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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.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.

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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Inhibition of proliferative response in mutant CTLA-4–transfected T cells. (A) Suppression of Ag-specific response in various forms of CTLA-4–transfected T cells and restoration by anti–CTLA-4 mAb. 105 T cells transfected with control vector and WT or mutant forms of CTLA-4 were stimulated with KLH (10 μg/ml) and 5 × 105 irradiated spleen cells in the presence of anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) at 50 μg/ml. Proliferation was measured by [3H]TdR uptake. Data are shown as mean ± SD of triplicates. (B) Suppression of T cell proliferation by CTLA-4 cross-linking in T cells expressing various forms of CTLA-4 upon TCR/CD28-mediated and FcR-dependent stimulation. 2 × 105 T cells were stimulated with 0.1 μg/ml anti-CD3∈ mAb alone (right) or in combination with 1 μg/ml soluble anti-CD28 mAb (left) in the presence of irradiated spleen cells as FcR+ cells. Anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) was added at 50 μg/ml. The data represent the mean ± SD of triplicates.
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Figure 2: Inhibition of proliferative response in mutant CTLA-4–transfected T cells. (A) Suppression of Ag-specific response in various forms of CTLA-4–transfected T cells and restoration by anti–CTLA-4 mAb. 105 T cells transfected with control vector and WT or mutant forms of CTLA-4 were stimulated with KLH (10 μg/ml) and 5 × 105 irradiated spleen cells in the presence of anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) at 50 μg/ml. Proliferation was measured by [3H]TdR uptake. Data are shown as mean ± SD of triplicates. (B) Suppression of T cell proliferation by CTLA-4 cross-linking in T cells expressing various forms of CTLA-4 upon TCR/CD28-mediated and FcR-dependent stimulation. 2 × 105 T cells were stimulated with 0.1 μg/ml anti-CD3∈ mAb alone (right) or in combination with 1 μg/ml soluble anti-CD28 mAb (left) in the presence of irradiated spleen cells as FcR+ cells. Anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) was added at 50 μg/ml. The data represent the mean ± SD of triplicates.

Mentions: Previous reports demonstrated that both intact and monovalent fragments of CTLA-4 augmented T cell proliferation in allogeneic MLR, whereas the same Ab inhibited proliferation under the condition where FcR cross-linking was provided 67. To investigate the region in the cytoplasmic tail of CTLA-4 responsible for delivering negative signals, we performed functional analyses using these T cells transfected with various mutant forms of CTLA-4. First, we examined the effect of CTLA-4 mutants in terms of Ag-specific proliferative response. Because 23-1-8 is a KLH-specific T cell clone, transfectants expressing various CTLA-4 mutants were stimulated with KLH and APCs in the presence or absence of anti–CTLA-4 mAb. As shown in Fig. 2 A, the proliferative response of CT T cells was not altered by the addition of anti–CTLA-4 mAb. However, proliferation of WT T cells was suppressed in comparison with CT T cells. The addition of anti–CTLA-4 mAb restored the response to a level almost equal to that in control cells. In T cells with high expression of surface CTLA-4, such as Y165G, Y165/182G, and ΔCP7 T cells, the suppression was more significant than in WT T cells, in accordance with their surface expression. For these cells, the addition of anti–CTLA-4 mAb induced dramatic restoration of the Ag-specific response. These results suggest that mutant forms of CTLA-4, with either the tyrosine mutation or deletion of the cytoplasmic region, could deliver a negative signal for Ag-specific proliferation. Alternatively, these molecules might inhibit the proliferation by merely competing for ligand binding with CD28.


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)

Inhibition of proliferative response in mutant CTLA-4–transfected T cells. (A) Suppression of Ag-specific response in various forms of CTLA-4–transfected T cells and restoration by anti–CTLA-4 mAb. 105 T cells transfected with control vector and WT or mutant forms of CTLA-4 were stimulated with KLH (10 μg/ml) and 5 × 105 irradiated spleen cells in the presence of anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) at 50 μg/ml. Proliferation was measured by [3H]TdR uptake. Data are shown as mean ± SD of triplicates. (B) Suppression of T cell proliferation by CTLA-4 cross-linking in T cells expressing various forms of CTLA-4 upon TCR/CD28-mediated and FcR-dependent stimulation. 2 × 105 T cells were stimulated with 0.1 μg/ml anti-CD3∈ mAb alone (right) or in combination with 1 μg/ml soluble anti-CD28 mAb (left) in the presence of irradiated spleen cells as FcR+ cells. Anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) was added at 50 μg/ml. The data represent the mean ± SD of triplicates.
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Related In: Results  -  Collection

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

Figure 2: Inhibition of proliferative response in mutant CTLA-4–transfected T cells. (A) Suppression of Ag-specific response in various forms of CTLA-4–transfected T cells and restoration by anti–CTLA-4 mAb. 105 T cells transfected with control vector and WT or mutant forms of CTLA-4 were stimulated with KLH (10 μg/ml) and 5 × 105 irradiated spleen cells in the presence of anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) at 50 μg/ml. Proliferation was measured by [3H]TdR uptake. Data are shown as mean ± SD of triplicates. (B) Suppression of T cell proliferation by CTLA-4 cross-linking in T cells expressing various forms of CTLA-4 upon TCR/CD28-mediated and FcR-dependent stimulation. 2 × 105 T cells were stimulated with 0.1 μg/ml anti-CD3∈ mAb alone (right) or in combination with 1 μg/ml soluble anti-CD28 mAb (left) in the presence of irradiated spleen cells as FcR+ cells. Anti–CTLA-4 mAb (▪) or anti-CD3ζ mAb as control (□) was added at 50 μg/ml. The data represent the mean ± SD of triplicates.
Mentions: Previous reports demonstrated that both intact and monovalent fragments of CTLA-4 augmented T cell proliferation in allogeneic MLR, whereas the same Ab inhibited proliferation under the condition where FcR cross-linking was provided 67. To investigate the region in the cytoplasmic tail of CTLA-4 responsible for delivering negative signals, we performed functional analyses using these T cells transfected with various mutant forms of CTLA-4. First, we examined the effect of CTLA-4 mutants in terms of Ag-specific proliferative response. Because 23-1-8 is a KLH-specific T cell clone, transfectants expressing various CTLA-4 mutants were stimulated with KLH and APCs in the presence or absence of anti–CTLA-4 mAb. As shown in Fig. 2 A, the proliferative response of CT T cells was not altered by the addition of anti–CTLA-4 mAb. However, proliferation of WT T cells was suppressed in comparison with CT T cells. The addition of anti–CTLA-4 mAb restored the response to a level almost equal to that in control cells. In T cells with high expression of surface CTLA-4, such as Y165G, Y165/182G, and ΔCP7 T cells, the suppression was more significant than in WT T cells, in accordance with their surface expression. For these cells, the addition of anti–CTLA-4 mAb induced dramatic restoration of the Ag-specific response. These results suggest that mutant forms of CTLA-4, with either the tyrosine mutation or deletion of the cytoplasmic region, could deliver a negative signal for Ag-specific proliferation. Alternatively, these molecules might inhibit the proliferation by merely competing for ligand binding with CD28.

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.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.

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