Exocytosis of CTLA-4 Is Dependent on Phospholipase D and ADP Ribosylation Factor-1 and Stimulated during Activation of Regulatory T Cells

Abstract

CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.

Abnormal CTLA‐4 function in T cells from patients with systemic lupus erythematosus

“…The bulk of CTLA-4 is maintained in intracellular stores and its surface expression is tightly regulated by a process of exocytosis and continuous endocytosis [34]. As well as an elevation in CTLA-4 expression in lupus T cells, we noted that CLTA-4 internalisation was also increased ex vivo, which would tend to limit its expression.…”

“…However, CTLA-4 expression was not increased in T cells from lupus patients as a consequence of anti-CD3/CD28 stimulation. When CTLA-4 expression is increased in activated T cells its surface expression is tightly regulated by a process of recycling to the plasma membrane surface [34], with the majority of CTLA-4 maintained in intracellular stores. To assess the recycling dynamics of CTLA-4, purified responder T cells were treated with PMA to induce its surface expression [34].…”

“…When CTLA-4 expression is increased in activated T cells its surface expression is tightly regulated by a process of recycling to the plasma membrane surface [34], with the majority of CTLA-4 maintained in intracellular stores. To assess the recycling dynamics of CTLA-4, purified responder T cells were treated with PMA to induce its surface expression [34]. Confocal microscopy analysis revealed that ex vivo PMA-treated T cells from both healthy and lupus T cells show a similar pattern for CTLA-4 staining with large aggregates at or near the cell surface (Fig.…”

TcR‐induced regulated secretion leads to surface expression of CTLA‐4 in CD4+CD25+ T cells

“…5) further strengthens the case that internal CTLA-4 is contained in a nonlysosomal compartment as previously described. 22,24 The functional mechanisms used by regulatory cells to suppress immune responses are unclear, and it seems likely that both suppressive cytokines and mechanisms involving direct cell-cell contact may contribute. The many proposed Treg cell subpopulations 4 may use different sets of effector mechanisms.…”

PD-1 and CTLA-4 Mediated Inhibitory Signaling for T cell Exhaustion during Chronic Viral Infections

“…Newly synthesized CTLA-4 is transported from Golgi to cell membrane that is dependent on ARF-ribosylation factor 1 and phospholipase D [29,30]. In resting T cells, the unphosphorylated YVKM motif in the cytoplasmic tail of CTLA-4 interacts with the clathrin adapter protein AP, leading to rapid internalization of CTLA-4 in a clathrin-dependent way [31,32].…”