A Peptide Binding Motif for I-Eg7, the MHC Class II Molecule That ...

A Peptide Binding Motif for I-Eg7, the MHC Class II Molecule That Protects Ea-Transgenic Nonobese Diabetic Mice from Autoimmune Diabetes1 Silvia Gregori,* Sylvie Trembleau,* Giuseppe Penna,* Fabio Gallazzi,* Juergen Hammer,* George K. Papadopoulos,† and Luciano Adorini2* The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due a deletion in the Ea gene promoter. Ea-transgenic NOD mice express the EaEbg7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7restricted T cell epitope corresponding to the sequence 4 –13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and Ea-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in Ea-transgenic NOD mice. The Journal of Immunology, 1999, 162: 6630 – 6640.

N

onobese diabetic (NOD)3 mice spontaneously develop autoimmune diabetes and are a model of the human insulin-dependent diabetes mellitus (IDDM) (1). At least 20 loci are now known to contribute to IDDM development in NOD mice and at least one, the MHC locus, is essential (2). NOD mice express a rare I-A allele, I-Ag7 (3) found only in NOD and in Biozzi AB/H mice (4), but fail to express I-E molecules on the cell surface due to a deletion within the first exon of the Ea locus (5). The Ebg7 chain is synthesized in NOD mice and its sequence has been found to be unique (6). Ea-transgenic NOD mice express the Ea:Ebg7 molecule and are protected from insulitis and IDDM (7– 9). Therefore, not only is the I-Ag7 molecule required for disease, but the lack of I-E expression is essential for IDDM development in NOD mice. This is paralleled by the positive, neutral, or negative association of particular class II molecules with human IDDM (10). Although expression of transgenic class II molecules in NOD mice has provided direct evidence for a protective effect of non

*Roche Milano Ricerche, Milan, Italy; and †Laboratory of Biochemistry and Biophysics, Technological Educational Institute of Epirus, Arta, Greece Received for publication November 30, 1998. Accepted for publication March 12, 1999. 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 European Community contract BIO4-CT960077. 2 Address correspondence and reprint requests to Dr. Luciano Adorini, Roche Milano Ricerche, Via Olgettina 58, I-20132 Milan, Italy. E-mail address: Luciano. [email protected] 3 Abbreviations used in this paper: NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; DOC, sodium deoxycholate; HEL, hen egg-white lysozyme; hsp, heat-shock protein; p, position; Mt, Mycobacterium tuberculosis; SC50, 50% of maximum stimulation.

Copyright © 1999 by The American Association of Immunologists

diabetogenic I-A or I-E molecules, their mechanism of action remains controversial. Several hypotheses have been proposed to explain the mechanism of protection afforded by expression of I-Eg7 molecules (11). Deletion of pathogenic autoreactive cells by the transgenic class II molecule was first proposed (12), then dismissed (8, 13), and recently reproposed (14). Other hypotheses formulated to explain protection from IDDM in class II transgenic mice include different possibilities for epitope stealing (1), determinant capture (15), and deviation of the immune response to the Th2 pathway by the protective class II molecules (16). Another proposed explanation for the protective effect of I-Eg7 molecules predicts that peptides derived from I-Eg7 can compete with diabetogenic peptides for binding to I-Ag7. Thus, both central and peripheral mechanisms have been considered and all, except the latter, require peptide binding to I-Eg7 molecules. However, the rules that govern peptide binding to I-Eg7 molecules have not yet been defined. In this study we have identified an I-Eg7-restricted T cell epitope in the sequence 4 –13 of Mycobacterium tuberculosis (Mt) 65-kDa heat shock protein (hsp). Interestingly, a similar epitope has been found to be recognized by DR3-restricted T cells and to bind selectively to DR3 molecules (17). The immune response to hsp Ags has been associated with T cell-mediated regulation of inflammatory diseases (18), in particular with induction as well as protection from adjuvant arthritis in rats (19) and autoimmune diabetes in NOD mice (20). In addition, a spontaneous T cell proliferative response to 65-kDa hsp could be induced in cultures established from 8-wk-old NOD females and was enhanced in 24-wk-old NOD mice, but was absent in other mouse strains, thus suggesting its possible role as an autoantigen recognized by diabetogenic T cells (21). We used hsp4 –13 as a template to analyze residues involved in binding to purified I-Eg7 molecules and in interaction with the 0022-1767/99/$02.00

The Journal of Immunology

6631

FIGURE 1. T cell proliferation to overlapping 65-kDa hsp peptides. T cells from NOD (filled bars) or Ea-transgenic NOD (open bars) mice immunized with recombinant Mt 65-kDa hsp protein were restimulated in vitro with 5 mM of 16-mer peptides of Mt 65-kDa hsp, overlapping by eight residues, or 5 mM of hsp protein or 10 mg/ml of PPD (purified tuberculin protein derivate). [3H]Thymidine incorporation was determined after 3 days of culture. The results are expressed as geometric mean of stimulation indices (ratio between cpm of triplicate wells from lymph node cells cultured with or without Ag) from three to four separate experiments.

TCR. A comparison of the peptide binding motif for I-Eg7 defined herein with the binding motif we have previously identified for I-Ag7 (22) reveals clear-cut differences. Analysis of overlapping peptides spanning the entire 65-kDa hsp sequence both for binding to I-Ag7 or I-Eg7 molecules and for proliferative responses in cells from NOD and Ea-transgenic NOD mice confirms the differences between the binding mode of I-Eg7 and I-Ag7 molecules and suggests that competition between these two MHC molecules for binding the same peptide is a very unlikely event. The definition of a motif for peptide binding to I-Eg7 should facilitate analysis of the mechanisms protecting Ea-transgenic NOD mice from IDDM.

Materials and Methods Mice NOD/Lt mice from The Jackson Laboratories (Bar Harbor, ME) were isolator-reared at Charles River Laboratories (Calco, Italy) and kept under specific pathogen-free conditions in our animal facility. Eak-transgenic NOD mice (8) were kindly provided by C. Benoist and D. Mathis (Strasbourg, France). Mice were used when 8 wk old.

Purification of I-Eg7 and I-Ag7 molecules I-Eg7 and I-Ag7 molecules were affinity-purified from detergent lysates of 4G4.7 B hybridoma cells by sequential desorption from 34.1.4 and 14.4.4S, or from OX-6 mAb respectively, as previously described (22). The 4G4.7 B cell hybridoma was derived by polyethylene glycol-induced fusion of NOD mouse T cell-depleted splenocytes with the HAT (hypoxanthine/ aminopterin/thymidine)-sensitive A20.2J lymphoma line (23). It expresses I-Ag7, I-Ad, I-Eg7 and I-Ed. The following mAbs were used for I-Eg7 purification: 14.4.4S, a mouse monoclonal IgG2a Ab recognizing the Eachain (24); 34.1.4, a mouse monoclonal IgG1 Ab against the Ebd chain that does not bind to I-Eg7 (24); and OX-6, a mouse monoclonal IgG1 Ab against an invariant chain determinant of rat Ia, which also recognizes I-Ag7 but not I-Ad (25). Approximately 20 mg of purified mAb was first bound to 5 ml of protein A-Sepharose 4 Fastflow (Pharmacia, Uppsala, Sweden) and then chemically cross-linked to the protein A with dimethyl pimelimidate dihydrochloride (Pierce, Rockford, IL) in sodium borate buffer (pH 9.0). After 40 min incubation at room temperature, the reaction

was quenched by adding in 0.2 M ethanolamine (pH 8.0) for 60 min. The suspension was thoroughly washed in PBS and stored in PBS containing 0.02% NaN3. 4G4.7 cells were harvested by centrifugation, washed in PBS, resuspended at 108 cells/ml in lysis buffer, and then allowed to stand at 4°C for 120 min. The lysis buffer was 0.05 M sodium phosphate (pH 7.5) containing 0.15 M NaCl, 1% (v/v) Nonidet P-40 detergent, and the following protease inhibitors: 1 mM PMSF (Sigma, St. Louis, MO), 5 mM e-amino-n-caproic acid (Sigma), and 10 mg/ml each of soybean trypsin inhibitor, antipain, pepstatin, leupeptin and chymotrypsin (Sigma). Lysates were cleared of nuclei and debris by centrifugation at 27,000 3 g for 30 min and stored as such if not immediately processed further. A total of 0.2 vol of 5% sodium deoxycholate (DOC) (Sigma) was then added to the postnuclear supernatant. After mixing at 4°C for 10 min, the supernatant was centrifuged at 100,000 3 g at 4°C for 120 min, carefully decanted, and filtered through 0.45-mm nylon membrane. The lysate of 1011 4G4.7 cells was recycled overnight at 4°C on a 34.1.4 protein A-Sepharose column and then on 14.4.4S protein A-Sepharose to obtain I-Eg7, and on OX-6 protein A-Sepharose to obtain I-Ag7. The columns were then washed with at least 20 vol of buffer A (0.05 M Tris (pH 8.0), 0.15 M NaCl, 0.5% NP40, 0.5% DOC, 10% glycerol and 0.03% NaN3), 5 vol of buffer B (0.05 M Tris (pH 9.0), 0.5 M NaCl, 0.5% NP40, 0.5% DOC, 10% glycerol, and 0.03% NaN3), and 5 vol of buffer C (2 mM Tris (pH 8.0), 1% octyl-b-D-glucopyranoside (OGP; Sigma), 10% glycerol, and 0.03% NaN3). Bound MHC molecules were eluted with 50 mM diethylamine HCl (pH 11.5) in 0.15 M NaCl, 1 mM EDTA, 1% OGP, 10% glycerol, and 0.03% NaN3, and immediately neutralized with 1 M Tris. Approximately 2 mg of protein was purified from 1011 4G4.7 cells. In SDS-PAGE, the majority (.95%) of the protein was resolved as two bands of m.w. ;33,000 and ;28,000 that correspond to the a and b subunits, respectively, of class II MHC molecules. Purity of Eg7 molecules was assessed by their lack of reactivity with the anti-Ebd mAb 34.1.4 in a peptide binding assay.

Peptide synthesis Peptides were synthesized with a multiple peptide synthesizer (model 396; Advance Chemtech, Louisville, KY) using Fmoc chemistry and solid phase synthesis on Rink Amide resin (Novabiochem, Laufelfingen, Switzerland). All acylation reactions were effected with 3-fold excess of activated Fmoc amino acids, and a standard coupling time of 20 min was used. Cleavage and side chain deprotection was achieved by treating the resin with 90% trifluoroacetic acid, 5% thioanisole, 2.5% phenol, and 2.5% water. The

I-Eg7 PEPTIDE BINDING MOTIF

6632 Table I. Truncation analysis of the I-Eg7-restricted hsp1–16 epitopea

hsp1–16 hsp2–16 hsp3–16 hsp4–16 hsp5–16 hsp6–16 hsp7–16 hsp1–15 hsp1–14 hsp1–13 hsp1–12 hsp1–11 hsp1–10 hsp1–9

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

M

A A

K K K

T T T T

I I I I I

A A A A A A

A A A A A A A

K K K K K K K

T T T T T T T

I I I I I I I

A A A A A A A

D D D D D D D D D D D D D D

E E E E E E E E E E E E E E

E E E E E E E E E E E E E

A A A A A A A A A A A A

R R R R R R R R R R R

R R R R R R R R R R

G G G G G G G G G

L L L L L L L L

E E E E E E E

M M M M M M M

Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Activation of T Cell Hybridoma (SC50, nM)

Binding to I-Eg7 (IC50, nM)

4BII

4HI

388 275 370 380 5,000 50,000 27,500 400 390 387 3,800 4,000 27,500 27,500

16 32 64 64 1,200 1,200 2,000 64 16 60 5,000 4,000 4,000 4,000

32 48 80 64 1,600 10,000 4,000 160 32 48 10,000 10,000 10,000 10,000

a Synthetic peptides representing sequential truncations of hsp1–16 were tested for binding to purified I-Eg7 molecules and for T cell hybridoma activation, as described in Materials and Methods. The results are shown as IC50 for binding and SC50 values for T cell activation.

indicator peptide for the binding assays was biotinylated before being cleaved from the resin by coupling two 6-aminocaproic acid spacers and one biotin molecule at the NH2 terminus sequentially, using the above described procedure. Peptides were routinely $85% pure as analyzed by reverse-phase HPLC.

Peptide binding assay Peptides were dissolved at 10 mM in DMSO and diluted into 25% DMSO/ PBS for assay. The indicator peptides hsp1–16 for I-Eg7, and HEL10 –23 for I-Ag7 were synthesized with two spacer residues and a biotin molecule at the NH2 terminus. Approximately 500 nM of biotinylated peptide and each test peptide diluted 10-fold from 50 mM to 50 pM were coincubated with 200 ng of MHC class II protein in U-bottom polypropylene 96-well plates (Costar Serocluster, Costar, Cambridge, MA) in binding buffer at room termperature. The binding buffer was 6.7 mM citric phosphate, pH 5.0, for I-Eg7, and pH 6.0 for I-Ag7, with 0.15 M NaCl, 2% NP40, 2 mM EDTA, and the protease inhibitors as used in the lysis buffer. After 48 h, each incubate was transferred to the corresponding well of an ELISA plate (Maxisorp, Nunc, Roskilde, Denmark) containing pre-bound 14.4.4S or OX-6 Abs (10 mg/ml overnight at 4°C followed by washing). After incubation at 37°C for 2 h and washing, bound biotinylated peptide-MHC complexes were detected colorimetrically at 405 nm with streptavidin-alkaline phosphatase and p-nitrophenylphosphate. Competition curves were plotted and the peptide affinity for MHC molecules was expressed as the peptide concentration required to inhibit the binding of biotinylated-peptide by 50% (IC50).

T cell proliferation Mice were immunized subcutaneously into the hind footpads with 50 mg 65-kDa hsp (a gift from Dr. Ruurd van der Zee, University of Utrecht, The Netherlands) emulsified in CFA containing H37Ra Mycobacteria (Difco,

Detroit, MI). Nine days later, popliteal lymph node cells were cultured (5 3 105 cells/well) in 96-well culture plates (Costar) in synthetic HL-1 medium (Ventrex Laboratories, Portland, ME) supplemented with 2 mM L-glutamine (Life Technologies, Grand Island, NY) and 50 mg/ml gentamicin (Sigma) with 10 mM of Ag. Purified tuberculin protein derivate (Statens Seruminstitut, Copenhagen, Denmark) was used as positive control for each culture at the final concentration of 10 mg/ml. Cultures were incubated for 3 days in a humidified atmosphere of 5% CO2 in air and pulsed 8 h before harvesting with 1 mCi [3H]thymidine (Amersham, Arlington Heights, IL). Thymidine incorporation was measured by scintillation spectrometry. The proliferative response was expressed as stimulation index, the ratio between cpm of triplicate wells from lymph node cells cultured with or without Ag.

T cell hybridoma activation The T cell hybridomas 4HI and 4BII were generated by polyethylene glycol-induced fusion of hsp1–16-immune lymph node cells from Ea-transgenic NOD mice with the TCR a/b-negative variant of the BW5147 thymoma, as previously described (26). Reactivity of 4HI and 4BII to hsp peptides was assayed by incubating 5 3 104 4G4.7 B hybridoma cells and hsp peptides (0.1 nM to 10 mM) with 5 3 104 T hybridoma cells/well. The response of T cell hybridomas was determined to be Eg7-restricted by the in vitro inhibition of IL-2 production by 14.4.4S but not 34.1.4 mAb. Culture medium was RPMI 1640 supplemented with 10% FCS, 2 mM Lglutamine (Life Technologies), 50 mg/ml gentamicin (Sigma), and 50 mM 2-ME (Fluka Biochemica, Buchs, Switzerland). After 24 h of culture, 50 ml of supernatant was transferred to culture wells containing 104 IL-2-responsive CTLL cells. During the final 5 h of a 24-h culture, CTLL cells were pulsed with 1 mCi [3H]thymidine (The Radiochemical Centre, Amersham,

Table II. Effect of alanine substitution at each position in hsp4 –13 on binding to I-Eg7 and T cell activationa

hsp4–13 hsp4–13 hsp4–13 hsp4–13 hsp4–13 hsp4–13 hsp4–13 hsp4–13 hsp4–13

(A4) (A5) (A7) (A8) (A9) (A10) (A12) (A13)

p21 4

p1 5

p2 6

p3 7

p4 8

p5 9

p6 10

p7 11

p8 12

p9 13

I-Eg7 Binding (IC50, nM)

T A T T T T T T T

I I A I I I I I I

A A A A A A A A A

Y Y Y A Y Y Y Y Y

D D D D A D D D D

E E E E E A E E E

E E E E E E A E E

A A A A A A A A A

R R R R R R R A R

R R R R R R R R A

400 600 10,000 1,500 9,000 300 400 700 1,500

4BII (SC50, nM)

30 80 700 ..10,000 ..10,000 ..10,000 70 ..10,000 200

4HI (SC50, nM)

80 200 1,000 ..10,000 ..10,000 ..10,000 60 ..10,000 .10,000

a Alanine (A) scan of hsp4 –13 was analyzed for binding to purified I-Eg7 molecules and in T cell hybridoma activation. The A substitutions are in bold. The results are shown as IC50 for binding and SC50 values for T cell activation.


6633

FIGURE 2. Effect of single amino acid substitutions in hsp4 –13 on binding to I-Eg7 and on T cell activation. A, Peptides with single amino acid substitutions at different positions in the hsp4 –13 sequence were tested for binding to purified I-Eg7 molecules by ELISA. Different concentrations (50 pM to 50 nM) of unlabeled peptide were coincubated with 200 nM biotinylated hsp4 –13 and with 200 ng of affinity-purified I-Eg7 molecules, as described in Materials and Methods. Results are expressed as ratio between the concentration of peptide required to inhibit the binding of biotinylated hsp4 –13 by 50% (IC50) relative to the IC50 of the unsubstituted hsp4 –13 peptide and represent the arithmetic mean of two to four experiments. B, Activation of T cell hybridomas by peptides with single amino acid substitutions at different positions in the hsp4 –13 sequence. 4G4.7 B cell hybridoma cells (I-Ag7, I-Eg7) were cocultured with the hsp4 –13-specific I-Eg7-restricted T cell hybridomas 4HI and 4BII and different concentrations of peptide (0.3 nM-10 mM). After 24 h of culture, 50 ml of supernatants were transferred to culture wells containing 104 CTLL-2 cells. Cells were pulsed with [3H]thymidine during the final 4 h of a 24-h culture. Results are expressed as ratio between the concentration of peptide that induced 50% maximum stimulation (SC50) relative to the SC50 of the unsubstituted hsp4 –13 peptide and represent the arithmetic mean of two to four experiments.

U.K.). Thymidine incorporation was measured by scintillation spectrometry. The concentration of peptide that caused 50% of maximum stimulation is referred to as SC50.

Structure of I-Eg7 and I-Es modeled on the I-Ek molecule The coordinates of the I-Ek homodimer of heterodimers complexed to the hemoglobin peptide (27) were obtained from the Protein Data Bank(Brookhaven National Laboratory, Upton, NY: access code 1iea.pdb). Because of the very high degree of homology between I-Ek, I-Eg7, and I-Es alleles, the alignment of these molecules presented no problems. Modeling of I-Eg7 and I-Es was performed using hsp4 –13 and b2-microglobulin 46 –56 (28) as antigenic peptide, respectively. Molecular modeling was performed on a Silicon Graphics Indy workstation using the program Insight II, version 95.0 (Biosym Technologies/Molecular Simulations, San Diego, CA). The individual amino acid conformations were automatically chosen from a library of rotamers provided by the program, using the most suitable rotamer for each case. An automatic energy minimization was performed after the replacement of each substituted amino acid residue. Interactions between identical amino acids in equivalent positions were preserved. The ionization state of amino acid side chains was that at pH 7.0. Energy minimization was accomplished by the steepest gradient method first, followed by the conjugate gradient method, using the program Discover (Biosym/ Molecular Simulations version 2.9.7/95.0/3.00). The minimization proce-

dure went through 1000 cycles for each method. The force field used included electrostatic terms for interactions up to 16 Å. For comparison purposes, the published structure of DR1 was subjected to the same minimization procedure, yielding an average root mean square deviation for all Ca atoms of 0.52 Å, and for all atoms of 0.76 Å.

Results hsp4 –13 is a dominant T cell epitope presented by I-Eg7 molecules To identify I-Eg7-restricted T cell epitopes, NOD and Ea-transgenic NOD mice (Ea16) were primed with Mt 65-kDa hsp. Nine days after priming, draining lymph node cells were restimulated in vitro with a panel of overlapping peptides spanning the entire hsp sequence. Several hsp peptides restimulated an I-Eg7-restricted T cell response, as demonstrated by the proliferative response in lymph node cells from NOD-Ea16 but not NOD mice (Fig. 1). Conversely, T cell proliferation in lymph node cells from both NOD and NOD-Ea16 mice indicates an I-Ag7-restricted response. The highest I-Eg7-restricted response was induced by the hsp1–16, and this immunodominant epitope was selected for further studies.


6634 Table III. Effect of single amino acid substitutions of hsp4 –13 on binding to I-Eg7 Relative Position in hsp4–13a 1

I

2 A

3 Y

4 D

5 E

6 E

7 A

8 R

9 R

A R N G H I L K M P S T W Y V D Q F

Q N A S T E D G I P V L

R N D E Q H I L K M F F S T

I L K F P Y V

K M F

Y M Y

C W E

N D Q Q G R M S T W

A N

Well-tolerated, IC50 ¶1 mM

L V

Q G H I L K F F S T W V

I L V

E

Weakly tolerated, IC50 1–10 mM

M F W

R N D E M Y

A R W D E Q G H K M F P S T W

S M V

Not tolerated, IC50 . 10 mM

A R N D E Q G H K P S T Y

A R N Q G H I L K F P T W Y

K W R H W

G I L K S T Y V

D E H P

a Amino acid residues in bold represent primary anchors; amino acid residues set roman and capitalized represent secondary anchors. Italics represent residues not directly involved in peptide binding to I-Eg7.

This peptide binds to purified I-Eg7 molecules with high affinity (Table I). Binding assays were conducted at pH 5, because at this pH binding was stronger than at pH 6 or 7 (data not shown). The competition assay with purified I-Eg7 is sensitive, specific and highly reproducible. In 15 experiments, the average IC50 for competition between biotinylated and unlabeled hsp1–16 was about 300 nM. Peptides representing sequential truncations of hsp1–16, from either the NH2 or COOH terminus, were each assayed for binding to I-Eg7 and for their ability to activate the hsp1–16-specific, I-Eg7restricted 4HI and 4BII T cell hybridomas, which express different Vb8 chains. Removal of T4 or R13 reduced by 10-fold or more the binding capacity of the peptide and reduced T cell activation by at least 20-fold (Table I). Binding and T cell activation data thus indicate that the minimum good binder to I-Eg7 and the minimum epitope for T cell activation is hsp4 –13. g7

hsp4 –13 residues involved in interaction with I-E

and TCR

Substitution of alanine (A) at each position, except A6 and A11, in hsp4 –13 revealed two primary anchors in I5 and D8, as substitution

at either of these positions nearly abolished peptide binding to the purified I-Eg7. Two secondary anchors were apparent in Y7 and R13, positions at which binding of the A-substituted peptides was 4-fold reduced (Table II). Conversely, while having no effect on binding, A substitutions at E9 and R12 abrogated T cell activation. Similarly, A substitution at Y7 had little effect on binding but abrogated T cell hybridoma activation. For the purpose of further analysis, the relative positions (p) of I, Y, D, E, R, and R in the epitope hsp4 –13 are designated p1, p3, p4, p5, p8, and p9. Therefore, analysis of A-substituted peptides indicates that the sequence hsp4 –13 contains two primary anchors, at p1 and p4, and two secondary anchors, at p3 and p9, involved in interaction with I-Eg7. Conversely, residues in p3, p5, and p8 appear to be primarily involved in interaction with the TCR. A motif for peptide binding to I-Eg7 To define a motif for peptide binding to the I-Eg7 molecule, we first investigated the effect on binding and T cell activation of all natural amino acid substitutions (except labile cysteine) at p1, p3, p4,


6635

FIGURE 3. Anchor positions and TCR contact sites in the hsp4 –13 peptide. A, The minimal T cell epitope hsp4 –13 shows TCR contact residues at the relative positions 3, 5, 8, 9. Primary anchors for binding to I-Eg7 are at the relative position 1 and 4, whereas secondary anchors are at positions 6 and 9. B, Molecular modeling of peptide hsp4 –13 bound to I-Eg7 as looked at from the side of the b1 helix, at the level of the b-sheet floor. The I-Eg7 molecule has been omitted for clarity. The anchoring residues into the groove are seen pointing downwards, whereas the putative TCR contact residues point upwards.

p5, p8, and p9. All substitutions were tolerated, as defined by a decrease in binding affinity of up to 10-fold, at p3, p5, and p8. Conversely, the binding affinity was decreased by more than 10fold by several substitutions at p1, p4, and p9 (Fig. 2A). In particular, at p1 and p4 most substitutions were not tolerated and only a few conservative substitutions failed to decrease peptide binding to I-Eg7, indicating that these two positions are primary anchors for peptide binding to purified I-Eg7. At p9, 5 of 18 substitutions were not tolerated, indicating this position as a secondary anchor. Analysis of the effect of single amino acid substitutions on T cell activation clearly demonstrates that p3, p5, and p8 are involved in interaction with the TCR (Fig. 2B). At p1, p4, and p9 only welltolerated substitutions are able to activate I-Eg7-restricted T cell hybridomas. We conclude that p1 and p4 are primary anchors and p9 is a secondary anchor for binding to I-Eg7 molecules. Residues at p3, p5, and p8 are primarily involved in interaction with the TCR, whereas p9 appears to represent a secondary position for interaction with the TCR. Although A substitution for E at p6 does not affect the binding of hsp4 –13 to I-Eg7 (Table II), this position has been found involved in peptide binding to other I-E alleles (see Table IV). Therefore, we analyzed all natural amino acid substitutions at p6 to verify its role in binding to I-Eg7. Inspection of these data reveals that, as in p9, a few substitutions were not tolerated at this position. Conversely, most of the substitutions tolerated for binding did not affect T cell activation (Fig. 2), suggesting that p6 is a secondary anchor for binding to I-Eg7. We also examined the effect of all natural amino acid substitutions at p2 and p7 for peptide binding to

FIGURE 4. Molecular modeling of pocket 4 in I-Eg7 and I-Es with a favored anchor residue. The p4Asp anchor residue of the hsp4 –13 peptide fits well in the p4 pocket of I-Eg7, with the two lysines (b28 and b71) forming salt bridges with each of its carboxylate oxygens. Only acidic residues (D, E) would fit well in this pocket. In I-Es, the bulky b71Arg points away from the p4 pocket due to the influence of the negatively charged b28Glu, thus favoring binding of large hydrophobic residues, such as Leu at p4 of the b2-microglobulin 46 –56 peptide (28).

I-Eg7 and found them either well or weakly tolerated, as in p5 (Table III). The effects of single amino acid substitutions on the binding of hsp4 –13 to I-Eg7 are summarized in Table III. Single residue substituted peptides were classified, according to their IC50 binding values, as well tolerated (#1 mM), weakly tolerated (1–10 mM), or nontolerated (.10 mM). Optimally, p1 is a large hydrophobic residue (I, L, V), whereas p4 is negatively charged (D, E). Most other amino acids are not tolerated at these positions. Specific amino acids are not tolerated at other positions, namely K, W, R, and H


6636 Table IV. I-E binding motifsa 1 k

2

3

Comparison between I-E binding motifs and nature of the residue-anchoring pockets 4

5

6

7

8

9

I-E

I L V F Y W

I L V F S A

Q N A G S T R

R K G

I-Ed

F Y W I L V

K R I V

I L V G

R K

I-Es

F Y W I L V

I L V S H Q

Q N A S T H R

R K

I-Eb

I L V F Y W

I L V F A

Q N A S T H R E

R K

I-Eg7

I L V F W M

D E S M V

Q N A S T E D G I P V L

R K M F

a Residues in bold represent well-tolerated amino acids. Binding motifs for I-Ek, I-Ed, I-Es, and I-Eb are from Reference 28. Only well-tolerated residues are indicated at p6 and p9 in I-Eg7.

at p6; D, E, H, P, and W at p9 (Table III). Accordingly, the minimum rules defining a motif for peptide binding to I-Eg7 could be described as follows: 1) good binders must have at least one welltolerated residue in p1 and/or in p4 and the other residue should be at least weakly tolerated, and 2) no nontolerated residues should be present in p6 and p9. Based on the above results, it appears that four residues in the hsp sequence 4 –13 interact with the TCR: p3, p5, and p8 correspond to primary interaction residues, whereas p9 represents a secondary interaction site. Four residues are involved in binding to I-Eg7: p1 and p4 are critical for binding and represent primary anchors, whereas p6 and p9 are less stringent in amino acid requirements and can be considered secondary anchors (Fig. 3A). Fig. 3B shows the orientation of the various residues of the hsp4 –13 peptide bound to I-Eg7 according to molecular modeling by energy minimization based on the peptide-Ek crystal structures (27). Residues p-1, p3, p5, and p8 point away from the groove and toward the solvent, whereas residues p1, p4, p6, and p9 point into the groove. Residue p7 is partly buried and partly exposed.

Comparison of the I-Eg7 binding motif with other defined I-E binding motifs (28) reveals shared anchor residues at p1 and p9, whereas the allele-specific residues are found at p4 and p6 (Table IV). In particular, negatively charged residues at p4 are unique in the I-Eg7 binding motif. Molecular modeling of I-Eg7 a and b side chains involved in interactions with anchor residues indicates that pockets 1, 6, and 9 are formed by the same residues as in the crystal structures of I-Ek molecules (27). This explains why in peptides that bind to I-Ek or I-Eg7 molecules the same residues are well tolerated at p1, p6, and p9 (Table IV). Conversely, the pocket 4 of the I-Eg7 allele appears to have the unique property of favoring an acidic residue (E or D), likely because of the b71Lys that allows for the formation of a salt bridge with the p4 acidic residue (Fig. 4). Comparison between pocket 4 of I-Eg7 and I-Es exemplifies the role of conservative substitutions in the MHC class II molecule on the accepted residues. I-Eg7 has Lys at b28 and b71, whereas I-Es has a Glu at b28 and an Arg at b71. According to molecular modeling, the two lysines of I-Eg7 point into the p4 pocket and establish two salt bridges, one with each of the carboxylate oxygens of the acidic p4 anchor residue (D or E). In I-Es the bulky b71Arg and the negatively charged b28Glu point toward each other, projecting the charged guanidine end of the b71Arg away from the p4 pocket. In addition, the b71Arg would point parallel to the b-pleated sheet floor unlike the downward pointing of b71Lys in the I-Eg7 molecule (data not shown). Therefore, the I-Eg7 pocket binds preferably negatively charged residues (D, E), whereas the enlarged pocket 4 in I-Es favors binding of larger hydrophobic residues (I, L, V). Presence of the I-Eg7 binding motif in overlapping peptides from 65-kDa hsp To validate the I-Eg7 binding motif, we tested 16-mer peptides overlapping by seven residues and spanning the entire sequence of Mt 65-kDa hsp protein for binding to I-Eg7, and inspected them for the presence of the binding motif (Table V). Eight of nine (89%) high-affinity binders (IC50 # 1 mM) contained a motif. Conversely, a motif was present only in 16 of 89 (18%) weak or nonbinders (data not shown). Clearly, the binding motif does not fully account for the effects of residue combinations or for flanking sequences, but could help in the identification of peptides binding with high affinity to I-Eg7. Binding to I-Eg7 or I-Ag7 and T cell proliferative responses of lymph node cells from NOD and NOD-Ea transgenic mice to overlapping hsp peptides Analysis of the primary anchor positions in peptides binding to I-Eg7 (present paper) and I-Ag7 (22) reveals major differences in the two binding motifs (Table 6). In the I-Eg7 binding motif the primary anchor residues are found at p1 and p4, whereas in the I-Ag7 binding motif they are found at p6 and p9. At two distinct primary anchor positions, p1 for I-Eg7 and p6 for I-Ag7, only large hydrophobic residues are well tolerated. The comparison between p4 in I-Eg7 and p9 in I-Ag7 binding motifs indicates that these two class II MHC molecules have different binding specificity. Only negatively charged residues are well tolerated at p4 in peptides binding to I-Eg7, whereas only positively charged or aromatic residues are well tolerated at p9 in peptides binding to I-Ag7. The secondary anchors are also at different positions, p6 and p9 in peptides binding to I-Eg7 vs p3 and p8 in peptides binding to I-Ag7. Using a panel of overlapping hsp peptides, both binding to purified I-Eg7 and I-Ag7 as well as proliferative responses of draining


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Table V. Presence of binding motif in Mt hsp 65-kDa high-affinity binders to I-Eg7 a 1

1–16 43–58 55–70 157–172 181–196 301–316 421–436 439–454 499–514

W

S

A

M G I

A

P

E T

L

Q

N

A A A

E L I A

K P K G F V D V A

T T E D G G E K S

I I I L L L L V I

4

A T E I Q T K A A

Y N L A L L L L G

D D E E E E E E L

6

E G D A L N G A F

E V P M T A D P L

9

A S Y D E D E L T

R I E K G L A K T

R A K V M S T Q

Motif

G K I G R L G I

L E G N F L

E A E D G

G K E

A

F

N

V G

Yes Yes Yes Yes Yes Yes Yes Yes No

a A panel of overlapping peptides encompassing the entire sequence of hsp 65 kDa was tested for binding to I-Eg7. Highaffinity binders (IC50 # 1 mM) were inspected for the presence of the I-Eg7 binding motif. Residues at p1, p4, p6, and p9 are anchor positions. Primary anchor positions are indicated in plain and secondary anchors in italic capitals. Well-tolerated residues are bold, and weakly tolerated residues are underlined.

lymph node cells from hsp-primed NOD and Ea-transgenic NOD mice were tested (Fig. 5). These results indicated that peptides capable of binding to I-Eg7 or I-Ag7 molecules with high affinity (IC50 # 1 mM) induced proliferative responses of lymph node cells (stimulation index .3). A few peptides were able to bind to I-Eg7 or to I-Ag7, but failed to induce proliferative responses. Interestingly, none of the 80 hsp peptides tested was able to bind with high affinity to both I-Eg7 and I-Ag7 molecules, as predicted from the major differences in the two binding motifs. This finding was paralleled by the lack of overlap between I-Eg7- and I-Ag7restricted T cell epitopes. Nine hsp epitopes were found to be I-Ag7-restricted: 13–28, 337–352, 343–358, 367–382, 373–388, 397– 412, 433– 448, 463– 478, 481– 496; and seven I-Eg7-restricted: 1–16, 55–70, 61–76, 301–316, 421– 436, 439 – 454, 457– 472 (Fig. 5).

Discussion The present study defines a motif for peptide binding to the I-Eg7 molecule, which is associated with protection from insulitis and IDDM in Ea-transgenic NOD mice. Using the I-Eg7-restricted T cell epitope hsp4 –13 as a template, we have identified an I-Eg7binding motif characterized by two primary anchors at the relative positions p1 and p4, and two secondary anchors at p6 and p9. At Table VI. Comparison between I-Eg7 and I-Ag7 binding motifsa I-Eg7 1

I L V F W M

2

3

4

D E S M V

5

I-Ag7 6

Q N T A E D G I P S T V L R H Y M F

7

8

9

K R M F A N Q G I L K S T W Y V

1

2

3

A Q L K Y D G P S W

4

5

6

I L M V N D H K F P T Y

7

8

9

A N G P S R D E Q L K F

R K F Y N D Q H L M P W

a Primary anchor positions are indicated in plain text and secondary anchor positions in italic capitals. Well-tolerated residues (IC50 # 1 mM) are in bold, and weakly tolerated residues (IC50, 1–10 mM) are in plain text capitals.

p1 a large hydrophobic or aromatic anchor residue is required for peptide binding to I-Eg7. These characteristics of the p1 anchor displayed by I-Eg7-binding peptides are shared by peptides binding to I-E molecules of k, d, s, and b haplotypes (28). The two secondary anchors at p6 and p9 are also similar in peptides binding to these I-E alleles and to I-Eg7. However, at p4 a negatively charged residue (D, E) is required for high-affinity peptide binding to I-Eg7 but not to other I-E molecules. This fine specificity makes the I-Eg7 binding motif unique among those reported for I-E molecules. Intriguingly, an hsp epitope similar to the I-Eg7-restricted T cell epitope hsp4 –13 was found to be recognized by HLA-DR3-restricted T cells (29). Binding of hsp3–13 to HLA-DR3 required a large hydrophobic residue at p1 and a negatively charged residue at p4 (17). The remarkable similarity of the motif for peptide binding to I-Eg7 and DR3 probably depends on the identical amino acid residues forming pockets 1 and 4 in these class II MHC molecules. The three-dimensional structure of I-Eg7 was modeled by energy minimization based on the crystal structure of peptide-Ek complexes (27). The I-Ek and I-Eg7 alleles are about 90% identical in the peptide binding a1b1 domain and .95% identical in the a2b2 homodimerization domain, thus facilitating homology modeling. According to molecular modeling, the I-Eg7 molecule possesses a unique pocket 4. Its propensity to accommodate acidic residues can be explained by the presence of Lys at b28 and b71, which appears to point into the p4 pocket and form two salt bridges, one with each of the carboxylate oxygens of the acidic p4 anchor residue (D or E). Pocket 6 is widely accommodating, except for basic residues, whereas pockets 1 and 9 accommodate aliphatic and basic residues, respectively. These binding preferences of p1, p6, and p9 are also found in all other I-E alleles analyzed (28), and the only residue differing in some (i.e., the b and d alleles) is b86Ser, which makes for a slightly larger pocket 1. All I-E alleles have a b9Glu and show only a Phe/Tyr dimorphism at b30 of all residues lining pocket 9, thus explaining the uniform preference for peptides containing basic residues at p9. A comparison between the motif for peptides binding to I-Ag7 that we previously defined (22) and to I-Eg7 described in the present study reveals that these two MHC molecules have different peptide binding specificity. In particular, the C-terminal primary anchors, p4 for I-Eg7 and p9 for I-Ag7 have opposite specificity: negatively charged residues are required for high-affinity peptide binding to I-Eg7 and positively charged or aromatic residues are required for high-affinity binding to I-Ag7 molecules. These distinct binding specificity are confirmed by the observation that, among a panel of hsp peptides, different peptides bind to I-Eg7 or to I-Ag7 and that the two binding repertoires do not overlap.

6638


FIGURE 5. Capacity of overlapping 65-kDa hsp peptides to bind to I-Ag7 or I-Eg7 and to restimulate T cell proliferation. A panel of overlapping peptides encompassing the entire sequence of Mt 65-kDa hsp was tested for binding to purified I-Ag7 and I-Eg7 molecules and for the capacity to induce proliferation in lymph node cells from hsp-primed NOD or Ea-transgenic NOD mice, as in Fig. 1. The results are expressed as IC50 for binding and as geometric mean of stimulation index (S.I., ratio between cpm of triplicate wells from lymph node cells cultured with or without Ag) for T cell proliferation. In dark gray are peptides with high affinity (IC50 # 1 mM) and S.I. . 3, in light gray peptides with weak affinity (IC50 1–10 mM). High-affinity binding to I-Ag7 and proliferation of NOD cells (S.I. . 3) are bordered.

I-Ag7 is a major susceptibility gene for IDDM development in NOD mice (30), and at least one dose of NOD MHC is required (31). In addition, the lack of I-E expression in NOD mice is essential for IDDM development. Thus, introduction of Ea genes in NOD mice, with resultant cell surface expression of EaEbg7 molecules, protects from insulitis and IDDM, as demonstrated by different groups using independent Ea transgenes (7–9, 16). Expression of other class II molecules can also have a protective effect. Transgenic introduction of I-Ag7 mutated in positions 56 and 57 (9) or of non-NOD I-A or I-E genes into the NOD background (32, 33), as well as conventional breeding (34), reduced or prevented insulitis and IDDM. However, not all MHC class II molecules protect NOD mice from IDDM development (35). Different hypotheses have been formulated to explain the mechanism of protection from IDDM in NOD-Ea transgenic mice. Thymic deletion of autoreactive T cells or inhibition of their selection was first proposed (12), but it became very unlikely when trans-

genic Ea expression only in the thymus was found to be ineffective in protecting NOD mice from insulitis or IDDM (8). Peripheral expression of I-Eg7 molecules thus appears necessary for protection, although this does not exclude their possible thymic role, for example, in the positive selection of regulatory T cells (8). I-Eg7restricted regulatory cells would require restimulation in the periphery by I-Eg7-positive APC, and this is currently being tested by their adoptive transfer in NOD-scid and NOD-Ea16-scid mice (S.T., S. G., G. P., and L.A., manuscript in preparation). It has been hypothesized that the protective class II molecules could favor the deviation of the autoreactive T cell response to the Th2 pathway (16, 36). This finding is consistent with the correlation between deviation to the Th2 phenotype and protection from IDDM in NOD mice treated with an IL-12 antagonist (37). In addition to induction of regulatory T cells, at least three different mechanisms based on competition for Ag presentation have been proposed to explain the peripheral protection observed in

The Journal of Immunology Ea-transgenic NOD mice. Epitope stealing predicts that I-Eg7 molecules have higher affinity for diabetogenic peptides than I-Ag7. I-Eg7 molecules would present these peptides to nonpathogenic T cells, thus avoiding pancreatic b cell destruction. This model implies that the diabetogenic peptides are relatively promiscuous in their binding to class II molecules and was proposed to explain dominant protection in human IDDM (38). However, we could not find evidence for high-affinity binding to both MHC molecules by the same peptide from a putative Ag in IDDM such as the 65-kDa hsp, as predicted from the very different peptide binding motifs for I-Ag7 and I-Eg7. It remains to be seen whether this applies to all autoantigen candidates, but this is likely based on the observation that only negatively charged residues are well tolerated at p4 in peptides binding to I-Eg7, whereas only positively charged or aromatic residues are well tolerated at p9 in peptides binding to I-Ag7. This motif for peptide binding to I-Ag7 (22) has recently been validated using a phage display library (S.G., H. Elisa Bono, F.G., J.H., Leonard C. Harrison, L.A., manuscript in preparation). The distinct motifs for peptide binding to I-Ag7 and I-Eg7 further argue against a motif for peptide binding to I-Ag7 which includes negatively charged residues at the C-terminal end (39, 40). Evidence supporting the determinant capture hypothesis as a protective mechanism afforded by I-Eg7 molecules is based on the observation that full-length unfolded proteins can bind to class II molecules (41). The expression of I-Ed molecules in NOD mice modified the processing of HEL via high-affinity binding of the dominant epitope 108 –116 to I-Ed, thus preventing the T cell response to an I-Ag7-binding subdominant determinant (15). This hypothesis, however suggestive, has not yet been confirmed in Ea-transgenic NOD mice with a candidate autoantigen for IDDM induction. The availability of a motif for peptide binding to I-Eg7 now renders the testing of this hypothesis feasible. Another possible explanation for the protective effect of I-Eg7 molecules predicts that peptides derived from I-Eg7 can compete with diabetogenic peptides for binding to I-Ag7 (1). However, this is unlikely because, at least in the response to hsp, the I-Ag7-restricted proliferation observed in Ea-transgenic NOD mice is only slightly reduced compared with NOD mice (S.T., unpublished data). It would be interesting to examine whether overlapping I-Eg7 peptides can bind to the I-Ag7 molecule. In conclusion, we have defined a motif for peptide binding to I-Eg7 molecules, characterized by a negatively charged amino acid residue at p4. The diversity between peptide binding motifs for I-Eg7 and I-Ag7 and the different binding specificity displayed by these two molecules indicate that epitope stealing does not account for protection from IDDM in Ea-transgenic NOD mice. The definition of a binding motif for I-Eg7 should help further analysis of the mechanisms which protect in NOD mice expressing transgenic Ea molecules from IDDM.

Acknowledgments

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5.

6. 7.

8.

9.

10. 11. 12.

13. 14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

We thank Dr. L. C. Harrison for critical reading of the manuscript and Dr. A. K. Moustakas for help in molecular modeling. 26.

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