Antibodies Directed Against the MHC-I Molecule H-2Dd Complexed with an Antigenic Peptide: Similarities to a T Cell Receptor with the Same Specificity1 Katarina Polakova,2,3* Daniel Plaksin,3,4* Doo Hyun Chung,* Igor M. Belyakov,† Jay A. Berzofsky,† and David H. Margulies5* ␣ TCRs, which use an Ab-like structure to form a combining site, recognize molecular complexes consisting of peptides bound to MHC class I (MHC-I) or class II (MHC-II) molecules. To explore the similarities and differences between Ab and T cell recognition of similar structures, we have isolated two mAbs, KP14 and KP15, that specifically bind H-2Dd complexed with an HIV envelope gp160-derived peptide, P18-I10. These Abs are MHC and peptide specific. Fine specificity of mAb binding was analyzed using a panel of synthetic peptides, revealing similarities between the mAb and a cloned TCR with the same specificity. These two mAbs used the same VH and JH gene segments, but different D, V, and J genes. Administered in vivo, mAb KP15 blocked the induction of CTL specific for recombinant vaccinia virus-encoded gp160, indicating its ability to bind endogenously generated MHC/peptide complexes. Analysis of the fine specificity of these mAbs in the context of their encoded amino acid sequences and the known three-dimensional structure of the H-2Dd/P18-I10 complex suggests that they bind in an orientation similar to that of the TCR. Thus, the plasticity of the B cell receptor repertoire and the structural similarities among BCR and TCR allow Abs to effectively mimic ␣ TCRs. Such mAbs may be useful in the therapeutic modulation of immune responses against infectious agents or harmful self Ags as well as in tracing steps in Ag processing. The Journal of Immunology, 2000, 165: 5703–5712.
T
cell receptors bind molecular complexes of MHC class I (MHC-I)6 or MHC class II (MHC-II) molecules as an initial step in the recognition of aberrant expression of proteins in somatic cells (1– 4). In the case of MHC-I-restricted responses, those Ags identified by TCR are usually peptidic products that result from degradation by proteasomes of molecules synthesized by the APC (5). For MHC-II-restricted Ags, the peptides usually derive from the endocytosis and proteolytic processing of extracellular proteins (6, 7). The ␣ receptor of the T cell recognizes such peptide Ags bound to an MHC-I or MHC-II molecule in a binding event dependent upon exposed amino acid residues of the peptide/MHC complex as well as those of the complementarity-determining regions (CDR) of the TCR. These CDR, originally defined by amino acid sequence similarity with Abs (8, 9), have
*Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases and †Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Received for publication May 2, 2000. Accepted for publication August 28, 2000. 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 in part by a fellowship (to K.P.) from the UIUC.
2
Visiting Scientist from Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic. 3
K.P. and D.P. contributed equally to this work.
4
Current address: Biotechnology General, Rehovot, Israel.
5
Address correspondence and reprint requests to Dr. David Margulies, Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, Building 10, Room 11N311, National Institutes of Health, Bethesda, MD 20892-1892. E-mail address:
[email protected] 6 Abbreviations used in this paper: MHC-I, MHC class I; ; MHC-II, MHC class II; P18-I10, HIV IIIB envelope glycoprotein 160-derived H-2Dd-restricted peptide, RGPGRAFVTI; CDR, complementarity-determining region(s); scTCR, single chain TCR; sH-2Dd, soluble H-2Dd; SPR, surface plasmon resonance; IMGT, ImMunoGeneTics database.
Copyright © 2000 by The American Association of Immunologists
recently been visualized in the x-ray crystallographic structures of TCR and peptide/MHC/TCR complexes (10 –14). In contrast to TCR that are obligate cell surface receptors, and function in a multivalent array, mature Ab IgG molecules usually exert their biological function by binding as soluble bivalent molecules. A major difference in Ag recognition by Abs as compared with TCR is that the former commonly function through high affinity interactions, whereas the strength of the TCR interaction with peptide/ MHC is weaker. These differences between Abs and TCR are in part dictated by the role of the TCR in sensing the presence of peptide/MHC complexes on the APC, and in coordinating signals required for the activation or tolerization of the T cell. In addition, the T cell integrates ␣ TCR-mediated signals at several stages in its life cycle, during T cell selection in the thymus, during tolerance induction in peripheral lymphoid tissues, and during the activation to various effector functions such as lymphokine release or cytolysis (15, 16). T cells that escape the selective environment of the thymus and preserve reactivity to self-peptide/MHC complexes in peripheral tissues may be chronically activated, leading to autoimmunity (17, 18). A complete understanding of the cellular and molecular process known as Ag presentation demands the ability to understand in detail the formation of the peptide/MHC complex and the nature of its binding to TCR. Two potent classes of reagents have been developed for visualizing specific peptide/MHC complexes. Specific TCR, engineered from cloned T cells of known peptide/MHC specificity, have been used to visualize cell surface peptide/MHC complexes (19, 20), and MHC-restricted, peptide-specific mAb, identified by various immunization and screening schemes, have been isolated and used similarly (21–28). Because we have already reported the detailed characterization of the interaction of a cloned, recombinant TCR for the peptide/MHC complex consisting of the HIV envelope peptide P18-I10 bound to H-2Dd, we set out to identify mAbs with the same specificity. Here, we report the 0022-1767/00/$02.00
5704 results of a novel strategy to identify peptide-specific MHC-restricted mAbs. These mAbs were produced by immunization of transgenic mice with soluble peptide/MHC complexes. The recipient mice were transgenic for, and partially tolerant to, the soluble MHC molecule complexed to its endogenous repertoire of peptides. Thus the resulting Abs were focused on differences between self-peptide/H-2Dd structures and the P18-I10/H-2Dd structure, and would be expected to recognize a conformation similar to that seen by the TCR. The detailed analysis reported here confirms this view. To explore the ability of these mAbs to bind peptide/MHC complexes generated via the endogenous MHC-I Ag presentation pathway during viral infection, we have studied the in vivo effect of one of them in the blocking of CTL priming. These results suggest both a general strategy for eliciting peptide-specific MHCrestricted mAbs as well as a potential therapeutic use of such reagents.
Materials and Methods Cell lines The following cell lines were used: LKD8 (an H-2Dd transfectant of a TAP-defective mouse embryonic cell; Refs. 29 and 30); P815 (a DBA/2 mouse mastocytoma); DAP3 (a thymidine kinase-negative L cell, H-2k, Ref. 31); SKT4.5 (DAP3 cells transfected with H-2Dd; Ref. 32); B4.2.3 (a T cell hybridoma with specificity for peptide P18-I10 bound to H-2Dd; Ref. 32); T2 (a human cell line deficient in TAP1 and TAP2 transporter proteins that expresses low levels of HLA-A2.1 at the cell surface; Refs. 33 and 34); EL4 (a murine lymphoma of C57BL origin; Ref. 35); EL4-Dd (EL4 cells transfected with H-2Dd); Jurkat (a human T cell leukemia line; Ref. 36); Jurkat-Dd (Jurkat cells transfected with H-2Dd); and Sp2/0 (a mouse plasmacytoma fusion partner cell line; Ref. 37). EL4-Dd and Jurkat-Dd were the gifts of Drs. S. Hansel and A. Rosenberg, Food and Drug Administration (Bethesda, MD). All cells were maintained in DMEM supplemented with 10% FCS, nonessential amino acids, and glutamine.
Antibodies The following mAbs were used: 34-5-8S, which recognizes a peptide-dependent conformational epitope on H-2Dd ␣1␣2 domains (38, 39) and W6/32 (40), which binds a conformational epitope on HLA class I molecules.
H-2Dd-restricted viral and self peptides The following peptides were synthesized by 9-fluoroenylmethoxylcarbonyl chemistry and purified to ⬎95% purity by HPLC at the Laboratory of Molecular and Structural Biology, National Institute of Allergy and Infectious Diseases: P18-I10, RGPGRAFVTI (the HIV IIIB gp160 envelope glycoprotein-derived H-2Dd-restricted peptide; Refs. 32 and 41); P18-I10 peptides substituted at different positions (as noted in the figures); H-2Dd motif peptide (42): AGPARAAAL; pD38B: AGPDRTEKAL; pD46A: SGPVALVNFI; pD47: IGPNRAFNF; pNA: CPIRGWAI (residues 77–94 of the influenza virus neuraminidase); and pNP: QPQNGQFIHFY (residues 397– 407 of the lymphocytic choriomeningitis virus nucleoprotein).
Proteins scTCR, an engineered, bacterially expressed, single chain TCR, V␣2.6V7C, with specificity for P18-I10 bound to H-2Dd, has been described in detail previously (19), and the crystal structure of its V␣ domain at 2.5 Å resolution has been reported (43). This protein was expressed as inclusion bodies in Escherichia coli strain BL21(DE3) containing the encoding plasmid, solubilized, refolded in vitro, and purified by ion exchange and size exclusion chromatography. Soluble H-2Dd (sH-2Dd) was produced by a transfected L cell line and immunoaffinity purified as described previously (32, 44). sH-2Dd was emptied of self-peptides and loaded with synthetic P18-I10 by a high pH treatment that has been described before (45).
Generation of mAbs specific to H-2Dd/P18-I10 complex C57BL/6 mice transgenic for a soluble, secreted analog of H-2Dd on a C57BL/6 background (B6.tDd(␣3)/Q10b, referred to here as DD; Refs. 46 and 47) were bred and maintained at the National Institute of Allergy and Infectious Diseases Transgenic Mouse Facility (Frederick, MD). Animals were immunized in one foot pad at 2-wk intervals with H-2Dd/P18-I10
MONOCLONAL ANTIBODIES THAT MIMIC A TCR peptide complex (20 g emulsified in Freund’s complete adjuvant). Following two immunizations, draining lymph node cells were isolated and were fused with SP2/0 cells at a ratio of 2:1 using PEG 4000 (Life Technologies, Grand Island, NY; Ref. 48). After fusion, the cells were resuspended in HAT (hypoxanthine, aminopterin, and thymidine) medium containing 20% FCS and recombinant mouse IL-6 (100 U/ml; Genzyme, Cambridge, MA) at a final concentration of 5 ⫻ 105 myeloma cells/ml and were plated at 100 l/well in 96-well plates. After 10 –14 days, supernatant from each well was screened separately by flow cytometry for reactivity with LKD8 cells (H-2Dd-transfected embryonic cells) pulsed with 10 g/ml P18-I10 peptide in the presence of 5 g/ml of human 2-microglobulin (h2m; Fitzgerald Industries International, Concord, MA). Of ⬃400 wells examined, five contained Abs that stained LKD8 cells pulsed with P18-I10 peptide. Two of these five Abs did not stain the unloaded LKD8 cells or those pulsed with the control motif peptide (AGPARAAAL). (The protocol in which lymph node rather than spleen cells were used for the fusion was developed after two fusions to spleen in which a total of 1500 hybridomas were screened without identifying any positive clones.) Hybridomas producing Abs specific for the P18-I10/H-2Dd complex were recloned by limiting dilution and retested. The isotyping of mAbs was performed by ELISA using the Mouse MonoAb ID kit (Zymed, San Francisco, CA). (KP14/1 is IgG2b, and KP15/11 is IgG1). The Abs were purified from hybridoma supernatants by affinity chromatography using protein A- and protein G-Sepharose 4 Fast Flow (Pharmacia, Piscataway, NJ) respectively.
Flow cytometry Approximately 2 ⫻ 105 cells were incubated with mAbs for 1 h at 4°C. After washing with PBS containing 2% BSA and 1% sodium azide, cells were incubated with FITC-conjugated goat anti-mouse IgG (1:100 dilution; Dako, Carpinteria, CA) for 40 min at 4°C. After washing in PBS, cell staining (with gating on viable cells using propidium iodide) was assessed with a FACScan flow cytometer, and data were analyzed using Cell Quest software (Becton Dickinson, San Jose, CA).
T cell activation assays mAbs KP14 and KP15 were tested for their ability to inhibit stimulation of B4.2.3 cells (H-2Dd-restricted T cell hybridoma specific for P18-I10 peptide; Ref. 32). In brief, 1 ⫻ 104 cells were stimulated with 2 ⫻ 103 SKT4.5 cells in the presence of graded concentrations of peptide P18-I10, with or without the indicated purified mAbs, for 16 h at 37°C. A portion of the culture supernatant was then removed and tested for IL-2 by ELISA with MiniKit mouse IL-2 (Endogen, Cambridge, MA). The remaining cells were provided with 1 Ci of [3H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA), and incorporation of radiolabel into insoluble material was assessed after a 4-h incubation at 37°C.
Surface plasmon resonance (SPR) Binding of scTCR (V␣2.6V7C) or purified mAbs (34-5-8S, KP14/1, or KP15/11) to the sH-2Dd complexed with different peptides was evaluated by SPR using a Pharmacia BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden). All binding experiments were performed at 25°C. mAbs were coupled to the biosensor surface through free amino groups at pH 4.5–5.5 according to the conventional method as described previously (45, 49). The scTCR was coupled to the biosensor chip via the thiol group of the free cysteine present in the C domain using a heterobifunctional cross-linking reagent (sulfo-succinimidyl 4[N-maleimido-methyl]cyclohexane-1-carboxylate) as described in detail elsewhere (19, 45). SPR was also used to estimate the affinity of mAb KP14/1 and KP15/11. To determine the kinetic association and dissociation rate constants of MHC/peptide complexes for individual mAbs (KP14/1 and KP15/11), sH-2Dd/P18-I10 complexes were first homogeneously loaded and purified by gel filtration and then injected at different concentrations ranging from 8 ⫻ 10⫺8 to 6.2 ⫻ 10⫺6 M over surfaces coupled to mAb KP14/1 or KP15/11. From these kinetic data, kinetic rate constants and the equilibrium constants for dissociation (Kd) were calculated as described previously (19). In addition, Kd values were determined from steady-state binding curves and analyzed according to Scatchard (50). Values obtained from kinetic measurements were consistent with those obtained from the steady-state curves.
Analysis of Ab-variable region gene and encoded protein sequences mRNA from hybridoma cells producing mAbs KP14/1 or KP15/11 was isolated using the Poly(A)Ttract System 1000 kit (Promega, Madison, WI).
The Journal of Immunology These mRNAs served as templates for synthesis of cDNA using hexadeoxyribonucleotides as primers and cloned murine reverse transcriptase. This cDNA was used as a template for preparation of Ab-variable (V) region genes in PCRs, for which heavy and light chain primers were purchased from Pharmacia Biotech and Taq polymerase was purchased from Perkin-Elmer (Norwalk, CT). Amplified heavy and light chain PCR products were purified by 1% agarose gel electrophoresis. For preparation of Fv gene fragments, we used reagents and a protocol provided by Pharmacia Biotech (Mouse ScFv Module). Purified cDNA corresponding to the Ab variable region genes (14VL, 14VH, 15VL, and 15VH) was inserted into the pCR 2.1 vector (Invitrogen, San Diego, CA) according to the procedure described in the Original TA Cloning Kit, and bacterial transformants were grown on Luria-Bertani agar plates containing kanamycin (50 g/ml) and X-Gal (5bromo-4-chloro-3-indolyl--D-galactopyranoside) as an indicator. White colonies were picked and grown overnight at 37°C in Luria-Bertani broth containing ampicillin, and plasmid DNA containing the V region fragments was purified (Wizard Minipreps DNA Purification System; Promega) and then sequenced with the Universal M13 Reverse and Forward (-20) Primers (Invitrogen) and the Perkin-Elmer DNA sequencing kit. Products of the sequencing reactions were electrophoresed and analyzed with an ABI 373 DNA sequencer. DNA sequences were analyzed with IgBlast (http://www. ncbi.nlm.nih.gov/igblast/) and the DNA plot analyses of the ImMunoGeneTics database (IMGT) (http://imgt.cines.fr:8104/dnaplot/) (51, 52). Nucleotide sequences have been deposited in GenBank under accession numbers AF261879, AF261880, AF261881, and AF261882. Display of the molecular structure of H-2Dd/P18-I10 using Protein Data Bank (53) coordinates 1DDH (54) was accomplished with GRASP 1.3.6 (55).
CTL induction in vivo and blocking with mAbs BALB/c mice were immunized i.p. with 5 ⫻ 106 PFU recombinant vaccinia virus (vPE16; Ref. 56), which expresses the HIV IIIB gp160 envelope glycoprotein. To block CTL induction against H-2Dd/P18-I10 complexes in vivo, BALB/c mice were injected with different concentrations of KP15 (1 day before, again 4 h before immunization with vPE16, and 4 h, and 1, 2, 3, and 4 days after immunization with vPE16). At each time point, BALB/c mice were injected simultaneously i.p. and i.v. with equal concentrations of KP15. Three weeks later, immune spleen cells were cultured at 5 ⫻ 106 per ml in 24-well culture plates in complete T cell medium (RPMI 1640 containing 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 g/ml), and 5 ⫻ 10⫺5 M 2-ME). These spleen cells from immunized mice were restimulated in vitro with 4 ⫻ 106 of 3300-rad irradiated syngeneic spleen cells pulsed with 1 M P18-I10 for 7 days before assay. (Additional details of the CTL assay are given elsewhere; Ref. 57.) The cytolytic activity of these CTL lines was measured in a 4-h 51Cr release assay. P815 (H-2d) targets were tested in the presence or absence of P18-I10 peptide (1 M). For testing the peptide specificity of CTL, 51Cr-labeled P815 targets were pulsed for 2 h with peptide (1 M) at the beginning of the assay. The percent specific 51Cr release was calculated as 100 ⫻ (experimental release ⫺ spontaneous release)/(maximum release ⫺ spontaneous release). Maximum release was determined from supernatants of cells that were lysed by the addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated without added effector cells.
Results Production of mAbs specific for P18-I10/H-2Dd To improve the likelihood of obtaining peptide/MHC-restricted mAbs, we developed a strategy based on mice transgenic for a soluble analog of H-2Dd, which we had previously shown to be partially tolerant to the H-2Dd molecule (47). These animals do not readily raise allo-Abs against H-2Dd molecules expressed on lymphoid cells. We prepared soluble H-2Dd molecules which were loaded in vitro with P18-I10 (45) and immunized the transgenic mice. Ab-producing cells were immortalized by somatic cell hybridization (see Materials and Methods), and hybridoma supernatants were screened by flow cytometry in three stages: first, to identify those that reacted with the TAP-deficient H-2Dd-expressing embryonic cell line, LKD8, upon exposure to P18-I10 and human 2-microglobulin; second, to eliminate those that bound LKD8 without addition of peptide; and third, to eliminate those that bound LKD8 when pulsed with an H-2Dd-motif peptide, a peptide known to bind H-2Dd well, with all nonanchor residues substituted with alanine. Efficient loading of the LKD8 indicator
5705 cells with peptide was confirmed by reactivity with 34-5-8S, a mAb that reacts with a structure that is peptide dependent, but not peptide specific (39). Two hybridomas that satisfied our criteria for peptide specificity were identified (KP14 and KP15) that were positive in the first and negative in the second and third flow cytometry screenings by indirect immunofluorescence. They were isolated and immediately cloned by limiting dilution. The mAbs KP14/1 and KP15/11 showed indirect immunofluorescent staining of high intensity only of LKD8 cells bearing H-2Dd/P18-I10 complexes (Fig. 1A). No reactivity was observed with cells not exposed to peptide or when LKD8 cells were loaded with a motif peptide or other H-2Dd-specific peptides derived from either endogenous proteins (pD38B, pD46A, pD47; Ref. 42) or from viral proteins such as influenza virus neuraminidase (pNA) or the nucleoprotein of lymphocytic choriomeningitis virus (pNP). The failure of the Abs to stain cells pulsed with peptide pD47 (IGPNRAFNF), which is identical in five of nine comparable positions to peptide P18-I10 (RGPGRAFVTI), confirmed the peptide specificity of both mAbs. To evaluate the possibility that the mAbs were specific for P18-I10 alone, even when bound to a different MHC-I molecule, we asked whether either KP14 or KP15 would show specific staining of cells that lacked H-2Dd, but that expressed cell surface MHC-I molecules capable of binding P18-I10. Using T2 cells, a human line expressing HLA-A2, a molecule known to bind P18-I10 (58), we asked whether either Ab bound the peptide/HLA-A2 complex under conditions that allowed a peptide-dependent increase in the binding of the conformation-dependent mAb, W6/32 (Fig. 1B). Although the level of peptide-dependent induction of W6/32 staining was twice the background level, there was no specific induction of reactivity with KP14 or KP15. This observation lends additional support to the view that KP14 and KP15 bind the H-2Dd/P18-I10 complex. Analysis of specificity of binding by SPR Although these experiments, using cell surface H-2 molecules exposed to P18-I10 and various other peptides, clearly demonstrated the peptide specificity of the Abs, it remained formally possible that the peptide-dependent complexes recognized by these Abs involved some other MHC molecule or even other unrelated molecules expressed on the plasma membrane. To confirm the H-2Dd dependence and the peptide specificity of these Abs, they were tested for their ability to bind purified soluble H-2Dd molecules containing either their pool of self-peptides or to bind preparations that were specifically loaded with P18-I10. These binding studies were performed using SPR as the detection method, and the results are summarized in Fig. 2. Although Ab 34-5-8S binds both the H-2Dd pool (i.e., those molecules complexed with a full repertoire of self peptides) and H-2Dd molecules loaded specifically with P18-I10, mAbs KP14/1 and KP15/11 fail to bind the H-2Dd pool and yet efficiently interact with those H-2Dd molecules specifically loaded with P18-I10. Thus, the purified sH-2Dd protein, in the absence of additional components other than P18-I10, binds these Abs. In addition, using SPR we determined the affinities of the two Abs for H-2Dd/P18-I10 complexes. KP14/1 and KP15/11 had affinity constants, Kd, of 7.14 ⫻ 10⫺7 and 8.47 ⫻ 10⫺7 M, respectively. Both mAbs KP14/1 and KP15/11 were obtained from the same fusion, in which lymph nodes from four immunized mice were pooled. Even though the clones producing these Abs were detected in two different 96-well plates, we could not exclude the possibility that these clones were siblings. The difference in Ab isotypes (KP14/1 is IgG2b and KP15/11 is IgG1) did not completely exclude this possibility because a common V region might switch independently to several C regions in the early stabilization of the
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MONOCLONAL ANTIBODIES THAT MIMIC A TCR
FIGURE 1. Monoclonal Abs KP14/1 and KP15/11 specifically recognize H-2Dd molecules complexed with peptide P18-I10 on LKD8 cells. A, LKD8 cells were pulsed with the indicated peptides and were then stained with mAbs KP14/1 or KP15/11 and then with FITC-conjugated rabbit anti-mouse Ig (bold lines). The cells incubated only with the FITC reagent served as controls (fine lines). LKD8 cells were loaded with the indicated viral (P18-I10, influenza virus neuraminidase, pNP), endogenous (pD38B, pD46A, pD47), or motif peptides and stained for flow cytometry as indicated in detail in Materials and Methods. B, T2 cells expressing HLA-A2.1 were incubated with the indicated mAbs following exposure to either motif or P18-I10 peptide as indicated. Baseline mean cell fluorescence of T2 as assessed with W6/32 was 112.01 and, upon addition of peptide, fluorescence of 174.15 (with P18-I10) and of 179.62 (with H-2Dd motif peptide), a result consistent with previously published findings (58, 79).
fusion (59). In addition, binding experiments using SPR revealed that KP14/1 and KP15/11 competed for the same binding site on H-2Dd complexes (data not shown). Therefore, we studied the fine specificity of both Abs using a panel of synthetic P18-I10-related peptides and several different H-2Dd-positive cell lines, including the TAP-negative H-2Dd transfectant LKD8. Immunofluorescence with the two Abs revealed that several different peptide/H-2Dd complexes were recognized distinctly. In the examples illustrated here (Fig. 3), the G6-substituted peptide formed a complex recognized better by KP14/1 than by KP15/11. The M1- and L1-peptide/ H-2Dd complexes were bound better by KP15/11 than by KP14/1, and in some experiments L1/H-2Dd complexes were not recognized at all by KP14/1. Despite the fine specificity differences between the two mAbs, the relative level of staining of cells pulsed with different peptides was independent of the cell line studied. We then surveyed of a panel of 27 different substituted peptides using the H-2Dd-transfected L cell SKT4.5 as the indicator (Fig. 4). As seen in Fig. 4A, with the exception of peptides M1, L1, G6 (as pointed out above), and A2, the majority of the synthetic peptide variants generate peptide/MHC complexes that are seen equivalently by both KP14/1 and KP15/11. The motif peptide (AG PARAAAL) failed to generate a complex capable of being
recognized well by either of the Abs, a behavior similar to that of peptides substituted at positions 5, 6, and 7 (with the exception of the strong recognition by KP14/1 of the G6 substitution). Peptides L1, M1, and A2 were seen better by KP15/11. The pattern of binding as revealed by immunofluorescence of live cells was closely mimicked by the pattern of binding as determined in a direct assay using purified proteins and SPR (Fig. 4B). We have previously described a direct assay in which the binding of a biotinylated recombinant three-domain TCR to cells exposed to peptides closely paralleled the pattern of T cell activation induced by the same panel of peptides (19). Here we compared the direct binding of Abs KP14/1 and KP15/11 to peptide complexed with H-2Dd with the binding of the scTCR (Fig. 4C). Like mAbs KP14/1 and KP15/11, the scTCR failed to bind to peptide/MHC complexes formed with variant peptides having amino acid substitutions in positions 5, 7, and 10. In addition, the scTCR was more sensitive than the mAbs to substitutions at positions 8 and 9. In addition, the scTCR bound well to the Y7 peptide/H-2Dd complex, whereas neither of the mAbs did. Thus, although the Abs bind the same peptide/MHC complex as the scTCR, their fine specificities differ from each other and from that of the scTCR. The suggestion that the two clones do not express identical Ab heavy
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FIGURE 2. Binding of sH-2Dd/P18-I10 complexes to mAb KP14/1 and KP15/11. Direct binding of these complexes to the indicated Abs was assessed by SPR. Purified Abs (KP14/1, KP15/11, and the anti-H-2Dd mAb, 34-5-8S), were coupled through amino groups to a biosensor chip as described in Materials and Methods. Purified sH-2Dd containing its natural repertoire of self-peptides as expressed by an L cell transfectant (Dd) and sH-2Dd loaded with P18-I10 (Dd⫹P18-I10) were exposed to separate biosensor surfaces coupled to the indicated Abs. The injection of the solution phase sH-2Dd/P18-I10 complexes was initiated at t ⫽ 80 s, and the buffer washout phase began at t ⫽ 210 s.
and light chain V regions has been confirmed by the analysis of their nucleotide sequences (see below). Inhibition of T cell recognition by mAbs KP14/1 and KP15/11 To compare the nature of the site to which the Abs and TCR bind, the two Abs were used to inhibit the peptide dose-dependent activation of the B4.2.3 T cell hybridoma (see Fig. 5). As shown in A, in the absence of added Ab, the hybridoma is stimulated to release IL-2 or to be inhibited in its constitutive proliferation at a dose of P18-I10 of ⬃1 nM. Ab KP15/11 (0.3 mg/ml) potently inhibits IL-2 secretion and constitutive proliferation, even at the highest doses of peptide tested (5 nM). The IL-2 secretion assay appears to be more sensitive than the proliferation inhibition one to the effect of the mAbs. Abs KP14/1 (0.3 mg/ml) and the anti-␣1␣2 domain-dependent mAb, 34-5-8S (0.3 mg/ml), inhibited less well. At a lower dose (0.06 mg/ml), KP14/1 showed no significant inhibition of the peptide-dependent activation. The above results are all consistent with the view that these two peptide-specific, MHC-
FIGURE 4. Different peptide/MHC complexes are bound distinctly by mAbs KP14/1 and KP15/11 as compared with a single chain TCR. A, SKT4.5 cells were pulsed with no peptide, P18-I10, or each of the indicated variant synthetic peptides and then analyzed by flow cytometry for mean fluorescence index (mean FI) with mAbs KP14/1 (open columns) or KP15/11 (filled columns). B, Binding was assessed by SPR for binding of the indicated peptide/H-2Dd complexes to either KP14/1 (open columns) or KP15/11 (filled columns); or SPR for binding of the indicated peptide/ MHC complexes to immobilized scTCR (C).
restricted mAbs bind a site that overlaps that seen by the TCR with the same specificity. Peptide/MHC-specific mAbs block the induction of CTL in vivo
FIGURE 3. Differences in fine specificities between mAbs KP14/1 and KP15/11 are reflected in differences in binding intensity to peptide/H-2Dd complexes made with some P18-I10 variants. LKD8, SKT4.5, or P815 cells were pulsed with P18-I10 variants (G6 (RGPGRGFVTI), M1 (MG PGRAFVTI), or L1 (LGPGRAFVTI)) followed by staining with mAb KP14/1 or KP15/11. The complex G6/H-2Dd is better recognized with mAb KP14/1 (dashed line) than with mAb KP15/11 (solid line). The opposite observation was made with variants M1 and L1. Cells stained with FITC-secondary Ab alone served as a negative control (dotted line).
The above assays indicated that KP14 and KP15 mAbs both recognize P18-I10/H-2Dd complexes produced from purified protein and synthetic peptide. However, they do not address the issue as to whether the mAbs bind peptide generated via the endogenous MHC-I presentation pathway. To demonstrate the effectiveness of the binding of such mAbs to peptides generated in this way, we administered KP15 mAb to BALB/c animals immediately before and during exposure to a dose of recombinant vaccinia virus encoding the entire HIV IIIB gp160 envelope glycoprotein. A schedule of seven injections of mAb was used, and the dose per injection was evaluated. Three weeks after in vivo priming, spleen cells were removed and restimulated in culture with the antigenic peptide for 1 wk. The resulting CTL were evaluated for lysis of peptide-pulsed target cells (Fig. 6). As shown in Fig. 6, exposure of the host animal during priming with a dose of 0.001 mg/ml of KP15 had no significant effect in reducing the activity of bulk CTL
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FIGURE 5. Inhibition of peptide presentation by MHC/peptide-specific mAbs. SKT4.5 H-2Dd transfectant cells were pulsed with the indicated concentrations of P18-I10 and then exposed to B4.2.3 cells in the presence of the indicated mAbs as described in detail in Materials and Methods. Stimulation was assessed by measurement of IL-2 produced or by [3H]thymidine incorporation.
as compared with an isotype-matched IgG1 control. However, doses of 0.01, 0.1, and 1 mg/ml showed significant inhibition of the resultant CTL. This experiment demonstrates that the in vivo use of the mAb can directly block the priming of CTL in vivo as elicited by infection with the recombinant vaccinia vector. Structural comparison of mAbs and TCR One of our initial goals in isolating these mAbs was to gain insight into the structural basis by which molecules of the Ig superfamily, mAb, and cloned TCR interact specifically with a peptide/MHC complex. To explore the relationship among the three molecules, KP14, KP15, and the V␣2.6V7C scTCR of hybridoma B4.2.3, we compared their amino acid sequences. We compared the VH sequences of KP14 and KP15 with the V sequence of the B4.2.3 TCR and the VL sequences of KP14 and KP15 with V␣ sequences
FIGURE 6. Inhibition of CTL priming by mAb KP15. BALB/c mice were immunized with recombinant vaccinia virus expressing HIV IIIB gp160 envelope glycoprotein (vPE16) and different doses of purified mAb KP15 or an isotype-matched control mAb according to the schedule given in Materials and Methods. A, Data expressed as a function of E:T ratio. Doses used were: f, 0.001 mg/ml; Œ, 0.01 mg/ml; 䡺, 0.1 mg/ml; ‚, 1.0 mg/ml. ⫻, P815 target cells not pulsed with peptide tested with vPE16primed effectors. E, Cytolysis of cells raised in the presence of 1.0 mg/ml of an isotype-matched control mAb. B, The same data plotted as a function of KP15 concentration for the different E:T ratios (E, 100:1; 䡺, 50:1; F, 25:1; and f, 12:1).
MONOCLONAL ANTIBODIES THAT MIMIC A TCR of the TCR (Fig. 7). Structural comparisons of V␣ and V have not yet definitively resolved the issue of which is more similar to VH or VL, but VH and V are both assembled at the DNA level from V, D, and J gene segments, and V␣ generally appears to provide a better fit when aligned with VL (13). For these TCR-like mAbs, the VH showed greater similarity to TCR V, and VL to V␣. Sequence comparisons reveal intriguing similarities among these molecules (see Fig. 7 and Table I). First, both VH, although they differed in CDR3, used the identical germline V gene. This is the mouse IGHV3 K01569 according to the IMGT nomenclature and is designated VH36-60* by IgBLAST (Table I). The CDR3 of the heavy chain of KP14 (labeled 14H) is 12 residues long and that of KP15 (15H) is 11, whereas that of the V of the B4.2.3 TCR is 10 (Fig. 7). The two mAb heavy chains use different IGHD segments (M35332 IGHD-FL16.1*02 for 14H and L32868 IGHDQ52*01 for 15H). The JH segments used for these two heavy chain genes seem to be the same (V00880 IGHJ2*01). These heavy chain CDR3s are similar in that they each contain three acidic residues and four aromatic ones. The amino acid sequence alignment of the heavy chains with the V (Fig. 7) is also remarkable for the identity of residues 50 –54 of the heavy chains with residues 47–51 of the V (amino acid residues YISYD). This sequence coincides with CDR2 and may be indicative of an important role CDR2 plays in a specific interaction with H-2Dd. Analysis of the two mAb light chains (Fig. 7B and Table I) reveals that they are distinct, though they both belong to the mouse light chain V family (60). They represent different germline genes: IGKV10S1 and IGKV6S6 according to the IMGT nomenclature (51, 52), and use different J segments. These two different light chains apparently represent structurally alternative solutions for the stringent dual requirements of productively interacting with their respective heavy chains and of binding the P18-I10/ H-2Dd complex.
Discussion Our understanding of the nature of Ag presentation by MHC-I and MHC-II molecules has improved steadily in recent years with the elucidation of the mechanisms by which antigenic peptides are generated and the rules that define the ability of peptides to bind to particular MHC molecules (1, 2, 61). In addition, high resolution crystallographic studies of peptide/MHC complexes and TCR molecules have provided formal evidence that the TCR exploits an Ab-like combining site for the specific recognition of peptide/ MHC complexes (13). In an effort to understand better the nature of the formation of peptide/MHC complexes in the course of Ag presentation and to produce reagents that may be useful in blocking the activation of specific T cells, a number of groups have explored strategies for producing reagents that specifically bind peptide/MHC complexes. Some of these reagents were Abs that were generated in an MHC-restricted fashion against tumor Ags (62, 63) or against the H-Y Ag (64). Other groups have demonstrated that particular mAbs recognize only a subset of the MHC molecules to which they were directed, and have suggested that such restricted reactivity was due to peptide specificity (65– 68). More recently a number of attempts have been made to identify MHC-restricted mAbs using a variety of immunization and screening methods (21–26, 69). Also, phage display methods have been exploited to identify Abs with peptide/MHC specificity (27). An alternative approach to the production of Abs has been to use recombinant TCR with defined peptide/MHC specificity to visualize particular MHC/peptide complexes on cell surfaces (19, 20). We have described here the use of a transgenic mouse that expresses a soluble analog of the MHC-I molecule H-2Dd that is partially tolerant to this molecule to generate specific Abs directed
The Journal of Immunology
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FIGURE 7. Comparison of amino acid sequences of mAb KP14 and KP15 heavy and light chains. The V regions of the two mAb heavy and light chains were cloned and sequenced as described in Materials and Methods. In A, the heavy chains are aligned with the V of clone B4.2.3 that was previously reported. In B, the light chains are compared with the V␣ sequence.
against a peptide/H-2Dd complex. These animals failed to generate an alloantibody response but were capable of rejecting H-2Dddisparate skin grafts, indicating partial tolerance (47). Therefore, we expected that such animals would produce highly specific Ab responses to the peptide/MHC complex. Immunization with peptide-loaded H-2Dd molecules induced an Ab response against the P18-I10/H-2Dd complex (data not shown), but a large number of hybrid clones still needed to be screened to identify positive hybridomas. Because of the possibility that some Abs might be peptide dependent and MHC specific without exhibiting both peptide and MHC specificity, our initial screen eliminated all Abs that reacted with H-2Dd when exposed to a motif peptide. With this strategy we isolated two mAbs directed against the peptide/MHC complex, and have reported here the characterization of these mAbs. They show peptide and MHC specificity—a specificity similar, but not identical, with that of a P18-I10/H-2Dd-specific TCR. In particular, the TCR shows great sensitivity to amino acid substitutions at positions 5, 6, 7, 8, and 9 of the decamer peptide, whereas the mAbs are most sensitive to substitutions at positions 6 and 7, but not positions 8 or 9. Because the high resolution crystal structure of H-2Dd complexed with P18-I10 is now known (54, 70, 71), visualization of the peptide residues of the complex helps to explain some of the differences in recognition between the B4.2.3 TCR and the two mAbs (Fig. 8). In particular, both the mAbs and the TCR seem to be focused on amino acid 7 of the bound peptide, phenylalanine, the side chain of which offers a prominent bulge to either TCR or mAb. All four substitutions at this position of this peptide tested (see Fig. 5) were not well tolerated by either of the
mAbs, whereas only the tyrosine substitution was acceptable to the TCR. We have determined the nucleotide sequence, and thus the encoded amino acid sequences, of the V regions of the two mAbs (see Fig. 7 and Table I). The sequences of the two mAb heavy chains are of the same VH family, but differ significantly at CDR3 due to usage of different D segments. As noted, a stretch of CDR2 is common among the two mAbs and the -chain of the TCR. A number of examples of structures of peptide/MHC-I/TCR complexes suggest a canonical orientation of TCR with respect to the p/MHC (13, 72), and the CDR2 of the TCR -chain would, by analogy, be expected to make major contacts with the carboxylterminal “right hand” side of the ␣1 helix of the MHC. This raises the possibility that the use of animals tolerant to H-2Dd for immunization with P18-I10/H-2Dd complexes might preferentially expand those B cells bearing Abs of this particular VH family. This particular VH may have a structure encoded in CDR2 that interacts strongly with an antigenic region of H-2Dd. More extensive analysis of both the B and T cell repertoires that emerge from exposure to P18-I10/H-2Dd complexes will be needed to understand this phenomenon better. Our mapping of the peptide residues that affect both TCR and mAb binding to the P18-I10/H-2Dd complex is complementary to the detailed analysis of two OVA peptide/H-2Kb-specific mAbs recently reported (73). In that study, the authors evaluated the reactivity of two peptide/H-2Kb- specific mAbs using a panel of variant peptides as well as several H-2Kb-mutant molecules. These
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MONOCLONAL ANTIBODIES THAT MIMIC A TCR
Table I. Comparison of junctional sequences of Absa
a Alignments and gene fragment assignments were performed by searching with the nucleotide sequences against IMGT, the international ImMunoGeneTics database, http://imgt.cines.fr:8104 (initiator and coordinator: Marie-Paule Lefranc, Montpellier, France; Refs. 51, 52). The best match IGHV, IGHD, and IGHJ families are indicated. The junctional sequence from the residue following the conserved cysteine (position 104 of the heavy chains) to the conserved tryptophan (position 117 or 116) is underlined. Dashes indicate the bases that align with the indicated V, D, and J segments. The best similarity scores for the indicated sequences as compared to the highest scoring germline sequences were 1108, 1098, 1251, and 1242, respectively, for 14H, 15H, 14L, and 15L. Scores are a function indicative of both similarity and length of homology (51, 52).
mAbs showed some fine specificity differences, but were very similar to each other and, based on the patterns of reactivity to different peptides and different H-2Kb-mutant presenting molecules, the authors concluded that the two mAbs were focused on the carboxyl-terminal “right hand” region of the bound peptide and of the adjacent carboxyl-terminal region of the ␣1 helix and the amino-terminal region of the ␣2 helix. The analysis of the CDR3 sequences of the mAb chains revealed that both mAbs used the same CDR3 and exploited a rare arginine-lysine sequence encoded at the JH/VH junction, possibly to accommodate an acidic glutamic acid at position 6 of the OVA, SIINFEKL peptide. In our studies of the binding of KP14 and KP15 to P18-I10/H-2Dd complexes, we have compared the binding specificity as determined in a direct binding assay to that of a recombinant TCR with the same specificity. The major focus of both mAbs, and of the TCR, seems to be the center of the peptide, residues 5 and 7, whereas the TCR is also particularly sensitive to peptide residues 8 and 9. Indeed, T cell functional studies showed that the T cells focus especially on the V at position 8 of P18-I10 (74 –76). mAb KP14 is also more sensitive to amino acid substitutions at peptide position 1, suggesting that its footprint may lie somewhat more to the “left hand side” of the peptide/MHC complex (encompassing the amino terminus of the ␣1 helix, the amino terminus of the bound peptide, and the carboxyl terminus of the ␣2 helix). As discussed in Results, the striking identity in sequence of the CDR2 region of the mAb and the TCR also suggests that the mAb VH align spatially with the V of the TCR. Although it is tempting to model the mAbs in complex with the peptide/MHC, as has been accomplished for a recombinant Ab that reacts with an influenza hemagglutinin peptide/H-2Kk complex (77), several considerations make this an uncertain undertaking: 1) only the structures of the P18-I10/H-2Dd complex and of the unliganded V␣ of the B4.2.3 TCR are now known; 2)
FIGURE 8. Location of exposed peptide residues of the P18-I10/H-2Dd complex. A molecular surface representation of the x-ray crystal structure of the P18-I10/H-2Dd complex (1DDH; Ref. 54) is shown in the “standard” orientation with the peptide amino terminus to the left and the carboxyl terminus to the right. ␣1 and ␣2 helices are labeled. H-2Dd is magenta and the solvent-exposed portions of the bound peptide are shown in the following colors: Arg (P1 and P5), blue; Gly (P2 and P4), white; Ala (P6), light blue; Phe (P7), yellow; Val (P8), red; Thr (P9), green; and Ile (P10), steel blue.
The Journal of Immunology although the accumulated structural evidence would suggest that the orientation of the TCR on the peptide/MHC is conserved among MHC-I molecules, with the V of the TCR directed more to the carboxyl half of the peptide and the V␣ aimed at the aminoterminal half of the peptide (13, 78), the recent description of an “orthogonal” orientation of an MHC-II-restricted TCR with respect to its peptide/MHC-II ligand (14) might suggest caution in broad generalizations at this time. Perhaps the most significant observation we report here concerns the potential value of peptide/MHC-specific mAbs in modulating the immune response in vivo. In particular, we have demonstrated that KP15, when given to animals during the priming stage of immunization against the HIV IIIB gp160 envelope glycoprotein using a recombinant vaccinia vector, specifically blocks the priming against the immunodominant antigenic peptide. The potential for exploiting this and other peptide/MHC mAbs to block or alter immune responses in vivo requires further exploration. In summary, we have demonstrated the effectiveness of a novel immunization strategy for generating peptide/MHC-specific mAbs, characterized the fine specificity of these mAbs, examined the relationship of these mAbs to a TCR with the same specificity, and revealed the efficacy of the administration of such mAbs in vivo in blocking the priming of a specific immune response. Further studies should allow more detailed understanding of the precise molecular footprint of these mAbs on the peptide/MHC complex, steps in the delivery of antigenic peptides to the MHC-I pathway, and the molecular and cellular details of the inhibition of in vivo priming.
Acknowledgments We thank R. Carey and L. Boyd for technical help, and are particularly indebted to M. Mage, R. Mage, K. Natarajan, and E. Shevach for their comments on the manuscript.
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