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By
From * the Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville
3050, Australia; and Roche Milano Ricerche, 1-20132, Milano, Italy
Summary
Materials and Methods
Results and Discussion
Footnotes
References
Summary 
The class II major histocompatibility complex molecule I-Ag7 is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice
and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally,
it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its 
chain. To identify the requirements for peptide binding to I-Ag7 and
thereby potentially pathogenic T cell epitopes, we analyzed a known I-Ag7-restricted T cell
epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-Ag7 M12-Y20/
K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions
(p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL
epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally,
p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y
or F) or positively charged (K, R). Specific residues were not tolerated at these and some other
positions. A motif for binding to I-Ag7 deduced from analysis of the model HEL epitope was
present in 27/30 (90%) of peptides reported to be I-Ag7-restricted T cell epitopes or eluted
from I-Ag7. Scanning a set of overlapping peptides encompassing human proinsulin revealed
the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.
Non obese diabetic (NOD)1 mice develop autoimmune, T cell-mediated destruction of pancreatic islet
Although amino acid motifs for peptides that bind to individual class I and some class II MHC molecules have been
well defined (5, 6), the rules that govern binding of peptides to I-Ag7 are still unclear. Reich et al. (7) eluted and sequenced several naturally processed peptides from I-Ag7
and concluded that binding may require an acidic residue
in the COOH terminus of the peptide. Carrasco-Marin et
al. (8) found that I-Ag7 either on the surface of antigen-presenting cells or in SDS-PAGE after its purification was unstable and that the binding of known I-Ag7-restricted T cell
epitopes or the peptides eluted by Reich et al. (7) was difficult or impossible to demonstrate. This led them to hypothesize that weak peptide binding by I-Ag7 militated against
elimination of autoreactive T cells in the NOD mouse.
Amor et al. (9) investigated the fine specificity of peptides
from myelin oligodendrocyte glycoprotein (MOG) or proteolipid protein (PLP) for the induction of experimental allergic encephalomyelitis (EAE) in Biozzi AB/H mice and
suggested a core motif for I-Ag7 binding peptides.
In this study, we used the I-Ag7-restricted T cell epitope,
hen egg white lysozyme (HEL) amino acids 9-27, as a template with which to analyze the amino acid sequence of
peptides that bind to purified, native I-Ag7 and activate a T
cell hybridoma. This has enabled us to define general rules
that identify most known I-Ag7 binding peptides.
Materials and Methods Purification of I-Ag7.
I-Ag7 protein was affinity-purified from
detergent lysates of 4G4.7 B cell hybridoma cells by desorption
from OX-6 mouse monoclonal antibody. The 4G4.7 B cell hybridoma was derived by polyethylene glycol (PEG)-induced fusion of
NOD mouse T cell-depleted splenocytes with the HAT-sensitive
A20.2J lymphoma line (10). OX-6 is a mouse monoclonal IgG1
antibody against an invariant determinant of rat Ia, which also recognizes I-Ag7 but not I-Ad (11, 12). Approximately 15 mg of OX-6 antibody was first bound to 4 ml of protein A-Sepharose 4 Fastflow
(Pharmacia, Uppsala, Sweden) and then chemically cross-linked to
the protein A with dimethyl pimelimidate dihydrochloride (Sigma
Chemical Co., St. Louis, MO) in sodium borate buffer, pH 9.0. After 60 min at room temperature (RT), the reaction was quenched
by incubating the Sepharose in 0.2 M ethanolamine, pH 8.0, for
60 min at RT. The suspension was washed thoroughly in PBS
and stored in PBS, 0.02% sodium azide (NaN3).
cells and are a model of human insulin-dependent diabetes mellitus (IDDM) (1). In common with humans who
develop IDDM, NOD mice have immune responses to islet autoantigens such as insulin and glutamic acid decarboxylase (GAD). In addition, they share a structurally similar
class II MHC molecule associated with disease susceptibility. This molecule, I-Ag7, has a chain sequence otherwise
found only in Biozzi AB/H mice that are susceptible to
chronic relapsing experimental allergic encephalomyelitis
(CR-EAE) (2). It is characterized by a non-Asp residue at
position 57 (3), as in the chain of the HLA-DQ molecules associated with human IDDM (4). The capacity of
these unique class II molecules to bind and present peptides
to autoreactive T cells could be critical in the development
of IDDM and CR-EAE.
-amino-n-caproic acid and
10 µg/ml each of soybean trypsin inhibitor, antipain, pepstatin,
leupeptin and chymotrypsin. Lysates were cleared of nuclei and
debris by centrifugation at 27,000 g for 30 min and stored as such
if not immediately processed further. To the postnuclear supernatant was added 0.2 vol of 5% sodium deoxycholate (DOC). After
mixing at 4°C for 10 min, the supernatant was centrifuged at
100,000 g at 4°C for 120 min, carefully decanted, and filtered
through a 0.45-µm nylon membrane. The lysate of 5 × 1010
4G4.7 cells was gently mixed overnight at 4°C with 4 ml of OX6-protein A-Sepharose, and the suspension then poured into a
column and washed with at least 50 vol each of buffers A, B, and
C. Buffer A was 0.05 M Tris, pH 8.0, 0.15 M NaCl, 0.5% NP40, 0.5% DOC, 10% glycerol, and 0.03% NaN3; buffer B was
0.05 M Tris, pH 9.0, 0.5 M NaCl, 0.5% NP-40, 0.5% DOC,
10% glycerol, and 0.03% NaN3; buffer C was 2 mM Tris, pH 8.0, 1% octyl--D-glucopyranoside (OGP), 10% glycerol, and 0.03%
NaN3. Bound I-Ag7 was 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.
Peptide Synthesis.
Peptides were synthesized with a multiple
peptide synthesizer (model 396; Advanced ChemTech, Louisville, KY) using Fmoc chemistry and solid phase synthesis on
Rink Amide resin. All acylation reactions were effected with a
threefold excess of activated Fmoc amino acids, and a standard
coupling time of 20 min was used. Each Fmoc amino acid was
coupled at least twice. Cleavage and side chain deprotection was
achieved by treating the resin with 90% trifluoroacetic acid, 5%
thioanisole, 2.5% phenol, 2.5% water. The indicator peptide for
the binding assay was biotinylated before being cleaved from resin
by coupling two 6-aminocaproic acid spacers on the NH2 terminus and one biotin molecule sequentially, using the above-described procedure. Individual peptides were analyzed by reverse-phase HPLC and those used in this study were routinely 
85% pure.
T Cell Hybridoma.
Hybridoma 2D12.1 was generated by PEG-
induced fusion of HEL-immune lymph node cells from a NOD
mouse with the TCR-
/-negative variant of the BW5147 thymoma, as described previously (13). Reactivity of 2D12.1 to HEL
peptides was assayed by incubating 2.5 × 105 NOD spleen cells
and HEL peptides (0.3 nM to 10 µm) with 5 × 104 T hybridoma
cells/well. Culture medium was RPMI 1640 supplemented with
10% FCS, 2 mM L-glutamine, 50 µg/ml gentamicin, and 50 µm 2-mercaptoethanol. After 24 h of culture, 50 µl of supernatants were transferred to culture wells containing 104 IL-2-
responsive CTLL-2 cells. During the final 4 h of a 24-h culture,
CTLL-2 cells were pulsed with 1 µCi [3H]thymidine. Thymidine
incorporation was measured by scintillation spectrometry. The
concentration of peptide that caused 50% of maximum stimulation is referred to as SC50.
I-Ag7 Peptide-binding Assay. Peptides were dissolved at 10 mM in DMSO and diluted into 20% DMSO/PBS for assay. Indicator I-Ag7 binding peptide, HEL 10-23, was synthesized with a biotin molecule and two spacer residues at the NH2 terminus. Approximately 200 nM of this biotinylated HEL peptide and each test peptide in seven concentrations ranging from 50 µM to 50 pM, were coincubated with ~200 ng of I-Ag7 protein in U-bottomed polypropylene 96-well plates (Costar Serocluster, Costar Corp., Cambridge, MA) in binding buffer at RT. The binding buffer was 6.7 mM citric phosphate, pH 7.0, with 0.15 M NaCl, 2% NP-40, 2 mM EDTA, and the protease inhibitors as used in the lysis buffer. After a minimum of 24 h, each incubate was transferred to the corresponding well of an ELISA plate (Nunc Maxisorp, Nunc, Roskilde, Denmark) containing prebound OX-6 antibody (5 µg/ml overnight at 4°C, followed by washing). After incubation at RT for at least 2 h, and washing, bound biotinylated peptide-I-Ag7 complexes were detected colorimetrically at 405 nm after reaction with streptavidin-alkaline phosphatase and paranitrophenolphosphate. Competition binding curves were plotted and the affinity of peptide for I-Ag7 was expressed as an inhibitory concentration 50 (IC50), the concentration of peptide required to inhibit the binding of bio-HEL 10-23 by 50%.
Results and Discussion
Approximately 2 mg
of protein, estimated by Coomassie blue binding (Bio Rad
Protein assay), was purified from 5 × 1010 4G4.7 cells. In
SDS-PAGE, the majority (>95%) of the protein was resolved as two bands of molecular weight ~33,000 and
~28,000 that correspond to the and subunits, respectively, of mouse class II MHC molecules (data not shown).
The competition binding assay with purified I-Ag7 was sensitive and specific (Fig. 1), and highly reproducible; in 15 separate assays the mean ± SD of the IC50 for competition between biotinylated and unlabeled HEL 10-23 was 295 ± 72 nM.
) was used as an internal control in each 96-well plate assay; 
, good
binder (IC50 100 nM); 
, weak binder (IC50 2000 nM); 
, non-binder
(IC50 50,000 nM).
Carrasco-Marin et al. (8) were unable to demonstrate direct binding of HEL 11-25 to purified I-Ag7 and proposed
that I-Ag7 was inherently unstable. We found that purified
I-Ag7 stored at 
70°C for more than 1 yr reproducibly
bound HEL 10-23 with high affinity. Therefore, our results do not support their hypothesis that I-Ag7 is inherently
unstable, which they postulated would impair its ability to
bind and induce tolerance to autoreactive peptides.
Peptides representing sequential truncations of HEL 9-27, from either the NH2 or COOH-terminus, were each assayed in parallel for binding to I-Ag7 and for their ability to activate the 2D12.1 hybridoma. Inspection of these data (Table 1) reveals that the minimum T cell epitope is M12-R21, and the minimum binder is M12-Y20 or K13-R21.
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Substitution of alanine (A) at each position in HEL 12-22 (Table 2) had no significant effect on binding, with the sole exceptions of positions L17 and Y20. Substitution at either of these two positions virtually abolished binding. On the other hand, while having no effect on binding, substitutions by A at K13, R14, H15, G16, and D18, and to a lesser extent at R21, abolished T cell activation. Removal of R21 (see Table 1) abolished T cell activation. Further substitutions of representative amino acids (D, K, P, Y, L, Q) at each position (Table 2) revealed varying levels of tolerance of specific residues/positions for binding (see below) and generally confirmed the results of the alanine substitutions on T cell activation. On the basis of these results, we can deduce that most residues in the minimal T cell epitope HEL 12-21 have TCR contacts and that two, L17 and Y20, are essential for binding to I-Ag7 (Fig. 2).
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