In this study, we show for the first time the membrane compartmentalization of CTLA-4 in T lymphocytes under conditions of CTLA-4–mediated inhibition of T cell activation. Our data demonstrate that a significant fraction of surface CTLA-4 compartmentalizes in the lipid rafts and accumulates at the IS with CD3 and GM1. Such compartmentalization is determined by the cytoplasmic tail of CTLA-4. From a functional point of view, coligation of CTLA-4 with the TCR is associated with an increase in the levels of CTLA-4 within lipid rafts. However, ligation of CTLA-4 within lipid rafts is not sufficient to inactivate T cells by CTLA-4-mediated signaling.
Compartmentalization of CTLA-4 on the T cell surface has been difficult to examine given the low level of CTLA-4 expression in primary, activated T cells. This forced us to generate a panel of Jurkat T cells expressing high levels of wild-type CTLA-4 upon doxycycline induction. In this way, we achieved sufficient surface CTLA-4 for biochemical, functional, and microscopy studies. More importantly, as Jurkat T cells do not express endogenous CTLA-4, these cells are ideal to assess the structural requirements for CTLA-4 compartmentalization using mutant molecules such as the ones reported here (tailless CTLA-4, GPI-anchored CTLA-4). An additional advantage of the system used in these studies is that we were able to examine the surface compartmentalization under conditions of APC-driven T cell stimulation, making the stimulation conditions more physiologically relevant than previously used monoclonal antibody stimulation.
The recently reported crystal structures of CTLA-4 have led to the proposal that, upon ligation by B7 homodimers, CTLA-4 could form a lattice-like structure whose intermembrane dimension (100–140 Å) might be accommodated within the IS (29
). Our finding of CTLA-4 in the IS is consistent with this model. Furthermore, our data indicate that this lattice does not grossly disrupt the morphology of the IS. This is particularly significant for the TCR complex, as one could claim that the negative function of CTLA-4 may be through interference with TCR oligomerization at the IS. Our findings stress the concept that CTLA-4 and CD3 have to be physically coligated for negative signaling to occur, consistent with the data indicating that such coligation is only functional when in cis, i.e., both ligands on the same surface (20
). Future studies will be required to determine the effect that CTLA-4 may have on the fine stoichiometry and organization of the IS.
We have also analyzed the compartmentalization of CTLA-4 with respect to lipid rafts. The majority of CTLA-4 was outside of lipid rafts. However, a significant fraction of total CTLA-4 was within lipid rafts. The cytoplasmic tail of CTLA-4 was required for its efficient localization within lipid rafts, a novel finding indicating that the cytoplasmic region and not the transmembrane region alone (which is present in the tailless CTLA-4) is critical for raft association. The exact residues within the tail responsible for the lipid raft localization are unknown. As expected from previous reports of GPI-anchored molecules being enriched in lipid rafts (38
), a GPI-anchored version of CTLA-4 was found mostly in these microdomains.
The physiological relevance of lipid raft-associated CTLA-4 is currently unknown. As CTLA-4 in lipid rafts can be found as homodimers and clusters to the IS, it is plausible to assume that the pool within lipid rafts is functional. More importantly, the observation that the content of CTLA-4 within lipid rafts increased under conditions of CTLA-4–mediated inhibition further supports this claim. Such an increase may be explained by retention of CTLA-4 on the cell surface upon TCR ligation or alternatively, by a net increase of CTLA-4 migration into lipid rafts.
The compartmentalization of CTLA-4 within lipid rafts has implications for the search of CTLA-4 targets and/or regulators since one would expect that such molecules are located in these microdomains. Our data are compatible with the model proposing that TCR-ζ is the target of CTLA-4 as the phosphorylated form of this chain has been found in lipid rafts (26
). However, it should be noted that in the T cell system described in this report, we have not been able to detect CTLA-4 associated with TCR-ζ or SHP-2 (unpublished data). We and others favor an effect of CTLA-4 on ERK activation and JNK signaling (23
), which is also consistent with the lipid raft compartmentalization of CTLA-4, as lipid rafts are enriched in LAT and ras (8
). Similar considerations apply to regulators of CTLA-4 function such as the regulatory subunit of the serine/threonine phosphatase PP2A (39a
). Therefore, the presence of the regulatory machinery of CTLA-4 function and of the potential targets of this molecule within lipid rafts argues that the lipid raft-associated fraction of CTLA-4 is biologically important.
As most of the lipid raft-associated CTLA-4 is on the cell surface, and this pool increases after coligation with the TCR, we propose that raft localization is necessary for CTLA-4–mediated negative signaling. In support of this claim is the data that tailless CTLA-4 that is mostly outside lipid rafts failed to inhibit T cell activation by negative signaling. However, raft localization of CTLA-4 is not sufficient for CTLA-4–mediated negative signaling, as the GPI-anchored CTLA-4 that is almost exclusively in lipid rafts and migrates to the IS did not inhibit T cell activation. We cannot exclude fine molecular differences in the lattice formed by the GPI-anchored CTLA-4 compared with that formed by the wild-type CTLA-4. However, the suggestion of lattice formation was made from crystals in which only the extracellular domain of CTLA-4 was used, and thus it is plausible to assume that lattice formation may be primarily determined by the extracellular portion of CTLA-4.
The results of this study can be incorporated into a broader model of CTLA-4 trafficking. The majority of CTLA-4 in a resting, previously activated T cell would be primarily in intracellular, detergent soluble compartments where it is constantly being shuttled to the surface, where it compartmentalizes in lipid rafts. This pool is reinternalized via an AP-2–dependent process (21
). The localization within lipid rafts may facilitate that process, as it is well established that lipid rafts are involved in internalization (40
). Upon TCR ligation, CTLA-4 is tyrosine phosphorylated in a lck and ZAP-70–dependent manner (24
). This phosphorylation event prevents AP-2–dependent internalization, and leads to retention of CTLA-4 on the cell surface. In addition, polarization of the intracellular stores of CTLA-4 toward the APC would facilitate its release into the IS (41
). The raft-associated CTLA-4 would then cluster in the IS, where it could potentially mediate the formation of extended lattice networks of B7 and CTLA-4 (29
). Further studies on the specific contribution of the cytoplasmic tail and lipid raft compartmentalization to CTLA-4 relocation into the IS should help us to identify the fine molecular arrangement within this highly organized interface.