Proc. Natl. Acad. Sci. USA Vol. 93, pp. 13108–13113, November 1996 Immunology Molecular basis for the recognition of two structurally different major histocompatibility complexypeptide complexes by a single T-cell receptor (allo-major histocompatibility complexyligand bindingymolecular modelingyreceptoryT cell) ROLAND BROCK*†,KARL-HEINZ WIESMU¨LLER‡,GU¨NTHER JUNG*, AND PETER WALDEN§¶ §Max-Planck-Institut fu¨r Biologie, Abteilung Immungenetik, D-72076 Tu¨bingen, Germany; *Institut fu¨r Organische Chemie, Universita¨t Tu¨bingen, D-72076 Tu¨bingen, Germany; and ‡Naturwissenschaftliches und Medizinisches Institut an der Universita¨t Tu¨bingen, D-72762 Reutlingen, Germany Communicated by Herman Eisen, Massachusetts Institute of Technology, Cambridge, MA, August 2, 1996 (received for review March 26, 1996) ABSTRACT 2C is a typical alloreactive cytotoxic T lym- different at the sequence level and so are the two MHC phocyte clone that recognizes two different ligands. These molecules (10, 11). To determine the molecular basis for the ligands are adducts of the allo-major histocompatibility com- observed cross-recognition of the two ligands by one single plex (MHC) molecule H-2Ld and an endogenous octapeptide, receptor, MHC binding and T-cell response experiments with and of the self-MHC molecule H-2Kb and another peptide. synthetic analogues of the two cognate peptides were com- MHC-binding and T-cell assays with synthetic peptides in bined with molecular modeling studies. Because the peptide combination with molecular modeling studies were employed constitutes an integral part of the protein structure (12, 13) this to analyze the structural basis for this crossreactivity. The approach is equivalent to mutagenesis studies of the MHC in molecular surfaces of the two complexes differ greatly in the vicinity of the peptide-binding groove but allows a much densities and distributions of positive and negative charges. larger set of analogues to be tested. Models for the two MHC However, modifications of the peptides that increase similar- peptide complexes were generated, whose peptide conforma- ity decrease the capacities of the resulting MHC peptide tions were in agreement with the binding experiments. In complexes to induce T-cell responses. Moreover, the roles of addition, the effects of conformational changes of the peptide the peptides in ligand recognition are different for self- and main chains and side chains on the characteristics of the allo-MHC-restricted T-cell responses. The self-MHC- molecular surfaces were investigated and additional structures restricted T-cell responses were finely tuned to recognition of were generated for each complex that partially violated the the peptide. The allo-MHC-restricted responses, on the other experimental constraints to explore the conformational space hand, largely ignore modifications of the peptide. The results available to exposed residues. The combination of the exper- strongly suggest that adaptation of the T-cell receptor to the imental and the modeling results allowed for the determina- different ligand structures, rather than molecular mimicry by tion of the particular requirements for self- versus allo- the ligands, is the basis for the crossreactivity of 2C. This restricted antigen recognition. conclusion has important implications for T-cell immunology and for the understanding of immunological disorders. MATERIALS AND METHODS The existence of allo-major histocompatibility complex Cell Culture. The transporter-deficient thymoma cell line (MHC)-specific T cells in immunocompetent animals that RMA-S and its parental RMA cell line (14) were used for have never encountered the allo antigen before is one of the stabilization and cytotoxicity assays. RLd-E2.1 is a subline of old unresolved mysteries of immunology (1, 2). If the forma- RLd-E2, which is a derivative of RMA-S transfected with the tion of the T-cell receptor (TCR) repertoire, i.e., the sum of all H-2Ld-gene (8). The cell line B2 was obtained from the mouse T-cell specificities of an individual, is controlled by that myeloma cell line Sp2y0 by transfection with the H-2Ld gene individual’s own MHC type (3–6), how then can the strong (8). The cells were maintained at 378C in a humidified allo-MHC-specific reactivity found regularly in all individuals atmosphere with 8% CO2 in DMEM supplemented with heat be rationalized? The most popular explanation for this phe- inactivated fetal calf serum (5%), 100 unitsyml penicillin, 100 nomenon is that the complex of the allo-MHC molecule with mgyml streptomycin, 2 mM glutamine, 5 mM Hepes, and 30 one peptide resembles a complex of a self-MHC molecule plus mM mercaptoethanol. In addition, the medium for RLd-E2 another peptide (2). This model implies that the two complexes and B2 contained Geneticin (200 mgyml). The cytotoxic T share structural features that make them look alike. Hence, lymphocyte clone 2C (7) was a generous gift from H. N. Eisen alloreactivity would be a case of molecular mimicry by the (Massachusetts Institute of Technology). 2C cells were main- different MHC peptide complexes. tained by biweekly stimulations with irradiated P815 cells 2C (7) is a murine cytotoxic T lymphocyte clone, which was (H-2d) in supplemented DMEM medium, containing 2–4% rat isolated from a BALB.B mouse (MHC haplotype: H-2b) that ConA supernatant. b had been injected with cells from a DBAy2 mouse (MHC Stabilization Assay. Stabilization assays (15, 16) for H-2K haplotype: H-2d). It recognizes the MHC class I molecule were carried out with RMA-S cells, those for H-2Ld with the H-2Ld together with the naturally occurring endogenous pep- cell line RLd-E2.1. The protocol was as described elsewhere tide LSPFPFDL (8). H-2Kb was suspected to be the self-MHC (16). The monoclonal antibodies B8.24.3 (17) and 30-5-7S (18), counterpart because it controls positive selection of the 2C which recognize the native conformation of the MHC mole- TCR specificity in the thymus (4). In fact, we were able to determine an epitope, SIYRYYGL, that is recognized to- Abbreviations: MHC, major histocompatibility complex; TCR, T-cell gether with H-2Kb by the 2C TCR (9). Both peptides are very receptor; rms, root-mean-square. †Present address: Max-Planck-Institut fu¨r Biophysikalische Chemie, Abteilung fu¨r Molekulare Biologie, D-37075 Go¨ttingen, Germany. The publication costs of this article were defrayed in part by page charge ¶To whom reprint requests should be addressed at: Dermatologische payment. This article must therefore be hereby marked ‘‘advertisement’’ in Klinik, Charite´,Humboldt-Universita¨tzu Berlin, Schumannstrasse accordance with 18 U.S.C. §1734 solely to indicate this fact. 21y22, D-10117 Berlin, Germany. 13108 Downloaded by guest on September 27, 2021 Immunology: Brock et al. Proc. Natl. Acad. Sci. USA 93 (1996) 13109 cules, were used for H-2Kb and H-2Ld, respectively. The levels 1.13 Å for backbone atoms only, and 1.35 Å for heavy atoms. of cell surface expression of the MHC molecules were mea- This structure served as the starting geometry for the gener- sured with a FACScan (Becton Dickinson). After transforma- ation of the H-2KbySIYRYYGL and the H-2LdyLSPFPFDL tion of the raw data to linear mean fluorescence intensities complexes. All minimizations started with a minimization of (MFI) using the MFI software (E. Martz, University of the peptide alone, followed by a minimization of the residues Massachusetts, Amherst), the peptide concentrations required of the peptide binding pocket [all residues with an atom within for half-maximal stabilization were calculated by employing 4 Å from the peptide (residues 5, 7, 9, 22, 24, 33, 45, 59, 63, 66, formalisms of the occupancy concepts (16) and linear regres- 70, 73, 74, 77, 80, 81, 84, 95, 97, 99, 114, 116, 123, 124, 143, 146, sions over plots with logit(p) 5 ln[p/(1 2 p)] versus log[peptide 150, 152, 155, 156, 159, 163, 167, and 171)]. Only then was the concentration] with p 5 (MFIexp 2 MFImin)y(MFImax 2 whole MHC free to move. During simulated annealing (24) MFImin). Results are expressed as the negative logarithm of only these residues, water molecules within the binding pocket, the concentration required for half-maximal stabilization [M]. and the peptide were allowed to move. All peptides were tested in duplicates and in two to three Generation of Three Alternative Structures for H-2Kb in independent experiments. Complex with SIYRYYGL. The peptide of the relaxed model Cytotoxicity Assay. The 51Cr release assay was employed to for the crystal structure of RGYVYQGLyH-2Kb was mutated measure cytolysis of targets cells by the T-cell clone 2C. The to SIYRYYGL. Except for isoleucine-2 and tyrosine-6, the target cell line RMA was used for the H-2Kb-dependent side-chain geometries were retained. Two of the seven water reaction and B2 for the H-2Ld-dependent lysis. Cytotoxicity molecules that were buried in the peptide binding cleft of the assays were conducted 4–6 days after stimulation. Target cells crystal structure were removed to avoid steric interference were labeled with 51Cr, washed, and incubated in DMEM with isoleucine-2. This assumption was later confirmed by the containing BSA (0.5%) with serial dilutions of peptides for 30 crystal structure of SIINFEKLyH-2Kb (25). All other water min at 378C in U-bottom microtiter plates. After addition of 2C molecules that were within7Åfromthepeptide were removed cells to an effector-to-target ratio of 5:1 and further incubation as well. One structure (see Fig. 3, structure a) was generated at 378Cfor5hrthe51Cr released from the cells was measured by minimization, followed by a molecular dynamic simulation with a g counter. Percent specific lysis was calculated as in water. Two more structures were obtained by using simu- (experimental release 2 spontaneous release)y(total re- lated annealing protocols with heating to 1000 K (26).