Limits...
Surface cytotoxic T lymphocyte-associated antigen 4 partitions within lipid rafts and relocates to the immunological synapse under conditions of inhibition of T cell activation.

Darlington PJ, Baroja ML, Chau TA, Siu E, Ling V, Carreno BM, Madrenas J - J. Exp. Med. (2002)

Bottom Line: Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface.However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling.These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.

View Article: PubMed Central - PubMed

Affiliation: The Biotherapeutics and Transplantation and Immunobiology Groups, The John P. Robarts Research Institute, and The Departments of Microbiology and Immunology, and Medicine, The University of Western Ontario, London, Ontario N6A 5K8, Canada.

ABSTRACT
T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.

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Wild-type CTLA-4, but not GPI-anchored CTLA-4, inhibits IL-2 production upon coligation with the TCR. (A) Wild-type CTLA-4 and GPI-anchored CTLA-4 were incubated with or without 100 ng/ml doxycycline, and incubated with anti-CD3 or anti-CD3 plus anti–CTLA-4 coated beads and soluble anti-CD28 for 48 h. IL-2 in the supernatant was determined by ELISA. The percentage change in IL-2 production in response to anti-CD3 plus anti-CTLA-4 is shown as a bar graph. The response to anti-CD3 beads was taken as 100% for each transfectant. (B) Doxycycline-induced Jurkat T cells were preincubated with or without blocking anti-CTLA-4 ScFv F′ab. After 30 min, beads coated with the indicated antibodies were added to the cells in the presence of soluble anti-CD28 antibodies. IL-2 production was measured after 48 h. Result were statistically significant as analyzed by one-way ANOVA (P < 0.05).
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fig7: Wild-type CTLA-4, but not GPI-anchored CTLA-4, inhibits IL-2 production upon coligation with the TCR. (A) Wild-type CTLA-4 and GPI-anchored CTLA-4 were incubated with or without 100 ng/ml doxycycline, and incubated with anti-CD3 or anti-CD3 plus anti–CTLA-4 coated beads and soluble anti-CD28 for 48 h. IL-2 in the supernatant was determined by ELISA. The percentage change in IL-2 production in response to anti-CD3 plus anti-CTLA-4 is shown as a bar graph. The response to anti-CD3 beads was taken as 100% for each transfectant. (B) Doxycycline-induced Jurkat T cells were preincubated with or without blocking anti-CTLA-4 ScFv F′ab. After 30 min, beads coated with the indicated antibodies were added to the cells in the presence of soluble anti-CD28 antibodies. IL-2 production was measured after 48 h. Result were statistically significant as analyzed by one-way ANOVA (P < 0.05).

Mentions: Next, we determined whether or not the ability of CTLA-4 to negatively signal correlated with its membrane compartmentalization. One could argue that localization of CTLA-4 in lipid rafts may be sufficient to interfere with TCR-mediated signaling upon coligation by causing physical disruption of the IS. To test this hypothesis we stimulated the different T cell transfectants with immobilized monoclonal antibodies against CD3 or against CD3 and CTLA-4 on beads in the presence of excess costimulation provided by soluble anti-CD28 mAbs. Our group has previously reported that coligation of TCR and wild-type CTLA-4 results in inhibition of ERK1/ERK2 phosphorylation and of IL-2 production, while tailless CTLA-4 does not cause such inhibition by negative signaling (20, 24). As expected, we confirmed that coligation of CD3 and wild-type-CTLA-4 caused significant inhibition of IL-2 production (Fig. 7 A). Blockade of CTLA-4 coligation prevented the inhibition of IL-2 production under these conditions, suggesting that CTLA-4 is responsible for this effect (Fig. 7 B). However, the GPI-anchored-CTLA-4, like we have previously reported for tailless CTLA-4 (20), failed to inhibit T cell activation by negative signaling upon coligation with CD3 and CD28 (Fig. 7 A), despite its almost exclusive localization within lipid rafts. Taken together these results demonstrate that while raft localization may be necessary for inhibition to occur, it is not sufficient to mediate negative signaling.


Surface cytotoxic T lymphocyte-associated antigen 4 partitions within lipid rafts and relocates to the immunological synapse under conditions of inhibition of T cell activation.

Darlington PJ, Baroja ML, Chau TA, Siu E, Ling V, Carreno BM, Madrenas J - J. Exp. Med. (2002)

Wild-type CTLA-4, but not GPI-anchored CTLA-4, inhibits IL-2 production upon coligation with the TCR. (A) Wild-type CTLA-4 and GPI-anchored CTLA-4 were incubated with or without 100 ng/ml doxycycline, and incubated with anti-CD3 or anti-CD3 plus anti–CTLA-4 coated beads and soluble anti-CD28 for 48 h. IL-2 in the supernatant was determined by ELISA. The percentage change in IL-2 production in response to anti-CD3 plus anti-CTLA-4 is shown as a bar graph. The response to anti-CD3 beads was taken as 100% for each transfectant. (B) Doxycycline-induced Jurkat T cells were preincubated with or without blocking anti-CTLA-4 ScFv F′ab. After 30 min, beads coated with the indicated antibodies were added to the cells in the presence of soluble anti-CD28 antibodies. IL-2 production was measured after 48 h. Result were statistically significant as analyzed by one-way ANOVA (P < 0.05).
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Related In: Results  -  Collection

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fig7: Wild-type CTLA-4, but not GPI-anchored CTLA-4, inhibits IL-2 production upon coligation with the TCR. (A) Wild-type CTLA-4 and GPI-anchored CTLA-4 were incubated with or without 100 ng/ml doxycycline, and incubated with anti-CD3 or anti-CD3 plus anti–CTLA-4 coated beads and soluble anti-CD28 for 48 h. IL-2 in the supernatant was determined by ELISA. The percentage change in IL-2 production in response to anti-CD3 plus anti-CTLA-4 is shown as a bar graph. The response to anti-CD3 beads was taken as 100% for each transfectant. (B) Doxycycline-induced Jurkat T cells were preincubated with or without blocking anti-CTLA-4 ScFv F′ab. After 30 min, beads coated with the indicated antibodies were added to the cells in the presence of soluble anti-CD28 antibodies. IL-2 production was measured after 48 h. Result were statistically significant as analyzed by one-way ANOVA (P < 0.05).
Mentions: Next, we determined whether or not the ability of CTLA-4 to negatively signal correlated with its membrane compartmentalization. One could argue that localization of CTLA-4 in lipid rafts may be sufficient to interfere with TCR-mediated signaling upon coligation by causing physical disruption of the IS. To test this hypothesis we stimulated the different T cell transfectants with immobilized monoclonal antibodies against CD3 or against CD3 and CTLA-4 on beads in the presence of excess costimulation provided by soluble anti-CD28 mAbs. Our group has previously reported that coligation of TCR and wild-type CTLA-4 results in inhibition of ERK1/ERK2 phosphorylation and of IL-2 production, while tailless CTLA-4 does not cause such inhibition by negative signaling (20, 24). As expected, we confirmed that coligation of CD3 and wild-type-CTLA-4 caused significant inhibition of IL-2 production (Fig. 7 A). Blockade of CTLA-4 coligation prevented the inhibition of IL-2 production under these conditions, suggesting that CTLA-4 is responsible for this effect (Fig. 7 B). However, the GPI-anchored-CTLA-4, like we have previously reported for tailless CTLA-4 (20), failed to inhibit T cell activation by negative signaling upon coligation with CD3 and CD28 (Fig. 7 A), despite its almost exclusive localization within lipid rafts. Taken together these results demonstrate that while raft localization may be necessary for inhibition to occur, it is not sufficient to mediate negative signaling.

Bottom Line: Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface.However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling.These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.

View Article: PubMed Central - PubMed

Affiliation: The Biotherapeutics and Transplantation and Immunobiology Groups, The John P. Robarts Research Institute, and The Departments of Microbiology and Immunology, and Medicine, The University of Western Ontario, London, Ontario N6A 5K8, Canada.

ABSTRACT
T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.

Show MeSH