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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CTLA-4 colocalizes in the IS with CD3 and GM1. Wild-type CTLA-4 transfected Jurkat T cells were induced with doxycycline or noninduced and incubated on poly-l-lysine coated confocal dishes with APC plus 100 ng/ml SEE where indicated. Similarly, tailless and GPI-anchored CTLA-4 in the absence of doxycycline (as these cells express significant amounts of CTLA-4 without it) were used. The samples were put on ice to prevent receptor internalization and immunostained for (A) CD3 (green) and CTLA-4 (red) or (B) GM1 (green) and CTLA-4 (red). The samples were then analyzed by confocal microscopy. Co-capping of CD3 and CTLA-4 was scored when both colocalized at the interface between T cell and APC and the cluster stained <50% of the cell surface. At least 100 doublets per group were counted and scored and results are shown at the bottom right of Fig. 2 A. APCs were identified based on their morphology and lack of staining for CTLA-4 and CD3. Light field pictures were taken simultaneously and merged with the color fields. Yellow indicates overlay of red and green signals.
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fig2: CTLA-4 colocalizes in the IS with CD3 and GM1. Wild-type CTLA-4 transfected Jurkat T cells were induced with doxycycline or noninduced and incubated on poly-l-lysine coated confocal dishes with APC plus 100 ng/ml SEE where indicated. Similarly, tailless and GPI-anchored CTLA-4 in the absence of doxycycline (as these cells express significant amounts of CTLA-4 without it) were used. The samples were put on ice to prevent receptor internalization and immunostained for (A) CD3 (green) and CTLA-4 (red) or (B) GM1 (green) and CTLA-4 (red). The samples were then analyzed by confocal microscopy. Co-capping of CD3 and CTLA-4 was scored when both colocalized at the interface between T cell and APC and the cluster stained <50% of the cell surface. At least 100 doublets per group were counted and scored and results are shown at the bottom right of Fig. 2 A. APCs were identified based on their morphology and lack of staining for CTLA-4 and CD3. Light field pictures were taken simultaneously and merged with the color fields. Yellow indicates overlay of red and green signals.

Mentions: Using these CTLA-4 transfectants, we then examined the distribution of surface CTLA-4 in relation to the IS by confocal microscopy (Fig. 2) . T cell transfectants were stimulated with the superantigen SEE and an APC expressing high levels of B7 and MHC class II molecules. In the absence of SEE stimulation, both CD3 and CTLA-4 were homogeneously distributed on the surface of the T cells (Fig. 2 A). As the T cells were not permeabilized, no intracellular CTLA-4 was detectable. Upon SEE stimulation, CD3 migrated to the interface between the T cell and APC (Fig. 2 A). LFA-1 and PKCθ also migrated to this area (data not shown), indicating that this interface is a putative IS. More importantly, after doxycycline induction, wild-type CTLA-4 migrated to this interface between T cell and APC and colocalized with CD3 upon stimulation with SEE and APC, conditions that correlate with inhibition of IL-2 production (see below). In addition, CD43 was excluded of the CTLA-4 cluster at the center of the T cell–APC interface (data not shown), supporting the idea that the CTLA-4 cluster is in the immunological synapse. Similar to the wild-type CTLA-4, GPI-anchored CTLA-4 was evenly distributed on the membrane in resting cells and migrated to the IS upon SEE stimulation, where it colocalized with CD3. Tailless CTLA-4 migrated to the IS although the frequency of colocalization with CD3 was lower than that seen with wild-type CTLA-4 as well as with GPI-anchored CTLA-4 as shown by quantification of CD3-CTLA-4 cocapping under conditions of doxycycline induction and coligation induced by SEE and APC (Fig. 2 A). For the GPI-anchored CTLA-4 transfectants and the tailless CTLA-4 transfectants, we show these experiments in the absence of doxycycline induction, as under these conditions, the level of CTLA-4 expression on the surface is more comparable.


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)

CTLA-4 colocalizes in the IS with CD3 and GM1. Wild-type CTLA-4 transfected Jurkat T cells were induced with doxycycline or noninduced and incubated on poly-l-lysine coated confocal dishes with APC plus 100 ng/ml SEE where indicated. Similarly, tailless and GPI-anchored CTLA-4 in the absence of doxycycline (as these cells express significant amounts of CTLA-4 without it) were used. The samples were put on ice to prevent receptor internalization and immunostained for (A) CD3 (green) and CTLA-4 (red) or (B) GM1 (green) and CTLA-4 (red). The samples were then analyzed by confocal microscopy. Co-capping of CD3 and CTLA-4 was scored when both colocalized at the interface between T cell and APC and the cluster stained <50% of the cell surface. At least 100 doublets per group were counted and scored and results are shown at the bottom right of Fig. 2 A. APCs were identified based on their morphology and lack of staining for CTLA-4 and CD3. Light field pictures were taken simultaneously and merged with the color fields. Yellow indicates overlay of red and green signals.
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Related In: Results  -  Collection

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fig2: CTLA-4 colocalizes in the IS with CD3 and GM1. Wild-type CTLA-4 transfected Jurkat T cells were induced with doxycycline or noninduced and incubated on poly-l-lysine coated confocal dishes with APC plus 100 ng/ml SEE where indicated. Similarly, tailless and GPI-anchored CTLA-4 in the absence of doxycycline (as these cells express significant amounts of CTLA-4 without it) were used. The samples were put on ice to prevent receptor internalization and immunostained for (A) CD3 (green) and CTLA-4 (red) or (B) GM1 (green) and CTLA-4 (red). The samples were then analyzed by confocal microscopy. Co-capping of CD3 and CTLA-4 was scored when both colocalized at the interface between T cell and APC and the cluster stained <50% of the cell surface. At least 100 doublets per group were counted and scored and results are shown at the bottom right of Fig. 2 A. APCs were identified based on their morphology and lack of staining for CTLA-4 and CD3. Light field pictures were taken simultaneously and merged with the color fields. Yellow indicates overlay of red and green signals.
Mentions: Using these CTLA-4 transfectants, we then examined the distribution of surface CTLA-4 in relation to the IS by confocal microscopy (Fig. 2) . T cell transfectants were stimulated with the superantigen SEE and an APC expressing high levels of B7 and MHC class II molecules. In the absence of SEE stimulation, both CD3 and CTLA-4 were homogeneously distributed on the surface of the T cells (Fig. 2 A). As the T cells were not permeabilized, no intracellular CTLA-4 was detectable. Upon SEE stimulation, CD3 migrated to the interface between the T cell and APC (Fig. 2 A). LFA-1 and PKCθ also migrated to this area (data not shown), indicating that this interface is a putative IS. More importantly, after doxycycline induction, wild-type CTLA-4 migrated to this interface between T cell and APC and colocalized with CD3 upon stimulation with SEE and APC, conditions that correlate with inhibition of IL-2 production (see below). In addition, CD43 was excluded of the CTLA-4 cluster at the center of the T cell–APC interface (data not shown), supporting the idea that the CTLA-4 cluster is in the immunological synapse. Similar to the wild-type CTLA-4, GPI-anchored CTLA-4 was evenly distributed on the membrane in resting cells and migrated to the IS upon SEE stimulation, where it colocalized with CD3. Tailless CTLA-4 migrated to the IS although the frequency of colocalization with CD3 was lower than that seen with wild-type CTLA-4 as well as with GPI-anchored CTLA-4 as shown by quantification of CD3-CTLA-4 cocapping under conditions of doxycycline induction and coligation induced by SEE and APC (Fig. 2 A). For the GPI-anchored CTLA-4 transfectants and the tailless CTLA-4 transfectants, we show these experiments in the absence of doxycycline induction, as under these conditions, the level of CTLA-4 expression on the surface is more comparable.

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
Related in: MedlinePlus