Identification of two distinct properties of class II major ...

Proc. Nati. Acad. Sci. USA

Vol. 90, pp. 1227-1231, February 1993 Immunology

Identification of two distinct properties of class II major histocompatibility complex-associated peptides (immunogenicity/antigen/T cel/peptide binding)

CHRISTOPHER A. NELSON, SHIRLEY J. PETZOLD, AND EMIL R. UNANUE Washington University, School of Medicine, Department of Pathology, St. Louis, MO 63110

Contributed by Emil R. Unanue, November 6, 1992

ABSTRACT We have examined the interactions of various peptides with the mouse class II major histocompatibility complex molecule I-Ak. The peptides were derived from the model protein hen egg white Iysozyme (HEL). The immunodominant peptide of HEL is a 10-mer, residues 52-61. Our previous work established that this sequence contains the key residues for binding and presentation to T cells. Now we show that the binding of this 10-mer sequence resulted in complexes of I-Ak and peptide that, in SDS/PAGE (without boiling the protein), rapidly dissociated from the component a and .8 chains. The binding interactions were studied in vitro, by incubating purified I-Ak and radiolabeled peptide, or ex vivo, by using antigen-presenting cells incubated with peptides. Peptides with additional residues at either the amino or carboxyl terminus behaved dramatically differently. Complexes of I-Ak with the longer peptides were stable to SDS/PAGE. Very few amino acid additions result in the change from unstable to stable complexes. The important issue here is that when cultured with HEL, antigen-presenting cells selected the HEL peptides containing the 52-61 sequences that favored stability [Nelson, C. A., Roof, R. W., McCourt, D. W. & Unanue, E. R. (1992) Proc. Nad. Acad. Sci. USA 89, 7380-7383]. Also, from other studies, such sequences correlate with a high immunogenicity of the peptide. We conclude that there are structural features of peptides that change the stability of the class II molecule and that are independent of the "core" peptide seen by the T cells. Recent studies have identified some of the characteristics of peptides associated with either class 1 (1-4) or 11 (5-7) major histocompatibility complex (MHC) proteins. MHC class I molecules without bound peptides are structurally unstable, under physiological conditions (8). Peptide binding to, and stabilization of, class I molecules assists both the assembly and the transport of nascent class I molecules (9). Current studies have indicated that peptide can also contribute to the stabilization of MHC class II structure (10-13). We recently identified the peptides isolated from the class II molecule I-Ak of antigen-presenting cells (APCs) cultured with the protein antigen, hen egg white lysozyme (HEL) (7). Previously our laboratory had identified an immunodominant sequence in HEL for I-Ak (14, 15), and this is precisely what was isolated from the APCs. The predominant peptides eluted from I-Ak (7) contained the lO-mer sequence of residues 52-61 (DYGILQINSR), the critical epitope for binding to I-Ak. Moreover, this sequence was contained in peptides that extended by four amino acids at the amino terminus and by one to three amino acids at the carboxyl terminus. We have now asked whether there is an advantage by the APCs to select peptides of length longer than the critical lO-mer sequence. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

MATERIALS AND METHODS Three sets of experiments were done. In each, peptides bound to I-Ak were examined by SDS/PAGE under reducing conditions but without boiling the immune precipitate. In the first set ofexperiments, radiolabeled peptides were incubated with purified I-Ak in detergent solution and then examined by SDS/PAGE. I-Ak was affinity-purified from CH27 B-lymphoma cells (16) by using the monoclonal antibody 10-3.6.2 (17), as reported (18). Synthetic peptides were labeled with 1251 by the chloramine-T method to a specific activity of 2-4 x 108 cpm/fug and then purified by C18 reverse-phase HPLC. Purified I-Ak was incubated with an excess of labeled peptide (30 pmol of I-Ak with 100 pmol of peptide) (19). After 72 hr, the newly formed complex was purified from unbound peptide by Sephadex G-50 chromatography (19, 20). The void volume was pooled and concentrated with a Centricon 10 (Amicon). The samples were resuspended in loading buffer (12) and analyzed by SDS/10%o PAGE. The amounts of peptide bound or dissociated were then quantitated. Two regions were excised from each lane: a region at the top of the gel where labeled peptide bound in the intact af3 dimers was found to migrate (12, 21), and a region at the bottom of the gel where free labeled peptide migrated. The incorporated radioiodine in each excised gel fragment was determined with a standard 'y counter. The total radioactive peptide remaining bound to I-Ak after SDS/PAGE was calculated as the ratio of counts found at the top position to the total counts in each lane and is given as a percentage. In the second set of experiments, the radiolabeled peptides were incubated with an I-Ak-bearing B-lymphoma cell line, from which the complexes of peptide-I-Ak were then isolated. M12.C3.F6 B-lymphoma cells (22), 2 ml at 108 cells per ml in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum and 20 mM Hepes, were incubated with 90-110 pmol of 125I-labeled peptide (-3 x 107 cpm) at 370C and 5% CO2 for 4 hr. The cells were harvested, washed, and then lysed in 5 ml of phosphate-buffered saline containing: 1% Triton X-100, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, and 20 ;Lg of leupeptin per ml. The lysate was incubated with the monoclonal anti-I-Ak antibody 10.3.62 and the immunoprecipitates were analyzed by SDS/ PAGE as above. The third approach consisted of metabolically labeling I-Ak of B lymphoma cells with [3H]leucine and then adding nonlabeled peptide. The M12.C3.F6 cells were cultured at 107 per ml in leucine-deficient Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum and, after 1 hr of incubation at 370C, were incubated in the same medium but containing [3H]leucine (800 ACi/ml; 1 ACi = 37 kBq) without or with peptide (100 AtM) or HEL (1 mg/ml). After 1 hr, nonradioactive leucine was added as a chase and the incubation continued for 4 hr. The metabolically labeled Abbreviations: HEL, hen egg white lysozyme; hsp70, 70-kDa heat shock protein; MHC, major histocompatibility complex.

1227

1228

Immunology: Nelson et al.

I-Ak molecules were immunoprecipitated, eluted at room temperature, and analyzed by SDS/PAGE. The ratio of I-Ak running in SDS/PAGE as a stable heterodimer or as dissociated a and P chains was then examined. To determine the amount of labeled class II at each position, excised gel fragments were dissolved in Solvable (DuPont/NEN) and mixed with Econofluor (DuPont/NEN) before liquid scintillation counting. A value of percent stable I-Ak was calculated for each peptide. The synthetic peptides were also examined for their relative binding to purified I-Ak. This was done by testing for their ability to inhibit a standard radiolabeled peptide-MHC binding reaction. Purified I-Ak was incubated with 125I1 labeled YE-52-61 peptide in the absence and presence of the unlabeled peptides. The concentration of each competing peptide required to inhibit 50% of the binding of the 1251labeled YE-52-61 peptide was determined. As a reference value, the concentration of unlabeled YE-52-61 needed to inhibit 50%o of the binding of the labeled YE-52-61 peptide was also determined. A value for the relative inhibitory capacity of each peptide was obtained by dividing the concentration of that peptide required for 50% inhibition by the concentration of unlabeled YE-52-61 required for 50%o inhibition. Therefore, by definition the relative inhibitory capacity of YE-52-61 is 1. In a typical experiment 3-10 pmol of peptide was incubated with 70-100 pmol of I-Ak at pH 5.5 for 48 hr, resulting in about 10-25% uptake of peptide (peptide bound varied from 50,000 to 130,000 cpm).

Proc. Natl. Acad. Sci. USA 90 (1993) RESULTS

We synthesized HEL peptides containing the 52-61 core sequence (Table 1), labeled them with 1251, and incubated them with affinity-purified I-Ak. The newly formed peptideMHC complex was isolated for analysis. In agreement with previous studies (19, 20), the complex of 52-61 with I-Ak was stable to dialysis in the detergent used to isolate I-Ak, with an average half-dissociation time of about 30 hr (determined in detergent solution over a range of pH from 5 to 7). Previous studies demonstrated that the stability of the MHC class II heterodimer could be measured as resistance to dissociation by SDS treatment during PAGE under conditions where the proteins were never boiled (10-13, 21). The peptide-MHC complex formed using the HEL core peptide, 52-61, completely dissociated during SDS/PAGE at room temperature (Fig. 1A), releasing the peptide, which was found near the front of the gel. In contrast, complexes formed with peptides longer than the lO-mer core of 52-61 were clearly resistant to dissociation by SDS/PAGE; these peptides migrated at the position expected for an assembled a/f3 heterodimer (Fig. 1A). The longer peptides were of two series. The first represented sequences of naturally occurring peptides isolated from I-Ak of APCs incubated with HEL. Peptide 48-62 corresponds to the predominant form of the physiologically processed HEL-derived material (accounting for about 70%), while 48-60 and 48-61 represent two minor forms (together accounting for about 10%) (7). In this case, all the peptides

Table 1. Summary of experiments using HEL peptides or peptides isolated from I-Ak of APCs Induction of stable Stability of labeled Relative I-Ak peptide, % Peptide inhibitory % increase n In vitro Ex vivo Name Composition capacity 10 11 3 -1.9 0.03 52-61 DYGILQINSR 74 63 3 16.2 0.11 T-52-61 51-61 51 3 K-52-61 43 -8.0 K-52-61 84 90 3 19.4 E-52-61 E-52-61 1.00* 87 2 28.4 89 YE-52-61 YE-52-61 1.00t 73 ST-52-61 83 50-61 53 31 0.15 52-61-W 52-62 86 9 29.4 96 NTDGST-52-61 -1.00* 46-61 97 2 28.5 DGST-52-60 0.48 48-60 2 86 30.5 0.77 48-61 DGST-52-61 2 94 48-62 DGST-52-61-W 20.9 16.70 DGST-52-61-WW 86 1 0.37 48-63 26.7 2 -5.2 1-17 KVFGRCELAAAMKRHGL None 2 34-45 19.3 0.01 FESNFNTQATNR 2 79-97 PCSALLSSDITASVNCAKK -6.3 0.06 2 115-129 -8.3