CD28 and CTLA-4 are members of the Immunoglobulin (Ig) superfamily and each forms a disulfide linked homodimer. Each CD28 homodimer is thought to interact with only one ligand molecule, whereas a difference in the angle between the Ig domains in the CTLA-4 dimer allows it to bind two ligands simultaneously and give it the potential to form elaborate oligomers. The Ig superfamily of proteins contains several bona fide adhesion molecules including the CD2-CD48/58 ligand pair, one of the first intercellular adhesion systems to be identified and fully reconstituted with purified molecules. The CD2-CD48/58 and CD28-CD80 interactions share similar affinities and kinetics. This suggests that CD28-CD80 has the potential to function as an adhesion molecule as well as a costimulatory one. Adhesion is a product of receptor-ligand interactions subject to the law of mass action and thus surface ex
Signaling pathways controlled by CD28
Two regions of CD80 cytoplasmic tail are necessary for full costimulation to occur. These regions have distinct functions that regulate different pathways in the CD28 signaling cascade. CD28 deficient mice reconstituted with wild-type or point mutation of Y170F of YMNM motif in the CD28 cytoplasmic domain had normal germinal center formation and CD4+ T cells that produced IL-2 and proliferated normally. However, these cells had impaired PKB phosphorylation and induction of BCL-xl ex
Signaling through the TCR is a very complex process involving several molecules. There are several avenues of “cross-talk” between different pathways and molecules of these pathways that can lead to increased activation or modulation of activation signals.
Upon cross-linking of the TCR complex by an APC, Lck is phosphorylated (and the inhibitory site is de-phosphorylated). Lck and Fyn can then phosphorylate the ITAMs (immuno-tyrosine-based activation motifs) of the TCR. The phosphorylated ITAMs then recruit Zap-70 via SH2 domain interactions. Lck then phosphorylates Zap-70 which phosphorylates the plasma membrane associated adapter protein, LAT. LAT is recruited to the complex with SLP-76. This then allows for the recruitment of Grb-2, which can then recruit sos, which in turn recruits the small G-protein ras. This can then activate the MAPKKK. MAPKKK activates MAPKK (MEK) which phosphorylates both the serine and threonine residues of ERK (MAPK). This leads the phosphorylation and degradation of IκK. These events then allow for the transport of NF-κB to the nucleus where it can activate transcription.
Another way in which TCR can regulate transcription is via the PLC-γ (phospholipase C-γ) pathway. PLC-γ is also recruited to the TCR activation complex, via SH2 domain interactions with LAT, where it becomes activated by phosphorylation. Phosphorylated PLC-γ can then cleave PIP2 (phosphotidylinositol-4,5-bisphosphate) into IP3 (inostol-1,4,5-triphosphate) and DAG (diacylglycerol). IP3 diffuses away and increases calcium in the endoplasmic reticulum. This causes increased intracellular calcium and the influx of calcium. This leads to calmodulin activation and binding to calcium. Calcinuerin is then activated and de-phosphorylates NFAT. NFAT is then able to translocated to the nucleus and activate transcription.
The YMNM motif of CD28, which has been shown to be important for PKC-θ recruitment and Bcl-xl ex
As mentioned above the VAV-1 PH domain can also bind to PIP2 and PIP3. VAV-1 serves as a GEF for Rho-family GTPases. Phosphorylation of VAV-1 allows the induction of guanine exchange, which triggers Rac and Rho function, thus reorganizing the active cytoskeleton. VAV-1 phosphorylation and membrane localization are increased with costimulation. Costimulation facilitates interaction of VAV-1 with SLP-76 at the membrane, where the YESP can bind SLP-76, whose phosphorylation is regulated by Zap-70 and Syk. The c-terminus of SLP-76 binds ADAP which can then bind Vasp and regulate actin filament growth. VAV-1 is also constitutively associated with IKKα and this association is increased upon CD28 ligation. The activation of NF-κB by VAV-1 is not dependent on TCR signaling, which is important for IL-2 production and IκB phosphorylation and degradation leading to NFκB activation, PKCθ recruitment and optimal phosphorylation and activation of PLCγ which in turn converts PIP2 to IP3.
Inhibition of PI3K by wortmanin has been shown to lead to T cell anergy. Without PI3K, PKCθ is not recruited and therefore NF-κB is not activated but IL-2 mRNA is still stabilized. PKCθ activation requires translocation to plasma membranes. Translocation of PKCθ is PLC-independent, but PI3K and Vav-dependent. PKCθ has been found to act at an intermediate step between TCR proximal events and the activation of transcription factors such as NF-κB and the activator protein-1 (AP-1). NFκB binds to CD28RE in the IL-2 promoter and stimulation of CD28 alone is sufficient to induce weak IKK phosphorylation of IκB and p65 PI3K-dependent NF-κB activation. PKCθ synergizes with calcineurin to activate NF-AT IL-2 transcription. PKCθ-deficient mice show a selective T-cell activation defect involving the transcription factors AP-1 and NF-κB and the production of IL-2.