Early observations in TCR signalling
- If inhibit try phoshatates, induce TCR phosphorylation and signalling without pMHC
- TCR is small compared with other surface molecules
=> kinetic segragation model (1990s/2000s)
Predictions of kinetic segregation model
- Tyr phosphatases segregate from TCR on ligand engagement
- Truncation of phosphatase enzymes inhibits their segregation and thus inhibits TCR triggering
- Elongation of pMHC decreases segregation of phosphatases
Varma et al 2006
Overlayed fluorescence of CD45 and TCR distribution at immunological synapse between T cell and artificial APC
Other NTR triggering mechanisms proposed
- Induced proximity/ aggregation
- Low surface density of pMHC and T cells only need a couple of pMHC to trigger makes this unlikely- how can a single TCR-pMHC induce TCR aggregation?
- Unlikely to be TCR/co-receptor heterodimerisation because not all TCR signalling requires co-receptor - Conformational change -> reveals tyr residue
- Allosteric? structural studies have not found conserved allosteric changes associated with binding
- Mechanical mechanism? Evidence of CD3 conformational change with binding but unclear how mechanical force would transfer. Also, if mechanical mechanism, accessory receptor-ligand interactions (CD2, CD58) would reduce force on TCR, but these interactions enhance signalling.
Kohler et al 2010
Studies NKCs and target cells. Introduced ligands for activator and inhibitory receptors into target cells -> affects cell lysis.
Elongation of inhibitory ligand decreased efficacy of inhibition.
When have longer activator ligands, shorter inhibitory ligands are less effective.
If same size, ligands colocalise => proximal membrane signal integration (allows localisation of abnormal cells and response polarisation)
Yokosuka et al 2012
Optimal PD1 inhibition of TCR requires matched size and colocalisation.
Authors varied size of PD1 and measured IL2 produced.
Long forms of PD1 lost colocalisation and inhibition; shortening restores.
Signal integration TCR and CD28
Can integrate in nucleus so do not need colocalisation
Arase et al 2002
Paired activator and inhibitory NTRs result from gene duplication, possibly due to host-pathogen arms race.
Authors studied mice (have Ly49Rs on NKCs rather than KIRs)
- inhibitory R Ly49I binds MHC-I
- murine CMV expresses decoy receptor m157 which engages the inhibitory receptor (so MHC-I can be down-regulated in infected cells without consequence, escaping NKCs and T cells)
- some mouse strains express a paired activator version of the inhibitory receptor (Ly49H) -> binds decoy receptor so activates NKC vs virally infected cells
Yang et al 2016
APCs only express a few copies of any given foreign pMHC.
Authors found 8-46 specific pMHCs in in vitro HIV infection
~10^12 T cells in periphery with ~10^8 unique TCRs
3 key studies in 90s
[Evavold 1991] [Hogquist 1994] [Lyons 1996]
T cells from mouse spleen -> mix with APCs loaded with different peptide concentrations (mixed lymphocyte reaction).
Good T cell response to wt Ag but abolished with single AA mutation
=> T cells very specific
Don Mason 1998
Theoretical calculations assuming ~10^8 unique TCRs can produce effective immunity.
Recognise peptide 8-14AA length, with 20 proteinogenic AAs.
Predicts ~10^5 to 10^6 pMHC per TCR
Ignatowicz 1996, 1997
Mice achieve comprehensive T cell repertoire with selection by a single peptide.
Their T cells respond to different, unrelated peptides.
Wooldridge et al 2012
Decamer CPLs. 9.36 x 10^12 peptides used to quantify degeneracy of patient derived CD8 TCR.
Subsampled likely peptide candidates from defamer CPLs, then incubate with T cells and C1R-A2 cells.
ELISA for MIP1beta.
Used mathematical algorithm weighting peptides based on circumstances of T cell activation.
~500 peptides within factor of 2 of optimal agonist
~60,000 within factor of 10
~1.3x10^6 within factor of 100
Functional consequences of TCR degeneracy
- solution to providing comprehensive immunity whilst conserving resources
- polyclonal responses
- molecular mimicry -> autoreactivity
T cell discrimination models
- Occupancy model
- assumes activation determined by number of bound TCRs, immediate signalling. pMHC potency depends on Kd. Most R-L interactions in body. - KPR model
- activation based on number of TCRs bound for sufficient duration
- due to proofreading time between binding and signalling (biochemical steps e.g. ITAM phosphorylation, binding and phosphorylation of ZAP70)
- unbinding reverses biochemical steps
- amplifies differences between Koff of different ligands => higher discrimination
Yousefi et al 2019
- Optogenetic system using phyB as a ligand, so TCR binding controlled by light
- red -> phyB cycles between bound and unbound (rate depends on intensity)
- mathematical modelling indicated that half life of ligand-TCR interactions determines signalling downstream of TCR
- artificial system not using pMHC, however their system did induce Ca and Erk MAPK signalling and led to up regulation of CD69 (T cell activation marker)
Pettmann et al 2021
- model of T cell discrimination and promiscuity
- T cells expressing 1G4 TCR (recognises peptides expressed in many cancers, Kd~8uM)
- altered peptide with series of point mutations to decrease affinity then plotted dose-response curve => gradual reduction in response as decrease affinity
- at 15% response cut-off, correlation between p15 (peptide conc) and Kd
- gradient gives index of discriminatory power, alpha
- for TCR, a = 2 whereas a = 1 for other receptors in body tested (e.g. IL13R, M3, c-Kit). a = 2 => 10x decrease affinity overcome by increase ligand conc by 100x. BCR a = 1.3 (intermediate) but more data needed.
=> evidence against occupancy model (which would have a = 1 due to linear relationship between affinity and concentration) - authors calculated 2.7 biochemical steps with proofreading time of 2.8s
- authors also did meta-analysis of existing murine and human data- the 3 90s studies showed a = 9 whereas all more recent data gave a ~ 2
- attributed to inaccurate measurements of binding using surface plasma resonance (which struggles to accurately measure very week TCR-pMHC interactions)
Kohanim et al 2020
- proposed an immune surveillance mechanism consistent with enhanced but imperfect discrimination
- auto reactive T cells kill abnormal hyper secreting endocrine cells to maintain homeostasis
- e.g. T cell may recognise unregulated proteins in the pathway connecting a mutation to hyper secretion of insulin in beta cells in T2DM, then kill those cells
- autoimmunity upon over expression of self-antigen
KPR to account for optimal affinity
- enhance TCR affinity for cancer therapeutics
- these receptors are actually less sensitive than WT
- once bind Ag, TCR could signal for a period of time then something limits it (e.g. internalised with ligand) => so ligands with supra-physiological affinity would prevent TCR returning to membrane
Huang et al 2013
T cell antigen discrimination is sensitive (able to respond to ~1-10 pMHCs on an APC)
- authors showed that a single pMHC can trigger cytokine secretion from T cells
- mixed CD4 T cells with APCs bearing qdot-labelled pMHC
- cytokine captured on T cell surface to allow detection by fluorescent antibodies
- T cells responded to even a single pMHC
- as increase the number of pMHCs, more T cells are able to respond on average
TCR sensitivity remarkable in light of two observations
- TCR/pMHC lifetime is short (seconds) and is subjected to molecular forces
- [Huppa et al 2010] - solution lifetime of recombinant proteins 1.24s; membrane lifetime with T cells 0.109s - Cytokine production requires TCR/pMHC interactions for several hours
- [Huppa et al 2003] - simultaneously tracked TCR-CD3 complex and PI3K activity in single T cells using 3D video microscopy
- despite rapid internalisation of most TCR-CD3, TCR-dependent signalling was still evident up to 10hrs after conjugate formation
- blocking this interaction caused dissolution of the synapse and proportional reduction in IL2 production and cellular proliferation
Molecular mechanisms of T cell Ag sensitivity
- microvilli-like protrusions (actin-mediated)
- adhesion receptors e.g. LFA1, CD2 (binds CD58 which is expressed on all cells in body)
- coreceptors (if block, need more Ag for same response)
Trade-off between T cell sensitivity and discrimination
^ KPR time increases discrimination (since increase fold-difference between pMHC) but decrease sensitivity (as probability of signalling for any pMHC is decreased)
Farfan & Dushek
T cell Ag sensitivity mechanisms
