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![Page 1: Structural and Molecular Investigations into Natural ... · Structural and Molecular Investigations into Natural killer T-cell (NKT) and CD1d glycolipid recognition Praveena Thirunavukkarasu](https://reader033.vdocuments.net/reader033/viewer/2022052313/5aede5977f8b9a572b8bf471/html5/thumbnails/1.jpg)
Structural and Molecular Investigations into Natural killer
T-cell (NKT) and CD1d glycolipid recognition
Praveena Thirunavukkarasu
Jamie Rossjohn lab
Monash University
Australia
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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
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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
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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
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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
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• 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
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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
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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
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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
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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
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� 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?
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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
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Experimental Flow
Clone and express protein
(Bacterial system)Protein CrystallizationProtein Purification
Diffraction patternElectron density map
Crystals
Protein Structure
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• 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
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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
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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
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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)
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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
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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’
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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
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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
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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
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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