only ligands with high affinity that bind the tcr for a long enough time can initiate a signal. all intermediate steps are reversible, such that upon ligand dissociation the receptor reverts to its original unphosphorylated state before a new ligand binds. this model predicts that maximum response of t cells decreases for pmhc with shorter lifetime. experiments have confirmed this model. however, the basic kinetic proofreading model has a trade-off between sensitivity and specificity. increasing the number of proofreading steps increases the specificity but lowers the sensitivity of the receptor. the model is therefore not sufficient to explain the high sensitivity and specificity of tcrs that have been observed. (altan bonnet2005) multiple models that extend the kinetic proofreading model have been proposed, but evidence for the models is still controversial. the antigen sensitivity is higher in antigen-experienced t cells than in naive t cells. naive t cells pass through the process of functional avidity maturation with no change in affinity. it is based on the fact that effector and memory (antigen-experienced) t cell are less dependent on costimulatory signals and higher antigen concentration than naive t cell. signaling pathway the essential function of the tcr complex is to identify specific bound antigen derived from a potentially harmful pathogen and elicit a distinct and critical response. at the same time it has to ignore any self-antigen and tolerate harmless antigens such as food antigens. the signal transduction mechanism by which a t cell elicits this response upon contact with its unique antigen is termed t-cell activation. upon binding to pmhc, the tcr initiates a signalling cascade, involving transcription factor activation and cytoskeletal remodelling resulting in t cell activation. active t cells secrete cytokines, undergo rapid proliferation, have cytotoxic activity and differentiate into effector and memory cells. when the tcr is triggered, t cells form an immunological synapse allowing them to stay in contact with the antigen presenting cell for several hours. on a population level, t cell activation depends on the strength of tcr stimulation, the dose–response curve of ligand to cytokine production is sigmoidal. however, t cell activation on a single cell level can be characterised by a digital switch-like response, meaning the t cell is fully activated if the stimulus is higher than a given threshold, otherwise the t cell stay in its non-activated state. there is no intermediate activation state. the robust sigmoid dose-response curve on population level results from individual t cells having slightly different thresholds. t cells need three signals to become fully activated. signal 1 is provided by the t-cell receptor when recognising a specific antigen on a mhc molecule. signal 2 comes from co-stimulatory receptors such as cd28, presented on the surface of other immune cells. it is expressed only when an infection was detected by the innate immune system, it is a "danger indicating signal". this two-signal system makes sure that t cells only respond to harmful pathogens and not to self-antigens. an additional third signal is provided by cytokines, which regulate the differentiation of t cells into different subsets of effector t cells. there are myriad molecules involved in the complex biochemical process (called trans-membrane signaling) by which t-cell activation occurs. below, the signalling cascade is described in detail. receptor activation the initial triggering follows the mechanism common for all ntr receptor family members. once the tcr binds a specific pmhc, the tyrosine residues of the immunoreceptor tyrosine-based activation motifs (itams) in its cd3 adaptor proteins are phosphorylated. the residues serve as docking sites for downstream signalling molecules, which can propagate the signal. phosphorylation of itams is mediated by the src kinase lck. lck is anchored to the plasma membrane by associating with the co-receptor cd4 or cd8, depending on the t cell subtype. cd4 is expressed on helper t cells and regulatory t cells, and is specific for mhc class ii. cd8, on the other hand, specific for mhc class i, is expressed on cytotoxic t cells. binding of the co-receptor to the mhc brings lck in close proximity to the cd3 itams. it has been shown that 40% of lck is active even before the tcr binds pmhc and therefore has the ability to constantly phosphorylate the tcr. tonic tcr signalling is avoided by the presence of phosphatase cd45 that removes phosphorylation from tyrosine residues and inhibits signal initiation. upon binding the balance of kinase activity to phosphatase activity is perturbed, leading to a surplus of phosphorylation and initiation of the signal. how such perturbation is accomplished by tcr binding is still debated. mechanisms involving conformational change of tcr, tcr aggregation and kinetic segregation have been suggested. tyrosine kinase fyn might be involved in itam phosphorylation but is not essential for tcr signalling. proximal tcr signaling phosphorylated itams in the cytoplasmic tails of cd3 recruit protein tyrosine kinase zap70 that can bind to the phosphorylated tyrosine residues with its sh2 domain. this brings zap70 into close proximity to lck which results to its phosphorylation and activation by lck. lck phosphorylates a number of different proteins in the tcr pathway. once activated, zap70 is able to phosphorylate multiple tyrosine residues of the transmembrane protein lat. lat is a scaffold protein associated with the membrane. it itself does not have any catalytic activity but it provides binding sites for signalling molecules via phosphorylated tyrosine residues. lat associates with another scaffolding protein slp-76 via the grap2 adaptor protein, which provides additional binding sites. together lat and slp-76 provide a platform for the recruitment of many downstream signalling molecules. by bringing these signalling molecules into close proximity, they can then be activated by lck, zap70 and others kinases. therefore, the lat/slp76 complex act as a highly cooperative signalosome. molecules that bind the lat/slp76 complex include: phospholipase cγ1 (plcγ1), sos via a grb2 adaptor, itk, vav, nck1 and fyb. signal transduction to the nucleus plcγ is a very important enzyme in the pathway as it generates second messenger molecules. it is activated by the tyrosine kinase itk which is recruited to the cell membrane by binding to phosphatidylinositol (3,4,5)-trisphosphate (pip3). pip3 is produced by the action of phosphoinositide 3-kinase(pi-3k), which phosphorylates phosphatidylinositol 4,5-bisphosphate (pip2) to produce pip3. it is not known that pi-3k is activated by the t cell receptor itself, but there is evidence that cd28, a co-stimulatory receptor providing the second signal, is able to activate pi-3k. the interaction between plcγ, itk and pi-3k could be the point in the pathway where the first and the second signal are integrated. only if both signals are present, plcγ is activated. once plcγ is activated by phosphorylation, it hydrolyses pip2 into two secondary messenger molecules, namely the membrane-bound diacyl glycerol (dag) and the soluble inositol 1,4,5-trisphosphate (ip3). these second messenger molecules amplify the tcr signal and distribute the prior localised activation to the entire cell and activate protein cascades that finally lead to the activation of transcription factors. transcription factors involved in t cell signalling pathway are the nfat, nf-κb and ap1, a heterodimer of proteins fos and jun. all three transcription factors are needed to activate the transcription of interleukin-2(il2) gene. nfat nfat activation depends on calcium signaling. ip3 produced by plc-γ is no longer bound to the membrane and diffuses rapidly in the cell. binding of ip3 to calcium channel receptors on the endoplasmic reticulum (er) induces the release of calcium (ca2+) into the cytosol. the resulting low ca2+ concentration in the er causes stim1 clustering on the er membrane, which in turn leads to activation of cell membrane crac channels that allows additional calcium to flow into the cytosol from the extracellular space. therefore, levels of ca2+ are strongly increased in the t cell. this cytosolic calcium binds calmodulin, inducing a conformational change of the protein such that it can then bind and activate calcineurin. calcineurin, in turn, dephosphorylates nfat. in its deactivated state, nfat cannot enter the nucleus as its nuclear localisation sequence (nls) cannot be recognised by nuclear transporters due to phosphorylation by gsk-3. when dephosphorylated by calcineurin translocation of nfat into the nucleus is possible. additionally, there is evidence that pi-3k via signal molecules recruits the protein kinase akt to the cell membrane. akt is able to deactivate gsk3 and thereby inhibiting the phosphorylation of nfat, which could contribute to nfat activation. nf-κb nf-κb activation is initiated by dag, the second, membrane bound product of plcγ hydrolysation of pip2. dag binds and recruits protein kinase c θ (pkcθ) to the membrane where it can activated the membrane bound scaffold protein carma1. carma1 then undergoes a conformational change which allow it to oligomerise and bind the adapter proteins bcl10, card domain and malt1. this multisubunit complex binds the ubiquitin ligase traf6. ubiquitination of traf6 serves as scaffold to recruit nemo, iκb kinase (ikk) and tak1. tak 1 phosphorylates ikk, which in turn phosphorylates the nf-κb inhibitor i-κb, leading to the ubiquitination and subsequent degradation of i-κb. i-κb blocks the nls of nf-κb therefore preventing its translocation to the nucleus. once i-κb is degraded, it cannot bind to nf-κb and the nls of nf-κb becomes accessible for nuclear translocation. ap1 activation of ap1 involves three mapk signalling pathways. these pathway use a phosphorylation cascade of three successive acting protein kinases to transmit a signal. the three mapk pathways in t cells involve kinases of different specificities belonging to each of the map3k, map2k, mapk families. initial activation is done by the gtpase ras or rac which phosphorylate the map3k. a cascade involving the enzymes raf, mek1, erk results in the phosphorylation of jun, conformational change allows jun to bind to fos and hence ap-1 to form. ap-1 then acts as transcription factor. raf is activated via the second messenger dag, sos, and ras. dag recruits among other proteins the ras guanyl nucleotide-releasing protein (rasgrp), a guanine nucleotide exchange factor (gef), to the membrane. rasgrp activates the small gtpase ras by exchanging guanosine diphosphate (gdp) bound to ras against guanosine triphosphate (gtp). ras can also be activated by the guanine nucleotide exchange factor sos which binds to the lat signalosom. ras then initiates the mapk cascade. the second mapk cascade with mekk1, jnkk, jnk induces protein expression of jun. another cascade, also involving mekk1 as mapk3, but then activating mkk3 /6 and p38 induces fos transcription. activation of mekk1, additionally to being activated by ras, involves slp-76 recruiting the gef vav to the lat signalosom, which then activates the gtpase rac. rac and ras activate mekk1 and thereby initiate the mapk cascade.