Adhesion?

Sticking &Signalling

E. Yvonne JonesErice 2006

T cell – target cell interactions, a particular form of cell-cell communication withspecificity contributed by pMHC – TCR binding.

Result: T cell receptor (TCR) signalling and formation of an ‘ImmunologicalSynapse’.

Immunodominant HLA-A2 tumour epitope NY-ESO-1157-165: SLLMWITQC

SLLMWITQV stimulates faster polarisation of lytic granules to the immunological synapse

Chen & Stewart-Jones et al (2005) J. Exp. Med. 201 1243-1255

But, all pMHCs are not equal…

Structure of the immunodominant V17-V10s2 T cell receptor with HLA A2/influenza matrix epitope

…..as used by >one billion humans!

and all TCRs are not equal…

Stewart-Jones et al (2003) Nature Immunol. 4 657-663

Vβ17 Flu Matrix T Cells- The Most Abundant T cell Clonotype on the Planet

• An estimated 3 billion people have HLA-A2• All have had Influenza• Flu matrix responses dominate the immune

reaction to infection• Vβ17 clonotype constitutes > 80% of that

response• 0.5 kg of this receptor circulating in the world• Stacking the receptors end-to-end would stretch

more than halfway to Neptune (2.6 billion km)

Guillaume Stewart-Jones, Jeffrey Ishizuka & John Bell, unpublished calculation

Why is a V17-V10s2 TCRso good at recognising HLA A2/influenza matrix epitope?

Kuby, 4th edition

T cell receptors

(TCRs)

Gene segment

s

Combinations

Vα 50

Jα 502.5 x 103 alpha chains

Vβ 20

Jβ 13

Dβ 2520 beta chains

Any alpha with any beta chain

1.3 x 106The potential TCR repertoire is further increased by the addition of N region nucleotides. The recombination process is not precise. • the exact points of splicing between V, D and J regions can vary over several nucleotides • extra nucleotides, called N regions, can also be inserted at these joints.

The number of TCRs that we make is ~2.5 x 107

Flu matrixpeptide presentedby HLA-A2 providesfew sidechainsfor TCR recognition

The challenge

TCR positioned to insert CRD3 between peptideand alpha 2 helix

The answer

Why is a V17-V10s2 TCRso good at recognising HLA A2/influenza matrix epitope?

Its because of a distinctive CDR3 motifand combination of residues unique to V17-V10s2 CDR1 and CDR2 loopsthat allow the CDR3 to be positioned optimally to sample a unique feature of the pMHC surface

Stewart-Jones et al (2003) Nature Immunol. 4, 657-663

sTCR mutants made and testedMutation (Vβ) Construct Produced? Expressed? Refolded and Purified? Measurable Binding to pMHC? Kinetic Data collected? Thermodynamic Data Collected?Wildtype Y Y Y Y Y YD32A Y Y Y Y Y ND32S Y Y Y N N NQ52A Y Y Y N N NQ52E Y Y Y N N NI53A Y Y Y N N NI53G Y Y Y N N NI53N Y Y Y N N NI53V Y Y Y Y Y YI53L Y Y Y Y Y NN55A Y Y Y Y Y YN55D Y Y Y Y Y YD56A Y Y Y Y Y YQ58A Y Y Y Y Y YQ58E Y Y Y Y Y YS99A Y Y Y Y Y YR98A Y Y Y N N NR98H Y Y Y N N NY101A Y Y Y Y Y YY101F Y Y Y Y Y Y

Mutation (Vα)S31A Y Y Y Y Y YS31T Y Y Y N N NS32A Y Y Y Y Y YS32R Y Y Y Y Y NQ34A Y Y Y Y Y YV51A Y N N N N NA93S Y Y Y Y Y NS95R/Q96E Y Y Y Y Y Y

Jeffrey Ishizuka & Guillaume Stewart-Jones

Vβ17 CDR2 Offers a Unique Sequence and Energetic Landscape

So we are still on target with our hypothesis for why a V17-V10s2 TCR is so good at recognising HLA A2/influenza matrix epitope.

But why is this TCR-pMHC immunodominant over all other TCR-pMHC interactions involving influenza virus epitopes presented by theMHC class I molecule HLA A2?

T cell – target cell interactions, a particular form of cell-cell communication withspecificity contributed by pMHC – TCR binding.

Result: T cell receptor (TCR) signalling and formation of an ‘ImmunologicalSynapse’.

SS

SS

SS

SS

SS

SS

CD

45

CR

YP

RP

TP

LA

RR

PT

P

1 2D

PT

P6 9

D

RP

TP

RP

TP

RP

TP

DP

TP

10D

CR

YP

2

DP

TP

99A

RP

TP

RP

TP

RP

TP

PT

P-

S

RP

TP

Ph

osp

hac

an

PT

P-I

A2

Type I IIA IIB III IV V VI VII

PT

P-B

R7/

SL

SS

SS

SS

SS

SS

SS

DL

AR

catalyticPTP domain

FN-III-likedomain

Ig-like domain

MAM domain

Highlyglycosilateddomain

carbonic anhydrase-related domain

Cys-rich domain

related to cadherinintracellular region

protease cleavage site

SS

Receptor protein tyrosine phosphatases: classification

A

B

C

D

RPTP expression on axonal growth cones (A and B, Ledig et al., 1999) and at the endothelial cell junctions (C and D, Bianchi et al., 1999).

RPTP is a cell-adhesion molecule involved in neural development (axon growth) and angiogenesis.

SS

RPTP

Crystal structure of the MAM-Ig unit Aricescu & Hon (2006) EMBO J. 25 701-712

RPTP

SS

N

C

for dimersneedMIg-FNIII

RPTP

SS

N

C

SS

MAM-Ig

RPTP

SS

N

C

and for adhesionneedMIg-2xFNIII

so…

*

structure

function?

NO

MIg structure plus biophysical and functional data generatedseveral possible models for RPTP adhesive interactions

****

monomer pH-dependent dimer (trans)

trans/trans zipper trans/cis zipper

Zipper model, consistent with the extended planarity of the plasma membrane observed at intercellular contact sites and with the multiple interactions observed between RPTP ectodomains

I recommend you go and see Radu Aricescu’s poster in the Thursday afternoon session

(also Christian Siebold’s poster on cell guidance signalling…)

Christian Siebold

Radu Aricescu

Weixian Lu

(Jeffrey Ishizuka) &Guillaume Stewart-Jones

In collaboration with: John Bell, Vincenzo Cerundolo,Andrew McMichael & Anton van der Merwe