International Immunology, Vol. 4, No. 7, pp. 773 - 777
High affinity for class II molecules as a necessary but not sufficient characteristic of encephalitogenic determinants Melissa Wall, Scott Southwood, John Sidney, Carla Oseroff, Marie-France del Guericio, Alan G. Lamont1, Sonia M. Col6n, Thomas Arrhenius, Federico C. A. Gaeta, and Alessandro Sette
Key words: autcxmmunity, class II affinity, experimental autoimmune encephalomyelitis, myelin basic protein
Abstract A direct binding assay specific for IA* molecules has been developed and Its immunological relevance validated by examining, for a panel of nine different synthetic peptJdes, the correlation between their capacity to bind purified IA* and to inhibit IA*-restrlcted antigen presentation. The IA* assay thus developed has then been used to study the IA* binding affinity of a set of overlapping peptJdes spanning the entire myelin basic protein (MBP). It was found that the encephalitogenic MBP region corresponds to peptides with high MHC binding affinities. Other regions of the MBP that have not been described as being pathogenic in the context of IA* molecules have also been found to be high IA* binders, suggesting that variables other than MHC affinity are also Involved in determining the pathogenic potential of self-derived determinants. Introduction Immune recognition of normal self components leading to tissue destruction and pathological abnormalities is underlying a group of apparently disparate diseases such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis, collectively known as autoimmune diseases (1,2) Susceptibility to these diseases is strongly associated with particular class II alleles, suggesting that class ll-restricted binding and presentation of specific autoantigens may be involved in the disease process. In humans, the exact molecular identity of the actual autoantjgen(s) recognized in the disease process is unknown, even though certain candidates have been implicated by several recent studies (3-8). By contrast, in a few animal disease models the actual molecular determinants (and the presenting class II alleles) involved in disease activation are well characterized (2,9-16). In particular, experimental autoimmune encephalomyelitis (EAE) is an experimental disease model of human multiple sclerosis. EAE is usually induced by injection with either whole spinal cord homogenate, purified myelin basic protein (MBP), or synthetic peptides derived from MBP or proteolipid protein (PLP). In mice, strains expressing certain MHC class II alleles, such as PLJ (expressing IA") or SJL (expressing IA8), appear to
be very susceptible to disease induction, while other strains are considered resistant (2). In particular, in H-2* mice (expressing IAd and IEd molecules), successful induction of EAE has been reported using only whole spinal cord homogenate (17), and IAd molecules have not thus far been reported as capable of acting as a restriction element for a pathological MBP-specific autoimmune response. A few different peptide epitopes from MBP and PLP have been identified and characterized. More specifically, MBP Ac 1-11 appears to contain the major encephalitogenic determinant for IAu-expressing mice (such as PLJ), while MBP 87-98 and PLP 139-151 appear to be dominant autoantigenic determinants for mice expressing the IAS molecules (such as SJL). An important point of debate has been the binding affinity of autoantigenic epitopes for their restricting class II epitopes. According to one theory, autoreactive pathogenic T cell clones could recognize low affinity binders, and have for that reason escaped deletion at the negative selection level (18,19). According to the opposite point of view, autoantigens are high affinity binders and thereby potentially highly immunogenic. When some as yet unspecified event, such as, for example, regulatory
Correspondence to: A. Sette Transmitting editor: G. Doria
Received 9 March 1992, accepted 6 April 1992
Downloaded from http://intimm.oxfordjournals.org/ at University of Birmingham on August 31, 2015
Cytel, 3525 John Hopkins Court, San Diego, CA 92121, USA 'Roche Products Ltd Research Centre, Broadwater Road, Welwyn Garden City, Hertfordshire, UK
774
Autoantigens are high affinity class II binders
imbalances (1,2) or molecular mimicry (20), leads to a breakdown of tolerance, their high immunogenic potential is then realized. In the present study, we took advantage of the delineation of MBP 87-98 and PLP 139-151 as encephalitogenic determinants presented by IAS class II molecules to address this question at the experimental level. For this reason, an IA"-specific binding assay has been developed and overlapping peptides spanning through the entre mouse MBP sequence have been synthesized. Methods Cells
Affinity purification of IAS molecules Since LS102.9 cells were derived by fusion of IAS spleen cells with A20 cells, they expressed both IAd and IA8. To avoid contamination with IAd molecules, the antibody Y3JP was used to purify IA3 molecules from the cell lysates. This antibody, in fact, does not cross-react with either IAd or mixed isotype lA"* molecules (21). IA8 molecules were purified as previously described (22) using the MAb Y3JP coupled to Protein A-Sepharose CL 4B beads. Lysates were filtered through 0.45 /iM filters and then passed over the affinity column, which was then washed with 20 column volumes of 0.5% NP-40 - 0 . 1 % SDS, 5 column volumes of PBS, and 2 column volumes of PBS containing 1 % n-octyglucoside. Finally, the IA* was eluted with 0.05 M diethylamine in 0.15 M NaCI containing 1 % noctyglucoside, pH 11.5. A 1 /20 volume of 1.0 M Tris, 1.5 M NaCI, pH 6.8, was added to the eluate to reduce the pH to ~ 7.5 and then concentrated by ultrafiltration in AMICON with YM30 membrane. Peptide synthesis Peptide syntheses were carried out by sequential coupling of A/a-r-Boc-protected amino acids on an Applied Biosystems (Foster City, CA, USA) 430A peptide synthesizer using standard f-Boc coupling cycles (software version 1.40) All amino acids, reagents, and resins were obtained from Applied Biosystems or Bachem (Torrance, CA, USA). Solvents were obtained from Burdick & Jackson (Muskegon, Ml, USA). Solid-phase synthesis was started from an appropriately substituted r-Boc-amino acid - phenylacetamidomethyl (PAM) resin. The loading of the starting resin was 0.5-0.7 mmol/g polystyrene and 0.5 meq were used in each synthesis. A typical reaction cycle proceeded as follows, (i) The N-terminal f-Boc group was removed with 33% trifluoroacetic acid (TFA) in dichloromethane (DCM) for 1.5 min, followed by another treatment with 50% for 18.5 min. (ii) The resin was washed 3 times with DCM and neutralized twice with 10% diisopropylethylamine (DIEA) in dimethylformamide (DMF) for 1 min each and then the resin was washed 5 times with DMF.
Class II-peptide
binding assays
Purified class II molecules were incubated with 5 nM 1 2 5 I radiolabeled ROIV peptide (sequence: YAHAAHAAHAAHAAHA) (23) for 48 h in PBS containing 5% DMSO in the presence of a protease inhibitor mixture. Purified peptides were iodinated using the chloramine-T method (24). The final concentrations of protease inhibitors were 1 mM PMSF, 1.3 mM 1.10 phenanthroline, 73 /iM pepstatin A, 8 mM EDTA, 6 mM Nethylmaleimide, and 200 pM /VQ-p-tosyl-L-lysine chloromethyl ketone. Final detergent concentration in the incubation mixture was 2.6% Digitonin. Class II - peptide complexes were separated from free peptide by gel filtration on TSK2000 columns and the fraction of peptide bound was calculated as previously described (22). In preliminary experiments, each of the IA* preparations was titered in the presence of fixed amounts of radiolabeled peptide to determine the concentration of class II molecules necessary to bind - 1 0 % of the total radioactivity. All subsequent inhibition and direct binding assays were then performed using these class II concentrations. In the inhibition assays, peptide inhibitors were typically tested at concentrations ranging from 120 nglm\ to 1.2 ng/ml. The data were then plotted and the dose yielding 50% inhibition measured. Each peptide was tested in two to four completely independent experiments.
Results Development of an IAs-specific binding assay In a previous study, several different synthetic peptides were tested for their capacity to inhibit antigen presentation in an IA8restricted system (12). Since a protocol involving a short pulse on fixed cells was used, their inhibitory capacity could be taken as an approximate measure of affinity for class II molecules. In the present study we have used that information to develop and validate a molecular binding assay. More specifically, one of the peptides which scored as highly inhibitory in the inhibition of antigen presentation assay, ROIV, was radiolabeled and incubated (as previously described for other IA alleles) with affinity
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The B cell hybrid LS102.9 was used as a source of IA». LS102.9 cells were maintained in vitro by culture in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 nM 2-ME, and 10% heat-inactivated FCS, and were also supplemented with 100 /ig/ml of streptomycin (Irvine Scientific, Santa Ana, CA, USA) and 100 U/ml of penicillin (Gibco, Grand Island, NY, USA). Cells were lysed at a concentration of 10s cells/ml in PBS containing 1 % NP-40,1 mM PMSF. The lysates were cleared of nuclei and debris by centrifugation at 15 000 g for 15 mm.
(iii) The DMF solution of a 2-fold excess of preformed symmetric anhydride generated from 2 mmol f-Boc-amino acid was added to the resin, and the mixture allowed to react for 1 5 - 4 5 min. Hydroxybenzotnazole (HOBt) esters (4-fold excess) were used for the coupling of arginine, asparagine, glutamine, and histidine residues, and the coupling step was repeated to ensure completeness, (iv) The resin was washed with DCM in preparation for the next elongation cycle. The fully protected, resin-bound peptide was subjected to a TFA cycle to remove the terminal f-Boc, and the product washed with DCM and dried. The resin was then treated with anhydrous HF in the presence of appropriate scavengers [e.g. 5% (v/v) p-cresol/15% (v/v) ethanedithiol] for 60 min at 0°C. After evaporation of excess HF, the crude peptide was washed with diethyl ether, extracted with aqueous acetic acid, and lyophilized. The crude peptides were purified to > 9 5 % homogeneity by reversed-phase HPLC using H2O/CH3CN gradients containing 0.2% TFA modifier on a Vydac, 300 A pore-size, C-18 preparative column. The purity of the synthetic peptides was assayed on an analytical reversephase column, and their composition ascertained by amino acid analysis and/or sequencing.
Autoantigens are high affinity dass II binders 775 8
purified IA molecules for 2 days at room temperature. The results obtained are shown in Fig. 1. Good binding activity (up to - 1 0 % of the input) was detected for IAS concentrations in the 0 . 1 - 1 /iM range (Fig. 1a). The binding was specific (Fig. 1b), in that it was inhibitable by excess unlabeled ROIV peptide (IC^, = - 1 5 nM). Scatchard analysis revealed that as previously shown for other IA alleles, only a minor fraction of the IAS molecules appeared to be available for binding (data not shown). Low levels of available binding sites are probably due to the presence of endogenously derived peptides in the IAS peptide binding site. Correlation between inhibition of antigen presentation and direct binding assay
8
12 i
IA$ and IAd capacity of a series of synthetic peptides spanning through the entire MBP sequence
10 8-
g
6H
CD
*
4
-
2-
.001
.01
.1 \iM
1
10
MHC
A set of overlapping 20-residue peptides spanning through the entire MBP sequence were synthesized and purified. They were then tested for their capacity to bind purified IA8 molecules. They were also tested for binding to IAd, chosen as a representative MHC molecule that does not, to the best of our knowledge (2), serve as a restriction element of MBP-specific EAE. The data obtained are shown in Table 2. It was found that the peptide 81 -100, which contains the MBP 8 7 - 9 8 region, was the highest affinity IAS binder of the entire MBP protein. The other peptide containing the MBP region, that has been described as being a dominant autoantigenic site in IAU mice (MBP Ac 1-11), also bound IA8 with similar high affinity. Six other peptides bound with lower affinities in the 1 - 1 0 nM range. Interestingly, three of them (peptides 121 -140,131 -150, and
Table 1. Correlation between direct binding and inhibition of antigen presentation data
Peptide KM core extension PLP 139-151 OVA 271-285. Nase 101-120 HA 307-319 OVA 1 5 - 2 9 .0001
Fig. 1. (a) Binding profile of 12Sl-radiolabeled ROIV peptide (5 nM) to purified IA* molecules, (b) Inhibition of the binding of radiolabeled ROIV peptides to purified IA8 molecules (1 *»M) by excess unlabeled ROIV peptide.
DE core extension X rep. 1 2 - 2 6 HEL 4 6 - 6 1
Direct binding assay OJM 50% dose) 0.71 0 46 0 43 3.3 42 8.9
Inhibition of antigen presentation assay8 OiM 75% dose) 4 4.1 7.6 10 56 200
_b
—
T h e inhibition of antigen presentation data has been published elsewhere (11) and is shown here only for the purpose of reference. b — indicates >200 /iM.
Downloaded from http://intimm.oxfordjournals.org/ at University of Birmingham on August 31, 2015
Having established this assay, we wished to obtain validation from the immunological point of view. To do so, we examined whether the data previously obtained in the inhibition of antigen presentation assay correlated with the data generated by the molecular assay. The rationale of such an experiment resides
in the fact that if correlation can be demonstrated, this would suggest that the radiolabeled ROIV peptide binds in the same binding site used by antigen peptides when recognized by T cells (23,25). More specifically, nine peptides previously tested for inhibition of antigen presentation were tested for their capacity to inhibit ROIV binding [Table 1; the data for inhibition of antigen presentation have already been published elsewhere (12), and are shown here only for reference purposes]. The three peptides [KM core extension PLP 139-151, chicken egg ovalbumin (OVA) 271 -285, and staphylococcal nuclease (Nase) 101-120] that scored as good inhibitors of antigen presentation (ICso 1 - 1 0 /iM) also were powerful inhibitors in the direct binding assay (IC50 < 5 /tM). Peptides HA 307 - 319 and OVA 15 - 29, which were less potent inhibitors of antigen presentation (IC50 10-200 /xM), were also less potent binders (ICSQ 5 - 5 0 ^M). Finally, the three peptides [DE core extension, X rep 12 - 26 and hen egg lysozyme (HEL) 4 6 - 6 1 ] that scored negative in the binding assay were also negative for IAS binding activity. Taken together, the data demonstrate that an excellent correlation exists between the data generated in the two assays, thus supporting the immunological relevance of the direct binding assay established.
776 Autoantigens are high affinity class II binders Table 2. IAS and IAd binding profile of a series of overlapping peptides spanning through the entire mouse MBP sequence 50%/ dose IAS
Sequences
Ac 1 - 20 11 •30 21- •40 31- •50 41 ••60 51 ••70 61- •80 7 1 • •90 81 •100 91- •110 101 •-120 111- •130 121 •140 131 •150 141 •160 151 •170
Ac-ASQKRPSQRSKYLATASTMD KYLATASTMDHARHGFLPRH HARHGFLPRHRDTGI LDSI G RDTGI LDSIGRFFSGDRGAP RFFSGDRGAPKRGSGKDSHT KRGSGKDSHTRTTHYGSLPQ RTTHYGSLPQKSQHGRTQDE KSQHGRTQDENPVVHFFKNI NPVVHFFKNIVTPRTPPPSQ VTPRTPPPSQGKGRGLSLSR GKGRGLSLRFSVVGAEGQKP FSVVGAEGQKPGFGYGGRASD GFGYGGRASDYKSAHI KGFKG YKSAHKGFKGAYDAQGTISK AYDAQGTLSKI FKLGGRDSR I FKLGGRDSRSGSPMARR
a
0.60 3.7 143
0.42 2.8 50
5.3 95 19 0.36 36 8.4 13 1.9 3 2.5 33
3.4 0.45 25 50 150
— indicates >20O ,
span through the 121-150 region that has also been demonstrated (26) to induce disease in IAS mice. Finally, five peptides bound in the 10 - 1 0 0 ^M range and no binding activity could be detected, up to the 100 pM level, for the remaining three epitopes. For IAd molecules, high binding is detected for Ac 1 - 2 0 and MBP 61 - 8 0 . Three other peptides (MBP 1 1 - 3 0 , 51 - 7 0 , and 101 -120) bound in the 1-10 yU range. Three peptides bound with weaker affinities (10-100 ^M). Finally, for the remaining eight peptides no binding was detected up to the 100 MM level. Discussion In the present report, a direct peptide-IA s binding assay is described. The assay established was validated at the biological level by examining, for a panel of nine different synthetic peptides, the correlation between data obtaned in the direct binding assay and data previously obtained (12) when the same set of peptides was tested for its capacity to inhibit antigen presentation to a myoglobin-specific, IAS restricted T cell hybridoma. The rationale for this experiment stems from the fact that if such a correlation can be demonstrated, this suggests that the binding site studied by the direct binding assay is the same site used by antigenic peptides when recognized by T cells. The good correlation observed supports the biological relevance of the assay established. The IA' and IAd binding pattern of a series of overlapping peptides was examined next. Two major points can be raised on the basis of the data obtained. Firstly, it can be observed that the major IAs-restricted, autoantigenic, and pathogenic MBP determinant is associated with high affinity binding and is, in fact, the highest binder of a whole series of overlapping peptides spanning through the entire MBP protein sequence. This result suggests that high affinity binding may be necessary for a self peptide in order to be a major autoantigenic or pathogenic determinant. In this respect, it is interesting that the same determinant binds with - 100-fold weaker affinity to IAd, a class
II molecule that has not, to the best of our knowledge (2), ever been reported as capable of acting as a restriction element for a pathological, MBP-specific autoimmune response. Secondly, the IA* and IAd binding profiles of the series of MBP-derived peptides suggest that high affinity binding may be a necessary condition, but not sufficient in itself for pathogemcity. In this respect, it is noteworthy that two other peptides (Ac 1 - 20 to both IAS and IAd, and 61 - 8 0 to IA01) bound with affinities similar to the one detected for the MBP 81 - 100/IA" pair. This result suggests that variables other than MHC affinities, such as perhaps the structure of T cell repertoire, selection by superantigens, or differential processing, may also be involved in determining the pathogenic potential of self determinants. We are now in the process of performing similar experiments using purified human class II molecules and human MBP peptides, to establish whether the observation that autoantigenic determinants which are high affinity class II binders can be reproduced in human systems, and for the peptide determinants and class alleles that have been implicated (5-8) in the pathogenesis of multiple sclerosis.
Acknowledgements The authors gratefully acknowledge the excellent assistance of Joyce Joseph and Grace Lapid in preparation of the manuscript. This work was supported in part by National Institutes of Health Grant AI18634.
Abbreviations DCM DIEA DMF EAE HA HEL HOBt MBP Nase OVA
dichloromethane diisopropylethytamine dimethytformamide experimental autoimmune encephalomyeiitis influenza hemagglutinin hen egg lysozyme hydroxybenzotriazole myelin basic protein staphyiococcal nuclease chicken egg ovalbumin
Downloaded from http://intimm.oxfordjournals.org/ at University of Birmingham on August 31, 2015
Residues
50%/ dose IAd
Autoantigens are high affinity class II binders 777 PAM PLP TFA
phenylactamidomethyl proteolipid protein trifluoroacetic acid
References
Downloaded from http://intimm.oxfordjournals.org/ at University of Birmingham on August 31, 2015
1 Nepom, G. T 1991. MHCclass-ll molecules and autoimmunity./4/inu. Rev. Immunol. 9:493. 2 Smilek, D. E , Lock, C. B., and McDevrtt, H. O. 1990. Antigen recognition and peptide-mediated immunotherapy in autoimmune disease. Immunol. Rev. 118:37. 3 Baekkeskov, S., Aanstoot, H. J , Christgau. S , Reetz, A , Sdimena, M , Cascalho, M., Folli. F., Richter-Olesen, H., and Camilli, P. D. 1990. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decartxixytase. Nature 347:151 4 Roep, B. O , Arden, S. D., DeVries, R. R P , and Hutton, J. C. 1990. T-cell clones from a type-1 diabetes patient response to insulin secretory gradule proteins. Nature 345632. 5 Martin, R., Howell, M. D., Jaraquemada, D., Flerlage, M., Richert, J , BrostoH, S , Long, E O , McFarlin, D. E., and McFarland, H. F 1991. A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis. J. Exp. Med. 173 19. 6 Martin, R., Jaraquemada, D., Rerlage, M., Richert, J., Whitaker, J., Long, E. O., McFarlin, D. E., and McFarland, H. F 1990. Fine specificity and HLA restriction of myelin basis protein-specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals J. Immunol. 145540 7 Pette, M., Fujita, K., Wilkinson, D., Altmann, D. M., Trowsdale, J , Giergerich, G., Hinkkanen, A , Epplen, J. T., Kappose, L , and Wekerle, H 1990. Myelin autoreactivity in multiple sclerosis, recognition of myelin basic protein in the context of HLA-DR products by T lymphocytes of multiple-sclerosis patients and healthy donors. Proc Natl Acad. Sd. USA 87:7968. 8 Ota, K., Malsui, M., Milford, E. L , Mackin, G A., Werner, H. L, and Hafler, D A. 1990. T-cell recognition of a immunodominant myelin base protetn eprtope in multiple sclerosis. Nature 346-183 9 Sakai, K., Zamvil, S. S., Mitchell, D. J , Lim, M., Rothbard, J. B , and Steinman, L. 1988 Characterization of a major encephalrtogenic T cell eprtope in SJUJ mice with synthetic oligopepbdes of myelin basic protein. J Neuroimmunol. 1921 10 Whitham, R. H , Bourdette, D. N., Hashim, G. A., Herndon, R. M., llg, R. C, Vandenbark, A. A , and Offner, H. 1991. Lymphocytes from SJUJ mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis. J Immunol. 146:101. 11 Clayton, J. P., Gammon, G. M , Ando, D. G., Kono, D. H., Hood, L, and Sercarz, E. E. 1989. Peptide-specific prevention of experimental allergic encephalomyelitis. Neonatal tolerance induced to the dominant T cell determinant of myelin basic protein. J. Exp. Med. 169:1681 12 Lamont, A. G., Sette, A., Fujinami, R., Col6n, S. M., Miles, C , and Grey, H. M 1990. Inhibition of experimental autoimmune
encephalomyetlitis induction in SJUJ mice by using a peptide with high affinity for IAS molecules. J. Immunol. 145:1687. 13 Zamvil, S. S., Mitchell. D. J., Moore, A. C , Kitamura, K., Steinman, L, and Rothbard, J. B. 1986. T-cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis. Nature 342:258. 14 Zamvil, S. S., Mitchell, D. J., Powell, M. B., Sakai, K., Rothbard, J. B., and Steinman, L. 1988. Multiple discrete encephalitogenic epitopes of the autoantigen myelin basic protein include a determinant for I-E class ll-restricted T cells. J. Exp. Med. 168:1181. 15 Sakai, K., Zamvil, S. S , Mitchell, D. J., Hodgkinson, S., Rothbard, J. B., and Steinman, L. 1989. Prevention of experimental encephalomyelitis with peptides that block interaction of T cells with major histocompatibinty complex proteins. Proc. Natl Acad. Sd. USA 86:9470. 16 Wraith, D. C , Smilek, D. E, Mitchell, D. J., Steinman, L, and McDevitt, H. O. 1989. Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy Cell 59:247. 17 Snnam, S., Topham, D. J., and Carroll, L 1987. Haplotype-specrfic suppression of experimental allergic encephalomyelitis with anti-IA antibodies J. Immunol. 139.1485. 18 Gammon, G and Sercarz, E. 1989. How some T cells escape tolerance induction. Nature 342:183. 19 Milich, D. R., Jones, J. E., McLachlan, A., Houghten, R., Thornton, G. B., and Hughes, J. L 1989. Distinction between immunogenicity and tolerogenicity among HBcAg T cell determinants. Influence of peptide-MHC interaction. J. Immunol. 1433148 20 Oldstone, M. B. 1989 Molecular mimicry as a mechanism for the cause and a probe uncovering etiologic agent(s) of autoimmune disease. Curr Top. Microbiol. Immunol. 145:127. 21 Janeway, Jr., C. A , Conrad, P. J., Lerner, E. A., Babich, J , Wettstein, P., and Murphy, D. B. 1984 Monoclonal antibodes specific for la glycoproteins raised by immunization wtth activated T cells: possble role of T cellbound la antigens as targets of immunoregulatory T cells. J. Immunol. 132.662 22 Sette, A., Southwood, S., O'Sullivan, D., Gaeta, F C. A., Sidney, J , and Grey, H. M. 1992. The effect of pH on class II-peptide interactions. J Immunol. 148:844 23 Sette, A., Sidney, J., Albertson, M., Miles, C , Col6n, S. M , Pedrazzini, T., Lamont, A. G , and Grey, H. M. 1990. A novel approach to the generation of high affinity class ll-binding peptides. J. Immunol. 145.1809. 24 Buus, S., Sette, A., Colon, S. M., Miles, C , and Grey, H. M. 1987. The relation between major histocompatibility complex (MHC) restriction and the capacity of la to bind immunogenic peptides. Science 235:1353. 25 Lamont, A. G , Powell, M F., Col6n, S. M., Miles, C, Grey, H M., and Sette, A. 1990. The use of peptide analogs with improved stability and MHC binding capacity to inhibit antigen presentation in vitro and in vivo J. Immunol. 144:2493. 26 Kono, D. H , Urban, J. L, Horvath, S. J., Ando, D. G., Saavedra, R. A., and Hood, L. 1988. Two minor determinants of myelin baste protein induce experimental allergic encephalomyelitis in SJUJ mice. J. Exp. Med. 168:213.
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High affinity for class II molecules as a necessary but not sufficient characteristic of encephalitogenic determinants Melissa Wall, Scott Southwood, John Sidney, Carla Oseroff, Marie-France del Guericio, Alan G. Lamont1, Sonia M. Col6n, Thomas Arrhenius, Federico C. A. Gaeta, and Alessandro Sette
Key words: autcxmmunity, class II affinity, experimental autoimmune encephalomyelitis, myelin basic protein
Abstract A direct binding assay specific for IA* molecules has been developed and Its immunological relevance validated by examining, for a panel of nine different synthetic peptJdes, the correlation between their capacity to bind purified IA* and to inhibit IA*-restrlcted antigen presentation. The IA* assay thus developed has then been used to study the IA* binding affinity of a set of overlapping peptJdes spanning the entire myelin basic protein (MBP). It was found that the encephalitogenic MBP region corresponds to peptides with high MHC binding affinities. Other regions of the MBP that have not been described as being pathogenic in the context of IA* molecules have also been found to be high IA* binders, suggesting that variables other than MHC affinity are also Involved in determining the pathogenic potential of self-derived determinants. Introduction Immune recognition of normal self components leading to tissue destruction and pathological abnormalities is underlying a group of apparently disparate diseases such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis, collectively known as autoimmune diseases (1,2) Susceptibility to these diseases is strongly associated with particular class II alleles, suggesting that class ll-restricted binding and presentation of specific autoantigens may be involved in the disease process. In humans, the exact molecular identity of the actual autoantjgen(s) recognized in the disease process is unknown, even though certain candidates have been implicated by several recent studies (3-8). By contrast, in a few animal disease models the actual molecular determinants (and the presenting class II alleles) involved in disease activation are well characterized (2,9-16). In particular, experimental autoimmune encephalomyelitis (EAE) is an experimental disease model of human multiple sclerosis. EAE is usually induced by injection with either whole spinal cord homogenate, purified myelin basic protein (MBP), or synthetic peptides derived from MBP or proteolipid protein (PLP). In mice, strains expressing certain MHC class II alleles, such as PLJ (expressing IA") or SJL (expressing IA8), appear to
be very susceptible to disease induction, while other strains are considered resistant (2). In particular, in H-2* mice (expressing IAd and IEd molecules), successful induction of EAE has been reported using only whole spinal cord homogenate (17), and IAd molecules have not thus far been reported as capable of acting as a restriction element for a pathological MBP-specific autoimmune response. A few different peptide epitopes from MBP and PLP have been identified and characterized. More specifically, MBP Ac 1-11 appears to contain the major encephalitogenic determinant for IAu-expressing mice (such as PLJ), while MBP 87-98 and PLP 139-151 appear to be dominant autoantigenic determinants for mice expressing the IAS molecules (such as SJL). An important point of debate has been the binding affinity of autoantigenic epitopes for their restricting class II epitopes. According to one theory, autoreactive pathogenic T cell clones could recognize low affinity binders, and have for that reason escaped deletion at the negative selection level (18,19). According to the opposite point of view, autoantigens are high affinity binders and thereby potentially highly immunogenic. When some as yet unspecified event, such as, for example, regulatory
Correspondence to: A. Sette Transmitting editor: G. Doria
Received 9 March 1992, accepted 6 April 1992
Downloaded from http://intimm.oxfordjournals.org/ at University of Birmingham on August 31, 2015
Cytel, 3525 John Hopkins Court, San Diego, CA 92121, USA 'Roche Products Ltd Research Centre, Broadwater Road, Welwyn Garden City, Hertfordshire, UK
774
Autoantigens are high affinity class II binders
imbalances (1,2) or molecular mimicry (20), leads to a breakdown of tolerance, their high immunogenic potential is then realized. In the present study, we took advantage of the delineation of MBP 87-98 and PLP 139-151 as encephalitogenic determinants presented by IAS class II molecules to address this question at the experimental level. For this reason, an IA"-specific binding assay has been developed and overlapping peptides spanning through the entre mouse MBP sequence have been synthesized. Methods Cells
Affinity purification of IAS molecules Since LS102.9 cells were derived by fusion of IAS spleen cells with A20 cells, they expressed both IAd and IA8. To avoid contamination with IAd molecules, the antibody Y3JP was used to purify IA3 molecules from the cell lysates. This antibody, in fact, does not cross-react with either IAd or mixed isotype lA"* molecules (21). IA8 molecules were purified as previously described (22) using the MAb Y3JP coupled to Protein A-Sepharose CL 4B beads. Lysates were filtered through 0.45 /iM filters and then passed over the affinity column, which was then washed with 20 column volumes of 0.5% NP-40 - 0 . 1 % SDS, 5 column volumes of PBS, and 2 column volumes of PBS containing 1 % n-octyglucoside. Finally, the IA* was eluted with 0.05 M diethylamine in 0.15 M NaCI containing 1 % noctyglucoside, pH 11.5. A 1 /20 volume of 1.0 M Tris, 1.5 M NaCI, pH 6.8, was added to the eluate to reduce the pH to ~ 7.5 and then concentrated by ultrafiltration in AMICON with YM30 membrane. Peptide synthesis Peptide syntheses were carried out by sequential coupling of A/a-r-Boc-protected amino acids on an Applied Biosystems (Foster City, CA, USA) 430A peptide synthesizer using standard f-Boc coupling cycles (software version 1.40) All amino acids, reagents, and resins were obtained from Applied Biosystems or Bachem (Torrance, CA, USA). Solvents were obtained from Burdick & Jackson (Muskegon, Ml, USA). Solid-phase synthesis was started from an appropriately substituted r-Boc-amino acid - phenylacetamidomethyl (PAM) resin. The loading of the starting resin was 0.5-0.7 mmol/g polystyrene and 0.5 meq were used in each synthesis. A typical reaction cycle proceeded as follows, (i) The N-terminal f-Boc group was removed with 33% trifluoroacetic acid (TFA) in dichloromethane (DCM) for 1.5 min, followed by another treatment with 50% for 18.5 min. (ii) The resin was washed 3 times with DCM and neutralized twice with 10% diisopropylethylamine (DIEA) in dimethylformamide (DMF) for 1 min each and then the resin was washed 5 times with DMF.
Class II-peptide
binding assays
Purified class II molecules were incubated with 5 nM 1 2 5 I radiolabeled ROIV peptide (sequence: YAHAAHAAHAAHAAHA) (23) for 48 h in PBS containing 5% DMSO in the presence of a protease inhibitor mixture. Purified peptides were iodinated using the chloramine-T method (24). The final concentrations of protease inhibitors were 1 mM PMSF, 1.3 mM 1.10 phenanthroline, 73 /iM pepstatin A, 8 mM EDTA, 6 mM Nethylmaleimide, and 200 pM /VQ-p-tosyl-L-lysine chloromethyl ketone. Final detergent concentration in the incubation mixture was 2.6% Digitonin. Class II - peptide complexes were separated from free peptide by gel filtration on TSK2000 columns and the fraction of peptide bound was calculated as previously described (22). In preliminary experiments, each of the IA* preparations was titered in the presence of fixed amounts of radiolabeled peptide to determine the concentration of class II molecules necessary to bind - 1 0 % of the total radioactivity. All subsequent inhibition and direct binding assays were then performed using these class II concentrations. In the inhibition assays, peptide inhibitors were typically tested at concentrations ranging from 120 nglm\ to 1.2 ng/ml. The data were then plotted and the dose yielding 50% inhibition measured. Each peptide was tested in two to four completely independent experiments.
Results Development of an IAs-specific binding assay In a previous study, several different synthetic peptides were tested for their capacity to inhibit antigen presentation in an IA8restricted system (12). Since a protocol involving a short pulse on fixed cells was used, their inhibitory capacity could be taken as an approximate measure of affinity for class II molecules. In the present study we have used that information to develop and validate a molecular binding assay. More specifically, one of the peptides which scored as highly inhibitory in the inhibition of antigen presentation assay, ROIV, was radiolabeled and incubated (as previously described for other IA alleles) with affinity
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The B cell hybrid LS102.9 was used as a source of IA». LS102.9 cells were maintained in vitro by culture in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 nM 2-ME, and 10% heat-inactivated FCS, and were also supplemented with 100 /ig/ml of streptomycin (Irvine Scientific, Santa Ana, CA, USA) and 100 U/ml of penicillin (Gibco, Grand Island, NY, USA). Cells were lysed at a concentration of 10s cells/ml in PBS containing 1 % NP-40,1 mM PMSF. The lysates were cleared of nuclei and debris by centrifugation at 15 000 g for 15 mm.
(iii) The DMF solution of a 2-fold excess of preformed symmetric anhydride generated from 2 mmol f-Boc-amino acid was added to the resin, and the mixture allowed to react for 1 5 - 4 5 min. Hydroxybenzotnazole (HOBt) esters (4-fold excess) were used for the coupling of arginine, asparagine, glutamine, and histidine residues, and the coupling step was repeated to ensure completeness, (iv) The resin was washed with DCM in preparation for the next elongation cycle. The fully protected, resin-bound peptide was subjected to a TFA cycle to remove the terminal f-Boc, and the product washed with DCM and dried. The resin was then treated with anhydrous HF in the presence of appropriate scavengers [e.g. 5% (v/v) p-cresol/15% (v/v) ethanedithiol] for 60 min at 0°C. After evaporation of excess HF, the crude peptide was washed with diethyl ether, extracted with aqueous acetic acid, and lyophilized. The crude peptides were purified to > 9 5 % homogeneity by reversed-phase HPLC using H2O/CH3CN gradients containing 0.2% TFA modifier on a Vydac, 300 A pore-size, C-18 preparative column. The purity of the synthetic peptides was assayed on an analytical reversephase column, and their composition ascertained by amino acid analysis and/or sequencing.
Autoantigens are high affinity dass II binders 775 8
purified IA molecules for 2 days at room temperature. The results obtained are shown in Fig. 1. Good binding activity (up to - 1 0 % of the input) was detected for IAS concentrations in the 0 . 1 - 1 /iM range (Fig. 1a). The binding was specific (Fig. 1b), in that it was inhibitable by excess unlabeled ROIV peptide (IC^, = - 1 5 nM). Scatchard analysis revealed that as previously shown for other IA alleles, only a minor fraction of the IAS molecules appeared to be available for binding (data not shown). Low levels of available binding sites are probably due to the presence of endogenously derived peptides in the IAS peptide binding site. Correlation between inhibition of antigen presentation and direct binding assay
8
12 i
IA$ and IAd capacity of a series of synthetic peptides spanning through the entire MBP sequence
10 8-
g
6H
CD
*
4
-
2-
.001
.01
.1 \iM
1
10
MHC
A set of overlapping 20-residue peptides spanning through the entire MBP sequence were synthesized and purified. They were then tested for their capacity to bind purified IA8 molecules. They were also tested for binding to IAd, chosen as a representative MHC molecule that does not, to the best of our knowledge (2), serve as a restriction element of MBP-specific EAE. The data obtained are shown in Table 2. It was found that the peptide 81 -100, which contains the MBP 8 7 - 9 8 region, was the highest affinity IAS binder of the entire MBP protein. The other peptide containing the MBP region, that has been described as being a dominant autoantigenic site in IAU mice (MBP Ac 1-11), also bound IA8 with similar high affinity. Six other peptides bound with lower affinities in the 1 - 1 0 nM range. Interestingly, three of them (peptides 121 -140,131 -150, and
Table 1. Correlation between direct binding and inhibition of antigen presentation data
Peptide KM core extension PLP 139-151 OVA 271-285. Nase 101-120 HA 307-319 OVA 1 5 - 2 9 .0001
Fig. 1. (a) Binding profile of 12Sl-radiolabeled ROIV peptide (5 nM) to purified IA* molecules, (b) Inhibition of the binding of radiolabeled ROIV peptides to purified IA8 molecules (1 *»M) by excess unlabeled ROIV peptide.
DE core extension X rep. 1 2 - 2 6 HEL 4 6 - 6 1
Direct binding assay OJM 50% dose) 0.71 0 46 0 43 3.3 42 8.9
Inhibition of antigen presentation assay8 OiM 75% dose) 4 4.1 7.6 10 56 200
_b
—
T h e inhibition of antigen presentation data has been published elsewhere (11) and is shown here only for the purpose of reference. b — indicates >200 /iM.
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Having established this assay, we wished to obtain validation from the immunological point of view. To do so, we examined whether the data previously obtained in the inhibition of antigen presentation assay correlated with the data generated by the molecular assay. The rationale of such an experiment resides
in the fact that if correlation can be demonstrated, this would suggest that the radiolabeled ROIV peptide binds in the same binding site used by antigen peptides when recognized by T cells (23,25). More specifically, nine peptides previously tested for inhibition of antigen presentation were tested for their capacity to inhibit ROIV binding [Table 1; the data for inhibition of antigen presentation have already been published elsewhere (12), and are shown here only for reference purposes]. The three peptides [KM core extension PLP 139-151, chicken egg ovalbumin (OVA) 271 -285, and staphylococcal nuclease (Nase) 101-120] that scored as good inhibitors of antigen presentation (ICso 1 - 1 0 /iM) also were powerful inhibitors in the direct binding assay (IC50 < 5 /tM). Peptides HA 307 - 319 and OVA 15 - 29, which were less potent inhibitors of antigen presentation (IC50 10-200 /xM), were also less potent binders (ICSQ 5 - 5 0 ^M). Finally, the three peptides [DE core extension, X rep 12 - 26 and hen egg lysozyme (HEL) 4 6 - 6 1 ] that scored negative in the binding assay were also negative for IAS binding activity. Taken together, the data demonstrate that an excellent correlation exists between the data generated in the two assays, thus supporting the immunological relevance of the direct binding assay established.
776 Autoantigens are high affinity class II binders Table 2. IAS and IAd binding profile of a series of overlapping peptides spanning through the entire mouse MBP sequence 50%/ dose IAS
Sequences
Ac 1 - 20 11 •30 21- •40 31- •50 41 ••60 51 ••70 61- •80 7 1 • •90 81 •100 91- •110 101 •-120 111- •130 121 •140 131 •150 141 •160 151 •170
Ac-ASQKRPSQRSKYLATASTMD KYLATASTMDHARHGFLPRH HARHGFLPRHRDTGI LDSI G RDTGI LDSIGRFFSGDRGAP RFFSGDRGAPKRGSGKDSHT KRGSGKDSHTRTTHYGSLPQ RTTHYGSLPQKSQHGRTQDE KSQHGRTQDENPVVHFFKNI NPVVHFFKNIVTPRTPPPSQ VTPRTPPPSQGKGRGLSLSR GKGRGLSLRFSVVGAEGQKP FSVVGAEGQKPGFGYGGRASD GFGYGGRASDYKSAHI KGFKG YKSAHKGFKGAYDAQGTISK AYDAQGTLSKI FKLGGRDSR I FKLGGRDSRSGSPMARR
a
0.60 3.7 143
0.42 2.8 50
5.3 95 19 0.36 36 8.4 13 1.9 3 2.5 33
3.4 0.45 25 50 150
— indicates >20O ,
span through the 121-150 region that has also been demonstrated (26) to induce disease in IAS mice. Finally, five peptides bound in the 10 - 1 0 0 ^M range and no binding activity could be detected, up to the 100 pM level, for the remaining three epitopes. For IAd molecules, high binding is detected for Ac 1 - 2 0 and MBP 61 - 8 0 . Three other peptides (MBP 1 1 - 3 0 , 51 - 7 0 , and 101 -120) bound in the 1-10 yU range. Three peptides bound with weaker affinities (10-100 ^M). Finally, for the remaining eight peptides no binding was detected up to the 100 MM level. Discussion In the present report, a direct peptide-IA s binding assay is described. The assay established was validated at the biological level by examining, for a panel of nine different synthetic peptides, the correlation between data obtaned in the direct binding assay and data previously obtained (12) when the same set of peptides was tested for its capacity to inhibit antigen presentation to a myoglobin-specific, IAS restricted T cell hybridoma. The rationale for this experiment stems from the fact that if such a correlation can be demonstrated, this suggests that the binding site studied by the direct binding assay is the same site used by antigenic peptides when recognized by T cells. The good correlation observed supports the biological relevance of the assay established. The IA' and IAd binding pattern of a series of overlapping peptides was examined next. Two major points can be raised on the basis of the data obtained. Firstly, it can be observed that the major IAs-restricted, autoantigenic, and pathogenic MBP determinant is associated with high affinity binding and is, in fact, the highest binder of a whole series of overlapping peptides spanning through the entire MBP protein sequence. This result suggests that high affinity binding may be necessary for a self peptide in order to be a major autoantigenic or pathogenic determinant. In this respect, it is interesting that the same determinant binds with - 100-fold weaker affinity to IAd, a class
II molecule that has not, to the best of our knowledge (2), ever been reported as capable of acting as a restriction element for a pathological, MBP-specific autoimmune response. Secondly, the IA* and IAd binding profiles of the series of MBP-derived peptides suggest that high affinity binding may be a necessary condition, but not sufficient in itself for pathogemcity. In this respect, it is noteworthy that two other peptides (Ac 1 - 20 to both IAS and IAd, and 61 - 8 0 to IA01) bound with affinities similar to the one detected for the MBP 81 - 100/IA" pair. This result suggests that variables other than MHC affinities, such as perhaps the structure of T cell repertoire, selection by superantigens, or differential processing, may also be involved in determining the pathogenic potential of self determinants. We are now in the process of performing similar experiments using purified human class II molecules and human MBP peptides, to establish whether the observation that autoantigenic determinants which are high affinity class II binders can be reproduced in human systems, and for the peptide determinants and class alleles that have been implicated (5-8) in the pathogenesis of multiple sclerosis.
Acknowledgements The authors gratefully acknowledge the excellent assistance of Joyce Joseph and Grace Lapid in preparation of the manuscript. This work was supported in part by National Institutes of Health Grant AI18634.
Abbreviations DCM DIEA DMF EAE HA HEL HOBt MBP Nase OVA
dichloromethane diisopropylethytamine dimethytformamide experimental autoimmune encephalomyeiitis influenza hemagglutinin hen egg lysozyme hydroxybenzotriazole myelin basic protein staphyiococcal nuclease chicken egg ovalbumin
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Residues
50%/ dose IAd
Autoantigens are high affinity class II binders 777 PAM PLP TFA
phenylactamidomethyl proteolipid protein trifluoroacetic acid
References
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