Structural and Molecular Investigations into Natural killer

T-cell (NKT) and CD1d glycolipid recognition

Praveena Thirunavukkarasu

Jamie Rossjohn lab

Monash University

Australia

Human Immune System

Innate Immunity Adaptive Immunity

Pathogens

• Bridge the gap between innate and adaptive immunity

• Function as `Innate-adaptive hybrids’

• Possess immunomodulatory potential

From Dranoff G. , Nature Review Cancer, 2004

Tcells

αβ TCR

NKcells

NK1.1NK1.1αβ TCR

NKTcells

Natural Killer T (NKT) cells

• Share properties of both conventional T cells and Natural Killer

(NK) cells

• Express a T cell receptor (TCR) that allows them to recognize

antigens

• Constitute ~ 0.1% of all peripheral blood T cells

cells

NKT cells recognize lipid antigens

Non Vα24

NKT

cells

TCRTNF-α

IL-4

IFN-γIL13

TGF-β

GMCSF

IL-17

IL-21Cytokines

Non Vα24

CD8+

T cells

TCR Non Vα24

CD4+

T cells

TCR Non Vα24

NKT

cells

TCR

CD1d

α β

Human APC

β2M

CD1dCD1d

Non Vα24

Human APC

α β

β2M

MHC - I

CD1d

Human APC

α β

β2M

MHC - II

CD1d

Human APC

α β

β2M

CD1d

Peptides Lipids

Vα Jα Cα

CDR1α CDR2α CDR3α

Vβ

α-chain

β-chain

T cell receptor (TCR)

Cα Cβ

α βDVβ Cβ

CDR1β CDR2β CDR3β

Jβ

CDR1α

α β

TCR

CDR2α CDR3α CDR1β CDR2β CDR3βCDR2α CDR1αCDR3α

CDR3β

CDR2β

CDR1β

Vα

Vβ

α βD

• Non-polymorphic antigen presenting molecule

• Heavy chain non-covalently associated with β2m

• Heavy chain is composed of three domains α1, α2 and α3

A’

F’

α1

α2

Structure of CD1d

α1• Heavy chain is composed of three domains α1, α2 and α3

• Antigen-binding cavity is formed from α1 and α2 domains

• The cleft is hydrophobic and has two pockets namely A’ and F’

α2

α3

β2mA’

F’

α2

Why do we care about NKT cells?

NKT cells

°°

°°

°°

°°°

°°

°°°°

°

°°

°°°°

IFN-γIL-13

IL-21IL-4IL-17

GMCSFTNF-α

NK cell

Direct activation

NKT cell Activation

Direct activation Indirect activation

Activation

NKT cell NK cell

AllergyInfection CancerAutoimmunity Microbial immunity

Killing

Killing

CD1d

Non Vα24α β

β2M

hCD1d

TCR

Tumor cell

Killing CD1d

Non Vα24

Human

APC

α β

β2M

hCD1d

TCR

Tumor cell

NKT cell

Types of NKT cells

Non Vα24

Type I

NKT

TCR

Vα24Jα18 Vβ11

Non Vα24

Type II

NKT

α β

TCR

Vα3.2Jα9 Vβ8

CD1d �2M

Human APC

α β

β2M

hCD1d

Vα24Jα18 Vβ11

CD1d �2M

Human APC

α β

β2M

hCD1d

Vα3.2Jα9 Vβ8

α-Galactosylceramide (α-GalCer)

Lipid antigen - α-Galactosylceramide

• Prototypic type I NKT cell antigen

• Marine sponge-derived glycolipid (non-mammalian, bacterial)

• Lipid portion interacts with the hydrophobic pocket of CD1d

• Carbohydrate head group interacts with TCR

• Currently in human phase I/II trials as anti-cancer agent

Vα

Vβ

Type I

Vα

Type II

Type I and Type II NKT TCRs docking modes

αβ TCR αβ TCR

α-GalCer

Vβ

Sulfatide

Borg et al., Nature, 2007 Patel et al., Nature Immun., 2012

CD1d

β2MCD1d

β2M

� TCRs dock on to peptide/MHC or lipid/CD1d

in a conserved orientation

� key aminoacids encoded by TCR and CD1d

What does docking orientation signify?

Type I Type II

Are there any other CD1d-restricted α-Galcer reactive

Human APCHuman APC

� key aminoacids encoded by TCR and CD1d

have been selected and maintained through

evolutionhCD1d hCD1d

β2Mβ2M

Are there any other CD1d-restricted α-Galcer reactive

NKT subsets in humans and do they dock in a conserved

manner?

New subset of NKT cells exist in humans

Donor 1

5.21

20.7

1.61

16.7

0.42

8.3

3.25

8.89Donor 2

hC

D1

d-a

Ga

lCe

r te

tra

me

r

CD1d

α−GalCer

Streptavidin

PE

NKTcells

FACS

Single cell sorting

Single cell sequencing

Clones identified

9B19B29B3

9B69B7

9B10

9B12

Vα24 Vβ11

hC

D1

d

Collaboration with University of Melbourne

Clones identified

Experimental Flow

Clone and express protein

(Bacterial system)Protein CrystallizationProtein Purification

Diffraction patternElectron density map

Crystals

Protein Structure

• Cloned into pET-30 vector

• Expressed in BL21 E.coli cells

• Inclusion body preparations were performed

Variable

DomainConstant

domainN-terminal C-terminal

Expression and Purification of TCR

Two injections of α and β

chain

Two injections of α and β

chain

Dialysed 3 daysDialysed 3 days

Refolding

Purification

Anion exchange chromatography (Hitrap Q)

Size exclusion chromatography

Hydrophobic interaction chromatography (HIC)

Ion exchange chromatography (DEAE)

Non-Reducing Reducing

αβ

Reducing

Heavy chain

32kDa

β2M

12kDa

Expression and purification of Human CD1d

� Cloned into a dual promoter baculovirus transfer vector pBacp10pH.

� Expressed in Hi5 insect cells.

Lipid loading

hCD1d

hCD1d

β2M

hCD1d

β2M

Endogenous lipid α-Galcer

Displacement

hCD1d + α-Galcer+ TCR

TCR

hCD1d + α-Galcer

Size exclusion chromatography

S200 16/60

9B2 TCR-hCD1d/α-Galcer co-complexation

� A Shift in peak of 5ml indicated complex formation

ReducingNon-Reducing

TCR

β2m

CD1dβα

hCD1d

hCD1d + α-Galcer

hCD1d endo + TCR

Structure of 9B2 ternary complex

Docking angle ~110°

Novel docking mode

α - chain

β - chain

α - Galcer

CD1d

20% PEG 8000

0.1M CHES pH 9.5

α-GalCer (2Fo-Fc electron

density map @ 0.8σ level)

CD1d

β2M α-GalCer (Fo-Fc electron

density map)

CαCβ

Vβ

Cα

Cβ

Vα

Vβ

Cα Cβ

Vα

Vβ

CαCβ

Vα

Vβ

Comparison of docking modes of different types of NKT TCRs

β2m

Vα

Vβ

hCD1d9B2Type II Type I

β2m

hCD1d

β2m

hCD1d

β2m

hCD1d

Borg et al., 2007 Nature Patel et al., 2012 Nat.Immunology

Interactions of 9B2 TCR with α-Galcer

CDR1αCDR1β

• Dominated by CDR3β loop

• Q99 interacts with O6 of galactose moiety by

Van der waals interaction.

• Dominated by CDR1α and CDR3α loops.

• G96, F29 and S30 are H-bonded to O2, O4 and O3

respectively.

Type I9B2 TCR

α1

α2

A’F’

CDR1α

CDR2α

CDR3α

2’3’

4’

CDR1βCDR2β

CDR3β

α1

α2

A’

F’

6’6’

19.6% 14.7%

12.1%5%

32.3%

6.8%

15.5%

9.5% 13.5%

3.5%

1.5%

39.5%

Buried surface area9B2 TCR Type I TCR

9B2 TCR-CD1d interactions

CD1d/α-Chain

Dominated by α-chain

α1

α2

A’F’

CDR3α

CDR1α CDR2α

CD1d/β-Chain

A’ F’

CDR2β

CDR1β

CDR3β

α2

α1

Affinity measurements of 9B2 TCR with CD1d-α-Galcer

Bin

din

g (

RU

)B

ind

ing

(R

U)

α-GalCer

KD: 4.0 µM

KD: 190 nM

‘Unloaded’

NKT15

(Type I) 66

’

4

’3

’2

’

Q99β

Surface plasmon resonance (SPR)

Bin

din

g (

RU

)

9B2 TCR

� Q99A TCR mutant showed 2-fold reduction in affinity compared with wild type

Collaboration with University of Melbourne

Summary

• A new subset of CD1d-restricted NKT TCRs were identified in humans and termed as

‘Atypical NKT cells’

• The ternary structure of 9B2 TCR revealed a novel docking mode (orthogonal) in clear

contrast to Type I but comparable with Type II TCR

• 6’-OH of galactose moiety interacted merely with Q99 residue of TCRβ chain

• SPR studies showed the affinity of interaction of 9B2 TCR (wild type) with hCD1d- α-

Galcer is 4.0μM and 2-fold reduction in affinity for Q99 mutant

• Diverse TCR repertoire broadens the spectrum of glycolipids recognised and thus

leading to stimulation of NKT cells

University of Melbourne

Dale Godfrey

Adam Uldrich

Daniel Pellicci

Monash University

Jamie Rossjohn

Jérôme Le Nours

Onisha Patel

Maria Sandoval

Acknowledgements

Maria Sandoval

Srinivasan Sundararaj