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Cytotoxic T lymphocyte antigen 4 (CTLA-4) interferes with extracellular signal-regulated kinase (ERK) and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphorylation of T cell receptor zeta and ZAP70.

Calvo CR, Amsen D, Kruisbeek AM - J. Exp. Med. (1997)

Bottom Line: We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK.In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells.Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, Amsterdam.

ABSTRACT
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an important regulator of T cell homeostasis. Ligation of this receptor leads to prominent downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK. In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells. Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

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CTLA-4 inhibits  ERK2 activity induced by anti-CD3 in preactivated T cells, but  not in naive T cells. (A) 2-d preactivated T cells were coated on  ice with the indicated combinations of anti-CD3, anti-CD28,  and anti–CTLA-4. 1 min after  addition of cross-linking anti– hamster antibody (10 μg/ml) in  warm (37°C) medium the cells  were lysed, ERK2 was immunoprecipitated, and in vitro kinase  reactions were performed using  MBP as substrate in the presence  of γ-[32P]ATP. Indicated by arrows are the bands representing  phosphorylated MBP (top). The  same lysates were tested for equal  protein abundance on immunoblot (bottom). (B) Naive purified  T cells were rested for 5 h and  coated with the different combinations of antibodies as indicated. Cells were lysed 1 or 5  min after addition of warm  cross-linking antibody and in  vitro kinase reactions were performed as in A.
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Figure 1: CTLA-4 inhibits ERK2 activity induced by anti-CD3 in preactivated T cells, but not in naive T cells. (A) 2-d preactivated T cells were coated on ice with the indicated combinations of anti-CD3, anti-CD28, and anti–CTLA-4. 1 min after addition of cross-linking anti– hamster antibody (10 μg/ml) in warm (37°C) medium the cells were lysed, ERK2 was immunoprecipitated, and in vitro kinase reactions were performed using MBP as substrate in the presence of γ-[32P]ATP. Indicated by arrows are the bands representing phosphorylated MBP (top). The same lysates were tested for equal protein abundance on immunoblot (bottom). (B) Naive purified T cells were rested for 5 h and coated with the different combinations of antibodies as indicated. Cells were lysed 1 or 5 min after addition of warm cross-linking antibody and in vitro kinase reactions were performed as in A.

Mentions: The ERK family of MAP kinases has been found to be required for stimulation of IL-2 transcription (19, 26). Therefore, we investigated whether the activity of ERK2 would be affected by CTLA-4 triggering. Because CTLA-4 has been reported to be expressed only after activation, peaking after 2 d (6), we first preactivated T cells purified from lymph nodes for 40 h on plates coated with anti-CD3 and anti-CD28. After harvesting, we incubated the preactivated cells in fresh medium for an additional 5 h, as we observed that immediately after the 40-h activation period, ERK2 and JNK were too strongly activated to allow visualization of induction by TCR triggering (data not shown). After this 5-h incubation, cells were stimulated with different combinations of antibodies as indicated, and lysed. In vitro kinase reactions were performed with anti-ERK2 immunoprecipitates from these lysates. As has been shown previously (20, 27), efficient ERK2 activation can be achieved by triggering the TCR alone (Fig. 1 A). However, in lysates derived from cells on which CTLA-4 is coengaged together with the antigen receptor, ERK2 activity is almost abrogated. Shown here are the results obtained with cells lysed 1 min after cross-linking the antibodies. In most experiments at this time point, anti-CD3–induced ERK2 activity has reached its maximum, being undetectable by 5 min after stimulation. However, in experiments in which anti-CD3– induced ERK2 activity persisted longer, this activity was still reduced by CTLA-4 engagement at 5 min after triggering (data not shown). The pronounced anti–CTLA-4–induced reduction in ERK2 activity in preactivated cells is contrasted with results in freshly isolated T cells in which no effect of addition of anti–CTLA-4 on ERK2 activity was observed (Fig. 1 B). These findings are consistent with the reported requirement for activation of CTLA-4 expression.


Cytotoxic T lymphocyte antigen 4 (CTLA-4) interferes with extracellular signal-regulated kinase (ERK) and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphorylation of T cell receptor zeta and ZAP70.

Calvo CR, Amsen D, Kruisbeek AM - J. Exp. Med. (1997)

CTLA-4 inhibits  ERK2 activity induced by anti-CD3 in preactivated T cells, but  not in naive T cells. (A) 2-d preactivated T cells were coated on  ice with the indicated combinations of anti-CD3, anti-CD28,  and anti–CTLA-4. 1 min after  addition of cross-linking anti– hamster antibody (10 μg/ml) in  warm (37°C) medium the cells  were lysed, ERK2 was immunoprecipitated, and in vitro kinase  reactions were performed using  MBP as substrate in the presence  of γ-[32P]ATP. Indicated by arrows are the bands representing  phosphorylated MBP (top). The  same lysates were tested for equal  protein abundance on immunoblot (bottom). (B) Naive purified  T cells were rested for 5 h and  coated with the different combinations of antibodies as indicated. Cells were lysed 1 or 5  min after addition of warm  cross-linking antibody and in  vitro kinase reactions were performed as in A.
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Related In: Results  -  Collection

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Figure 1: CTLA-4 inhibits ERK2 activity induced by anti-CD3 in preactivated T cells, but not in naive T cells. (A) 2-d preactivated T cells were coated on ice with the indicated combinations of anti-CD3, anti-CD28, and anti–CTLA-4. 1 min after addition of cross-linking anti– hamster antibody (10 μg/ml) in warm (37°C) medium the cells were lysed, ERK2 was immunoprecipitated, and in vitro kinase reactions were performed using MBP as substrate in the presence of γ-[32P]ATP. Indicated by arrows are the bands representing phosphorylated MBP (top). The same lysates were tested for equal protein abundance on immunoblot (bottom). (B) Naive purified T cells were rested for 5 h and coated with the different combinations of antibodies as indicated. Cells were lysed 1 or 5 min after addition of warm cross-linking antibody and in vitro kinase reactions were performed as in A.
Mentions: The ERK family of MAP kinases has been found to be required for stimulation of IL-2 transcription (19, 26). Therefore, we investigated whether the activity of ERK2 would be affected by CTLA-4 triggering. Because CTLA-4 has been reported to be expressed only after activation, peaking after 2 d (6), we first preactivated T cells purified from lymph nodes for 40 h on plates coated with anti-CD3 and anti-CD28. After harvesting, we incubated the preactivated cells in fresh medium for an additional 5 h, as we observed that immediately after the 40-h activation period, ERK2 and JNK were too strongly activated to allow visualization of induction by TCR triggering (data not shown). After this 5-h incubation, cells were stimulated with different combinations of antibodies as indicated, and lysed. In vitro kinase reactions were performed with anti-ERK2 immunoprecipitates from these lysates. As has been shown previously (20, 27), efficient ERK2 activation can be achieved by triggering the TCR alone (Fig. 1 A). However, in lysates derived from cells on which CTLA-4 is coengaged together with the antigen receptor, ERK2 activity is almost abrogated. Shown here are the results obtained with cells lysed 1 min after cross-linking the antibodies. In most experiments at this time point, anti-CD3–induced ERK2 activity has reached its maximum, being undetectable by 5 min after stimulation. However, in experiments in which anti-CD3– induced ERK2 activity persisted longer, this activity was still reduced by CTLA-4 engagement at 5 min after triggering (data not shown). The pronounced anti–CTLA-4–induced reduction in ERK2 activity in preactivated cells is contrasted with results in freshly isolated T cells in which no effect of addition of anti–CTLA-4 on ERK2 activity was observed (Fig. 1 B). These findings are consistent with the reported requirement for activation of CTLA-4 expression.

Bottom Line: We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK.In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells.Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, Amsterdam.

ABSTRACT
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an important regulator of T cell homeostasis. Ligation of this receptor leads to prominent downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK. In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells. Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

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