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.