4.3.1 Molecular and Cell Biology of the CTLA-4 Pat...
Cytotoxic T Lymphocyte antigen 4 (CTLA-4) (CD152) and CD28 are homologous receptors expressed by both CD4+ and CD8+ T cells, which mediate opposing functions in T cell activation.Both receptors share a pair of ligands expressed on the surface of antigen presenting cells (APCs).CD28 interacts with the CD80 dimer with relatively high affinity and the CD86 monomer with lower affinity, mediating T cell co-stimulation in conjunction with T cell receptor (TCR) signals.In contrast, interactions of the ligands with CTLA-4 serve to inhibit T cell responses, although the precise mechanisms are not fully understood.CTLA-4 interacts with both ligands with higher affinity and avidity than CD28 with CTLA-4-CD80 forming the highest avidity interaction and CD28-CD86 the weakest (Figure 4.4A).Amongst several possibilities, this raises the concept that CTLA-4 can compete with CD28 for ligand binding and thereby act as an antagonist of CD28-mediated co-stimulation.These interactions are thought to take place at the immune synapse between T cells and APCs where CTLA-4 has been shown to recruit CD80 thereby limiting its interactions with CD28.Once internalised, CTLA-4 molecules appear to be either recycled to the plasma membrane or degraded in lysosomal compartments (Figure 4.4B), however, the details of this process and its functional significance are not yet fully understood [10].
Figure 4.4 Schemati c of CT LA-4 cell biology.(A) CTLA-4 and CD28 receptors share two ligands CD80 and CD86.CD80 is a dimeric high affinity ligand and CD86 is a monomeric lower affinity ligand for both receptors.CTLA-4 has a higher affinity and avidity for CD80 than CD86.The relative affinities go from high to low from left to right.(B) CTLA-4 expressed in T-cells is highly endocytic.CTLA-4 is constitutively expressed in Treg or induced following T cell activation via CD28 and TCR signaling.In the absence of the ligand, CTLA-4 is mainly found in intracellular compartments following clathrin-mediated endocytosis mediated through CTLA-4 interaction with the AP2 molecule.The AP2(μ2 subunit) binds to the tyrosine-based (YVKM) motif of the cytoplasmic domain of CTLA-4 and mediates rapid internalization.LRBA and AP1 proteins have also been found to bind to the YVKM motif on CTLA-4, which appear to impose different fates on CTLA-4.LRBA may mediate recycling of CTLA-4 to the plasma membrane, whereas AP1 may mediate CTLA-4 trafficking to lysosomal compartments resulting in subsequent degradation.
Source: Rowshanravan B, Halliday N, Sansom D M.“CTLA-4: A Moving Target in Immunotherapy”.Blood,2018, 131(1):58-67.
Whilst the biophysical characteristics of CD80 and CD86 are well defined, their functional differences are less clear and the ligands are often referred to together as B7 molecules or CD80/CD86.Both ligands are found on antigen presenting cells such as dendritic cells and B cells,although they have different expression patterns, inducibility and kinetics.Knockout mice show impaired immune responses consistent with reduced CD28 co-stimulation, with CD86 deficiency arguably demonstrating a more severe phenotype.There is considerable variation in ligand expression making generalisation difficult.CD86 is often expressed constitutively on dendritic cells(DC) and is induced to high levels following inflammatory stimuli.In contrast CD80 is thought to be upregulated later by DC.Human peripheral blood monocytes express CD86 and not CD80,whereas some human T cells can express both CD80 and CD86 under different conditions of activation.Thus, at present, differences between the two ligands revolve mostly around different expression patterns, whereas clear functional distinctions have yet to emerge [10, 11].
Whilst CD28 is constitutively expressed on the plasma membrane and co-stimulates T cells,CTLA-4 is predominantly found in intracellular vesicles in FoxP3+ Treg cells or activated conventional T cells.This localization is due to the constitutive endocytosis of CTLA-4 from the plasma membrane and results in approximately 90% of CTLA-4 being intracellular.The endocytosis of CTLA-4 is extremely rapid, with more than 80% of surface CTLA-4 being internalised within five minutes.Once internalised, CTLA-4 molecules appear to be either recycled to the plasma membrane or degraded in lysosomal compartments, however, the details of this process and its functional significance are not yet fully understood [10, 12].
The post-endocytic fate and control of CTLA-4 trafficking is still poorly characterized, although experiments show that CTLA-4 lacking its 36-amino acid cytoplasmic tail is predominantly located at the cell surface.A number of studies have therefore focused on identifying partners that interact with the cytoplasmic tail of CTLA-4 and on defining the role of its cytoplasmic domain.CTLA-4 endocytosis is dependent on clathrin due to its interaction with the μ2 subunit of the clathrin adaptor protein complex AP2 and is also dependent upon dynamin.AP2 targets the cytoplasmic tyrosine containing YVKM motif of CTLA-4 and can be disengaged when this motif is tyrosine-phosphorylated upon T cell activation.However, the immunological settings where the CTLA-4-AP-2 interaction is disrupted are unclear since activated T cells and Treg continue to endocytose CTLA-4.The cytoplasmic domain sequence and trafficking behavior of CTLA-4 are highly conserved in mammals whereas some animals such as fish lack this endocytic motif resulting in predominantly surface expression.This suggests that endocytosis may be a later adaptation of CTLA-4.Overall, CD28 and CTLA-4 differ profoundly in sub-cellular location despite binding to the same ligands and, given the conservation of these features, it seems likely they are pivotal to the functioning of the system [10, 13].(https://www.daowen.com)
The significance of CTLA-4 trafficking was highlighted in patients with Lipopolysaccharideresponsive and beige-like anchor protein (LRBA) deficiency.This BEACH domain containing protein appears to regulate CTLA-4 turnover and it co-localizes predominantly with recycling(Rab11+) endosomes.Loss of LRBA leads to increased CTLA-4 degradation with associated autoimmunity, indicating that LRBA may inhibit CTLA-4 trafficking to lysosomes and promote its recycling.This may be achieved through LRBA binding to the YVKM consensus sequence of CTLA-4.Additional control of CTLA-4 trafficking and cellular distribution may also be mediated via interaction with the TRIM/LAX/Rab8 complex, which is involved in the post-Golgi transport of CTLA-4 to the cell surface.CTLA-4 interactions involving PKC-eta and PIX-PAK pathway have also been reported.These interactions appear to modulate Treg-APC interactions: PKC-eta deficient Treg are associated with impaired depletion of CD86 from APCs by Treg and increased Treg motility [10, 14].
One molecular mechanism that is consistent with a dominant cell-extrinsic role for CTLA-4 is the physical capture of CD80 and CD86, and their subsequent removal from APCs: a process known as trans-endocytosis (Figure 4.5A).Trans-endocytosis requires T cell recognition of peptide and represents an antigen specific mechanism for controlling CD80 and CD86 expression levels on APC.Whilst the molecular mechanisms underlying trans-endocytosis itself are still largely unknown, the CTLA-4 pathway is not the only receptor-ligand pair that utilizes such a pathway.Other examples include the Notch-Delta pathway, where binding of Delta (ligand) allows subsequent removal of Notch (receptor) from the adjacent cell.Similar examples operating in the absence of ectodomain cleavage include the gap junction protein connexin, adhesion junctions via VE-cadherin receptor tyrosine-kinases Eph-ephrin interaction and CD47-SHPS-1.Although these examples are present in varied biological systems, their overall similarity suggests that a conserved molecular machinery capable of removing membrane bound molecules from neighboring cells exists.Since these ligands are up-regulated by a variety of inflammatory signals, as shown in Figure 4.5B, CD28 signaling provides information on the inflammatory context or “danger”accompanying TCR recognition [10, 15].
Figure 4.5 CT LA-4 funct ion and the impa ct of immune ch eckpoint blockade.(A) In health,regulatory T cells express CTLA-4, which binds CD80 and CD86 expressed on antigen presenting cells(APCs).CTLA-4 binds to CD80 and CD86 with higher affinity and avidity than does CD28, preventing Tcon stimulation through CD80/CD86 interaction with CD28.Removal of CD80/CD86 ligands by trans-endocytosis results in impaired co-stimulation of T cells via CD28, resulting in immune regulation.(B) When the immune system is stimulated in the presence of inflammatory/innate immune stimuli,APCs up-regulate the expression of CD80/CD86 overcoming their control by Treg, enabling co-stimulation and the proliferation of T cells.(C) In the tumor microenvironment, CD80/CD86 are controlled by Treg and the abundance of Treg leads to the suppression of immune responses.(D)Anti-CTLA-4 antibodies bind to CTLA-4 molecules with high affinity leading to Treg depletion or functional blockade resulting in enhanced T cell activation and immunological responses to cancer.The impacts of CTLA-4 blockade can be mediated by a variety of mechanisms: prevention of trans-endocytosis increasing CD80/CD86 levels on APCs, direct Treg cytotoxicity and antibody dependent cellular cytotoxicity mediated by FcR-IV expressing intra-tumoral macrophages.NB only CD80 is shown here for clarity.
Source: Rowshanravan B, Halliday N, Sansom D M.“CTLA-4: A Moving Target in Immunotherapy”.Blood,2018, 131(1):58-67.
In summary, CTLA-4 is a highly endocytic molecule that binds to two distinct ligands, CD80 and CD86, leading to their removal from opposing cells.By utilizing trans-endocytosis CTLA-4 can act as an immune regulatory mechanism by directly reducing the ability of APC to stimulate via CD28.This concept helps explain why a stimulatory and an inhibitory receptor share the same ligands and embraces the endocytic nature of CTLA-4.Taken together, this model suggests that the CD28/CTLA-4 system functions as a rheostat that can tune T cell activation up or down [10].