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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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Anti-CD3–induced phosphorylation of TCR-ζ and ZAP70 is not affected by CTLA-4. (A) Preactivated T cells were coated with different  combinations of antibodies and lysed 1, 5, or 10 min after addition of warm cross-linking antibody. TCR-ζ was immunoprecipitated and immunoblotted  with antiphosphotyrosine antibody 4G10. Indicated with arrows are the p21 and p23 isoforms of tyrosine phosphorylated TCR-ζ. (B) Lysates prepared as  in A from cells stimulated for 1 min were immunoprecipitated with anti-ZAP70 antibody and immunoblotted with antiphosphotyrosine antibody 4G10.  Indicated with an arrow is phosphorylated ZAP70. Similar results were also obtained on lysates from 5 and 10 min–stimulated cells (not shown).
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Figure 4: Anti-CD3–induced phosphorylation of TCR-ζ and ZAP70 is not affected by CTLA-4. (A) Preactivated T cells were coated with different combinations of antibodies and lysed 1, 5, or 10 min after addition of warm cross-linking antibody. TCR-ζ was immunoprecipitated and immunoblotted with antiphosphotyrosine antibody 4G10. Indicated with arrows are the p21 and p23 isoforms of tyrosine phosphorylated TCR-ζ. (B) Lysates prepared as in A from cells stimulated for 1 min were immunoprecipitated with anti-ZAP70 antibody and immunoblotted with antiphosphotyrosine antibody 4G10. Indicated with an arrow is phosphorylated ZAP70. Similar results were also obtained on lysates from 5 and 10 min–stimulated cells (not shown).

Mentions: The TCR-ζ chain contains three so-called ITAMs (immunoreceptor tyrosine based activation motif) sequences that are necessary for coupling the TCR to the intracellular signaling machinery (30–32). Upon TCR activation, these motifs become phosphorylated on tyrosine residues as a consequence of which the electrophoretic mobility of the protein changes (33, 34). We immunoprecipitated TCR-ζ from lysates obtained as described above using an antibody specific for phosphorylated TCR-ζ. After immunoblotting the precipitates with an antibody against phosphotyrosine, bands of 21 kD and p23 kD were visualized, representing different phosphorylated isoforms of TCR-ζ (Fig. 4 A; reference 34). Although the 21-kD band was also present in unstimulated cells as previously reported for fresh lymph node T cells (35, 36), the p23 band specifically appeared after CD3 triggering. Importantly, at none of the time points examined was the intensity of either one of these two phospho-ζ bands altered upon coengagement of CTLA-4 with CD3 (Fig. 4 A). Yet, in these same cells, ERK2 activity was again abrogated by CTLA-4 engagement (data not shown). We conclude that ζ-phosphorylation is not affected by CTLA-4.


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)

Anti-CD3–induced phosphorylation of TCR-ζ and ZAP70 is not affected by CTLA-4. (A) Preactivated T cells were coated with different  combinations of antibodies and lysed 1, 5, or 10 min after addition of warm cross-linking antibody. TCR-ζ was immunoprecipitated and immunoblotted  with antiphosphotyrosine antibody 4G10. Indicated with arrows are the p21 and p23 isoforms of tyrosine phosphorylated TCR-ζ. (B) Lysates prepared as  in A from cells stimulated for 1 min were immunoprecipitated with anti-ZAP70 antibody and immunoblotted with antiphosphotyrosine antibody 4G10.  Indicated with an arrow is phosphorylated ZAP70. Similar results were also obtained on lysates from 5 and 10 min–stimulated cells (not shown).
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Related In: Results  -  Collection

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Figure 4: Anti-CD3–induced phosphorylation of TCR-ζ and ZAP70 is not affected by CTLA-4. (A) Preactivated T cells were coated with different combinations of antibodies and lysed 1, 5, or 10 min after addition of warm cross-linking antibody. TCR-ζ was immunoprecipitated and immunoblotted with antiphosphotyrosine antibody 4G10. Indicated with arrows are the p21 and p23 isoforms of tyrosine phosphorylated TCR-ζ. (B) Lysates prepared as in A from cells stimulated for 1 min were immunoprecipitated with anti-ZAP70 antibody and immunoblotted with antiphosphotyrosine antibody 4G10. Indicated with an arrow is phosphorylated ZAP70. Similar results were also obtained on lysates from 5 and 10 min–stimulated cells (not shown).
Mentions: The TCR-ζ chain contains three so-called ITAMs (immunoreceptor tyrosine based activation motif) sequences that are necessary for coupling the TCR to the intracellular signaling machinery (30–32). Upon TCR activation, these motifs become phosphorylated on tyrosine residues as a consequence of which the electrophoretic mobility of the protein changes (33, 34). We immunoprecipitated TCR-ζ from lysates obtained as described above using an antibody specific for phosphorylated TCR-ζ. After immunoblotting the precipitates with an antibody against phosphotyrosine, bands of 21 kD and p23 kD were visualized, representing different phosphorylated isoforms of TCR-ζ (Fig. 4 A; reference 34). Although the 21-kD band was also present in unstimulated cells as previously reported for fresh lymph node T cells (35, 36), the p23 band specifically appeared after CD3 triggering. Importantly, at none of the time points examined was the intensity of either one of these two phospho-ζ bands altered upon coengagement of CTLA-4 with CD3 (Fig. 4 A). Yet, in these same cells, ERK2 activity was again abrogated by CTLA-4 engagement (data not shown). We conclude that ζ-phosphorylation is not affected by CTLA-4.

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.

Show MeSH