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Cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement delivers an inhibitory signal through the membrane-proximal region in the absence of the tyrosine motif in the cytoplasmic tail.

Nakaseko C, Miyatake S, Iida T, Hara S, Abe R, Ohno H, Saito Y, Saito T - J. Exp. Med. (1999)

Bottom Line: Here, we report a new mechanism of negative signaling based on the analysis of murine T cell clones transfected with various mutants of CTLA-4.Furthermore, the mutant CTLA-4 lacking most of the cytoplasmic region strongly suppressed interleukin 2 production as well.These data suggest that negative signals by CTLA-4 could be mediated through the membrane-proximal region of CTLA-4 but not through the YVKM motif and that the association of CTLA-4 with SHP-2 is not required for CTLA-4-mediated suppression of T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.

ABSTRACT
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell costimulation receptor that delivers inhibitory signals upon activation. Although the tyrosine-based motif ((165)YVKM) within its cytoplasmic tail has been shown to associate in vitro with Src homology 2 domain-containing tyrosine phosphatase (SHP-2) and phosphatidylinositol 3 kinase upon phosphorylation, the mechanism of negative signaling remains unclear. Here, we report a new mechanism of negative signaling based on the analysis of murine T cell clones transfected with various mutants of CTLA-4. Upon T cell activation by cross-linking with anti-CD3 and anti-CD28 antibodies, CTLA-4 engagement inhibited both proliferation and interleukin 2 production in tyrosine mutants as well as in wild-type CTLA-4 transfectants. Furthermore, the mutant CTLA-4 lacking most of the cytoplasmic region strongly suppressed interleukin 2 production as well. These data suggest that negative signals by CTLA-4 could be mediated through the membrane-proximal region of CTLA-4 but not through the YVKM motif and that the association of CTLA-4 with SHP-2 is not required for CTLA-4-mediated suppression of T cell activation.

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Surface expression of transfected CTLA-4 on resting T cell clones. (A) Schematic structure of WT and mutant forms of CTLA-4. These constructs were subcloned into the retrovirus vector pMXneo and transfected into murine Th1 cell clone 23-1-8. Black boxes represent the transmembrane region. EX, extracellular; TM, transmembrane; CP, cytoplasmic domains; a.a., amino acid. (B) Surface expression of CTLA-4 on resting T cell clones transfected with control vector, WT, or CTLA-4 mutants. ΔCP7-low T cells were isolated from ΔCP7 T cells as T cells expressing low levels of the cell surface CTLA-4 by cell sorting. T cells were stained with biotinylated anti–CTLA-4 mAb and PE–streptavidin and analyzed by FACS™. (C) Northern blot analysis of WT and various mutant CTLA-4–transfected T cells. 10 μg of total RNA was analyzed using a full length CTLA-4 cDNA as a probe. Quantitation of each band showed the following relative values when normalized by the vector-transfectant cells as 1: vector, 1.0; WT, 33.4; Y165G, 55.3; Y182G, 45.8; Y165/182G, 33.8; ΔCP7, 26.5; and ΔCP7-low, 6.6. (D) Total protein levels of CTLA-4 in WT and various mutant CTLA-4–transfected T cells. T cells expressing control vector, WT CTLA-4, mutant CTLA-4 (Y165G, Y182G, Y165/182G, ΔCP7), and ΔCP7-low T cells were metabolically labeled with [35S]methionine and lysed in 1.0% NP-40 lysis buffer. The lysates were immunoprecipitated with anti–CTLA-4 mAb or anti-CD3∈ mAb as control. Immunoprecipitates (IP) by anti–CTLA-4 mAb were treated with N-glycosidase F and then resolved on 13% SDS-PAGE. Molecular size is indicated, right (kD).
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Figure 1: Surface expression of transfected CTLA-4 on resting T cell clones. (A) Schematic structure of WT and mutant forms of CTLA-4. These constructs were subcloned into the retrovirus vector pMXneo and transfected into murine Th1 cell clone 23-1-8. Black boxes represent the transmembrane region. EX, extracellular; TM, transmembrane; CP, cytoplasmic domains; a.a., amino acid. (B) Surface expression of CTLA-4 on resting T cell clones transfected with control vector, WT, or CTLA-4 mutants. ΔCP7-low T cells were isolated from ΔCP7 T cells as T cells expressing low levels of the cell surface CTLA-4 by cell sorting. T cells were stained with biotinylated anti–CTLA-4 mAb and PE–streptavidin and analyzed by FACS™. (C) Northern blot analysis of WT and various mutant CTLA-4–transfected T cells. 10 μg of total RNA was analyzed using a full length CTLA-4 cDNA as a probe. Quantitation of each band showed the following relative values when normalized by the vector-transfectant cells as 1: vector, 1.0; WT, 33.4; Y165G, 55.3; Y182G, 45.8; Y165/182G, 33.8; ΔCP7, 26.5; and ΔCP7-low, 6.6. (D) Total protein levels of CTLA-4 in WT and various mutant CTLA-4–transfected T cells. T cells expressing control vector, WT CTLA-4, mutant CTLA-4 (Y165G, Y182G, Y165/182G, ΔCP7), and ΔCP7-low T cells were metabolically labeled with [35S]methionine and lysed in 1.0% NP-40 lysis buffer. The lysates were immunoprecipitated with anti–CTLA-4 mAb or anti-CD3∈ mAb as control. Immunoprecipitates (IP) by anti–CTLA-4 mAb were treated with N-glycosidase F and then resolved on 13% SDS-PAGE. Molecular size is indicated, right (kD).

Mentions: Although it has been shown that Y-165 of CTLA-4 is crucial for association with AP-2 complex and endocytosis from the cell surface in a model system using nonlymphoid cells 12131415, the regulation of the cell surface expression of CTLA-4 in normal T cells has not been analyzed. Therefore, we examined which of the two tyrosines in the cytoplasmic tail of murine CTLA-4 (Y-165, Y-182, or both) are important for the regulation of the cell surface expression and inhibitory signals of CTLA-4 in normal T cells. For this purpose, we generated various forms of mutant CTLA-4 with a single tyrosine→glycine mutation (Y165G and Y182G), double mutations (Y165/182G), and a deletion of most of the cytoplasmic tail except for the membrane-proximal 7 amino acids (ΔCP7) (Fig. 1 A). These constructs were transfected into a murine T cell clone, 23-1-8, by retrovirus-mediated gene transfer, and the G418-resistant bulk population of transfected T cells was analyzed for the surface expression of CTLA-4 (Fig. 1 B). CTLA-4 was hardly detected on the surfaces of resting T cells transfected with the vector alone (CT T cells). T cells transfected with the WT CTLA-4 (WT T cells) showed only a dull expression of CTLA-4 on the cell surface, as did T cells transfected with the Y182G mutant CTLA-4 (Y182G T cells). In contrast, the mutation of 165Tyr→Gly (Y165G), which blocks the association with AP-2, induced constitutively high expression of CTLA-4 on the surfaces of resting T cells. Interestingly, Y165/182G mutants exhibited a higher expression of surface CTLA-4 than Y165G mutants, and ΔCP7 mutants showed even greater expressions than Y165/182G mutants.


Cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement delivers an inhibitory signal through the membrane-proximal region in the absence of the tyrosine motif in the cytoplasmic tail.

Nakaseko C, Miyatake S, Iida T, Hara S, Abe R, Ohno H, Saito Y, Saito T - J. Exp. Med. (1999)

Surface expression of transfected CTLA-4 on resting T cell clones. (A) Schematic structure of WT and mutant forms of CTLA-4. These constructs were subcloned into the retrovirus vector pMXneo and transfected into murine Th1 cell clone 23-1-8. Black boxes represent the transmembrane region. EX, extracellular; TM, transmembrane; CP, cytoplasmic domains; a.a., amino acid. (B) Surface expression of CTLA-4 on resting T cell clones transfected with control vector, WT, or CTLA-4 mutants. ΔCP7-low T cells were isolated from ΔCP7 T cells as T cells expressing low levels of the cell surface CTLA-4 by cell sorting. T cells were stained with biotinylated anti–CTLA-4 mAb and PE–streptavidin and analyzed by FACS™. (C) Northern blot analysis of WT and various mutant CTLA-4–transfected T cells. 10 μg of total RNA was analyzed using a full length CTLA-4 cDNA as a probe. Quantitation of each band showed the following relative values when normalized by the vector-transfectant cells as 1: vector, 1.0; WT, 33.4; Y165G, 55.3; Y182G, 45.8; Y165/182G, 33.8; ΔCP7, 26.5; and ΔCP7-low, 6.6. (D) Total protein levels of CTLA-4 in WT and various mutant CTLA-4–transfected T cells. T cells expressing control vector, WT CTLA-4, mutant CTLA-4 (Y165G, Y182G, Y165/182G, ΔCP7), and ΔCP7-low T cells were metabolically labeled with [35S]methionine and lysed in 1.0% NP-40 lysis buffer. The lysates were immunoprecipitated with anti–CTLA-4 mAb or anti-CD3∈ mAb as control. Immunoprecipitates (IP) by anti–CTLA-4 mAb were treated with N-glycosidase F and then resolved on 13% SDS-PAGE. Molecular size is indicated, right (kD).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195638&req=5

Figure 1: Surface expression of transfected CTLA-4 on resting T cell clones. (A) Schematic structure of WT and mutant forms of CTLA-4. These constructs were subcloned into the retrovirus vector pMXneo and transfected into murine Th1 cell clone 23-1-8. Black boxes represent the transmembrane region. EX, extracellular; TM, transmembrane; CP, cytoplasmic domains; a.a., amino acid. (B) Surface expression of CTLA-4 on resting T cell clones transfected with control vector, WT, or CTLA-4 mutants. ΔCP7-low T cells were isolated from ΔCP7 T cells as T cells expressing low levels of the cell surface CTLA-4 by cell sorting. T cells were stained with biotinylated anti–CTLA-4 mAb and PE–streptavidin and analyzed by FACS™. (C) Northern blot analysis of WT and various mutant CTLA-4–transfected T cells. 10 μg of total RNA was analyzed using a full length CTLA-4 cDNA as a probe. Quantitation of each band showed the following relative values when normalized by the vector-transfectant cells as 1: vector, 1.0; WT, 33.4; Y165G, 55.3; Y182G, 45.8; Y165/182G, 33.8; ΔCP7, 26.5; and ΔCP7-low, 6.6. (D) Total protein levels of CTLA-4 in WT and various mutant CTLA-4–transfected T cells. T cells expressing control vector, WT CTLA-4, mutant CTLA-4 (Y165G, Y182G, Y165/182G, ΔCP7), and ΔCP7-low T cells were metabolically labeled with [35S]methionine and lysed in 1.0% NP-40 lysis buffer. The lysates were immunoprecipitated with anti–CTLA-4 mAb or anti-CD3∈ mAb as control. Immunoprecipitates (IP) by anti–CTLA-4 mAb were treated with N-glycosidase F and then resolved on 13% SDS-PAGE. Molecular size is indicated, right (kD).
Mentions: Although it has been shown that Y-165 of CTLA-4 is crucial for association with AP-2 complex and endocytosis from the cell surface in a model system using nonlymphoid cells 12131415, the regulation of the cell surface expression of CTLA-4 in normal T cells has not been analyzed. Therefore, we examined which of the two tyrosines in the cytoplasmic tail of murine CTLA-4 (Y-165, Y-182, or both) are important for the regulation of the cell surface expression and inhibitory signals of CTLA-4 in normal T cells. For this purpose, we generated various forms of mutant CTLA-4 with a single tyrosine→glycine mutation (Y165G and Y182G), double mutations (Y165/182G), and a deletion of most of the cytoplasmic tail except for the membrane-proximal 7 amino acids (ΔCP7) (Fig. 1 A). These constructs were transfected into a murine T cell clone, 23-1-8, by retrovirus-mediated gene transfer, and the G418-resistant bulk population of transfected T cells was analyzed for the surface expression of CTLA-4 (Fig. 1 B). CTLA-4 was hardly detected on the surfaces of resting T cells transfected with the vector alone (CT T cells). T cells transfected with the WT CTLA-4 (WT T cells) showed only a dull expression of CTLA-4 on the cell surface, as did T cells transfected with the Y182G mutant CTLA-4 (Y182G T cells). In contrast, the mutation of 165Tyr→Gly (Y165G), which blocks the association with AP-2, induced constitutively high expression of CTLA-4 on the surfaces of resting T cells. Interestingly, Y165/182G mutants exhibited a higher expression of surface CTLA-4 than Y165G mutants, and ΔCP7 mutants showed even greater expressions than Y165/182G mutants.

Bottom Line: Here, we report a new mechanism of negative signaling based on the analysis of murine T cell clones transfected with various mutants of CTLA-4.Furthermore, the mutant CTLA-4 lacking most of the cytoplasmic region strongly suppressed interleukin 2 production as well.These data suggest that negative signals by CTLA-4 could be mediated through the membrane-proximal region of CTLA-4 but not through the YVKM motif and that the association of CTLA-4 with SHP-2 is not required for CTLA-4-mediated suppression of T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.

ABSTRACT
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell costimulation receptor that delivers inhibitory signals upon activation. Although the tyrosine-based motif ((165)YVKM) within its cytoplasmic tail has been shown to associate in vitro with Src homology 2 domain-containing tyrosine phosphatase (SHP-2) and phosphatidylinositol 3 kinase upon phosphorylation, the mechanism of negative signaling remains unclear. Here, we report a new mechanism of negative signaling based on the analysis of murine T cell clones transfected with various mutants of CTLA-4. Upon T cell activation by cross-linking with anti-CD3 and anti-CD28 antibodies, CTLA-4 engagement inhibited both proliferation and interleukin 2 production in tyrosine mutants as well as in wild-type CTLA-4 transfectants. Furthermore, the mutant CTLA-4 lacking most of the cytoplasmic region strongly suppressed interleukin 2 production as well. These data suggest that negative signals by CTLA-4 could be mediated through the membrane-proximal region of CTLA-4 but not through the YVKM motif and that the association of CTLA-4 with SHP-2 is not required for CTLA-4-mediated suppression of T cell activation.

Show MeSH
Related in: MedlinePlus