Structural Basis of Specificity and Degeneracy of T Cell Recognition: Pluriallelic Restriction of T Cell Responses to a Peptide Antigen Involves Both Specific and Promiscuous This information is current as Interactions Between the T Cell Receptor, of September 26, 2021. Peptide, and HLA-DR Derek G. Doherty, Julie E. Penzotti, David M. Koelle, William W. Kwok, Terry P. Lybrand, Susan Masewicz and

Gerald T. Nepom Downloaded from J Immunol 1998; 161:3527-3535; ; http://www.jimmunol.org/content/161/7/3527 http://www.jimmunol.org/ References This article cites 60 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/161/7/3527.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Structural Basis of Specificity and Degeneracy of T Cell Recognition: Pluriallelic Restriction of T Cell Responses to a Peptide Antigen Involves Both Specific and Promiscuous Interactions Between the T Cell Receptor, Peptide, and HLA-DR1

Derek G. Doherty,2* Julie E. Penzotti,*† David M. Koelle,†‡ William W. Kwok,* Terry P. Lybrand,† Susan Masewicz,* and Gerald T. Nepom3*†

TCR engagement of peptide-MHC class II ligands involves specific contacts between the TCR and residues on both the MHC and Downloaded from peptide molecules. We have used molecular modeling and assays of peptide binding and T cell function to characterize these interactions for a CD4؉ Th1 cell clone, ESL4.34, which recognizes a peptide epitope of the herpes simplex type 2 virus virion protein, VP16 393–405, in the context of several HLA-DR alleles. This clone responded to VP16 393–405 in proliferation and cytotoxicity assays when presented by DRB1*0402, DRB1*1102, and DRB1*1301, which share a common amino acid sequence, ILEDE, at residues 67–71 in the ␣-helical portion of the DR␤ polypeptide, but not when presented by other DR4, DR11, and DR13

alleles that are negative for this sequence. Using a panel of APCs expressing DR4 molecules that were mutagenized in vitro at http://www.jimmunol.org/ individual residues within this shared epitope and using peptide analogues with single amino acid substitutions of predicted MHC and TCR contact residues, a unit of recognition was identified dependent on DR␤ residues 67–71 and relative position 4 (P4) of the VP16 393–405 peptide. The interactions of this portion of the peptide-DR ligand with the ESL4.34 TCR support a structural model for MHC-biased recognition in some Ag-specific and alloreactive T cell responses and suggest a possible mechanism for autoreactive T cell selection in rheumatoid arthritis. The Journal of Immunology, 1998, 161: 3527–3535.

he ligand recognized by TCR on CD4ϩ T cells consists of ␣-helical regions of the MHC class II molecules. Two recent crys- a class II MHC molecule complexed with a peptide frag- tal structures of class I MHC-peptide-TCR complexes (18–20) T ment of a protein Ag on the surface of an APC. The struc- have demonstrated that the ␣␤ TCR recognizes its ligand through by guest on September 26, 2021 ture of MHC molecules imposes constraints on the nature of pep- three variable loops (complementarity-determining regions tides that they can bind, and therefore the selection of antigenic (CDRs4)) on each polypeptide that form a relatively flat surface determinants that can be presented to T cells (1). Each MHC class that fits diagonally over the MHC-peptide complex. All three II molecule can bind and present an extremely large number of CDRs of the TCR ␣-chain and CDR3 of the ␤-chain contact the structurally diverse peptides based on motif residues which are MHC ␣ helices, while the CDR1 and CDR3 loops of both the TCR anchored by complementary pockets within the MHC molecule ␣- and ␤-chains contact the bound peptide. Based on sequence and (2–7). Similarly, a given peptide can be bound and presented by structure comparisons between peptides complexed with MHC many class II molecules with varying efficiencies for recognition class I and class II molecules, a similar mode of TCR interaction by T cells (2, 7, 8). with peptide-MHC class II ligands has been predicted (19, 21). The interaction between ␣␤ TCRs and peptide-MHC class II While the overall conformation of the peptide-MHC-TCR ter- ligands involves direct contact between the TCR and residues on nary complex is thought to be similar for all MHC class I and class both the MHC molecule and the bound peptide (9, 10). Analyses II molecules and all ␣␤ TCRs (19, 21), the nature of the signal of the crystal structures of MHC class II molecules complexed generated upon TCR ligation can vary greatly, leading to activa- with antigenic peptides (11–15) and studies using substituted pep- tion, apoptosis, or nonresponsiveness of the T cell and positive or tides and mutated MHC molecules (6, 16, 17) have identified pu- negative selection in the thymus (22, 23). TCR activation can, in tative TCR contact residues on both the antigenic peptides and the turn, result in a range of different effector functions, including T cell proliferation, cytolysis, and cytokine secretion of the Th0, *Virginia Mason Research Center, †University of Washington School of Medicine, Th1, or Th2 profiles. Factors that contribute to the outcome of and ‡Fred Hutchinson Cancer Research Center, Seattle, WA 98101 TCR ligation include the nature and cell surface density of the Received for publication April 27, 1998. Accepted for publication June 5, 1998. peptide-MHC ligand, the avidity of its interaction with the TCR, The costs of publication of this article were defrayed in part by the payment of page the presence or absence of other costimulatory molecules, and the charges. This article must therefore be hereby marked advertisement in accordance local cytokine milieu (22, 24–26). Specific contacts between with 18 U.S.C. Section 1734 solely to indicate this fact. amino acid residues on the peptide, MHC, and TCR molecules 1 This work was supported by Grants AR37296, DK02319, and AI34616 from the have profound effects on the nature of T cell stimulation. Single National Institutes of Health. 2 Current address: Education and Research Centre, St. Vincent’s Hospital, Elm Park, Dublin 4, Ireland. 4 Abbreviations used in this paper: CDR, complementarity-determining region; HVR, 3 Address correspondence and reprint requests to Dr. Gerald T. Nepom, Virginia hypervariable region; RA, rheumatoid arthritis; HSV-2, herpes simplex type 2 virus; Mason Research Center, 1000 Seneca Street, Seattle, WA 98101-2744. HSV-1, herpes simplex type 1 virus; LCL, lymphoblastoid cell lines.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 3528 PLURIALLELIC T CELL RECOGNITION substitutions of MHC or peptide residues predicted to contact the ␮M L-glutamine, 50 ␮g/ml streptomycin, 50 U/ml penicillin, and 10% TCR can enhance, antagonize, or anergize a T cell response or heat-inactivated human male serum). Acyclovir (50 ␮M) was added for the selectively inhibit some but not all effector functions (27–32). first 2 weeks of culture to prevent viral replication (38). Human natural ϩ IL-2 (50 U/ml; Schiaperelli Biosystems, Columbia, MD) was added on day The TCRs of human CD4 T cells primarily recognize antigenic 3, and the cells were subsequently fed with IL-2 every 2 to 3 days. After peptides presented by MHC class II (HLA-DR, DQ, or DP) mole- 16 days of growth, 5 ϫ 105 cells were restimulated with crude HSV-2 Ags cules. HLA-DR molecules are heterodimeric glycoproteins composed and 5 ϫ 105 autologous irradiated PBMC as APC and maintained as above. of a nonpolymorphic DR␣ and a polymorphic DR␤ chain. The poly- After 12 days, the cells were cloned at 1 cell/well using PHA, IL-2, and allogeneic feeders and subsequently restimulated every 12 to 14 days. morphic amino acid residues in DR␤ polypeptides are mostly clus- tered in three hypervariable regions (HVRs) which line the peptide Analysis of TCR genes binding groove (33). HVR1 (residues 9–14) and HVR2 (residues 26– mRNA was prepared from T cell clones that were restimulated with lym- 33) are located on the floor of the groove and predominantly influence phoblastoid cell lines (LCLs) as APC instead of PBMC using the Quick- the specificity of peptide binding, while HVR3 (residues 67–74) is Prep Micro RNA Purification Kit (Pharmacia Biotech, Alameda, CA) and located on the ␣-helical portion of the DR␤ molecule and can affect used for first strand cDNA synthesis using the Superscript Preamplification ␣ ␤ both peptide binding and TCR interaction through direct contacts with system (Life Technologies, Gaithersburg, MD). TCR V and V gene segment usage was determined by PCR amplification of first strand cDNA both molecules (11, 13, 14). HVR3 amino acid sequences can vary (RT-PCR) using a set of subfamily specific 5Ј-primers (V␣1–29 and V␤1– greatly within serologically related DR types but are conserved 23) and constant region 3Ј-primers (kindly provided by Dr. Christine Viss- among serologically distinct alleles. For example, the ILEDE amino inga, Virginia Mason Research Center, Seattle, WA) essentially as de- acid sequence at DR␤ positions 67–71 is present on some but not all scribed by Choi et al. (39). V␤ typing was confirmed using the Clontech allelic variants of the DR1, DR4, DR11, and DR13 specificities. An- (Palo Alto, CA) TCR Amplimer Kit. Nucleotide sequences of the V-(D)-J Downloaded from junctions were determined by cycle sequencing of both strands of the PCR other sequence at this position, LLEQRRAA, which is encoded by products obtained as above using the Prism Ready Reaction DyeDeoxy certain DR1, DR4, and DR14 alleles, constitutes the “shared epitope” Terminator Cycle Sequencing Kit (Applied Biosystems) and analyzed on thought to play a role in the development of rheumatoid arthritis an Applied Biosystems 373 DNA Sequencer. Two different V␣7 genes ␣ (RA) (34, 35). were detected by sequence analysis; therefore, the V 7 PCR products were cloned into the pCRII vector (Invitrogen, San Diego, CA), used to trans- In this study, we examined the molecular basis of the interac- form One Shot (INV␣FЈ) cells (Invitrogen) and selected on antibiotic- tions between the TCR, MHC class II, and peptide molecules, containing L-agar plates. Plasmid DNA was prepared from overnight cul- http://www.jimmunol.org/ using a herpes simplex type 2 virus (HSV-2)-specific human tures of isolated colonies using the QIAprep Spin Miniprep Kit (Qiagen, CD4ϩ T cell clone, ESL4.34, which recognizes a peptide deter- Santa Clarita, CA) and amplified as above by PCR. The nucleotide se- minant corresponding to residues 393–405 of the virion protein quences of both strands of the PCR products were determined by cycle sequencing as above. VP16, in the context of several HLA-DR alleles. This T cell, which elicits strong proliferative, cytolytic, and cytokine secretion re- Lymphoblastoid cell lines and Abs sponses, recognizes the VP16 393–405 peptide with a pattern of The EBV-transformed LCLs YAR (DRB1*0402), MT (*0404), HHKB restriction that correlates with a specific amino acid sequence at (*1301), JVM (*1102), TISI (*1103), and MAT (*0301) were obtained positions 67, 70, and 71 of HVR3 of the DR␤ polypeptide. By a from the VIIIth and Xth International Histocompatibility Workshop Panels. combination of molecular modeling and assays of peptide binding The LCL 8854 (*1303) was generated by EBV transformation of locally by guest on September 26, 2021 characterized PBMC. The MHC class II-deficient BLS-1 line was kindly and T cell stimulation using in vitro mutagenized DR molecules donated by Dr. Janet Lee, Sloan-Kettering Memorial, New York, NY. and substituted peptide analogues, we have dissected the structural LCLs were cultured in RPMI 1640 supplemented with 10% heat-inacti- interactions between DR␤ HVR3, the peptide, and the TCR. The vated FCS (HyClone, Logan, UT) and sodium pyruvate, HEPES, L-glu- DR␤ HVR3 was found to have a direct effect on both peptide tamine, streptomycin, and penicillin as above. mAbs used were L243 binding and TCR recognition, suggesting an important role for this (anti-DR; American Type Culture Collection, Rockville, MD), SPV-L3 (anti-DQ; kindly provided by Dr. Hans Yssel, DNAX, Palo Alto, CA), epitope in alloreactivity, in T cell repertoire selection, and in au- B7/21 (anti-DP; Becton Dickinson, San Jose, CA), anti-␣␤ TCR and anti- toimmune disease. V␤8 (PharMingen, San Diego, CA), NFLD.D1, and NFLD.D11 (anti-DR4 and -DR4w4; kindly provided by Dr. Sheila Drover, University of St. Johns, St. Johns, Canada). Cell surface expression of HLA-DR and TCR Materials and Methods was assayed by mAb staining as described previously (40) and analyzed on Viruses and viral antigens a Becton Dickinson FACSort flow cytometer. HSV-1 strain E115, HSV-2 strain 333, and a recombinant virus consisting Retroviral gene transfer of VP16 of HSV-2 in an HSV-1 background, RP-2, were grown in human diploid cells and titered by plaque assay on Vero cells as previously de- HLA-DR genes and mutagenized constructs were introduced into MAT scribed (36). Crude viral Ags were prepared from virus stocks by exposure LCLs by retroviral infection using the methods of Kwok et al. (41). The to UV light for 30 min at 10 cm from a GT038 bulb (General Electric, construction of the retroviral vectors containing wild-type and mutagenized Cleveland, OH), eliminating infectious virus, and used at a final dilution of DRB1*0404 genes which were altered at codons 67 (L to I), 70 (Q to D), 1:100 which corresponds to 106 to 107 plaque-forming units per ml before 71 (R to E), and both 67 and 71 (L67 to I67 and R71 to E71) has been UV treatment. Peptides corresponding to amino acids 1–409 of VP16 of described previously (40). For the purpose of clarity, these mutants are HSV-2, 13 amino acids long and overlapping by 9 amino acids, were named according to their differences from the DRB1*0402 amino acid obtained from Chiron Mimotopes, Clayton, Australia. The VP16 393–405 sequence in this report, i.e., DRB1*0402-Q70,R71, DRB1*0402-L67,R71, peptide (LVAPRMSFLSAGQ) and its analogues (see legend to figures and DRB1*0402-L67,Q70, and DRB1*0402-Q70, respectively. Surface ex- tables) were synthesized with an Applied Biosystems 432 Peptide Synthe- pression of the transgenes was monitored by flow cytometry using the sizer (Foster City, CA). Peptides were biotinylated by amino-terminal ad- NFLD.D1 and NFLD.D11 mAbs. dition of biotin onto ⑀-aminocaproic acid, as described by Kwok et al. (37), and used at the concentrations shown in the figure legends. T cell proliferation assays T cell cloning Stimulator cells (PBMC irradiated with 3,300 rads or LCL irradiated with 20,000 rads ␥-irradiation) were pulsed with Ag (peptide concentrations

Mononuclear cells were prepared from the herpetic vesicle fluid from a shown in figure legends) for 2 to4hat37°C, 5% CO2, followed by three DRB1*0402/*1301-positive patient with culture-proved recurrent HSV-2 washings with supplemented RPMI medium. PBMC (105) or LCL (2.5 ϫ infection by Ficoll-Hypaque density gradient centrifugation. The cells were 104) were plated in 96-well U-bottom plates in T cell medium containing stimulated with PHA (0.4 ␮g/ml PHA-P; Murex Diagnostics, Dartford, 104 cloned T cells or bulk lesion-derived cells as responders in a final U.K.) and an equal number of irradiated (3300 rads ␥-irradiation) alloge- volume of 150 ␮l. All assays were performed in triplicate. After incubation neic PBMC in T cell medium (RPMI 1640 containing 25 ␮M HEPES, 2 for 48 h, [3H]thymidine (1 ␮Ci/well; NEN, Boston, MA) was added for 12 The Journal of Immunology 3529

FIGURE 1. Specificity of the proliferative response of T cell clone ESL4.34 to autologous PBMC as APCs. The ordinate lists the viral Ags used as described in Materials and Methods. Numbers correspond to amino acid positions of 13-mer peptides corresponding to virion protein VP16 of HSV-2. Pool 10 denotes a mixture of 10 overlapping peptides correspond- ing to VP16 361–409. Pools 1 to 9, corresponding to VP16 1–369, had no significant stimulatory activity. Proliferation assays were performed using Downloaded from 2.5 ␮M peptide concentrations. to 16 h, the cells were harvested using a Tomtec 96 Mach III Harvester (Hamden, CT), and 3H incorporation was measured on a 1450 Microbeta Plus Liquid Scintillation Counter (Wallac, Gaithersburg, MD). Stimulation indices were calculated as (cpm of sample Ϫ cpm in absence of respond- ers)/cpm in absence of stimulators. To determine restricting HLA mole- http://www.jimmunol.org/ cules, mAbs L243, SPV-L3, and B7/21, which recognize HLA-DR, DQ, and DP, respectively, were used as previously described (38). T cell cytotoxicity assays Target LCLs (5 ϫ 105) were pulsed with peptide (concentrations shown in figure legends) and simultaneously loaded with 51Cr (25 ␮Ci; NEN) for 8 to 12 h at 37°C, 5% CO2, followed by gentle washing three times with supplemented RPMI medium. Labeled targets (1–2 ϫ 103) were incubated with effector T cells at various E:T ratios in a final volume of 150 ml in T FIGURE 2. Proliferative and cytotoxic responses of T cell clone by guest on September 26, 2021 cell medium in 96-well U-bottom plates. After 4 h, cell supernatants (50 ESL4.34 to peptide VP16 393–405 presented by YAR (DRB1*0402), ␮ 51 l) were assayed for Cr release in a 1450 Microbeta Plus Liquid Scin- HHKB (DRB1*1301), and MT (DRB1*0404) cells. E:T ratios of 15:1 tillation Counter. Percent specific lysis was expressed as (cpm of sample Ϫ were used in cytotoxicity assays. Error bars represent the SD of triplicate cpm of spontaneous release)/(cpm of maximum release Ϫ cpm of sponta- neous release). results. Peptide binding assays Binding of biotinylated peptides to HLA-DR on whole cells, with subse- quent capture of the peptide-class II complex, was performed using a eu- in proliferation assays (Fig. 1), indicating a specificity for VP16 of ropium-labeled streptavidin assay as described previously (37), except that HSV-2 but not HSV-1. The fine specificity of ESL4.34 was de- the L243 Ab was used to immobilize the HLA-DR molecules. A 10 ␮M peptide concentration was used in all experiments, this concentration hav- termined by testing its proliferative response to a set of 10 pools of ing been previously determined to be submaximal for binding. The class overlapping peptides corresponding to amino acids 1–409 of VP16 II-deficient LCL, BLS-1, served as a negative control for nonspecific of HSV-2. Only a pool spanning amino acids 361–409 (pool 10) binding. gave a positive response (Fig. 1). Assay with individual peptides of Modeling of peptide-HLA-DR complexes this pool identified the epitope as one contained in overlapping peptides 389–401 and 393–405 (Fig. 1). Peptide VP16 393–405 Molecular models of DRB1*0402, *1102, and *1301 were constructed was used in subsequent studies to characterize the residues re- from the crystal structure coordinates of the HLA-DR1 molecule com- plexed with the influenza hemagglutinin peptide 306–318 (11) as previ- quired for MHC binding and T cell recognition. ously described (42). Using the interactive graphics program PSSHOW The HLA locus that serves as the restriction element for (43), amino acid side chains were replaced and assigned to the preferred ESL4.34 was determined in proliferation assays using HSV-2 and conformer from the Ponder and Richards rotamer database (44) that was VP16 393–405, presented by autologous PBMC, in the presence closest to the side chain conformer in the crystal structure template. The and absence of mAbs to HLA-DR, -DQ, and -DP. Inhibition of HSV VP16 393–405 peptide was constructed from the hemagglutinin pep- tide coordinates (11), and several different alignments of the anchor resi- proliferation was found in the presence of the anti-DR mAb only dues with the VP16 393–405 sequence were examined to predict the bind- (data not shown), indicating that ESL4.34 is DR restricted. Oligo- ing motif. Peptide binding experiments were designed to test the predicted nucleotide typing of autologous PBMC revealed positivity for binding motif. DRB1*0402 and DRB1*1301. To determine which allele is the restriction element for ESL4.34 recognition of VP16 393–405, Results DR-homozygous LCLs expressing DRB1*0402 (YAR) and Characterization of T cell clone ESL4.34 DRB1*1301 (HHKB) were tested as APCs for the peptide. Figure Recovery of cells from herpetic vesicle fluid and subsequent stim- 2 shows that both DRB1*0402 and DRB1*1301 presented VP16 ulation with whole HSV-2 Ag followed by cloning yielded a T cell 393–405 to ESL4.34, eliciting both proliferative and cytolytic clone, ESL4.34, that responded to HSV-2 and RP-2 but not HSV-1 responses. 3530 PLURIALLELIC T CELL RECOGNITION

ESL4.34 TCR ␣- and ␤-chain gene usage was determined by PCR amplification of cDNA using primers specific for V␣1–29 and V␤1–23. PCR products corresponding to V␣7 and V␤8 only were obtained. Flow cytometric analysis of ESL4.34 using anti-␣␤ TCR and anti-V␤8 mAbs confirmed that all ␣␤ TCRϩ cells ex- pressed V␤8. Nucleotide sequence analysis of the V␤8 PCR prod- uct in both directions revealed a unique V-D-J junctional sequence that encodes a TCR ␤-chain CDR3 (residues 92–117) amino acid sequence CASSERGDTDTQYFGPGTRLTVLEDL, which corre- sponds to V␤8, D␤ (ERGD), J␤2.3 (TDTQYFGPGTRLTV LEDL), and C␤2. Sequencing of the V␣7 PCR product revealed two V␣7 TCR gene sequences, and cloning and sequencing of these two products identified distinct V␣7J␣ junctional sequences. One of these containedaGtoCnucleotide substitution which, if translated, would result in the substitution of the conserved cys- teine residue at position 90 with a serine residue, resulting in a nonfunctional TCR ␣-chain (45). The viable TCR ␣-chain CDR3 region was encoded by V␣7.2, J␤2.3, and a V-J junctional se- quence (residues 90–106), which code for the amino acid sequence Downloaded from CAPRGAGRRALTFGSGT. Pluriallelic restriction of ESL4.34 responses to VP16 393–405 The above experiments indicate that ESL4.34 is a V␣7ϩV␤8ϩ T cell clone specific for VP16 393–405 presented by both autolo- gous DR alleles, DRB1*0402 and DRB1*1301, in both prolifer- http://www.jimmunol.org/ ation and cytotoxicity assays (Fig. 2). This clone, however, does not respond to the peptide presented by the allogeneic DRB1*0404 FIGURE 3. Proliferative and cytotoxic responses of ESL4.34 T cell allele, which differs from *0402 only at three amino acid positions ␤␣ clone to VP16 393–405 presented by various LCLs and relative binding of (residues 67, 70, 71) in the DR helix (Fig. 2). To further in- this peptide to their cell surface DR molecules. LCLs used are YAR vestigate the pluriallelic restriction of ESL4.34, a set of LCLs ex- (DRB1*0402), MT (*0404), HHKB (*1301), 8854 (*1303), JVM (*1102), pressing DRB1 allelic variants of three serologically defined DR and TISI (*1103). A, Proliferative responses using 1 ␮M peptide, expressed types, DR4, DR11, and DR13, which differ in their ␣-helical as stimulation indices; B, cytolysis of target cells pulsed with 1 ␮M peptide amino acid sequences, were used as APCs for VP16 393–405 in using E:T ratios of 20:1; C, relative binding of peptide VP16 393–405 to proliferation and cytotoxicity assays. Figure 3, A and B, shows that cell surface HLA-DR expressed as units of europium fluorescence, using by guest on September 26, 2021 VP16 393–405 was recognized by ESL4.34 in proliferation and 1.5 ϫ 106 cells and 10 ␮M peptide. The class II-deficient BLS-1 cell line cytotoxicity assays, when presented on LCL expressing was used as a negative control. Error bars represent the SD of triplicate DRB1*0402, DRB1*1301, and DRB1*1102. Each of these DRB1 results. alleles encode the same sequence at DR␤ residues 67–71 (HVR3), namely ILEDE, whereas the other DR4, DR13, and DR11 alleles the involvement of the peptide in ESL4.34 responses, since no that did not support ESL4.34 responses had different HVR3 se- proliferation or cytotoxicity was observed in the absence of VP16 quences, as summarized in Table I. Even the single isoleucine to 393–405, and the responses increased proportionately with peptide phenylalanine substitution at DR␤ position 67, that distinguishes concentration (Fig. 2). DRB1*1102 and DRB1*1103, is sufficient to abolish ESL4.34 re- sponses. Further analyses of ESL4.34 responses VP16 393–405- Specific HVR3 residues on HLA-DR␤ control ESL4.34 responses pulsed LCLs expressing other DR␤ 67–71 ILEDE-negative DR4 (DRB1*0401 and *0405) and DR11 (DRB1*1101) alleles (data The three DRB1 alleles (DRB1*0402, DRB1*1102, and not shown) were consistent with the hypothesis that this motif at DRB1*1301) that could present VP16 393–405 to ESL4.34 in HVR3 is required for ELS4.34 recognition. The qualitative differences in the proliferative and cytolytic re- Table I. DR␤ specificities of LCLs used as APC for VP16 393–405, sponses of ESL4.34 to VP16 393–405 restricted by the different their position 67 to 71 amino acid sequences, and responses of T cell DRB1 alleles might be explained either by differences in the avid- clone ESL4.34 in proliferation and cytotoxicity assaysa ity of the TCR for the peptide-MHC complexes or by structural interactions between the peptide and MHC molecule alone. Figure Position 67–71 Relative Peptide ESL4.34 3C shows that the relative binding of VP16 393–405 to DR ex- DRB1 Allele Amino Acids Binding Responses pressed on the various APCs varied greatly for the different DRB1 *0402 ILEDE 167 ϩ alleles and showed no correlation with the proliferative and cyto- *0404 L––QR 15 Ϫ lytic responses of ESL4.34 to VP16 393–405. While DRB1*1303 *1301 ––––– 30 ϩ and DRB1*1103 bound the peptide as well as, or better than, their *1303 ––––K 39 Ϫ ϩ stimulatory DRB1*1301 and DRB1*1102 counterparts, no prolif- *1102 ––––– 69 *1103 F–––– 94 Ϫ erative or cytolytic responses were observed using these ligands. *0402-Q70 –––Q– 86 Ϫ This suggests that the ability of ESL4.34 to respond to its peptide- *0402-L67,Q70 L––Q– 74 Ϫ MHC ligand is controlled in part by the presenting MHC molecule *0402-L67,R71 L–––R 18 Ϫ Ϫ itself, through specific interactions between residues on the TCR *0402-Q70,R71 –––QR 23 and the DR molecule. This hypothesis, however, does not exclude a Peptide concentrations of 10 ␮M were used in binding assays. The Journal of Immunology 3531

FIGURE 4. Flow cytometric analysis of DR4 expression by lymphoblastoid cells YAR (DRB1*0402), MT (DRB1*0404), and MAT (DRB1*0301) expressing mu- tagenized DRB1*0402 genes, using DR4- specific mAb NFLD.D1, which recognizes all DR4 subtypes. NFLD.D1 staining of untransfected MAT cells was negative. Downloaded from http://www.jimmunol.org/

proliferation and cytotoxicity assays are serologically and struc- HSV-2 VP16 393–405 binding motif turally distinct but share a common amino acid sequence, ILEDE, Molecular modeling of the VP16 peptide 393–405 complexed with at DR␤ positions 67 to 71, whereas the other DRB1 alleles tested

DRB1*0402, DRB1*1102, and DRB1*1103 suggested a binding mo- by guest on September 26, 2021 in the present study encode different amino acid sequences at this tif such that V394 occupies pocket 1 and R397 occupies pocket 4. region. This suggests a role for this “shared epitope” in association This predicted binding motif was tested by synthesizing peptides with with VP16 393–405 in eliciting ESL4.34 responses. To test this nonconservative substitutions at these anchor positions and measuring hypothesis, we generated a panel of LCLs expressing mutagenized their relative binding to LCLs expressing DRB1*0402, DRB1*0404, DRB1 molecules that distinguish the stimulatory DRB1*0402 al- DRB1*1301, DRB1*1303, DRB1*1102, and DRB1*1103 (Fig. 6). lele from the nonstimulatory DRB1*0404 molecule, which differ Substitution of valine at position 394 with lysine (P1 [V3K]) dra- only at the three polymorphic residues in this epitope, residues 67, matically decreased the binding of this peptide to DRB1*0402, 70, and 71. Surface expression of these mutant DR4 molecules was DRB1*1102, and DRB1*1103, whereas replacing leucine at position comparable with that of wild-type DRB1*0402 and DRB1*0404 393 with lysine (P-1 [L3K]) slightly increased the level of binding in YAR and MT LCLs, respectively (Fig. 4). Figure 5 shows the to these alleles (Fig. 6). This indicates that V394 is the first anchor proliferative and cytolytic responses of ESL4.34 to VP16 393–405 residue. The increase in binding observed for L393K (P-1 [L3K]) presented by these mutant APCs and the relative binding affinities may be due to favorable interactions between K393 and DR␣ E53. of this peptide for the mutant DR molecules. Binding was drasti- Substitution of arginine at VP16 393–405 position 397 with the neg- cally reduced by substitutions at 67 and 71 (DRB1*0402-L67, atively charged residue glutamic acid (P4 [R3E]) reduced peptide R71) and substitutions at 70 and 71 (DRB1*0402-Q70, R71), but binding to the DR␤ alleles containing negatively charged residues at changes at residues 67 and 70 (*0402-L67,Q70, and *0402-Q70) positions 70 or 71, whereas binding to DRB1*0404 which has argi- had less significant effects on VP16 393–405 binding (Fig. 5C). In nine at DR␤ position 71 was increased by this substitution (Fig. 6). contrast, substitution of any of the DRB1*0402 residues 67, 70, or This clearly demonstrates the importance of the negatively charged 71 completely abrogated ESL4.34 proliferative and cytolytic re- DR␤ residues D70 and E71 of DRB1*0402, DRB1*1301, sponses to VP16 393–405. These data, together with the responses DRB1*1102, and DRB1*1103 in influencing the nature of pocket 4. of ESL4.34 to the DR4, DR11, and DR13 allelic variants (Fig. 3), Substitution of this P4 residue with a nonpolar alanine (P4 [R3A]) confirm that DR␤ residues I67, D70, and E71 are essential (and resulted in a less dramatic reduction in binding to DRB1*0402, and possibly sufficient) for presentation of VP16 393–405 to ESL4.34. surprisingly, an increase in binding to DRB1*0404 compared with The involvement of HVR3 in peptide binding and ESL4.34 re- that of the P4 (R3E) analogue peptide. The minimum binding sponses does not exclude less significant contributions by the first epitope of VP16 393–405 was tested using truncated analogues with and second HVRs of DR␤ and/or the V/G dimorphism at position two, three, or four residues removed from the C terminus. The VP16 86. Significant influences of the DRB3 and DRB4 gene products, peptides 393–401 and 393–402 did not bind or bound very poorly to which are encoded on DR11, DR13, and DR4 haplotypes, how- DRB1*0402 (data not shown), but VP16 393–403 did bind although ever, are unlikely, because these molecules have distinct HVR3 with a lower affinity than the wild-type VP16 393–405 peptide (Fig. sequences. 6). These observations suggest that the two C-terminal residues of 3532 PLURIALLELIC T CELL RECOGNITION Downloaded from http://www.jimmunol.org/

FIGURE 5. Proliferative and cytotoxic responses of ESL4.34 T cell clone to VP16 393–405 presented by LCLs expressing wild-type and mu- tagenized DR4 molecules and relative binding of this peptide to their cell FIGURE 6. Relative binding of peptide VP16 393–405, substituted an- surface DR molecules. LCLs used are YAR (DRB1*0402), MT (*0404), alogue peptides, and truncated analogue peptide (VP16 393–403) to and MAT (*0301) expressing retroviral constructs encoding mutagenized HLA-DR expressed on YAR (DRB1*0402), MT (*0404), HHKB (*1301), ␤ by guest on September 26, 2021 DRB1*0404 genes that encode DRB1*0402 molecules substituted at DR 8854 (*1303), JVM (*1102), and TISI (*1103). Substituted peptide ana- positions 70 (DRB1*0402-Q70), 67, and 70 (DRB1*0402-L67,Q70), 67, logues are named according to the relative DR binding positions (P) of the and 71 (DRB1*0402-L67,R71), and 70 and 71 (DRB1*0402-Q70,R71). A, peptides. Relative binding is expressed as units of europium fluorescence, ␮ Proliferative responses using 1 M peptide; B, cytolysis of target cells using 1.5 ϫ 106 cells and 10 ␮M peptide. pulsed with 1 ␮M peptide using E:T ratios of 20:1; C, relative binding of peptide VP16 393–405 to LCL surface HLA-DR expressed as units of europium fluorescence, using 1.5 ϫ 106 cells and 10 ␮M peptide. Peptide binding to the untransfected MAT cells was similar to that for the class with that of the VP16 393–403 peptide. However, the responses of II-deficient BLS-1 cell line used as a negative control. Error bars represent ESL4.34 to these APCs were greatly diminished or completely the SD of triplicate results. abrogated (Table II), indicating that these peptide residues are TCR contact residues that can modulate T cell recognition while playing only a minor role in binding to DR. VP16 393–405 are not anchor residues but that they may fur- ther stabilize the association by interacting with residues on the Discussion DR molecule. Despite the high degree of specificity of ligand recognition by To further analyze this binding motif, VP16 393–405 analogues TCRs, several reports have described T cells that can recognize with amino acid substitutions of predicted TCR contact residues were synthesized and tested for their capacity to bind to DRB1*0402 and to stimulate ESL4.34 in proliferation and cytol- Table II. Relative DRB1*0402 binding affinity of peptide VP16 393- ysis assays. Table II compares the DRB1*0402-binding and 405, the truncation form VP16 393–403, and VP16 393-403 analogs ESL4.34-stimulatory capacities of the minimum VP16-binding with a phenylalanine to isoleucine substitution at position 400 (P7 3 epitope VP16 393–403 and two analogues of this peptide with (F I)) or a methionine to norleucine substitution at position 398 (P5 (M 3NL)), and proliferative and cytolytic responses of ESL4.34 to substitutions at the predicted TCR contact positions 5 (P5 these ligandsa [M3NL]) and 7 (P7 [F3I]) of the truncated peptide. Although VP16 393–403 bound to DRB1*0402 more weakly than the full Peptide Binding Proliferation Cytotoxicity length VP16 393–405 peptide, the proliferation and cytolytic re- Peptide (Fluorescence Units/1000) (Stimulation Index) (% Specific Lysis) sponses of ESL4.34 to the two peptides were similar, indicating VP16 393-405 157.7 186.4 88.4 that the two C-terminal residues of VP16 393–405 are not in- VP16 393-403 39.8 196.4 89.2 volved in T cell responses. The binding of VP16 393–403 ana- P7 (F3I) 62.4 17.3 15.4 logues with a phenylalanine to isoleucine substitution at position P5 (M3NL) 23.9 0 ND 3 400 (P7 [F I]), or a methionine to norleucine substitution at po- a Peptide concentrations used were 10 ␮M in binding assays and 1 ␮M in pro- sition 398 (P5 [M3NL]), was better or slightly reduced compared liferation and cytotoxicity assays. E:T ratios in cytotoxicity assays were 20:1. The Journal of Immunology 3533 and respond to multiple peptide-MHC complexes. Such multiple “up” toward the TCR and into the peptide binding cleft interacting ligands can be provided by the presentation of multiple peptides by with peptide position 4, residue 70 pointing up, and residue 67 a single MHC molecule (46), presentation of a single peptide by pointing up and away from the peptide binding cleft (11, 14). multiple MHC molecules (47, 48), or ligands that are distinct in There is precedent for the importance of HVR3 in both peptide both the peptide and MHC portions (21, 49). TCR recognition binding and Ag-specific T cell responses to DRB1*0402 (51, 52), involves specific molecular contacts with residues on both the DRB1*0401 (17, 50, 53, 54), DR13 (55), and DR11 (47, 56) and MHC molecule and the bound antigenic peptide. Although the in alloreactive T cell responses (40, 57, 58). overall conformation of the peptide-MHC-TCR ternary complex is The peptides present in the five MHC class II molecules for thought to be similar for all MHC and ␣␤ TCR molecules (19, 21), which crystal structures have been determined to date, which in- the relative contributions of peptide and MHC to T cell specificity clude DR1, DR3, DR4, and I-Ek, have remarkably similar confor- can vary greatly. We therefore have investigated the structural in- mations (11–15). In every case, the peptide binds in a extended teractions between the TCR, MHC, and peptide for a CD4ϩ T cell conformation with residues at relative peptide positions P1, P4, P6, clone, ESL4.34, which recognizes the VP16 393–405 peptide P7, and P9 projecting into pockets in the MHC class II molecule, epitope of HSV-2 in the context of multiple distinct HLA-DR al- and P-2, P-1, P2, P3, P5, P8, P10, and P11 exposed to the solvent. leles. This clone, which was isolated from a DRB1*0402/*1301 Molecular modeling of VP16 393–405 complexed with heterozygous individual, responds to the peptide presented by both DRB1*0402 predicts a binding motif such that V394 occupies parental DR alleles, as well as the allogeneic DR11 allele, pocket 1 and R397 occupies pocket 4, as follows: DRB1*1102, in proliferation and cytotoxicity assays. These three

LVAPRMSFLSAGQ Downloaded from alleles are serologically and structurally distinct, but they share a common amino acid sequence motif, ILEDE, at positions 67–71 of 14679 the DR␤ polypeptide. The role of this sequence motif in supporting ESL4.34 stimulation was confirmed by testing an extended panel This motif was confirmed using substituted peptide analogues in of LCLs expressing DR4, DR13, and DR11 variants which were peptide binding and T cell stimulation assays. Substitutions of negative for this sequence and by site-directed mutagenesis exper- V394 (P1) and R397 (P4) and removal of the C-terminal residues iments in which DRB1*0404-specific substitutions were intro- S402 (P9) substantially reduced peptide binding, while substitu- http://www.jimmunol.org/ duced into the DRB1*0402 gene at codons 67, 70, and 71. We tion of L393 (P-1) or removal of the last two C-terminal residues found that proliferative and cytolytic responses of ESL4.34 to (G404 and Q405; P11 and P12) did not. Substitutions of M398 VP16 393–405 exhibited an absolute dependence on the presence (P5) and F400 (P7) had small effects on peptide binding but sub- of DR␤ I67, D70, and E71 (summarized in Table I). This restric- stantially reduced ESL4.34 T cell responses, indicating that these tion of T cell recognition appears to be due to a direct interaction changes influence TCR contact sites. The crystal structure of between these residues and the TCR, since no correlation was DRB1*0401 complexed with a human collagen II peptide (14) has found between the relative binding affinities of VP16 393–405 for confirmed previous predictions that the residue at DR␤ position 71 HLA-DR and ESL4.34 reactivity, and some DR␤ 67–71 ILEDE- influences the peptide that can be bound by forming a salt bridge negative alleles strongly bound the peptide but failed to stimulate with the side chain of the residue at peptide position P4 (5, 17, 50). by guest on September 26, 2021 the T cells. Thus, amino acid substitutions that had little or no Thus, while peptides with negatively charged P4 residues can bind effect on relative peptide binding, such as the single I67 to F67 to DRB1*0401 and other alleles with lysine or arginine at DR␤ 71, substitution that converts DRB1*1102 to *1103; mutations that peptides with positively charged P4 residues (lysine, arginine, or change DRB1*0402 to DRB1*0402-Q70 and DRB1*0402- histidine) can bind to alleles expressing the ILEDE epitope at DR␤ L67,Q70; and substitutions that distinguish DRB1*1301 and 67–71, such as DRB1*0402, DRB1*1102, and DRB1*1301. How- DRB1*1303, totally abolished ESL4.34 recognition. This suggests ever, our observation that a nonpolar alanine residue at position P4 that the responses of ESL4.34 are solely directed against HVR3 of also supported good binding to DRB1*0404 suggests that size con- the DR␤ molecule, similar to allospecific responses, but our ob- straints are also important for this interaction. servations that no T cell recognition occurred in the absence of In the diagonal binding mode observed in the class I MHC- VP16 393–405 and the responses increased proportionately with peptide-TCR crystal structures, the CDR1 loops interact with the peptide concentration (Fig. 2) confirm the Ag-specific nature of peptide termini, the CDR2 loops interact predominantly with the ESL4.34 stimulation. MHC ␣ helices, and the CDR3 loops cover the central portion of Numerous studies using allelic variants of class II and in vitro the MHC-peptide ligand (18–20). In the absence of crystallo- mutagenized class II molecules with substituted peptide analogues graphic data for class II MHC-peptide-TCR complexes, a similar or using sequence analysis of naturally processed peptides pre- diagonal binding orientation has been predicted based on shared sented by class II, have identified residues on MHC molecules that properties of the class I and class II MHC molecules (19, 21). It is are critical for peptide binding (2, 5–7) and T cell activation (9, 16, interesting to note the presence of three arginine residues in the 17, 50) and have shown that TCR recognition is dependent both on CDR3 sequence of the ESL4.34 TCR ␣ chain and an arginine at residues necessary for peptide binding and residues with little or position 97 in CDR3 of the TCR ␤ chain. A binding configuration no effect on binding. Our results indicate that DR␤ E71 is critical analogous to the crystal structures of the class I MHC-peptide- for the binding of VP16 393–405, since substitution of this residue TCR complexes would position the ESL4.34 CDR3 loops such with R in DR4 variants and in DRB1*0402 mutants (DRB1*0402- that these arginines could potentially interact with residues of the Q70,R71, and DRB1*0402-L67,R71) drastically reduced binding negatively charged ILEDE epitope. of the peptide. Less significant reductions in relative peptide bind- Our data indicate that although ESL4.34 displays promiscuous ing were associated with substitutions of DR␤ D70, while changes recognition of MHC class II molecules, its activation requirements at position 67 alone had little or no effect on binding. This hier- are highly specific and depend on both the presence of the DR␤ archy of peptide binding dependence in the order E71 Ͼ D70 Ͼ 67–71 ILEDE sequence and the VP16 393–405 peptide. Although I67 is consistent with the location of these residues in the crystal multiple DR alleles expressing the ILEDE epitope were able to structures of HLA-DR molecules, which place residues 67, 70, and present this peptide to ESL4.34, conservative substitutions at this 71 at the highest point of the DR␤␣helix, with residue 71 pointing HVR eliminated T cell reactivity. Although the presence of the 3534 PLURIALLELIC T CELL RECOGNITION

DR␤ 67–71 ILEDE epitope has an influence on peptide binding, 5. Hammer, J., P. Valsasnini, K. Tolba, D. Bolin, J. Higelin, B. Takacs, and the relative affinity of the peptide-DR interaction does not have a F. Sinigaglia. 1993. Promiscuous and allele-specific anchors in HLA-DR-binding peptides. Cell 74:197. major effect on the capacity of the ligand to stimulate ESL4.34. 6. Sette, A., J. Sidney, C. Oseroff, M.-F. del Guercio, S. Southwood, T. Arrhenius, Instead T cell recognition appears to depend on specific interac- M. F. Powell, S. M. Colon, F. C. A. Gaeta, and H. M. Grey. 1993. HLA- DR4Dw4-binding motifs illustrate the biochemical basis of degeneracy and spec- tions between the TCR and DR residues 67, 70, and 71, and pep- ificity in peptide-DR interactions. J. Immunol. 151:3163. tide residues such as methionine at P5 and phenylalanine at P7, as 7. Chicz, R. M., R. G. Urban, J. C. Gorga, D. A. Vignali, W. S. Lane, and discussed above. Other workers have also found that T cell re- J. L. Strominger. 1993. Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles. J. Exp. Med. 178:27. sponses do not depend on the affinity of peptide for MHC (59) and 8. Hill, C. M., J. D. Hayball, A. A. Allison, and J. B. Rothbard. 1991. Conforma- conservative substitutions in antigenic peptides and MHC mole- tional and structural characteristics of peptides binding to HLA-DR molecules. cules can have more dramatic effects on T cell responsiveness than J. Immunol. 147:189. 9. Allen, P. M., G. R. Matsueda, R. J. Evans, Jr., J. B. Dunbar, G. R. Marshall, and nonconservative substitutions or unrelated peptides (27–32). In E. R. Unanue. 1987. Identification of the T-cell and Ia contact residues of a T-cell this respect, the pluriallelic T cell recognition by clone ELS4.34 is antigenic epitope. Nature 327:713. reminiscent of recognition patterns of alloreactive T cells, in which 10. Sette, A., S. Buus, S. M. Co´lon, J. A. Smith, C. Miles, and H. M. Grey. 1987. Structural characteristics of an antigen required for its interaction with Ia and specific recognition of particular DR␤ HVR3 epitopes has been recognition by T cells. Nature 328:395. shown to be essential for the activation of many alloreactive T cell 11. Stern, L. J., J. H. Brown, T. S. Jardetzky, J. C. Gorga, R. G. Urban, J. L. Strominger, and D. C. Wiley. 1994. Crystal structure of the human class II clones (40, 58, 59). MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368: The peptide-self-MHC ligands recognized by developing thy- 215. mocytes have an important influence on the selection of the pe- 12. Jardetzky, T. S., J. H. Brown, J. C. Gorga, L. J. Stern, R. G. Urban,

␤ J. L. Strominger, and D. C. Wiley. 1996. Crystallographic analysis of endogenous Downloaded from ripheral T cell repertoire. In particular, the DR HVR3 residues peptide associated with HLA-DR1 suggests a common, polyproline II-like con- have been shown to determine the specificity of positive selection formation for bound peptides. Proc. Natl. Acad. Sci. USA 93:734. and exert a strong bias on the nature of ligands recognized by T 13. Ghosh, P., M. Amaya, E. Mellins, and D. C. Wiley. 1995. The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature cells selected in an individual (60, 61). Promiscuous DR-restricted 378:457. T cell recognition of Ag has been seen in the context of both 14. Dessen, A., C. M. Lawrence, S. Cupo, D. M. Zaller, and D. C. Wiley. 1997. X-ray parental alleles (48), consistent with the finding that ESL4.34 rec- crystal structure of HLA-DR4 (DRA*0101, DRB1*0401) complexed with a pep- tide from human collagen II. Immunity 7:473. ognizes VP16 393–405 in the context of the parental alleles, 15. Fremont, D. H., W. A. Hendrickson, P. Marrack, and J. Kappler. 1996. Structures http://www.jimmunol.org/ DRB1*0402 and *1301. The ILEDE epitope, present on both al- of an MHC class II molecule with covalently bound single peptides. Science 272:1001. leles, likely amplified the selection bias for this (and related) 16. Krieger, J. I., R. W. Karr, H. M. Grey, W.-Y. Yu, D. O’Sullivan, L. Batovsky, specificities. Z.-L. Zheng, S. M. Colo´n, F. C. A. Gaeta, J. Sidney, M. Albertson, M.-F. del This observation may have important implications for the se- Guercio, R. W. Chesnut, and A. Sette. 1991. Single amino acid changes in DR ␤ and antigen define residues critical for peptide-MHC binding and T cell recog- lection of autoreactive T cells. The third HVR of DR is thought nition. J. Immunol. 146:2331. to have a central role in susceptibility to autoimmune diseases 17. Fu, X.-T., C. P. Bono, S. L. Woulfe, C. 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