Binding stability was based on calculated half-life (predicted t1/2 in minutes at 37°C) of the dissociation of peptide-HLA class I complexes.

Binding stability was based on calculated half-life (predicted t1/2 in minutes at 37°C) of the dissociation of peptide-HLA class I complexes.

4. Resuspend cells in 250 ^L PBS.

5. Detect tetramer-stained fluorescent cells using appropriate flow cytometry fluorescent channel (i.e., FL-2 for phycoerythrin) (see Fig. 6).

6. During tetramer/CD8 double staining of cells, some anti-CD8 Abs may interact with the tetramer to decrease or inhibit antigen-specific tetramer binding.

4. Notes

1. TAL-H enzyme activity was tested in the presence of 3.2 mMD-fructose-6-phos-phate, 0.2 mM erythrose-4-phosphate, 0.1 mM nicotinamide adenine dinucleotide hydrogen (NADH), 10 ^g a-glycerophosphate dehydrogenase/triosephosphate isomerase at a 1:6 ratio at room temperature by continuous absorbance reading at 340 nm for 20 min (4).

Fig. 5. Schematic diagram of a tetrameric MHC-antigen complex or tetramer. Four antigen-bearing MHC molecules (heavy-chain subunits with p2-microglobulin) are attached via biotin to a single molecule of phycoerythrin (PE)-labeled streptavidin.

Highly specific polyclonal Abs against rTAL-H were raised in New Zealand white rabbits immunized on two separate occasions 3 wk apart with 500 mg of gel-purified rTAL-H protein (7). Specific reactivity of immune sera was evaluated by Western blot analysis using preimmune rabbit sera as a negative control (7). The Abimed (Langenfeld, Germany) automated solid-phase peptide synthesizer uses N-terminal Fmoc (9-fluorenylmethoxycarbonyl) amino acid protection chemistry (based on the Merrifield method) for assembling spot-synthesized peptides (19) directly on the surface of a polyethylene glycol (PEG)-modified and (P-ala-nine)2-functionalized cellulose membrane (30) (e.g., GRVSTEVDARLSFDK- p alanine-PEG-cellulose). The cellulose membrane's porous and hydrophilic qualities allow measurement of aqueous solutions (31). PEG and (P-Ala)2 act as spacers, improving peptide accessibility and preserving the natural conformation of the C-terminus attached peptide. The (P-Ala)2 anchor provides a uniform start for synthesis, following the removal of its Fmoc group, allowing receipt of the incoming protected and activated amino acids to be spotted by the Abimed ASP 422 robotic arm (31).

Fig. 6. Flow cytometry analysis of PBLs of control and patients with MS as well as T-cell line (TCL) from patient MS 2. Control cells were unstained, and tetramer-stained cells were stained with TAL-H 168-176/HLA*0201 tetramer for 30 min at 4°C.

4. Cutoff levels are set at 3 SDs over reactivity of negative control sera. Reactivities at or below cutoff levels are set at 0. Maximum reactivity with a single peptide is set at 2.0 for each Ab. Immunodominant epitopes with high binding affinity to Ab were defined as having a binding intensity of half-maximal reactivity (1.0) or above, that is, 10-fold or more over background. Repeat experiments should give variations of less than 10% for peptide-binding values (18).

5. TAL-H peptides not recognized by the polyclonal rabbit sera (raised against full-length TAL-H) but recognized by MS sera may indicate nonimmunogenic pep-tides in the rabbit. However, this may indicate a cryptic epitope, perhaps resulting from proteolytic degradation after TAL-H release during demyelination in the human MS brain (8,18).

6. Previously, concentration ranges of 1-30 ^g/mL for TAL-H and 1-100 ^g/mL for MBP were tested to find the optimal concentration for inducing cell proliferation (8). MBP was purified from neurologically normal human brain according to the procedure of Deibler et al. (32).

7. SI = (Measurement for target material)/(Measurement for control material). An SI value of 3.5 (i.e., an average of 3.5-fold increase of 3H-TdR incorporation in peripheral blood mononuclear cells in response to a single in vitro exposure to antigen) is considered highly significant.

8. Initially, determine optimal tetramer concentration during incubation (e.g., 100fold dilution, 1000-fold dilution). Also, staining should be tried at 4°C, room temperature, and 37°C. To optimize the signal-to-noise ratio, incubate for 15-60 min, decreasing the time used for staining as the temperature increases (28).


This work was supported by grant RG 2466 from the National Multiple Sclerosis Society.


1 Oldstone, M. B. A. (1998) Molecular mimicry and immune-mediated diseases. FASEB J. 12, 1255-1265.

2 Wucherpfennig, K. W. and Strominger, J. L. (1995) Molecular mimicry in T cellmediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80, 695-705.

3 Martin, R., McFarland, H. F., and McFarlin, D. E. (1992) Immunological aspects of demyelinating diseases. Annu. Rev. Immunol. 10, 153-187.

4 Banki, K., Colombo, E., Sia, F., Halladay, D., Mattson, D., Tatum, A., et al. (1994) Oligodendrocyte-specific expression and autoantigenicity of transaldolase in multiple sclerosis. J. Exp. Med. 180, 1649-1663.

5 Roder, J. and Hickey, W. F. (1996) Mouse models, immunology, multiple sclerosis and myelination. Nat. Genet. 12, 6-8.

6. Lees, M. B. and Brostoff, S. W. (1984) Proteins of myelin, in Myelin (Morell, P., ed), Plenum Press, New York, pp. 197-224.

7 Banki, K., Halladay, D., and Perl, A. (1994) Cloning and expression of the human gene for transaldolase: a novel highly repetitive element constitutes an integral part of the coding sequence. J. Biol. Chem. 269, 2847-2851.

8 Colombo, E., Banki, K., Tatum, A. H., Daucher, J., Ferrante, P., Murray, R. S., et al. (1997) Comparative analysis of antibody and cell-mediated autoimmunity to transaldolase and myelin basic protein in patients with multiple sclerosis. J. Clin. Invest. 99, 1238-1250.

9 Devereux, J., Haeberli, P., and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395.

10. Parker, K. HLA peptide binding predictions. NIH BIMAS website: http:// Accessed April, 2003.

11. Smith, D. B. and Johnson, K. S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31-40.

12. Banki, K. and Perl, A. (1996) Inhibition of the catalytic activity of human transaldolase by antibodies and site-directed mutagenesis. FEBSLett. 378, 161-165.

13. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.

14. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81, 3998-4002.

15. Geysen, H. M. (1990) Molecular technology: peptide epitope mapping and the pin technology. Southeast Asian J. Trop. Med. Public Health 21, 523-533.

16. Williams, R.C., Jr., Staud, R., Malone, C. C., Payabyab, J., Byres, L., and Underwood, D. (1994) Epitopes on proteinase-3 recognized by antibodies from patients with Wegener's granulomatosis. J. Immunol. 152, 4722-4737.

17. Lang, E., Szendrei, G., Lee, V. M., and Otvos, L., Jr. (1994) Spectroscopic evidence that monoclonal antibodies recognize the dominant conformation of medium-sized synthetic peptides. J. Immunol. Methods 170, 103-115.

18. Esposito, M., Venkatesh, V., Otvos, L., Weng, Z., Vajda, S., Banki, K., et al. (1999) Human transaldolase and cross-reactive viral epitopes identified by autoantibodies of multiple sclerosis patients. J. Immunol. 163, 4027-4032.

19. Frank, R. (1992) Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217-9232.

20. Fleiss, J. L. (1981) Statistical Methods for Rates and Proportions, Wiley, New York.

21. Germain, R. N. (1994) MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76, 287-299.

22. Sette, A., Vitiello, A., Reherman, B., Fowler, P., Nayersina, R., Kast, W. M., et al. (1994) The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol. 153, 5586-5592.

23. Tsuchida, T., Parker, K. C., Turner, R. V., McFarland, H. F., Coligan, J. E., and Biddison, W. E. (1994) Autoreactive CD8+ T-cell responses to human myelin protein-derived peptides. Proc. Natl. Acad. Sci. U S A 91, 10,859-10,863.

24. Parker, K. C., Bednarek, M. A. and Coligan, J. E. (1994) Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152, 163-175.

25. Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., et al. (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274,94-96. Published erratum appears in Science (1998) 280, 1821.

26. Bodinier, M., Peyrat, M. A., Tournay, C., Davodeau, F., Romagne, F., Bonneville, M., et al. (2000) Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding. Nat. Med. 6, 707-710.

27. Kuroda, M. J., Schmitz, J. E., Barouch, D. H., Craiu, A., Allen, T. M., Sette, A., et al. (1998) Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J. Exp. Med. 187, 1373-1381.

28. National Institutes of Allergy and Infectious Diseases (NIAID), Tetramer Facility Guide, McKesson Biosciences Corp. index.html. April 2003

29. McMichael, A. J. and O'Callaghan, C. A. (1998) A new look at T cells. J. Exp. Med. 187, 1367-1371.

30. Rudiger, S., Germeroth, L., Schneider-Mergener, J., and Bukau, B. (1997) Substrate-specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501-1507.

31. Otvos, L., Jr., Pease, A. M., Bokonyi, K., Giles-Davis, W., Rogers, M. E., Hintz, P. A., et al. (2000) In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen. J. Immunol. Methods 233, 95-105.

32. Deibler, G. E., Martenson, R. E., and Kies, M. W. (1972) Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep. Biochem. 2, 139-165.

Animal Models of Autoimmunity

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