Organization of plasma membrane functional rafts upon T cell activation

Abbreviation:

1 Introduction

Cell membranes are composed of a complex mixture of cholesterol and various glycerolphospholipids and sphingolipids. Glycerolphospholipids and sphingolipids exhibit distinct biophysical properties and behave in a different way when forming a monolayer, with glycerolphospholipids showing tendency to adopt a mobile fluid phase and sphingolipids showing a more tigthly packed organization 1. Simons and Ikonen 2 formulated a general "raft hypothesis" that predicts that in all cell types attractive forces between sphingolipids and cholesterol mediate the formation of lateral lipid clusters in an unsaturated glycerolphospholipid environment. According to this hypothesis, rafts serve as platforms were particular molecules are enriched while others are excluded.

A variety of signaling molecules are accumulated in raft domains, including the two Src family kinases Lck and Fyn, both implicated in T cell activation, the T cell linker molecule LAT, monomeric and heterotrimeric G proteins, G-coupled protein receptors and molecules involved in Ca^2 + responses 3, 4. Posttranslational addition of lipids (myristylation, palmitoylation and farnesylation) is a critical requirement for the targeting of many of these molecules to the membrane as well as for their functions 5.

When clustered by antibodies, many glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to transduce intracellular signals leading to tyrosine phosphorylation, Ca^2 + flux or cell proliferation 6. This signaling depends on the raft-associated kinases and can be suppressed by reduction of the membrane cholesterol level or by changing the membrane lipid composition, indicating that integrity of raft domains is required 7.

In T cells it has been shown that engagement of TCR induces its recruitment to rafts and that raft integrity is required for efficient T cell activation 8, 9. Upon TCR triggering, supramolecular signaling complexes containing hyperphosphorylated ζ chains, activated ZAP-70 and many other signaling molecules are accumulated in raft domains. These events, as well as T cell activation, can be suppressed by disrupting raft structure, indicating that membrane compartmentation is a prerequisite for TCR signal transduction. It has been recently shown that CD28 engagement promotes an impressive redistribution of rafts at the TCR contact site, thus amplifying and sustaining TCR-induced signaling 10. Thus, if the recruitment of TCR into rafts may induce its triggering and may start the signaling cascade, the active recruitment and clustering of raft microdomains to the site of TCR triggering may represent a general mechanism by which co-stimulation can amplify the signaling process 11.

While the outcome of naive T cell activation is strongly influenced by CD28 co-stimulation, effector T cells exhibit a lower dependence on co-stimulation and a faster response than naive cells 12, 13. Recently, it has been shown that effector and memory T cells have a more efficient TCR signaling machinery than naive T cells. In murine naive cytotoxic T cells only few CD8 molecules are associated with Lck and the kinase is homogeneously distributed inside the cell, while in vivo priming of naive T cells induces the targeting of Lck to the CD8 coreceptor in the cell membrane 14.

The present study was undertaken to determine whether T cell activation results in reorganization of rafts at the cell membrane. We found that in vitro activation of human resting T cells induced rapid targeting of the cytosolic Lck and GM1 to the plasma membrane. Moreover, activation of resting T cells induced synthesis of the raft lipid GM1 which is highly expressed in effector cells.

2 Results and discussion

Raft microdomains are known to be enriched for glycosphingolipids, and the ganglioside GM1, which is recognized by cholera toxin, has been used in several studies as a raft marker. We analyzed the expression of GM1 in human resting peripheral blood CD4⁺ T cells and activated CD4⁺ T cells by FACS analysis and immunoblotting. Activated T cells always showed higher levels of GM1 expression than resting T cells, while expression of the CD3 complex was unaffected (Fig. 1 A, B). The higher level of GM1 in activated T cells was due to de novo synthesis induced by T cell activation, as shown by the kinetics of GM1 up-regulation and its inhibition by fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase) (Fig. 1 C). The inhibition of GM1 synthesis by fumonesin B1 was not due do inhibition of T cell activation, as demonstrated by the fact that control and treated cells produced the same amount of IL-2 (not shown).

To quantify the increase in GM1 levels in activated as compared to resting T cells, we performed GM1 ELISA. In all the experiments, activated T cells showed ∼ 350-fold increase in total GM1 amount as compared to resting T cells (Fig. 1 D). This impressive difference was not due to the bigger size of activated T cells, as shown by the fact that the total protein content was only ∼ 5 times higher in activated than in resting T cells (Fig. 1 D).

These results show that resting T cells have very low levels of the raft marker GM1 and that T cell activation induces synthesis of this lipid which is then expressed at very high levels in the membrane of activated / effector cells.

To study localization of rafts in T cells, we performed GM1 intracellular staining of naive and activated T cells, and analyzed the cells by confocal microscopy. As shown in Fig. 2 A, while in naive T cells the FITC-cholera toxin-B (CT-B) staining was diffuse inside the cell showing a clear intracelular distribution of GM1, in activated T cells the staining was restricted to the membrane, indicating that all the synthesized GM1 is not stored inside the cells, but directly targeted to the plasma membrane. Thus, while in resting T cells rafts are mainly intracellular and present only at low levels on the cell surface, in effector T cells they are present at higher levels and only on the cell surface. The Src-family kinase Lck is developmentally regulated in murine CD8 T cells 14. While in naive cytotoxic T cells Lck has a cytosolic distribution and is weakly associated to the coreceptor, in effector and memory CD8 T cells, the kinase is targeted to the membrane and associates with the coreceptor. To investigate whether this is true also in the case of human CD4⁺ T cells, we performed an anti-Lck staining of naive and activated T lymphocytes, and analyzed the cells by confocal microscopy. We found that in naive cells the Lck staining was cytoplasmic, while the kinase was predominantly plasma membrane-associated in cells activated by PHA (not shown). The translocation of Lck from the intracellular compartment to the membrane was followed at the microscope by looking at cells stimulated by anti-CD3 + anti-CD28-coated beads (Fig. 2 B). The fraction of Lck which was not plasma membrane associated was however raft associated (Fig. 3), suggesting that in naive T cells vesicles containing pre-formed rafts are retained in the cytoplasm and exported to the plasma membrane upon TCR triggering. We also found higher levels of Lck in activated versus resting T cells. This result fits with the higher expression of rafts observed in activated cells.

Together, these results show that in human naive T cells, raft microdomains are not organized at the cell plasma membrane, but retained in the cytoplasm. Upon T cell stimulation, these domains are targeted to the membrane and assembled to create signaling compartments. Moreover, T cell priming induces neo-synthesis of raft components, allowing effector cells to be more responsive than naive T cells. The fact that the up-regulation of GM1 in activated / effector T cells is related to the signaling capacity of these cells was proved by the higher co-stimulation offered by GM1 cross-linking to activated than to naive T cells (not shown).

Finally, we tried to investigate whether, among the resting peripheral blood T cells, there was a difference in the GM1 levels of naive (CD45RA^high, CD45RO^low) and memory cells (CD45RO^high, CD45RA^low). We sorted these populations and found that memory cells showed constantly higher levels of GM1 staining than naive cells (Fig. 4). However, the GM1 staining in resting memory cells was always much lower than in activated / effector cells, indicating that rafts are regulated during the different stages of T cell life.

The rapid interaction between triggered receptors and signaling molecules requires availability of components that depends on their concentration and distribution. The organization of the plasma membrane in distinct lipid microdomains which concentrate specific molecules represents a strategy to assemble or separate receptors and signaling molecules. Here we showed that the availability of the signaling components can vary in different cells or under different conditions, because the local concentration of the various components can be developmentally regulated. Naive T cells have few raft domains organized on the membrane, and in order to be activated they need CD28 co-stimulation which amplifies the TCR signaling by recruiting more rafts to the triggered TCR. Upon activation, the T cell signaling machinery is optimized, with the de novo synthesis of GM1 and Lck, and the organization of signaling microdomains at the plasma membrane. Indeed, effector T cells respond to lower doses of antigen than naive cells and do not require the CD28-mediated recruitment of rafts. In this way, by up-regulating or down-regulating the local concentration of signaling molecules, the T cell can modulate its responsiveness to antigens during different phases of its life.

3 Materials and methods