binding properties and stoichiometry of the t cell receptor · binding properties and stoichiometry...

Binding Properties and Stoichiometry of the T Cell

Receptor

Jennifer StoneSummer School on Theoretical and

Experimental ImmunologySeptember 2010

Outline

• T Cell Receptor Complex− Components, Assembly, and Organization

• Ligand binding measurements− Surface Plasmon Resonance− Peptide-MHC Multimers− In Situ 2D Binding Measurements

Outline

• T Cell Receptor Complex− Components, Assembly, and Organization

• Ligand binding measurements− Surface Plasmon Resonance− Peptide-MHC Multimers− In Situ 2D Binding Measurements

Components of the TCR Complex

α βε γδ ε

CD4

LCK

APC

Class II MHC-peptide

α βε γδ ε

CD4

LCK

APC

Class II MHC-peptide

α βε γδ ε

CD8

APC

Class I MHC-peptide

LCK

α βε γδ ε

CD8

APC

Class I MHC-peptide

α βε γδ ε

CD8

APC

Class I MHC-peptide

LCK

T cell recognition of peptide-MHC ligands

Rudolph et. al. Annu Rev Biophys Biomol Struct 2002

Structures of MHC, TCR, and co-receptors

Cochran et. al. Trends Biochem Sci 2001

The T Cell Receptor Complex

No complete structure of the TCR complex has been solved

Stoichiometry and surface expression/distribution of subunits still a subject of study

Assembly of the TCR-CD3 complex.

Wucherpfennig K W et al. Cold Spring Harb Perspect Biol 2010©2010 by Cold Spring Harbor Laboratory Press

Alarcon et. al. EMBO Rep 2006

Characterization of TCR-CD3 complex from T cells

Early evidence for a Bivalent TCR

Fernandez-Miguel et. al. PNAS 1999

Double TCR-Tg mice

145

83

6050

35 kDa

Early evidence for a Bivalent TCR

Fernandez-Miguel et. al. PNAS 1999

Double TCR-Tg mice

Donor quencing of FITC detection

Model of Bivalent TCR

Fernandez-Miguel et. al. PNAS 1999

MβCD

Schamel et. al. J Exp Med 2005

Cholesterol-dependent TCR multimers

BN-PAGE : proteins labeled with Coomassie Blue G-250

Doesn’t disrupt membrane protein interactions like SDS

Campi et. al. J Exp Med 2005

TCR microclusters are present before activation

Kuhns et. al. PNAS 2010

TCR complex dimerization

Intracellular ErythropoetinReceptor Signaling Domain Dimerization Assay

Outline

• T Cell Receptor Complex− Components, Assembly, and Organization

• Ligand binding measurements− Surface Plasmon Resonance− Peptide-MHC Multimers− In Situ 2D Binding Measurements

de Mol and Fischer, & Schuck and Zhao, Methods Mol Biol, 2010

A+B AB

Issues to be concerned about:

Temperature

Mass Transport artifacts

Surface heterogeneity

Decay of immobilized analyte

Multivalency or aggregation of solution-phase ligand

Affinity ranges:

very high (pM) affinities can be difficult to measure

estimate only above ~50 µM

t½ ranges

qualitative only under 5-10 seconds

extremely long dissociation can be problematic

Surface Plasmon Resonance

][])[]]([[][max

ABkABABAkdtABd

offon−−=

0 10 20 30 40 50 60 70 80 90-7

-6

-5

-4

-3

t1/2 (s)

KD (M

)

Agonist ▲ Weak Agonist▼ Antagonist

Stone et. al. Immunology 2009

Correlation of binding parameters with stimulatory capacity of interaction

Relatively few peptide-MHC/TCR

interactions measured by SPR

Furthermore, most SPR measurements are taken at 25°C,

while T cell activation occurs at

37°C

T cell activation correlates with KDand t½

Chervin, Stone et. al. J Immunol 2009

Antagonist threshold

1

10

100

0.1

0.01

dEV8

SIYAffi

nity

(KD, µ

M)

Agonist threshold

CD4/CD8 requirement threshold

Optimal activity plateau

dEV8

SIY

Null peptides

Wild-type TCR (2C) High Affinity TCR (2Cm33)

Null peptides

Different Similar

Spectrum of peptide similarity to wild type:

Antigen Specificity Fine Specificity

Antagonist threshold

1

10

100

0.1

0.01

dEV8

SIYAffi

nity

(KD, µ

M)

Agonist threshold

CD4/CD8 requirement threshold

Optimal activity plateau

dEV8

SIY

Null peptides

Wild-type TCR (2C) High Affinity TCR (2Cm33)

Null peptides

Different Similar

Spectrum of peptide similarity to wild type:

Antigen Specificity Fine Specificity

Different Similar

Spectrum of peptide similarity to wild type:

Antigen Specificity Fine Specificity

Stone et. al. Immunology 2009

Correlation of KD with T cell stimulation

Comparison of wild-type and high-affinity TCR

Kinetic Proofreading model

Short t½ would not be predicted to allow full zeta phosphorylation

Longer t½ would be predicted to be fully phosphorylated and cause T cell activation

Jones et. al., J Immunol, 2008

Kersh et. al., Science, 1998

0 10 20 30 40 50 60 70 80 90-7

-6

-5

-4

-3

t1/2 (s)

KD (M

)

Agonist ▲ Weak Agonist▼ Antagonist

Stone et. al. Immunology 2009

Longer t½ does not always result in better stimulation

Extended t½ results in less potent

stimulation in the case of the OT-1 TCR binding to

OVA(G4)/Kb

Optimal Dwell-time model

Kalergis et. al., Nat Immunol, 2001

Peptide-MHC/TCR interactions with intermediate t½ are the most potent

Based on the hypothesis of serial triggering Valitutti et. al., Nature, 1995

*Cells with engineered TCRs with t½ 100- to 1000-fold longer than wild-type are efficiently and sensitively triggered

Molecular Flexibility (∆Cp)

Rigid body adjustments

Alterations to reach transition state

Alterations to attain fully bound complex

Qi et. al., PNAS, 2006

Molecular Flexibility (∆Cp)

Krogsgaard et. al., Mol Cell, 2003

A

OOO KRTSTHG ln−=∆−∆=∆

aassockRTEa ln−=

ddisskRTEa ln−=

Measure ∆Cp by SPR or Isothermal Titration Calorimetry

)ln()(O

O

p

O

TO

O

p

O

T

O

T TTCTSTTTCHG

OO∆−∆−−∆+∆=∆

Boniface et. al., PNAS, 1999

Molecular Flexibility (∆Cp)

]exp[3

2/1

2

2/1CpBAtt DD ∆−=

Qi et. al., PNAS, 2006

Peptide-MHC Confinement Time

Aleksic, Dushek, et. al. Immunity 2010

Peptide-MHC Confinement Time

Aleksic, Dushek, et. al. Immunity 2010

)k(kkk

T1k

*

on

off

C

*

off

−

−

+==*

*-

-

Peptide-MHC Confinement Time

Aleksic, Dushek, et. al. Immunity 2010

correlation KD-koff

koff

KD

molecular flexibility

molecular flexibility+confinement time

confinement time

Outline

• T Cell Receptor Complex− Components, Assembly, and Organization

• Ligand binding measurements− Surface Plasmon Resonance− Peptide-MHC Multimers− In Situ 2D Binding Measurements

Casalegno-Garduño et. al. Cancer Immunol Immunother 2009

Peptide-MHC Multimers

IgG or Fcfusion (dimer)

Ultimer(hexamer)

Pentamer

X-Link (dimer-octamer)

Streptavidin-linked Tetramer

Cochran et. al. Trends Biochem Sci 2001

Stone et. al. Immunology 2009

KX KX KXKD

Complications:

Relatively high threshold of sensitivity (up to 10% receptors bound)

“Steady state” measurements do not represent a true equilibrium

Binding affected by multiple factors, including co-receptor

*this may be an advantage

Binding of peptide-MHC multimers to T cells

Chervin, Stone et. al. J Immunol 2009

TCR panel with various binding parameters

TCR t½ (s) KD (nM) S51 A 86 15

m33 46 16 Y26 A 50 17 N27βA 33 40 Y49 A 58 47

S51 A/Y48βA 11 540 Y48βA 2 2900 N30βA 3 8200 Y50 A 0.5 7000

2C 0.9 36,000

Chervin, Stone et. al. J Immunol 2009

Peptide-MHC tetramer t½,tet values

Chervin, Stone et. al. J Immunol 2009

Are KD,tet values valid?

Chervin, Stone et. al. J Immunol 2009

Are KD,tet values valid?

Wooldridge et. al. Immunology 2009

TCR KD: 30 80 >250µM

Co-receptor and peptide-MHC tetramers

The effect of CD8 co-

receptor binding on

MHC tetramer

dissociation

Wooldridge et. al. J Biol Chem 2005

Outline

• T Cell Receptor Complex− Components, Assembly, and Organization

• Ligand binding measurements− Surface Plasmon Resonance− Peptide-MHC Multimers− In Situ 2D Binding Measurements

Toletino et. al. Biophys J 2008

2D Rates of binding and diffusionFRAP: Fluorescence Recovery After Photobleaching

Wu et. al. Biophys J 2008

lipid only

CD2: CD58

CD16aGPI

-RbIgG

2D Rates of binding and diffusion

Wu et. al. Biophys J 2008

2D Rates of binding and diffusion

Rapid initial phase of recovery due to diffusion of unbound molecules

Slower second phase of recovery due to unbinding, diffusion, and re-binding for reaction-limited kinetics

1.4x1041.4x1061.20.1405In vitroIn situIn vitroIn situIn vitroIn situ

KD (µM) t½ (s-1) kon (M-1s-1)

Huppa et. al. Nature 2010

2D Rates of binding and diffusionIn Situ FRET: Fluorescence Resonance Energy Transfer

Huang et. al. Nature 2010

mrmlAcKa and koff Ackon = AcKa×koff

2D Rates of binding and diffusionIn Situ Cell Adhesion Assay – controlled contact time and area

Huang et. al. Nature 2010

Correlation of 2D Binding Parameters with T Cell

Recognition

2D off-rates up to 8300-fold

faster than 3D off-rates

Broader dynamic range of values seen

Reference List• Rudolph MG, Luz JG, Wilson IA. Structural and thermodynamic correlates of T cell signaling. Annu Rev Biophys Biomol

Struct. 2002;31:121-49.

• Cochran JR, Aivazian D, Cameron TO, Stern LJ. Receptor clustering and transmembrane signaling in T cells. Trends BiochemSci. 2001 May;26(5):304-10.

• Wucherpfennig KW, Gagnon E, Call MJ, Huseby ES, Call ME. Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling. Cold Spring Harb Perspect Biol. 2010 Apr 1;2(4):a005140.

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• Fernández-Miguel G, Alarcón B, Iglesias A, Bluethmann H, Alvarez-Mon M, Sanz E, de la Hera A. Multivalent structure of an alphabetaT cell receptor. Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1547-52.

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• Krogsgaard M, Prado N, Adams EJ, He XL, Chow DC, Wilson DB, Garcia KC, Davis MM. Evidence that structural rearrangements and/or flexibility during TCR binding can contribute to T cell activation. Mol Cell. 2003 Dec;12(6):1367-78.

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