T cells are a subset of lymphocytes that play a crucial role in immune response. T-cell receptor (TCR) is a complex of integral membrane proteins on the surface of T cells, which takes part in the activation of T-cells in response to an antigen. Activation of TCR is triggered by major histocompatibility complex (MHC) molecules on antigen presenting cells (APC) that present antigen peptides to TCR complexes and promote a series of cellular signaling cascades, which eventually result in cellular proliferation, differentiation, cytokine production, and/or activation-induced cell death.
TCR consists of six different polypeptide chains that form the TCR heterodimer responsible for ligand recognition.CD3 molecules (CD3ε, CD3γ, CD3δand CD3ζ), assembled together with the TCR heterodimer, possess a characteristic sequence motif for tyrosine phosphorylation, known as immuno-receptor tyrosine-based activation motifs (ITAMs). The TCR polypeptides themselves have very short cytoplasmic tails, and CD3 molecules are responsible for all the proximal signaling events mediation. TCR/CD3 complex interaction plays a central role in mediating cell recognition events ranging from receptor assembly, cell-surface expression, and signaling.
TCR activation is regulated by various co-stimulatory receptors. Many transmembrane receptors modulate specific elements of TCR signaling. CD28 is found to enhance TCR-induced proliferation and differentiation of naive T cells. Once binding with B7-1 or B7-2, CD28 provides the T-cell with an initial adhesion ability of approximating the T-Cell and APC membranes. Besides, CD45 regulates TCR signaling by modulating the phosphorylation state of the tyrosine kinases like Lck and Fyn, and antagonizing the inhibitory impact of inhibitory proteins, thereby favoring T-Cell activation.
TCR Signaling Cascades
Upon engagement of the TCR by antigen presented on MHC molecules, the Src family kinase Lck is activated by the interaction of MHC-II and CD4 or CD8, and proceeds to ITAMs on the ε, δ, γ and ζ subunits of the TCR/CD3 complex. Phosphorylated ITAMs recruit zeta-chain associated protein kinase (ZAP-70) to the TCR/CD3 complex where it becomes activated, promoting recruitment and phosphorylation of downstream adaptor or scaffold proteins. The linker for activation of T cells (LAT) and leukocyte protein SLP-76 are two main substrates of ZAP-70. Phosphorylated LAT and SLP-76 result in recruitment of a number of other proteins involved in activation of the calcium mobilization, Ras pathway and cytoskeletal reorganization.
1. Calcium Mobilization
One critical protein that is recruited to tyrosine-phosphorylated LAT upon TCR stimulation is phospholipase Cγ1 (PL-Cγ1). Activated PL-Cγ1 is responsible for the production of the second messenger diacylglycerol (DAG) and inositol triphosphate (IP3) by hydrolysis the phosphatidylinositol-4, 5-bisphosphate (PIP2) at the plasma membrane. DAG activates a number of proteins, such as the various isoforms of protein kinase Cθ (PKCθ), and the MAPK/Erk pathways, both promoting transcription factor NF-κB activation, whereas IP3 binds to IP3 receptors (IP3Rs) on the surface of the endoplasmic reticulum (ER) and releases Ca2+ from ER. This event triggers the opening of Ca2+release activated Ca2+(CRAC) channels, allowing influx of extracellular Ca2+. The calcium-bound calmodulin (Ca2+/CaM) activates the protein phosphatase calcineurin by disrupting the inhibitory effects of calmodulin. Activation of calcineurin leads to the dephosphorylation of nuclear factor of activation T cell (NFAT), allowing it to enter the nucleus, where it cooperates with other transcription factors to promote IL-2 gene transcription.
2. Ras Activation
Ras activation occurs through recruitment of its exchange factors sos and RasGRP to the membrane. Tyrosine-phosphorylated LAT binds multiple members of the growth factor receptor-binding protein 2 (GRB2) family of adaptor proteins, such as GRB2, GRB2- related adaptor protein (GRAP) and GRB2 -related adaptor protein-2 (GADS), to facilitate the assembly of macromolecular signaling complexes that are required for efficient T-Cell activation. The interaction of tyrosine-phosphorylated LAT with GRB2 provides a mechanism by which GRB2 and GRAP-associated sos are recruited to the plasma membrane and potentially activate Ras. Ras activation results in the activation of a number of serine/threonine kinases: Raf1, MEK (MAPK/ERK Kinase) and dual-specificity kinases that are responsible for the eventual activation of the mitogen activated protein kinases (MAPKs) superfamily. The MAPKs activate kinases (and other) signaling cascades that result in the activation of the transcription factors NF-κB and AP-1, both of which are necessary for the transcription of several key genes involved in the T cell immune response.
3. Cytoskeletal Reorganization
TCR signaling induces dramatic changes in cytoskeletal architecture. Antigen recognition by the T cell stimulates a burst of actin polymerization at the immunological synapse, generating a lamellipodial sheet structure that spreads over the surface of the APC. After TCR triggering, cytoskeleton modifications are regulated by two pathways that are organized by the LAT–SH2-domain-containing SLP76 complex. One pathway depends on the adaptor–scaffold complex Src kinase-associated phosphoprotein of 55 kDa (SKAP55)–adhesion and degranulation promoting adaptor protein (ADAP) that controls T-cell adhesion to the APC by upregulating leukocyte function-associated antigen 1 (LFA1) integrin avidity. The other depends on VAV1, through binding of its Src-homology 2 (SH2) domain to phosphorylated SLP76. Tyrosine phosphorylated VAV1 stimulates the Rho family GTPases, preferentially Rac1 and Rac2. This leads to a signaling pathway that controls actin polymerization through the Wiskott-Aldrich syndrome protein (WASP) and the actin nucleating ARP2–ARP3 complex, clustering of TCRs and coalescence of glycolipid-enriched membrane microdomains (GEMs). This pathway, therefore, has a role in the organization of the ‘signalosome’ by concentrating receptors and signaling components.
Negative regulation of TCR signaling is also important to keep the hyperactivation of immune response associated with the pathway, which is achieved through the intervention of several proteins and receptors.
SHP2-interacting transmembrane adaptor protein (SIT), a transmembrane adaptor protein, interacts with the SH2-containing protein tyrosine phosphatase-2 (SHP2) via an ITIM, and the complex plays a critical role in negative regulation of TCR-mediated signaling. Phosphoprotein associated with glycosphingolipid microdomains (PAG), a transmembrane adaptor molecule in resting human T-cells, is tyrosine phosphorylated and associated with C-Src Tyrosine Kinase (CSK), an inhibitor of Scr-related protein tyrosine kinases. As a result, the overexpression of PAG inhibits TCR-mediated signaling. Additionally, cytotoxic T-lymphocyte antigen-4(CLTA4), a transmembrane protein, serves as a natural inhibitor. When a need to regulate TCR signaling, ZAP-70 activates the process of active release and translocation of CTLA4 to the membrane. At the membrane, CTLA4 interacts with the SHP2 and inhibits the phosphorylation of TCR. Another mechanism by which CTLA4 might antagonize T-Cell function is through inhibition of CD28 signaling by competing for their shared ligands B7-1 and B7-2. When CTLA4 is not required, it is endocytosed, to maintain a fast T-cell activation response.
Relationship with Diseases
T cells are especially significant in cell-mediated immunity, the defense against tumor cells, pathogenic organisms inside cells, and the rejection reactions. Deregulation of T-cell function, whether by defect or by excess, results in serious consequences for the organism including immunodeficiency and autoimmunity.
TCR is an extremely sensitive system. Very few peptide-MHC complexes on APC are needed to trigger a T-cell response. TCR signaling pathway helps us have a deep understanding of what faults in immune regulation leads to immune related diseases and of how the immune system could be better manipulated to overcome afflictions such as cancer, infection and autoimmune diseases.
Classically, T cell receptor signaling is described as going down from the TCR, via LAT, to activate the transcription factors that are required for T cell proliferation and the differentiation of effector function. It is now clear that signaling also branches sideways via multiple adaptor domains and incorporates actin polymerization causing cytoskeletal rearrangements. Signals are propagated to membrane associated molecules, such as integrins, via the recruitment of small GTPases to the plasma membrane. In turn, integrin engagement influences the persistence of microclusters containing signaling molecules and, consequently, the extent of signal propagation. The regulation of these signaling cascades sets the threshold of activation of naive T cells so that they are not activated by self-peptide–MHC complexes but respond only to foreign peptide–MHC complexes. Effector T cells are more sensitive to stimulation than naive T cells and, therefore, have an increased potential for self-reactivity and to cause immune pathology. Negative regulators are upregulated in effector T cells to counteract this increased sensitivity to activation and to maintain immune tolerance.