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ProImmune Pentamer Day
Pro5® MHC Pentamers and their
Applications
Dr. Helen Barnby-Porritt
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Overview
• Antigen-presentation / MHC function
• Pro5® MHC Class I Pentamers
• Primary Applications
– Flow cytometry
– Antigen-specific cell separation
– Intracellular Cytokine Staining
• CD1d tetramers
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MHC Molecules Enable T Cells to Recognize and Eradicate
Foreign Material in Tissues
MHC molecules present
intracellular material to CD8+ T
cells
APC
peptides from virus
particles, genes causing
cancer, etc.
MHC class I +
peptide
CD8+ T cells are activated by
foreign, unrecognized peptides
TCR
co-stimulators
Cytotoxic immune
response
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CD8+
T cell
• Proliferation
• Cytokine production
• Cytotoxic effector proteins
(Granzyme B, Perforin)
Why use Pro5® MHC Pentamers?
• Fluorescently-labeled soluble proteins that mimic
surface MHC-peptide complexes
• Bind specifically to the TCR
• Sensitive detection of T cells reactive to a single MHCpeptide complex
• Flexible-labeling technology
• Most commonly cited commercial MHC multimer
technology
• Consistent and reproducible results
• Rapid delivery (stock and custom synthesis)
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Pro5® MHC Pentamers
5 MHC-peptide complexes
Coiled-coil domain
5 Fluorescent tags
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Primary Applications of Pro5® MHC Pentamers
 Flow cytometry
 Antigen-specific T cell isolation
 Intracellular Cytokine Staining
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Flow Cytometry
• Frequency determination of rare, antigen-specific
T cell responses
• Freshly isolated or frozen cells and T cell lines
• Combine with antibody staining for activation
markers or intracellular proteins such as cytokines
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Labeled Pentamer Staining Protocol
+
1 x 106 cells in 50ul
per tube
+
1 test MHC
Pentamer
(e.g. R-PE, APC
or biotin)
10 minutes
room temp.
then wash
anti-CD8 mAb
(e.g. FITC)
Fluorescentlabeled
Streptavidin
Pentamer
20 minutes
on ice
2 x washes
CD8
Analyze by flow cytometry
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Unlabeled Pentamer Staining Protocol
+
+
10 minutes
room temp.
then wash
1 x 106 cells in 50ul
per tube
1 test unlabeled
MHC Pentamer
Pro5 Fluorotag
(e.g. R-PE)
anti-CD8 mAb
(e.g. FITC)
Pentamer
20 minutes
on ice
2 x washes
CD8
Analyze by flow cytometry
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Cells Stained for Flow Cytometry
CD8
T cell receptor
T cell
R-PE-labeled Pentamer
anti-CD8-FITC
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Flow Cytometry - Sample Intake
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Flow Cytometry - Optical Design
PMT 5
PMT 4
Sample
PMT 3
Flow cell
Dichroic
Filters
Scatter
Sensor
PMT 2
PMT 1
Bandpass
Filters
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Laser
Flow Cytometry Data
SSC-H
Pentamer R-PE
CD8+ Pentamer+
(Antigen-specific)
population of interest
FSC-H
CD8 FITC
• Lymphocyte population is determined and gated upon
• Plot of CD8 vs. Pentamer drawn based on this gate
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Staining on Human Influenza-Specific Peripheral Blood Cells
A*02:01 / GILGFVFTL
(FluMP)
Irrelevant control
B*08:01 / RAKFKQLL
(EBV BZLF-1)
0.0%
0.0%
Pentamer
1.4%
CD8
Human PBMCs
15,000 live events
10ul Pentamer
CD8 clone LT8
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0.0%
A*02:01 Negative Control Pentamer
A*02:01 / NLVPMVATV
(CMV pp65)
A*02:01 / Negative Pentamer
0.00%
0.05%
A2 Neg Pentamer
A2/CMV Pentamer
0.04%
CD8 FITC
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CD8 FITC
1.06%
A*02:01 / ELAGIGILTV (Melan-A/MART-1)
A*02:01 / ELAGIGILTV
Stronen et al. (2009). Scand J. Immunol. 69: 319-328
CD8 APC
A*02:01 / ELAGIGILTV staining
immediately after isolation)
A*02:01 / ELAGIGILTV staining
after 12 days of co-culture with ELAGIGILTV
peptide-pulsed DCs
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Case Study: Validation of Trovax® epitopes using ProVE®
Pentamers
Harrop et al (2008) Cancer Immunol. Immunother. 57: 977-86
• Modified Vaccinia Ankara (MVA) expressing tumour antigen, 5T4
• Administration by intramuscular injection
• Induces humoral and cellular immune response to 5T4
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Stage IV Colorectal Cancer – TroVax® + Chemotherapy
Phase II Clinical Trial Design
• Cycle of TroVax® injections
• Accompanied by chemotherapy
• Last TroVax® injections following end of chemotherapy
Immune monitoring & 5T4 epitope discovery
• Good 5T4 responses detected, e.g. by ELISPOT
• Analysis of immune responses with PEPscreen®
• MHC peptide binding assay
• Validation with ProVE® MHC Pentamers
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Staining on Cancer-Specific PBMCs Following Trovax®
Vaccine Delivery
Harrop et al (2008) Cancer Immunol. Immunother. 57: 977-86
Irrelevant Pentamer
HLA-A2 5T4 Pentamer
0.18%
0.22%
5T4 Pentamer
0.00%
Baseline
CD8+
X+2wk
X+14wk
ELISPOT
Analysis
Baseline
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X+2wk
X+14wk
Staining on Murine Cells
Splenocytes from Ovalbumin peptide-immunized mice
H-2Kb/SIINFEKL
(Ovalbumin)
0.39%
Pentamer
0.03%
CD8
Immunized Murine splenocytes
50,000 live events
10ul Pentamer
CD8 clone KT15
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Negative control
H-2Kb/SSIEFARL
(HSV-1 gp B)
0.01%
0.00%
Staining with multiple Pentamers
• Precious cell samples, e.g. derived from childhood diseases
• Many different specificities to test
• Library of MHC Pentamers
−
Use 3 different pre-labeled Pentamers
−
Screen with up to 6 unlabeled Pentamers
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0.00%
0.04%
0.02%
CD8 FITC
0.00%
0.02%
A2/CMV Bio + SA-PerCP
B7/CMV Pentamer APC
A2/flu Pentamer R-PE
CD8 FITC
0.01%
B7/CMV Pentamer APC
0.03%
0.27%
0.00%
0.02%
A2/CMV Bio + SA-PerCP
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0.25%
CD8 FITC
A2/flu Pentamer R-PE
0.01%
A2/CMV Bio + SA-PerCP
A2/flu Pentamer R-PE
Triple Staining on Human PBMCs
0.04%
0.00%
0.27%
B7/CMV Pentamer APC
Rapid Screening with a Pool of Unlabeled Pentamers
Pentamer
(a) Pool of 6 unlabeled
Pentamers
0.11%
0.19%
(b)
A*01:01/Flu
(CTELKLSDY)
0.07%
0.01%
A*02:01/Flu
(GILGFVFTL)
0.02%
0.06%
A*24:02/CMV-1
(VYALPLKML)
0.01%
0.00%
CD8 FITC
A*24:02/CMV-2
(QYDPVAALF)
Human PBMCs
100,000 live events
2ul Pentamer
8ul R-PE Fluorotag
CD8 clone LT8
•
0.05%
0.00%
B*07:02/CMV
(TPRVTGGGAM)
0.06%
0.01%
B*08:01/EBV
(FLRGRAYGL)
0.02%
0.11%
Pentamer pools that yield positive Pentamer staining can be dissected to resolve the
antigen-specific populations
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Phenotypic Characterization
Stain for additional markers in conjunction with Pentamer staining to
obtain as much information as possible from one batch of cells
• Detailed Phenotype of the cell
• Memory / activation status
• Cytokine / Chemokine receptor expression
• Homing characteristics
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Phenotypic Characterization
ProVE® Pentamer
versus CD8
(naïve T cell marker)
(memory T cell marker)
0.95%
Pentamer
CD8
0.11%
0.20%
Pentamer
1.19%
ProVE® Pentamer
versus CD45RO
Pentamer
0.04%
ProVE® Pentamer
versus CD45RA
CD45RA APC
Vaccinated Human PBMCs
500,000 live events
0.5ug ProVE™ Pentamer
CD8 clone RPA-T8
CD45RA clone HI100
CD45RO clone UCHL-1
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CD45RO APC
1.00%
Applications of Pro5® MHC Pentamers
 Flow cytometry
 Antigen-specific T cell isolation
 Intracellular Cytokine Staining
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Antigen-Specific T Cell Isolation
(1) Fluorescence-Activated Cell Sorting (FACS)
(2) Magnetic Bead Separation
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Fluorescence-Activated Cell Sorting (FACS)
• Fluorescently labeled MHC Pentamers bind antigen-specific T
cells
• FACS instrument used to sort cells according to fluorescent
properties
• Used to isolate a pure population of live T cells (highly specific)
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Principles of FACS
Laser
analysis point
charge
delay
= 10 l
break-off point
Charged deflection plate
l
Population 2
Population 1
Waste
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FACS data
A*02:01/GLCTLVAML (EBV) Pentamer-specific cells sorted using a BD
FACS Aria
Pre-sort
Post-sort
0.4%
Pentamer
86.4%
CD8
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Magnetic Bead Separation
• Cells stained with Pentamer
• Cells incubated with magnetic beads that recognize Pentamer
• Antigen-specific cells isolated by magnetic separation
• Two methods :
− Column based
− Tube based
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Magnetic Bead Separation (Column-based)
Wash
T cell
Place on column.
Flow through
collected as
negative fraction
R-PE labeled Pentamers are bound to T
cells then detected by anti-R-PE
microbeads
Enriched cells labeled with
anti-CD8 mAb (e.g. FITC)
for analysis
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Column removed from
magnetic field and retained
target cells flushed out as
positively selected cells
Column-based Magnetic Bead Enrichment
MACS® anti-R-PE microbeads and single-step purification
Pre-enrichment
Post-enrichment
0.1%
0.6%
6.6%
51.4%
B*07:02/TPRVTGGGAM
(HCMV pp65)
0.2%
0.8%
4.3%
38.9%
Pentamer
B*08:01/RAKFKQLL
(EBV BZLF-1)
CD8
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Column-based Magnetic Bead Separation
A*02:01/GLCTLVAML (EBV BMLF-1) Pentamer-specific cells separated with
MACS® anti-R-PE microbeads in two steps
Pre-sort
0.30%
8.63%
Pentamer
0.08%
Positive fraction
after 1 round
CD8
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38.85%
Positive fraction
after 2 rounds
14.7%
79.08%
Magnetic Bead Separation (Tube-based)
Wash
T cell
Biotin-labeled Pentamers are bound to T
cells then detected by Streptavidin
microbeads
Enriched cells labeled with
anti-CD8 mAb (e.g. FITC)
for analysis
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Place on magnet.
Target cells attracted
to side of tube.
Supernatant collected
as negative fraction.
Tube removed from
magnetic field and
remaining positivelyselected cells washed.
Tube-based Magnetic Bead Separation
B*08:01/RAKFKQLL (EBV BZLF-1) Pentamer-specific cells separated with
LodeStars™ 2.7 Streptavidin beads (Varian Inc.)
Pre-depletion
Post-depletion
B8/EBV Pentamer
1.53%
CD8
97.4% of antigen-specific cells removed
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0.04%
Column-based Magnetic Bead Enrichment
in a life-saving treatment for PTLD
Uhlin, M. et al. (2009). A novel haplo-identical adoptive CTL therapy as a treatment for
EBV-associated lymphoma after stem cell transplantation. Cancer Immunol. Immunother.
59(3):473
The Patient
• 18 year old woman with acute leukaemia.
• Limited or no response to chemo and other drugs.
• Received cord blood transplant (similar to bone marrow transplant).
• Following transplant patient developed EBV linked Post-transplant
lymphoproliferative disorder (PTLD): this is a disease caused by a common
virus infection (EBV) and can occur after transplantation due to immune
suppressants that have to be given after transplantation.
• PTLD is like cancer and if not treated successfully is lethal like cancer.
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Column-based Magnetic Bead Enrichment
in a life-saving treatment for PTLD
• Blood donated by mother; EBV-specific cells
were stained with A*02:01 / GLCTLVAML or
A*02:01 / CLGGLLTMV Pro5® MHC Pentamer
• 600,000 cells were isolated with CliniMACS® and
adoptively transferred to the patient from the
mother
• 189 days later, EBV lymphoma regression
confirmed by CT scan
Please note that ProImmune Products are sold for Research
Use Only (RUO)
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The Result
• Donor-derived EBV-specific T cells expanded in vivo
• Complete elimination of EBV virus from the patient
• Patient currently alive 3 years after treatment and cured of PTLD
Before treatment
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After treatment
The Result
• Donor-derived EBV-specific T cells expanded in vivo
• Complete elimination of EBV virus from the patient
• Patient currently alive 3 years after treatment and cured of PTLD
Before treatment
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After treatment
Applications of Pro5® MHC Pentamers
 Flow cytometry
 Antigen-specific T cell isolation
 Intracellular Cytokine Staining
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MHC Pentamer &
Intracellular Cytokine Staining (ICS)
• Determine the frequency and effector function of antigen-specific
T cells simultaneously
• Co-staining of CD8+ T cells with MHC multimers and antibodies
against intracellular cytokines
• ICS relies upon stimulation of T cells in presence of an inhibitor of
protein transport, thus retaining cytokines inside the cell
• ICS staining on customer samples available as service from
ProImmune
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Intracellular Cytokine Staining Protocol (1)
Stain cells with Pentamer
Stimulate 1 hour with specific peptide / alternative
Add Inhibitor of protein transport, e.g. Brefeldin A
Culture 37°C for 5-24 hours
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Intracellular Cytokine Staining Protocol (2)
Stain cells with any additional
cell surface markers (optional)
Fix with 4% Paraformaldehyde
Permeabilize cells
Stain with antibodies to
Intracellular markers, e.g. antiIFNg
Fix with formaldehyde for analysis
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ICS / Pentamer Staining Data
Activation Controls
Gated on Live, CD8+ cells
B*08:01/RAKFKQLL
(EBV BZLF-1)
Stimulation with PMA
Pentamer R-PE
1.79%
2.01%
20.99%
IFNg - FITC
30,000 live Human PBMCs
10ul Pentamer
CD8 clone HIT8a
IFNg clone 4S.B3
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B*08:01/RAKFKQLL
(EBV BZLF-1)
No stimulation
3.39%
0.04%
0.01%
ICS / Pentamer Staining Data
Stimulation with peptide Gated on Live, CD8+ cells
B*08:01/RAK Pentamer
(RAKFKQLL peptide)
Pentamer R-PE
1.48%
0.52%
0.05%
IFNg - FITC
30,000 live Human PBMCs
10ul Pentamer
CD8 clone HIT8a
IFNg clone 4S.B3
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Negative control
A*02:01/GLC Pentamer
(RAKFKQLL peptide)
0.06%
0.01%
0.58%
Importance of Staining with Pentamer Prior to Activation
B*08:01/RAK Pentamer
(RAKFKQLL peptide)
Staining pre-activation
Pentamer R-PE
1.48%
0.52%
0.05%
IFNg - FITC
30,000 live Human PBMCs
10ul Pentamer
CD8 clone HIT8a
IFNg clone 4S.B3
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B*08:01/RAK Pentamer
(RAKFKQLL peptide)
Staining post-activation
1.10%
0.28%
0.46%
MHC Pentamer &
Intracellular Cytokine Staining (ICS)
Tellam et al. (2007) J. Exp. Med 204: 525-532
Protein translation affects
the molecular dynamics of
antigen presentation
B*08:01 / FLRGRAYGL
Pentamer - R-PE
EBNA1
EBNA1-DGA
35.1%
50.3%
IFNg - FITC
•
Comparison of immune responses to EBV encoded nuclear antigen with (EBNA1)
and without (EBNA-DGA) internal Gly-Ala repeat sequence
•
EBNA-DGA has more rapid protein translation efficiency
•
Increased frequency of EBNA-1 specific and functional T cells were observed in
the EBNA-DGA version
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Conclusions
• Most consistent MHC Multimer Technology
• Pentamer staining is highly flexible
• Pentamers can be used in combination with other cell surface and
intracellular markers
• Pentamers can be used to isolate antigen-specific cell populations
• Results of Pentamer staining can be combined with other
techniques, (e.g. ELISPOT) to gain a full picture of an antigenspecific response
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CD1d Tetramers - Introduction
• Natural killer T (NKT) cells implicated in regulation of immune
responses associated with a broad range of diseases
• Unique lymphocyte population that co-express NK cell markers and
a semi-invariant T cell receptor
• When stimulated with CD1d-restricted glycolipid antigen, NKT cells
produce Th1-type and/or Th2-type cytokines
• CD1d are non-classical MHC molecules, characterized as nonpolymorphic and conserved among species
• Possess binding pockets capable of presenting glycolipids and
phospholipids to Natural Killer T (NKT) cells
• Best characterized CD1d ligand is α-Galactosyl Ceramide* (α-GalCer)
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CD1d Tetramers
• CD1d-lipid complexes bind to T cell receptors of NKT cells
• Allow identification and enumeration of NKT cells by flow cytometry
• Excellent brightness and experimental reproducibility
• Several options regarding fluorescence and loading
• Only commercial source worldwide for fluorescently labeled human
and mouse CD1d tetramers
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CD1d Tetramers – Available Options
• Loading options
− Pre-loaded with alpha-Galactosyl Ceramide* for convenience
− Negative Control (mock-loaded with carrier only)
− Also supplied empty for loading with the ligand of your choice
• Mouse or human CD1d
• R-PE or APC labeled
* The alpha-GalCer molecule and derivatives thereof are covered by US Patent No.5,936,076, which is owned by Kirin
Pharma. The alpha-GalCer molecule is licensed to Funakoshi Co. Ltd. for research use worldwide.
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Primary Applications of CD1d Tetramers
 Flow cytometry
 Antigen-specific CD1d-restricted NKT cell isolation
 Intracellular Cytokine Staining
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Staining on Human Peripheral Blood Cells
Non-specific staining was eliminated by gating on CD19 negative cells before plotting CD1d tetramer vs CD3.
Human CD1d APC
a-Gal Cer-loaded
CD1d tetramer
CD3
Human PBMCs
50,000 live events
0.5ul Tetramer
CD3 clone UCHT1
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Negative control
tetramer
Staining on Murine Cells
Splenocytes from a naïve B6 mouse depleted of B cells. Data shown is gated on live, CD3 positive cells.
Empty tetramer
a-Gal Cer loaded
Murine CD1d R-PE
0.11%
CD4-Alexa 488
Data courtesy of Markus Skold and Sam Behar, Harvard Medical School
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2.5%
ProImmune’s Human CD1d tetramer can detect NKT cells
from non-human primates
R-PE labeled human CD1d tetramers used to stain PBMCs from rhesus macaque monkeys.
PBMCs were pre-stimulated with RGI-2001 (liposomal α-GalCer, a ligand for NKT cells) for 3 days.
Data shown is gated on live, CD3 positive cells.
Stimulated
97.1%
Untreated
2.93%
Human CD1d R-PE
Data courtesy of Omar Duramad, RegImmune Inc.
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99.7%
0.31%
CD1d Tetramers - Conclusions
• Consistent, reliable technology
• Only available from ProImmune
• Flexible labeling options
• CD1d tetramers can be used in combination with other cell surface
and intracellular markers
• CD1d tetramers can be used to isolate antigen-specific CD1drestricted NKT cell populations
• Results of CD1d staining can be combined with other
techniques, (e.g. ELISPOT) to gain a full picture of an NKT cell
specific response
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