The T cell receptors - University of Leeds web... · The T cell receptors E. J. Evans et al. The T...
Transcript of The T cell receptors - University of Leeds web... · The T cell receptors E. J. Evans et al. The T...
QuanTI’ Marie Curie Initial Training Network “Quantitative T-cell Immunology” Summer School, 23-24 June & Annual Meeting
NUI Galway, Ireland
The T cell receptors
Oreste Acuto University of Oxford
The T cell receptors
E. J. Evans et al. The T cell surface - How do we know it well? Immunity. 2003. 19:2130223
Many help identify with high sensitivity, specificity and rapidity antigens of microbial pathogens and prepare adequate countermeasures to fend them off, while protecting from attacks against our normal tissues.
They act as receptors to execute and finely regulate immune responses and tolerance. However, the T cell antigen receptor (TCR) holds a decisive hegemony in these tasks by steering T cell fate and function.
Many are partially dispensable or redundant. However, T cells cannot develop, survive or acquire and maintain their functionality without the TCR
More than 100 different ones !!
This supremacy ensures that the immune system successfully controls and outpaces constantly mutating microbial pathogens while keeping in check the auto-destructive potential of adaptive immunity.
Vast structural diversity of TCR a/b variable (V) regions
Somatic rearrangements of the gene segments coding for TCR variable regions generates a potential stunning number (>1015) of diverse receptors
Each T cell bears a unique TCR (hence clonotypic) displaying a binding site likely to be different from that of a TCR expressed on another T cell.
Each T cell expresses on their plasma membrane (PM) ~ 50,000 molecules of TCR
TCR-pMHC juxtacrine interaction
2D affinity in the 1-3 digit mM range (fast on- and off-rates) with dwelling times of seconds
Binding relies on a poorly co-evolved (i.e., conserved) structural complementarity that forces only relative orientation by very weak affinity between TCR and MHC (“TCR-MHC bias”).
TCR-pMHC recognition (i.e., the encounter of variable receptors and ligands) is not governed by classic “lock-and-key” fit and high affinity recognition.
The peptide and specialised parts of the TCR variable regions (CDR3) are then crucial to achieve agonist affinities
…. upredictible encounters between variable receptors and variable ligands...
Peptide:MHC (pMHC) complexes are displayed in large numbers (105-106) on antigen presenting cells (APCs. i.e., dendritic cells, DC), but are dominated by self-pMHC over foreign pMHC, by orders of magnitude
It is not absolutely guarantied for a given T cell to find a TCR “cognate” ligand of sufficient affinity to produce cellular activation
The fast on-and off-rates of the TCR-pMHC reaction, coupled to the relatively high speed at which T cell swarm through intricate cellular networks in lymphoid organs favour a high scanning frequency by the TCR of many pMHC molecules on the same or different APC
Once a few agonists are found intracellular signalling ensues in a few seconds
T cell clonal expansion upon TCR- agonist pMHC recognition generates functional diversification
Immature thymocytes and T cell respond to weak pMHC by surviving or die if exposed to strong agonists (or for too long)
Reconcile high specificity, sensitivity and speed in such a noisy environment poses challenges to established notions of ligand-receptor interaction and signalling (how to create sharp thresholds in this environment?)
TCR discriminates engagement with and signalling from a few agonist pMHC in a plethora of poorly-stimulatory self-pMHC
The TCR conandrums….
McKeithan. Kinetic proofreading in T-cell receptor signal transduction. PNAS 1995 Altan-Bonnet and Germain. PLoS Biol. 2006 Dushek, Raibatak and Coombs. PLoS Comp. Biol. 2009 Dushek and van der Merve. Trends Immunol. 2014
723 × 261 - brewer.me.uk
723 × 261 - brewer.me.uk
Canonical kinetic proofreading model for TCR signaling
McKeithan. Kinetic proofreading in T-cell receptor signal transduction. PNAS 1995
1 – Structural features of the TCR (and co-receptors) (structural cooperativity) 2 – TCR and signalling machinery membrane organisation (membrane lipids, cytoskeleton) (lateral segregation, structural cooperativity, convoluted multi-step signaling ) 3 – negative and positive feedback mechanisms (negative and positive structural cooperativity; negative and positive feedback of enzymatic activities)
The TCR conandrums….
T-cell antigen receptor triggering mechanism
TCR triggering by
pMHC binding to a/b chain KD = 5-10 mM
Lck
P P
+
>1015 a/b dimer
ZAP-70
Kd = 30 nM
Acuto O., Di Bartolo V., Michel F. Nat. Rev. Immunol. 2008
No intrinsic kinase activity
Dushek, Raibatak and Coombs. PLoS Comp. Biol. 2009
Other versions of the proofreading models
TCR topology
Reinherz and co-workers
Wucherpfennig and co-workers
CD3d CD3g
CD3e
TCRa TCRb
Antreas Kalli and Andre Cohnen
Molecular dynamics modeling of TCR complex transmembrane regions
Nanodomain
a b
g
e e d
TCR
Nanodomain
The TCR ITAMs are bound to the lipid bilayer
Wucherpfennig and co-workers. Cell. 2008
Independent signalling module at steady-state organised in dynamic nanoclusters
Lipid-dependent temporal lateral confinement
Douglass and Vale and co-workers. Cell. 2005; Gauss and co-workers. Nat. Immunol. 2013
Lck dynamic membrane organisation
Src-PTKs
module
CD45
Csk
lipid mdomain lipid mdomain
Lck
Y394
CD4
Y394
lipid mdomain
Y505 Y505
?
?
?
- - + + kinase activity
Active Lck in T cells at steady-state
Nika et al. Immunity. 2010
~ 50%
~ 40%
Activated Lck found in isolated cells and in situ in all thymocyte subsets, mature T cells
Special regulation of SFKs in lymphocytes
Csk
CD45
Lck
a b
g
e e d
TCR
SH3
SH2 Y505
Y394 SH3
SH2 Y505
Y394 SH3
SH2 Y505
Y394
Nanodomain Nanodomain
Csk
?
Dynamics of Lck membrane localisation at steady-state
1 – “sufficient” proximity to CD45: to dephosphorylate pY505
2 – “sufficient” protection from CD45: to allow auto-transphosphorylation and to protect pY394 from dephosphorylation by CD45
3 – “sufficient” distance from the TCR: to avoid unsolicited ITAMs phosphorylation
Positive regulation
Negative regulation
K. Nika et al. 2014 (unpublished)
a b
g e e d
TCR
Lck
ZAP-70
CD45
actin
cytoskeleton cell
adhesion
gene expression
IP3
DAG
CaN
PKCs
RasGRP
MAPKs
NF-B
AP-1
Ca2+
NFAT
LAT
Vav-1
Nck
Adap
PLCg1
SLP-76
Itk
PAK
Cdc42/Rac
WASp
Arp2/3
SKAP55
Rap1
integrins
Nanodomains
pMHC
Membrane localization of Lck after TCR engagement
Baoyu Liu , Wei Chen , Brian D. Evavold , Cheng Zhu
Accumulation of Dynamic Catch Bonds between TCR and Agonist Peptide-MHC Triggers T Cell Signaling
Cell, Volume 157, Issue 2, 2014, 357 - 368
http://dx.doi.org/10.1016/j.cell.2014.02.053
A. Cohnen and A. Kalli (unpublished)
1 – Structural features of the TCR (and co-receptors) (structural cooperativity) 2 – TCR and signalling machinery membrane organisation (membrane lipids, cytoskeleton) (lateral segregation, structural cooperativity, convoluted multi-step signaling ) 3 – negative and positive feedback mechanisms (negative and positive structural cooperativity; negative and positive feedback of enzymatic activities)
The TCR conandrums….
diversification/regulation
module
Src-PTKs
module
triggering
module
CD45
Csk
a b
g e e d
actin
cytoskeleton
remodelling cell
adhesion
gene expression
IP3
DAG
Ca
N
PKCs
RasGRP
MAPKs
NF-B
AP-1
Ca2+
NFA
T
ZAP-70
TCR
LAT
lipid mdomain
Vav-1
Nck
Adap
PLCg1
SLP-76
Itk
Gads
PAK
Cdc42/Rac
WASp-WIP
Arp2/3
lipid mdomain
SKAP55
Rap1
integrins “Independent” signaling modules at steady-state
Adapted from Acuto O., Di Bartolo V., Michel F. Nat. Rev. Immunol. 2008
Lck
Y394
CD4
Y394
lipid mdomain
TCR signalosome: an unusual receptor signaling machine……
Y505 Y505
?
Nanocluster arrangement of TCR and LAT scaffold protein
E. Sherman et al. Immunity. 2011; 35: 705–720 Alarcon, Schamel and co-workers (EM analysis of TCR clusters
clonotypic TCRs that appear to be pre-organised into highly dynamic molecular nano-clusters (5-20 TCR/cluster).
Nanocluster arrangement of TCR and LAT scaffold protein
- Do they increase TCR sensitivity?
- Do they increase favour TCR re-binding to same MHC and contribute to ligand discrimination?
- Do they “shield” the TCR from CD45 and favour triggering?
- Do they exist at steady-state ?
1 – Structural features of the TCR (and co-receptors) (structural cooperativity) 2 – TCR and signalling machinery membrane organisation (membrane lipids, cytoskeleton) (lateral segregation, structural cooperativity, convoluted signaling ) 3 – negative and positive feedback mechanisms (negative and positive structural cooperativity; negative and positive feedback of enzymatic activities)
The TCR conandrums….
McKeithan. Kinetic proofreading in T-cell receptor signal transduction. PNAS 1995 Altan-Bonnet and Germain. PLoS Biol. 2006 Dushek, Raibatak and Coombs. PLoS Comp. Biol. 2009 Dushek and van der Merve. Trends Immunol. 2014
Themis binds to LAT and recruits SHP to the TCR: a negative feedback required for ligand discrimination
during thymocyte selection
pY
540
pY
541
THEMIS
LAT
a b
g e e d
Lck
Lipid binding
Wolfgang Paster et al. 2014 (in revision)
Fu. G. et al. Nature 2013
Images of a Jurkat E6.1 T cell expressing LAT-PAmCherry and spread on a TCR-stimulating coverlsip with (A) brightfield microscopy (left) or epiillumination (right), (B) TIRF microscopy (left) and zoomed region (right), and (C) PALM microscopy (left) and zoomed region (right). (D) Different study regions of the cell shown in panels A–C and an example of pixelization of one of the study regions. (E) Univariate pair-correlation function, g(r), (PCF) of the identified molecules within the study regions in panel D. (F) Heterogeneity in the density of molecules within one of the study regions in panel D. (G) PCF ,g(r), of the study region considering either a homogeneous Poisson process for calculating the 95% confidence interval (between the upper and lower gray dotted lines) or a heterogeneous Poisson process (between the upper and lower red dotted lines) corresponding to the density map in panel F. (H) Ripley's second order statistics, L(r)-r, with a similar study region and null models as in panel (G). Bars - 2 μm for whole cell images and 200nm for zoomed images.