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Molecular Basis of T Cell Inactivation by CTLA-4

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Science  18 Dec 1998:
Vol. 282, Issue 5397, pp. 2263-2266
DOI: 10.1126/science.282.5397.2263

Figures

  • Figure 1

    Anti-CD3–induced LAT and TCRζ tyrosine phosphorylation is inhibited by anti-CTLA-4 engagement. Activated T cells (5 ×106) (19) were admixed with 293 cells (2.5 × 106) transiently expressing a membrane-bound single-chain mAb to CD3 in the presence or absence of a membrane-bound single-chain mAb to CTLA-4 (20). The cells were incubated at 37°C for the time indicated and then subjected to lysis in buffer (LB). Immunoprecipitates (IPs) were prepared with mAb FB2 to phosphotyrosine (pTyr), rabbit antiserum to LAT (3), or mAb H146-968 to TCRζ (21, 22). IPs separated on a reducing SDS–12% polyacrylamide gel were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) and subsequently immunoblotted (IB) with mAb 4G10 to pTyr (Upstate Biotechnology Inc., Lake Placid, NY). Bound proteins were detected by chemiluminescence (Pierce, Rockford, IL). CTLA-4 cross-linking resulted in a consistent but incomplete reduction of tyrosine phosphorylated p23TCRζ and LAT. Results are representative of three independent experiments.

  • Figure 2

    (A) CTLA-4 coprecipitation of metabolically labeled proteins. Activated T cells (20 × 106 cells) (23) were labeled with35S-methionine and 35S-cysteine (Amersham, Arlington Heights, IL). CTLA-4 was immunoprecipitated from cell lysates with mAb UC10-4F10 to CTLA-4 (15). Immune complexes were subjected to two-dimensional SDS-PAGE analysis with 10% acrylamide gels in both the first (nonreduced) and second (reduced) dimensions (5). Arrows indicate proteins coprecipitated with UC10-4F10. (B and C) Identification of p16 TCRζ by immunoblotting. Activated T cells (100 × 106 cells per condition) (23) were lysed and subjected to immunoprecipitation with mAb UC10-4F10 to CTLA-4 or mAb H146-968 to TCRζ. (B) IPs run on two-dimensional SDS–polyacrylamide gel (10% nonreduced, 12% reduced) were subjected to immunoblotting with a rabbit antiserum to TCRζ (Ab 387) (24). The bound proteins were visualized by chemiluminescence. Upper bands in the anti-ζ IP (right) represented higher phosphorylated forms of TCRζ and TCRη. (C) Anti-CTLA-4 IPs prepared from CTLA-4 wild-type (WT) or knockout (KO) mice were separated by one-dimensional reducing SDS-PAGE and immunoblotted with rabbit antiserum to ζ (Ab 387). Ab(L) represents antibody light chain. Independent experiments with similar results were performed five times.

  • Figure 3

    Analysis of CTLA-4-TCRζ association in 293 cell transfectants. (A) Coimmunoprecipitation of CTLA-4 with TCRζ. 293 cells were transiently transfected with cDNA constructs encoding murine CTLA-4 WT, cytoplasmic domain-deficient (tailless) (12) CTLA-4, or CTLA-4 tyrosine double mutant (Y201F/Y218F) (25) in the absence or presence of WT p56lck and murine TCRζ (25). Cells were incubated for 40 hours and lysates were prepared. CTLA-4 was precipitated from an equal amount of protein [700 μg per IP (left); 300 μg per IP (right)]. IPs were analyzed by reducing SDS-PAGE and immunoblotted with the antibodies listed. The expression vectors used were pCDNA3 (CTLA-4 and TCRζ, 2.5 μg each) and pEF (p56lck, 1.25 μg). (B) GST–(CTLA-4)3 fusion protein binds TCRζ. 293 cells were transiently transfected with cDNA encoding GST–(CTLA-4)3(12) in the presence or absence of p56lck and TCRζ. GST proteins were precipitated from the lysates (200 μg per sample) with glutathione Sepharose beads (12.5 μl, Pharmacia) and analyzed by electrophoresis and immunoblotting with either rabbit antiserum to TCRζ (Ab 387), or GST mAb (Santa Cruz, Santa Cruz, CA). GST and GST–(CTLA-4)3 proteins migrated at 25 and 40 kD, respectively. (C) CTLA-4 coprecipitates Tac–ζ in 293 transfectants. 293 cells were transiently transfected with cDNAs for Tac–ζ chimera (7) in the absence or presence of WT CTLA-4. CTLA-4 IPs were subjected to SDS-PAGE and immunoblotted with Ab 387. Chimeric Tac–ζ, migrated as a doublet when detected with Ab 387. Ab(H) represents antibody heavy chain. (D) Kinase-defective lck (KA) failed to enhance TCRζ association. 293 cells were transiently transfected with cDNAs for WT CTLA-4, murine TCRζ, and either WT or kinase-defective p56lck(26) and analyzed as described above.

  • Figure 4

    SHP-2 associates with CTLA-4–TCRζ complexes and regulates binding of TCRζ to CTLA-4. (A) Equal numbers (100 × 106) of activated T cells (23) were lysed and subjected to IP with mAbs to CTLA-4 (UC10-4F10), rabbit antiserum against SHP-2 (27), and mAb to TCRζ (H146). IPs were analyzed by SDS-PAGE and immunoblotted with a rabbit antiserum to SHP-2 (27), goat polyclonal antibodies to CTLA-4 (Q20, Santa Cruz, Santa Cruz, CA), or rabbit antiserum to TCRζ (Ab 387). (B) 293 cells were transiently transfected with either WT CTLA-4 or mutated CTLA-4 (Y201F/Y218F) and TCRζ in the presence or absence of p56lck or SHP-2. CTLA-4 IPs prepared from lysates (400 μg per sample) or whole cell lysates (35 μg per lane) were electrophoresed and immunoblotted with rabbit antibody to SHP-2 (27) or antiserum to TCRζ (Ab 387). Results are representative of three separate experiments.

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