CTLA4 Signaling Pathway
The T-cell compartment of adaptive immunity provides vertebrates with the potential to survey for and respond specifically to an incredible diversity of antigens. In the periphery, one important level of regulation is the action of costimulatory signals in concert with TCR (T-Cell antigen Receptor) signals to promote full T-cell activation (Ref. 1). Stimulation of TCR is triggered by MHC (Major Histocompatibility Complex) molecules on antigen presenting cells that present antigen peptides to TCR complexes and induce a series of intracellular signaling cascades that regulate T-cell development, homeostasis, activation, acquisition of effector’s functions and apoptosis. All these functions are regulated through the action of various costimulatory receptors. Costimulation involves an integration of activating signals and inhibitory signals from CD28 and CTLA4 (Cytotoxic T-Lymphocyte Antigen-4) molecules, respectively, with TCR signals to determine the outcome of a T-cell's encounter with an antigen (Ref. 2).
CTLA4 (CD152) is a member of a class of cell surface molecules capable of terminating early events in the receptor-mediated signaling cascade. It is a 41–43 kDa, type 1 transmembrane glycoprotein of the immunoglobin superfamily, 223 amino acids in length. The extracellular architecture of CTLA-4 is characterized by a single IgV-like domain containing the B7-1 (CD80)/B7-2 (CD86) ligand-binding site. Expression of CTLA4 is dependent both on TCR stimulation by the antigens and CD28-B7 engagement. The costimulatory CTLA4 pathway, attenuates or downregulates T-cell activation, and CTLA4 is designed to remove body cells displaying a foreign epitope, such as virus-infected cells, cells containing intracellular bacteria, and cancer cells with mutant surface proteins. The CTLs (Cytotoxic T-Lymphocytes) are able to kill these cells by inducing a programmed cell death known as apoptosis (Ref. 3 & 4).
CTLA4 is an essential negative regulator of T-cell activation. It is not constitutively expressed but is upregulated in a manner dependent on TCR stimulation, such that greater stimulation produces more CTLA4 at the cell plasma membrane (Ref. 5 & 4). Accumulation of CD28 occurs during T-cell activation and has also been shown to induce expression of CTLA4 and increase stability of CTLA4 mRNA. Expression of CTLA4 is up-regulated within 1 hour following T-cell activation, where it is trafficked by reorganization of the microtubule-organizing center to the cell surface. At the cell membrane, CTLA4 undergoes dimerization, and each CTLA4 dimer can bind two independent B7-1/B7-2 homodimers, forming a linear zipper-like structure between B7-1/B7-2 and CTLA-4 homodimers (Ref. 6 & 7). Activated CTLA4 binds to PI3K (Phosphatidylinositol 3-Kinase), the tyrosine phosphatases SHP1 and SHP2, and the serine/threonine phosphatase PP2A. SHP1 and SHP2 dephosphorylate TCR-signaling proteins, whereas PP2A targets phosphoserine/threonine residues and is known to interfere with the activation of Akt. Binding of CTLA4 to PI3K indicates that the co-receptor can generate positive signals in common with CD28. But in the context of an additional negative-signaling protein, such as PP2A, SHP1 and SHP2 would be dominant by interfering with T-cell function (Ref. 8, 6 & 1).
CTLA4 can inhibit T-cell responses by various mechanisms
- One mechanism involves antagonism of B7-CD28–mediated costimulatory signals by CTLA4, which occurs because CTLA4 has a much higher affinity for B7 than does CD28. CTLA4 binds CD80/86 500 to 2500 times more avidly than CD28 does. Signaling through CD28 promotes cytokine IL-2 mRNA production and entry into the cell cycle, T-cell survival, T-helper cell differentiation and immunoglobulin isotype switching. Signaling through CTLA4 inhibits IL-2 mRNA production and inhibits cell cycle progression. This mechanism is only dependent on the extracellular domain of CTLA4 and is operational in a manner directly proportional to the level of surface expression (Ref. 9, 7 & 4).
- A second mechanism by which CTLA4 can inactivate T-cells involves the delivery of a negative signal. In contrast to CTLA4–mediated sequestration of B7, negative signaling by CTLA4 requires the cytoplasmic tail of CTLA4 and is operational at low levels of surface expression (Ref. 4).
- Another mechanism for the inhibitory activity of CTLA-4 involves direct interactions with TCR-CD3 complex at the immunological synapse and also the proteins involved in downstream signaling after TCR activation. The activation of T-cells by Antigen–MHC-II complex carried on antigen presenting cells is a complex process involving a cascade of events, the first of which is phosphorylation of the PTKs (Protein Tyrosine Kinases) belonging to the Src and SYK ZAP70 (Zeta-Chain-Associated Protein Kinase) families. Initiation of T-cell activation is mediated by phosphorylation of the ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) on the TCR-CD3 complex by Lck (attached to CD4 or CD8), and Fyn, both of which are members of the Src family of kinases. The phosphorylated ITAMs then bind the SH2 domains of ZAP70 SYK. This in turn results in phosphorylation and activation of ZAP70 and SYK, which amplify signals from the TCR through the activation of the adaptor proteins: LAT (Linker Activator for T-Cells), SLP76 (SH2 Domain-Containing Leukocyte Protein-76), GADS (Growth Factor Receptor-Bound Protein-2-Related Adaptor Protein-2), TRIM (T-Cell Receptor Interacting Molecule) and enzymatic effectors such as PLC-Gamma1 (Phospholipase-C-Gamma1) in order to trigger immune response. CTLA4 interacts with the ITAMs present on the TCR and CD3 and essentially disrupts the cascade of biochemical signals that lead to activation of the T-cell. ZAP70, SYK and Fyn are also potential targets of direct inhibitory interaction of CTLA4.
- In most cases, the inhibitory actions of CTLA4 are brought about through its association with the tyrosine phosphatases: SHP1, SHP2 and PP2A (Ref. 10 & 11).
After T-cell activation, CTLA4 is rapidly endocytosed, thus removing it rapidly from the cell surface. The significance of the apparent tight control of CTLA4 expression is due to the fact that CTLA4 has a greater affinity for its CD80/86 ligands. Thus, when CTLA4 is not required, it is removed, to maintain a fast T-cell activation response. Second, CTLA4 might apply its inhibitory effect by acting on downstream signaling pathways at activation. CTLA4 is unique in binding to the Clathrin adaptor complexes, such as AP2 (Adaptor Protein-2) and AP1 (Adaptor Protein-1) through its nonphosphorylated Tyr-Val-Lys-Met motif. AP2 regulates endocytosis of CTLA4, whereas AP1 controls the amount of intracellular CTLA4. AP1 mediates the lysosomal degradation of CTLA4. The rapid endocytosis that is controlled by AP2 ensures that the cell surface expression of CTLA4 is tightly regulated, and most reside in intracellular compartments (Ref. 12 & 6).
CTLA4 function only in cis-association with TCR engagement, and, unlike CD28, will not function in trans when B7 is expressed on an antigen-deficient antigen presenting cells. Thus, CTLA4 may regulate signal transduction in the cells, which leads to differentiation into regulatory T-cells; or alternatively, CTLA4 engagement on the effector cells may alter signal transduction and subsequent cytokine production (Ref. 13 & 1). CTLA4 may also function by physically disturbing the assembly or organization of molecules in the synapse. This occurs by sequestration of proteins involved in signal transduction away from the immunological synapse, thereby reducing the resultant signaling.
CTLA4 is involved in the maintenance of tolerance in autoimmune diseases such as diabetes and thyroiditis as well as a predisposition toward spontaneous abortion (Ref. 14). A SNP (Single-Nucleotide Polymorphism) in exon 1 of CTLA4 is associated with susceptibility to several autoimmune diseases, including MS (Multiple Sclerosis) (Ref. 15). Antibody-mediated blockade of CTLA4 prevents development of tolerance, augments anti-tumor responses, and exacerbates autoimmune disease (Ref. 16).
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