rent studies provide a conceptual framework for developing approaches to regulate T cell function through CTLA-4

References and Notes 1. J. A. Bluestone, J. Immunol. 158, 1989 (1997); A. I. Sperling and J. A. Bluestone, Immunol. Rev. 153, 155 (1996). 2. C. R. Calvo, D. Amsen, A. M. Kruisbeek, J. Exp. Med. 186, 1645 (1997), P. S. Linsley et al., Immunity 4, 535 (1996); M. Griffin and J. A. Bluestone, unpublished observations. 3. W. Zhang et al., Cell 92, 83 (1998). 4. K.-M. Lee et al., unpublished observation. 5. A. M. Weissman, L. E. Samelson, R. D. Klausner, Nature 324, 480 (1986); M. Baniyash et al., J. Biol. Chem. 263,18225 (1988). 6. P. W. Wallace et al., Transplantation 58, 602 (1994). 7. F. Letourneur and R. Klausner, Science 255, 79 (1992). 8. J. D. Bradshaw et al., Biochemistry 36, 15975 (1997); E. Chuang et al., J. Immunol., in press. 9. L. E. Marengere et al., Science 272, 1170 (1996). 10. P. J. Blair et al., J. Immunol. 160, 12 (1998). 11. A. Kupfer and S. J. Singer, Proc. Natl. Acad. Sci. U.S.A. 85, 8216 (1988); C. B. Thompson and J. P. Allison, Immunity 7, 445 (1997). 12. E. Chuang et al., J. Immunol. 159, 144 (1997); Y. Zhang and J. P. Allison, Proc. Natl. Acad. Sci. U.S.A. 94, 9273 (1997). 13. G. Feng et al., Science 259, 1607 (1993); W. Vogel et al., ibid., p. 1611; T. Tauchi et al., J. Biol. Chem. 269, 15381 (1994). 14. E. N. Kersh, A. S. Shaw, P. M. Allen Science 281, 572 (1998). 15. T. L. Walunas et al., Immunity 1, 405 (1994). 16. O. Leo et al., Proc. Natl. Acad. Sci. U.S.A. 84, 1374 (1987). 17. R. Abe et al., J. Immunol. 154, 985 (1995). 18. B. A. Houlden, R. Q. Cron, J. E. Coligan, J. A. Bluestone, ibid. 141, 3753 (1988). 19. T cells from BALB/c lymph nodes were purified (15) and cultured for 60 hours in a dish coated with mAb to CD3 (anti-CD3) (145-2C11; 2 mg/ml) (16) and mAb to CD28 (anti-CD28) (PV-1; 2 mg/ml) (17). Cells were then washed three times to remove any residual antibodies present on the cell surface and rested at 37oC for additional 4 hours. 20. M. Griffin et al., unpublished data. 21. Immunoprecipitations were performed with lysates prepared in LB [1% Nonidet P-40, 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 20 mM EDTA (pH 8.0), 1 mM sodium vanadate, leupeptin (10 mg/ml), 10 mM aprotinin, 1 mM phenylsulfonylsulfoxide]. Lysates were precleared twice with protein A beads and once with protein A beads coated with a mAb from a control hamster (UC3-10A6) (18) before precipitating antibodies were added. Immunoprecipitation was performed overnight at 4oC. Immune complexes were washed five times with LB and subjected to SDSPAGE. 22. M. M. Rozdzial, R. T. Kubo, S. L. Turner, T. H. Finkel J. Immunol. 153, 1563 (1994). 23. BALB/c whole lymph node and spleen T cells were activated with mAb 145-2C11 (1 mg/ml) plus mAb PV-1 (1 mg/ml) for 60 hours. Cells were washed and rested as described (19). Viable cells were enriched by passage over Ficoll/Hypaque. Over 90% of viable cells were CD31 by fluorescence-activated cell sorter analysis. 24. D. G. Orloff et al., J. Biol. Chem. 264, 14812 (1989). 25. The CTLA-4 tyrosine mutant ( Y201F/Y218F) was generated by site-directed mutagenesis (Chameleon sitedirected mutagenesis kit, Stratagene) and the presence of mutations was verified by DNA sequencing. A cDNA sequence incorporating the coding region of murine TCRz was generated by reverse transcription and polymerase chain reaction from RNA extracted from activated murine T cells and cloned into the mammalian expression vector pCDNA3.1(1). 26. A. C. Carrera et al., Proc. Natl. Acad. Sci. U.S.A. 90, 442 (1993). 27. T. Yin, R. Shen, G. S. Feng, Y. C. Yang, J. Biol. Chem. 272, 1032 (1997). 28. Supported by the National Institutes of Health (PHS PO1 AI35294-6). Additional support was provided by a Howard Hughes Medical Institute Medical Student Research Training Fellowship (D.K.H.) and a Mayo Foundation Scholarship (M.G.). We thank G. Feng for the reagents; A. Sharpe for CTLA-4 knockout mice; N. Patel and C. Long for technical assistance; S. Thomas, M. Fonstein, and W. Buikema for services provided at the Cancer Research Center (CRC) DNA Sequencing Facility; and M. Clark for critical reading of the manuscript.