Diverse mechanisms regulate the surface expression of immunotherapeutic target ctla-4

Abstract

T-cell co-receptor cytotoxic T-cell antigen-4 (CTLA-4) is a critical inhibitory regulator of T-cell immunity and antibody blockade of the co-receptor has been shown to be effective in tumor immunotherapy. Paradoxically, the majority of CTLA-4 is located in intracellular compartments from where it is transported to the cell surface and rapidly internalized. The intracellular trafficking pathways that control transport of the co-receptor to the cell surface ensures the appropriate balance of negative and positive signaling for a productive immune response with minimal autoimmune disorders. It will also influence the degree of inhibition and the potency of antibody checkpoint blockade in cancer immunotherapy. Current evidence indicates that the mechanisms of CTLA-4 transport to the cell surface and its residency are multifactorial involving a combination of immune cell-specific adapters such as TRIM and LAX, the small GTPase Rab8 as well as generic components such as ARF-1, phospholipase D, and the heterotetrameric AP1/2 complex. This review covers the recent developments in our understanding of the processes that control the expression of this important co-inhibitory receptor for the modulation of T-cell immunity. Interference with the processes that regulate CTLA-4 surface expression could provide an alternate therapeutic approach in the treatment of cancer and autoimmunity.

Keywords: CTLA-4; LAX; Rab8; TRIM; trafficking.

Figure 1

Structure of co-receptors. Left panel: CTLA-4 and CD28 bind to the same natural ligands CD80/CD86 via the MYPPPY motif, whereas ICOS binds to ICOSL via the FDPPPF motif. Right panel: structure of the cytoplasmic domains of human CTLA-4, CD28, and ICOS. The cytoplasmic domains of these co-receptors have a common YxxM motif, which binds to the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). CTLA-4 has a unique YVKM motif, which binds to the SH2 domain of the tyrosine phosphatase SHP-2. In its non-phosphorylated form, it associates with the clathrin adapters AP-1 and AP-2. The serine/threonine phosphatase PP2A binds to the lysine rich motif and the tyrosine 218 (Y₂₁₈FIP). The asparagine in the YMNM motif of CD28 is needed for Grb-2 SH2 domain binding, whereas the distal proline motif allows for binding of the SH3 domains of Grb-2, the protein tyrosine kinase p56lck, and Filamin A.

Figure 2

Schematic structure of the transmembrane adapters TCRζ, LAT, TRIM, LAX, and SIT with their binding motifs for Grb-2, Gads, PI3K, and PLCγ.

Figure 3

Mechanisms of CTLA-4 trafficking. Left panel: CTLA-4 forms a multimeric complex composed of TRIM, LAX, and Rab8 for post-Golgi transport to the cell surface. TRIM and LAX bind to the cytoplasmic tail of CTLA-4, while LAX binds via its N-terminus to active GTP-Rab8 (inset). TRIM requires LAX for binding to Rab8 in a complex. LAX functions as a central coordinator by bridging Rab8 with the other LAX-associated proteins TRIM and CTLA-4. This multimeric complex facilitates the transport of newly translated CTLA-4 to the cell surface. The transmembrane adapters TRIM and LAX play a role for CTLA-4 transport similar to the TCRzeta chain, which resides in the TGN and surrounding vesicles and is needed for the association with the hexameric TCR/CD3 in the TGN for proper and efficient cell surface expression. Right panel: non-phosphorylated CTLA-4 associates with the clathrin adapter complex AP-1 and AP-2. AP-1 regulates trafficking of CTLA-4 by shuttling the receptor from the TGN to lysosomes. In activated cells, newly synthesized CTLA-4 is transported to the cell surface, which is facilitated by the TRIM/LAX/Rab8 complex (left panel) and by more generic factors such as ARF-1, PLD, and calcium. Phosphorylation of CTLA-4 by Fyn and Lck recruits PI3K and SHP-2. Dephosphorylated CTLA-4 binds to AP-2 leading to the internalization of CTLA-4 to intracellular compartments such as endosomes and lysosomes from where it can recycle to the cell surface.