MHC-transfected cells, the fraction of the response relative to the immunogen or wild-type MHC was determined.

	Results

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TNP peptides

Several factors were taken into consideration in the process of choosing the structure of the TNP peptides to be tested for immunogenicity. The first factor considered was peptide length. K^b binding peptides are usually octapeptides with major MHC anchors at positions 5 and 8. Nonapeptides can also bind, in which case the first anchor is shifted to position 6, and as suggested by the structural analysis of peptide-K^b complexes, positions 4 and 5 may form a bulge that extends up from the peptide binding groove toward the solvent 12 . Because of this feature, it was considered of interest to compare the specificity of the response to hapten when conjugated to octapeptides vs nonapeptides. Another factor that was taken into consideration was the determination of the importance of other amino acid side chains in the anti-hapten response. Designer poly(A)-containing peptides with the appropriate MHC anchor residues were compared with known immunodominant K^b-restricted viral epitopes. Finally, the crystal structure of an MHC/peptide/TCR complex indicated that a deep pocket might be formed between the CDR3 regions of the - and ß-chain of the TCR that can engage peptide TCR contact residues at position 4 (of an octamer) or 5 (of a nonamer). Because of these considerations, the TNP peptides analyzed in this study consisted of octamers and nonamers in which the TNP was conjugated to a lysine at position 4 or 5, respectively. The peptide backbones were either derived from SEV nucleoprotein 324–332 (9 mer), VSV nucleoprotein 52–59 (8 mer), or a poly(A) 8 mer or 9 mer that contained the major anchors F 5 (or 6) and L 8 (or 9), together with I at positions 2 and 3, which were found to be important for high-affinity binding to K^b. The amino acid sequences of these peptides and their MHC binding affinities are shown in Table I.

To generate hapten-specific CTL, each of the four haptenated peptides listed in Table I was used to immunize H-2^b mice (C57BL/6). Two weeks after priming, splenocytes were cultured with Ag for 6 days, and CTL activity was measured in a standard ⁵¹Cr release assay. Hapten specificity was investigated by comparing the response to the haptenated peptide used as immunogen with the response to the same peptide nonhaptenated. Fig. 1 shows representative data from a CTL line derived from mice primed with TNP-poly(A)9. Good CTL activity to the haptenated immunogen was observed, and no activity above the control was observed against the nonhaptenated peptide. These initial data clearly demonstrated that it was possible to generate a hapten-specific T cell response using as immunogen a high-affinity MHC binding peptide that was haptenated at a central position and that the predominant CTL population elicited was hapten-specific.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. TNP-specificity of CTL culture derived from TNP peptide-immunized mice. EL-4 cells (H-2b) were 51Cr-labeled and incubated with different numbers of T cells in the presence or absence of 1 µg/ml of TNP-poly(A)9 peptide (), the nonhaptenated peptide (•), or no peptide (). Cell culture supernatants were harvested 4 h later and 51Cr release calculated as [(sample-spontaneous release)/(maximum release-spontaneous release)] x 100.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Cross-reactive recognition by TNP-specific CTL of TNP conjugated to different peptide backbones. T cell lines were derived from mice immunized with the following TNP conjugated peptides: a, poly(A)9; b, poly(A)8; c, VSV; and d, SEV. The T cell lines were tested for their capacity to lyse the immunizing peptide and four other haptenated peptides: poly(A)8 (), poly(A)9 (), VSV (), SEV (•), and OVA ().

Cross-reactivity between the immunogen and the other haptenated peptides was calculated as a ratio by dividing the LU obtained with the immunogen into the LU obtained with the other peptides tested. For instance, lysis of VSV targets by anti-poly(A)8 CTL resulted in 15.9 LU of activity, giving a LU ratio relative to the anti-VSV activity of 0.14 (15.9 ÷ 116). The data represented in this manner in Table II suggest the following conclusions. Whereas the two octapeptides, VSV and poly(A)8, generated relatively highly cross-reactive CTL responses, with average cross-reactivity indices of 0.37 and 0.33, the indices of the two nonapeptides, SEV and poly(A)9, were only 0.13 and 0.08. Furthermore, when the data were analyzed with respect to which of the peptides tested for their capacity to cross-react, it was evident that the two poly(A) peptides were the most cross-reactive, with average cross-reactive indices of 0.35 for the poly(A)8 and 0.54 for the poly(A)9. In contrast, VSV had an average index of 0.06, SEV of 0.12, and OVA of 0.11.

The fine specificity of the hapten-specific response was further analyzed at the clonal level with a panel of T cell clones derived from three T cell lines specific for TNP-VSV, TNP-SEV, and TNP-poly(A)9. When analyzed collectively, the data obtained were consistent with the data described above for the T cell lines. As shown in Table III, T cell clones generated against TNP-VSV were more highly cross-reactive than the clones generated against TNP-SEV or TNP poly(A)9. Also, TNP-poly(A)8 and TNP-poly(A)9 were more often recognized as cross-reactive Ags than TNP-SEV, TNP-VSV, or TNP-OVA. However, when individual clones specific for a given TNP peptide were analyzed, a great deal of variation in their cross-reactivity patterns was observed. Examples of this heterogeneity are shown in Fig. 3, which illustrates the cross-reactivity pattern of three TNP-VSV-specific clones. Of the 12 anti-TNP-VSV clones analyzed, 2 gave a pattern, as shown in Fig. 3a, that indicated good CTL activity with the immunogen and no cross-reactivity with any of the other TNP peptides. At the other extreme, 2 of 12 clones showed extensive cross-reactivity with all the other TNP peptides tested, as exemplified by the data in Fig. 3c. Most of the clones exhibited intermediate patterns of cross-reactivity, reacting with 1 (2 of 12 clones), 2 (4 of 12 clones) or 3 (2 of 12 clones) of the 4 heterologous TNP peptides tested. One such intermediate pattern of cross-reactivity is shown in Fig. 3b. It is to be noted that in no instance was there any significant cross-reactivity with the corresponding nonhaptenated peptides.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Different profiles of cross-reactive recognition by TNP-specific T cell clones of four different haptenated peptides. A profile of three representative T cell clones generated using the TNP-VSV peptide as immunogen are shown. All T cell clones studied were hapten-specific, as demonstrated by the lack of killing of peptide backbone-pulsed targets (). a, A T cell clone that recognized only the haptenated immunogen (). b, A T cell clone that cross-reacted with the TNP-OVA peptide sequence (), but not with the other three TNP peptides. c, A T cell clone that cross-reacted extensively with all haptenated peptides tested. Symbols for the different TNP peptides are the same as in Fig. 2.

Haptenated peptides present a unique topography compared with conventional peptides bound in the Ag binding groove (the hapten is predicted to protrude further away from the helices that make up the walls of the Ag binding groove compared with the side chains of the natural amino acids). Thus, it was of interest to determine the extent of MHC restriction demonstrated by the cytotoxic T cells specific for the TNP hapten. To study this, two kinds of experiments were performed: 1) The capacity of CTL to recognize K^b mutant molecules presenting TNP peptides; and 2) The capacity of hapten to be recognized by CTL when presented by MHC alleles other than the original MHC.

Ag presentation by mutant K^b molecules

A series of transfected cells containing mutant K^b molecules (and wild-type K^b) were previously generated 10, 11 . A subset of these mutants, which were predicted to have a mutation at a position in the 1 or the 2 helix that pointed up toward the TCR rather than into the peptide binding groove, were selected to assess the relative importance of MHC recognition by anti-TNP-specific CTL. Table IV lists the mutants studied and the position of the mutation in the K^b -chain. These cells were used as targets for killing by TNP-specific CTL in the experiments described below.

Hapten recognition in the context of other MHC specificities

To further evaluate the role of MHC in the recognition of TNP, we investigated the possibility that CTL could be generated that recognized haptenated peptides presented by MHC molecules other than the MHC molecules used to present the immunizing peptide. The first set of experiments determined whether a TNP peptide capable of binding D^b (but not K^b) could be recognized by CTL generated against a TNP-conjugated K^b binding peptide. D^b was chosen for these experiments because it, like K^b, binds peptides with primary anchor residues at position 5 and the C terminus (although K^b preferentially binds octamers, whereas D^b binds nonamers) 13 . To generate a TNP-D^b binding peptide, the poly(A) sequence AAAK*NAAAM was selected. Binding experiments indicated high-affinity binding to D^b (IC₅₀ = 1 nM) and no appreciable binding to K^b molecules (IC₅₀ > 50,000 nM). After an initial immunization with the TNP-K^b binding peptide poly(A)9* (IC₅₀ K^b = 4 nM; IC₅₀ D^b > 50,000 nM), a very weak but detectable response was observed when target cells were incubated with the TNP-D^b binding peptide (Fig. 4a). However, following limiting dilution cloning with the TNP-D^b binding peptide, T cell clones could be generated that responded equivalently to the TNP-D^b binding peptide and the TNP-K^b-binding peptide (Fig. 4b).

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Cross-reactive TNP peptide/MHC recognition of haptenated peptides presented by Kb, Db, and Kk MHC molecules. a, Bulk CTL culture. C57BL/6 (H-2b) mice were immunized with the haptenated Kb binding poly(A)9, and, after 10 days, lymphocytes were obtained and cultured for 7 days with the same peptide. CTL analysis was performed with the immunogen () and with a TNP-Db binding haptenated peptide (). b, The ability of a T cell clone derived from the bulk T cell line shown in a to kill EL-4 target cells in the presence of haptenated peptides either presented by Kb molecules () or by Db molecules (). The response to nonhaptenated poly(A)9 () is also shown. c, Bulk CTL culture of C57BL/6 (H-2b) spleen cells from mice immunized with the TNP-Kb binding poly(A)8 and cultured 7 days with the same peptide. The ability of the T cell line to kill H-2k (RDM4) targets presenting a TNP-Kk binding peptide () was compared with its ability to present the TNP-Kb binding poly(A)8 ().

Comparison of TCR avidity between TNP-specific and peptide-specific T cell clones

The data obtained comparing the capacity of anti-TNP and anti-peptide-specific clones to recognize Ag presented by the K^b mutants suggested that the anti-TNP clones were less dependent on the integrity of the potential TCR contact residues in the K^b molecule than the anti-peptide clones. One possible explanation for this observation is that TNP peptide/MHC has a higher binding affinity for their respective TCRs than does peptide/MHC for their TCRs. If this were the case, then anti-TNP CTL would be expected to tolerate MHC mutations that affect the affinity of binding to TCR to a greater extent than anti-peptide CTL. To obtain information concerning this possibility, several anti-TNP-SEV clones and anti-SEV peptide clones were studied to determine the amount of Ag required to sensitize target cells for an equivalent degree of lysis. The data shown in Fig. 5 indicate a striking difference between these two sets of CTL clones, exemplified by the fact that the anti-TNP-specific clones required an average Ag concentration of 0.0263 ± 0.0145 nM to achieve 20% lysis, whereas the anti-SEV peptide-specific clones required 3.2898 ± 3.205 nM, a 125-fold difference (p < 0.027).

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Peptide dose-response analysis of five anti-SEV/TNP CTL clones (solid lines) and four antibackbone SEV clones (dashed lines). The data were generated with an E:T ratio of 10:1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The strategy we used for the positioning of the hapten and the choice of the peptide backbone was influenced by immunochemical studies showing that P4 and P6 of an octamer VSV peptide were immunodominant for TCR recognition 19 . For instance, Fig. 6 shows a model of TNP-VSV peptide bound to H-2K^b based on the previous reports of the H-2K^b/VSV structure 12 . Further, the published crystal structure of peptide/MHC complexes and the more recent reports on the structure of the ternary complex of TCR peptide/MHC 20, 21 suggest the presence of a deep pocket formed by the apposition of the CDR3 regions of - and ß-chains of the TCR that could engage bulky side chains present at the P4 position (of an octamer) or P5 position (of a nonamer). We chose to compare peptides of 8 and 9 residues in length because of the crystallographic analysis that indicates nonapeptides (with the anchor at P6 rather than P5) could bulge out of the binding groove at P5. We also wished to compare peptide backbones that had minimal capacity to interact with TCR (polyalanine backbones) to backbones derived from previously described dominant T cell epitopes containing effective TCR-contact residues. Our a priori prediction was that the TNP polyalanine nonapeptide would be the best Ag for eliciting a hapten-specific CTL response, due to the lack of potent TCR-contact residues other than the hapten itself, as well as having the TNP moiety in a position that could bulge out to optimally interact with a TCR. The experimental data we obtained, however, did not confirm this prediction. All four of the TNP peptides tested elicited similar levels of CTL activity as measured by LU in bulk T cell cultures (data not shown). The degree to which the anti-TNP CTL cross-reacted with other TNP-K^b binding peptides varied in the following order: VSVpoly(A)8 > SEVpoly(A)9. This variation in cross-reactive recognition occurred despite the fact that in all instances there was little or no detectable recognition of the peptides’ backbones per se. Two possible explanations exist for the lack of more extensive cross-reactivity. First, detrimental residues in the peptide backbone of some of the potentially cross-reactive TNP peptides interfered with the effective engagement of the TNP peptide/MHC complexes with some TCRs. This explanation is supported by the finding that the two TNP poly(A) peptides were, in general, more cross-reactive than the two TNP peptides derived from known viral epitopes, since the alanine backbone would be unlikely to contain detrimental residues 22 . Another explanation is that although the peptide backbone is not recognized in the absence of the hapten, amino acid side chains do contact the TCR and contribute to the overall binding energy. Regardless of the mechanism, the failure of some TNP peptides to elicit a broadly cross-reactive CTL response is an important consideration if an anti-hapten response is to be considered as part of a prophylactic or therapeutic vaccine strategy (for instance, in the generation of a glycopeptide-specific CTL response).

View larger version (65K): [in this window] [in a new window]   FIGURE 6. Model of H-2Kb/TNPylated VSV peptide. Model created using coordinates of H-2Kb/VSV (12). The final coordinates underwent 300 cycles of conjugate gradient minimization to optimize the geometry. Computational results obtained using software programs from Molecular Simulations (San Diego, CA) using the CVFF forcefield. A, Ag presentation domain of H-2Kb covered in molecular surface. TCR binding region tilted so that 2 helices are at front. Except for position 4, peptide residues in purple, with N terminus at left. Position 4 has been changed from the original valine to a TNPylated lysine and colored by atom type: C, green; N, blue; O, red. B, Side view of Ag-presentation domain; N terminus of peptide at left, 2 helices at front. C trace of H-2Kb in grey. Peptide atoms colored by type.

	Acknowledgments

 
We thank Joyce Joseph for assistance in preparation of the manuscript.

	Footnotes

 
¹ This work was funded by National Institutes of Health Grant CA71541 (to H.M.G.) and National Institutes of Health Grant CA78657 (to A.F.). This is publication no. 249 from the La Jolla Institute for Allergy and Immunology.

² Address correspondence and reprint requests to Dr. Howard M. Grey, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.

³ Abbreviations used in this paper: TNP, trinitrophenyl; VSV, vesicular stomatitis virus; SEV, Sendai virus; IC₅₀, 50% inhibition.

Received for publication August 24, 1998. Accepted for publication December 7, 1998.

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