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Tetramer Staining Guide
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Content
Reagents for antigen-specific T cell detection 6
Detection of antigen-specific T cells by MHC Tetramers 7
Preparation of MHC class I Tetramer reagents 8
Greater specificity: The patented α3 mutation 9
HLA-A*02:01 CMV pp65 Tetramer 10
HLA-A*24:02 EBV BRLF1 Tetramer 10
MHC Tetramer staining method 12
Procedure for whole blood samples 12
Procedure for Peripheral Blood Mononuclear Cells (PBMC) samples 13
Procedure for Mouse spleen samples 13
Induction of CTL from peripheral blood using the Mixed Lymphocyte-PeptideCulture method 14
Induction and detection of cancer antigen specific CTL 15
Identification of virus-specific T cells using MHC Tetramers 16
EBV 16
CMV 17
Influenza M1 20
HTLV-1 20
Measurement methods for antigen-specific CTL using MBL Kits 22
IMMUNOCYTO IFN-γ ELISPOT Kit 22
IMMUNOCYTO CD107a Detection Kit 23
IMMUNOCYTO Cytotoxity Detection Kit 24
MHC Tetramer related products 26
Clear Back blocking reagent for tetramer staining 26
Peptides for antigen-specific CTL induction 26
Induction method for antigen-specific murine CTL 27
Induction of antigen-specific murine CTL by in vitro stimulation (influenza virus) 27
Staining with Mouse MHC class I Tetramer 30
Induction of murine WT1 Tetramer-specific CTL 34
H-2Kb OVA Tetramer 36
H-2Kb OVA Tetramer 36
Effect of CD8 antibody clones on H-2Kb OVA Tetramer staining: Cells from peptide-immunized mice 38
Staining using cancer-related MHC Tetramers 41
Mouse MHC class II Tetramer 42
I-Ab OVA323-339 Tetramer-PE 43
I-Ab ESAT-61-20 Tetramer-PE 44
Reagents for studying OVA specificity in the context of I-Ad murine class II molecules 47
CD1d Tetramer 48
Dissolving and loading α-GalCer on CD1d Tetramer 48
Detection of human NKT cells with CD1d Tetramer 49
Detection of murine NKT cells with CD1d Tetramer 49
iTAgTM MHC Tetramer and MBL 50
Tetramer References 51
Support 55
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Th2
Th1
Treg
NKTTarget cells
Cell death
MDSC
B cell
CD4+ T
Dendritic cells CD8+T
IL-2IFN-γTNF-α
IL-4IL-5IL-10IL-13
IL-17
IL-4IL-12
Antibodies
IL-10TGF-β
IL-6+TGF-β
TGF-β
S100A8/A9 PerforinGranzymeIFN-γTNF-α
Pathogens
TLR
CTLMHC Class II
CD1d
MHC Class I
Th17MHC
Peptide
Fluorochrome
TCR CTL
MHC Tetramer
Streptavidinβ2m
MHC Class I
Peptide
Fluorochrome
CTLβ2m
Reagents for antigen-specific T cell detection Detection of antigen-specific T cells by MHC Tetramers
T cell receptors (TCRs) expressed on T cells specifically recognize and bind to
complexes of major histocompatibility complex (MHC) molecules and peptide
fragments. CD8-positive T cells, also called cytotoxic T lymphocytes (CTL),
recognize complexes of MHC class I molecules and peptide. CTL directly kill
target cells including virus-infected and cancer cells. CD4-positive T cells recognize
complexes of MHC class II molecules and peptide fragments. CD4-positive T cells
are mainly helper T (Th) cells and are classified into Th1 and Th2 cells. Th1 cells
affect CTL and macrophages, which are involved in cell-mediated immunity. Th2
cells act on dendritic cells and B cells to activate humoral immunity. In 1996,
Altman et al., successfully detected antigen-specific T cells at a single cell level
by flow cytometry using MHC Tetramer reagents (Science 1996, 274: 94). MHC
Tetramers enable direct detection of antigen-specific T cells, which previously
had been analyzed indirectly based on cytokine production or cytotoxic
activity. MHC Tetramer reagents enable detailed analysis of function and
phenotype of antigen-specific T cells. Since their discovery, MHC Tetramers
have become accepted as essential tools for measuring T cell responses in basic
research, clinical development, and immune monitoring in the fields of infectious
disease, cancer vaccine therapy, cellular immunotherapy, transplantation tolerance,
and autoimmune disease.
TCRs recognize and bind to complexes composed of MHC molecules and specific
peptides expressed on the surface of antigen-presenting cells. While it was
believed that that antigen-specific T cells could be detected using soluble MHC/
peptide complexes, monomeric MHC/peptide complexes proved to be impractical
due to their instability and low affinity to the TCR. To overcome this difficulty, MHC/
peptide monomers are biotinylated and tetramerized with streptavidin to maintain
stable binding to multiple TCR, enabling MHC/peptide Tetramers to be used as
detection tools. MHC Tetramers are labeled with fluorescent molecules including
phycoerythrin (PE) or allophycocyanin (APC) and thus allow detection of antigen-
specific T cells by flow cytometry or fluorescence microscopy.
Tetramerization increases the avidity of MHC/peptide complexes binding to TCR, allowing for detection of antigen-specific T cells.
Monomeric MHC/peptide complexes cannot efficiently detect antigen-specific T cells due to low affinity binding to the TCR.
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Fluorescent labeled Streptavidin
MHC Tetramer
MHC Class I
Peptide
β2mMonomer d-Biotin
MHC class I heavy chain, β2m, and a peptide are folded to generate a MHC class I/peptide complex (monomer).
The MHC class I heavy chain is biot inylated and the monomers are purified by column chromatography.
Folding Biotinylation
Tetramerization 245
Preparation of MHC class I Tetramer reagents Greater specificity: The patented α3 mutation
MHC class I Tetramer reagents are generated primarily using the method
developed by Altman et al., who demonstrated that Tetramers of MHC class I/
peptide complexes could be used as probes for detection and quantitation of
antigen-specific CTL (Science 1996, 274: 94−96).
MHC class I heavy chain, β2m, and peptide are folded to generate a MHC class I/
peptide complex (monomer), a process that is monitored daily for several days,
depending on the particular peptide/MHC combination. The lysine residue in the
C-terminus of MHC class I heavy chain is biotinylated using the BirA enzyme,
and the monomers are purified by column purification. E. coli-expressed recombinant
MHC class I heavy chain and β2-microglobulin (β2m) are folded in the presence
of a peptide antigen to generate a soluble monomeric MHC class I/peptide complex
(monomer). The monomer is then biotinylated by the biotin ligase enzyme BirA
enzyme at a lysine residue present in the biotinylation sequence that has been
added to the C-terminus of MHC class I heavy chain of the complex. The biotinylated
monomer is purified by column chromatography. The purified biotinylated monomers
are tetramerized by mixing with a fluorescently-labeled streptavidin to make MHC
Tetramer reagents.
The human leukocyte antigen (HLA) system is the name of the major histocompatibility
complex in humans. CD8 molecules are known to assist binding of HLA to CTL
in vivo and thus HLA molecules have binding sites for CD8 molecules. Bodinier et
al. reported that introducing a mutation (A245V) in the HLA class I heavy chain α3
domain minimized unwanted binding to CD8 molecules and dramatically improved
specificity (Nat. Med. 2000, 6: 707). MBL has incorporated the patented
mutation of the α3 domain in their HLA class I Tetramers and is the only
manufacturer of MHC multimers with this technology to enhance specificity
for the antigen-specific T cell receptor.
French Application Number; FR9911133*1) Gao GF, et al., Nature 387: 630−634 (1997)*2) Bodinier M, et al., Nat. Med. 6: 707−710 (2000)
α3 domain of HLA-A2.1 *
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Figure 2
CD8-FITC
With α3 mutation Without α3 mutationNegative Tetramer
EBV BRLF1 TetramerT
etra
mer
-PE
Non-mutated tetramers can bind CTLs via CD8, independent of TCR, leading to detection of antigen-non-specific CTLs (false positives) and poor resolution of true positives.
Antigen-specific TCR can be detected selectively.
iTAg HLA class I Tetramer
Wild type MHC/peptide complex
Cell population containingHLA-A*02:01 CMVpp65 specific CTL
Cell population NOT containingHLA-A*02:01 CMVpp65 specific CTL
Cell population containingHLA-A*02:01 CMVpp65 specific CTL
Cell population NOT containingHLA-A*02:01 CMVpp65 specific CTL
Tet
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Tet
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Tet
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Tet
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HLA/peptide Complex
Irrelevant TCR
CD8
HLA/peptide Complex
Specific TCR
CD8
Conventional non-mutated Tetramer
α3-mutated Tetramer
Figure 1a
Figure 1b
CD8-FITC CD8-FITC
CD8-FITC CD8-FITC
Non-mutated tetramers can bind CTLs via CD8, independent of TCR, leading to detection of antigen-non-specific CTLs (false positives) and poor resolution of true positives.
Antigen-specific TCR can be detected selectively.
iTAg HLA class I Tetramer
Wild type MHC/peptide complex
Cell population containingHLA-A*02:01 CMVpp65 specific CTL
Cell population NOT containingHLA-A*02:01 CMVpp65 specific CTL
Cell population containingHLA-A*02:01 CMVpp65 specific CTL
Cell population NOT containingHLA-A*02:01 CMVpp65 specific CTL
Tet
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Tet
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E
Tet
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Tet
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HLA/peptide Complex
Irrelevant TCR
CD8
HLA/peptide Complex
Specific TCR
CD8
Conventional non-mutated Tetramer
α3-mutated Tetramer
Figure 1a
Figure 1b
CD8-FITC CD8-FITC
CD8-FITC CD8-FITC
Peripheral blood of HLA-A*02:01 donors
positive or negative for CMV was stained
with HLA-A*02:01 CMV pp65 Tetramer
reagent. A dramatic improvement in
specificity was observed in samples stained
with the MHC Tetramer containing the α3
mutation (Figure 1b) compared with those
stained with the conventional Tetramer
lacking the mutation (Figure 1a).
PBMC were separated from peripheral blood of HLA-A*24:02 positive healthy
subjects (Figure 2). EBV BRLF1-specific CTL were induced by the MLPC method
(see page 14) and stained with PE-labeled HLA-A*24:02 Negative (MBL code no.
TS-M007-1), or EBV BRLF1 Tetramers with (MBL code no. TS-M002-1) or without
the α3 mutation. Non-specific staining of CD8 positive cells was observed in the
sample stained with the conventional, but not the α3-mutated, Tetramer.
HLA-A*24:02 EBV BRLF1 Tetramer
This data was analyzed within the lymphocyte gate and 7-AAD negative gate.
Impact of the α3 mutation
HLA-A*02:01 CMV pp65 Tetramer
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Bloodcollection
HemolysisFixation
Pellet,resuspend
in PBS
Analysis byFlow Cytometry
Tetramer reactionIncubation
MHC Tetramer-PE 10 μLCD8-FITC 20 μL
200 μL whole blood
MHC Tetramer staining method
1 ) Collect blood by venipuncture into a blood collection tube containing an
appropriate anti-coagulant.
2 ) To each 12x75 mm test tube add 10 μL of MHC Tetramer and any additional
antibodies (e.g. anti-CD8).
3 ) Add 200 μL of whole blood into each test tube.
4 ) Vortex gently.
5 ) Incubate for 30 minutes at room temperature protected from light.
6 ) Lyse red blood cells using 2 mL of Lyse Reagent* supplemented with 50 μL
Fixative Reagent** per tube.
7 ) Vortex for 5 seconds immediately after the addition of the Lyse/Fixative solution.
8 ) Incubate for a minimum of 10 minutes at room temperature protected from light.
9 ) Centrifuge tubes at 150 x g for 5 minutes.
10) Aspirate or decant the supernatant.
11) Add 3 mL of PBS.
12) Centrifuge tubes at 150 x g for 5 minutes.
13) Aspirate or decant the supernatant.
14) Resuspend the pellet in 500 μL of PBS with 0.1% formaldehyde.
(12.5 μL Fixative Reagent**/1 mL PBS).
15) Store prepared samples at 4°C protected from light for a minimum of 1 hour
(maximum 24 hours) prior to analysis by flow cytometry.
If hemolysis is incomplete, a non-specific staining pattern caused by diffuse
reflection from erythrocytes may be observed. Inclusion of a CD45 antibody can
help identify the true lymphocyte population.
*Samples should be stored in a dark room at 4°C and analyzed within 24 hours.
■Procedure for whole blood samples
1 ) Prepare peripheral blood mononuclear cells (PBMC) according to established
procedures. Cells should be resuspended at a concentration of 5 x 106 cells/mL.
200 μL of sample is recommended for each Tetramer determination.
2 ) To each 12x75 mm test tube add 10 μL of MHC Tetramer and any additional
antibodies (e.g. anti-CD8).
3 ) Add 200 μL PBMC into each test tube (e.g. 1 x 106 cells per tube).
4 ) Vortex gently.
5 ) Incubate for 30 minutes at room temperature protected from light.
6 ) Add 3 mL of PBS.
7 ) Centrifuge tubes at 400 x g for 5 minutes.
8 ) Aspirate or decant the supernatant.
9 ) Resuspend the pellet in 500 μL of PBS with 0.5% formaldehyde.
(62.5 μL Fixative Reagent**/1 mL PBS).
10) Store prepared samples at 4°C protected from light for a minimum of 1 hour
(maximum 24 hours) prior to analysis by flow cytometry.
1 ) Collect lymph node, spleen or thymus and prepare a single-cell suspension
according to an established protocol. Cells should be resuspended at a
concentration of 5 x 106 cells/mL. 200 μL of sample is recommended for each
Tetramer determination.
2 ) To each 12x75 mm test tube add 10 μL of MHC Tetramer and any additional
antibodies (e.g. anti-CD8).
3 ) Add 200 μL cell suspension into each test tube (e.g. 1 x 106 cells per tube).
4 ) Vortex gently.
5 ) Incubate for 30 minutes at room temperature protected from light. If red
blood cell lysis is necessary, proceed to step 6-15 in the PROCEDURE FOR
WHOLE BLOOD section. If red blood cell lysis is not necessary, continue to
step 6 below.
6 ) Add 3 mL of PBS.
7 ) Centrifuge tubes at 400 x g for 5 minutes.
8 ) Aspirate or decant the supernatant.
9 ) Resuspend the pellet in 500 μL of PBS with 0.5% paraformaldehyde or formalin.
10) Store at 4°C protected from light for a minimum of 1 hour (maximum 24 hours)
prior to analysis by flow cytometry.
■Procedure for Peripheral Blood Mononuclear Cells (PBMC) samples
■Procedure for Mouse spleen samples
*VersaLyseTM, Beckman Coulter A09777 **IO Test®3 10X Concentrate, Beckman Coulter A07800
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PBMC were separated from peripheral blood of healthy subjects and cancer
antigen-specific CTL were induced by the 96-well MLPC method. Fourteen days
after peptide stimulation, the cells were stained with Tetramer as follows: First, an
aliquot of cells from each of 8 wells in a column of a 96-well plate were collected to
make 12 pooled samples. These samples were stained separately with Tetramer.
Pools yielding antigen-specific CTL were further deconvoluted by testing for
Tetramer binding to cells from individual wells that contributed to the positive pool.
Based on the number of positive wells, the frequency of antigen-specific CTL at
blood collection was calculated using the following equation.
(Number of Tetramer positive wells)
(Cell number/well) × (Total well number) × (CD8 positive cell rate in day 0)
Induction of CTL from peripheral blood usingthe Mixed Lymphocyte-Peptide Culture method Induction and detection of cancer antigen specific CTL
MHC Tetramers are used as detection reagents for viral or cancer antigen-specific
CTL. Because of their relative rarity compared with viral antigen-specific CTL,
cancer antigen-specific CTL can be difficult to detect immediately following
blood collection. Using the Mixed Lymphocyte-Peptide Cultures (MLPC) method,
Karanikas et al. found that increased specific CTL in peripheral blood resulted
from vaccine therapy in melanoma patients (J. Immunol, 2003, 171:4898). The
MLPC method expands antigen-specific CTL via culture of PBMC with a
stimulating peptide, thus allowing for the enumeration of low frequency CTL,
which are below detection in freshly isolated blood. Using the following modified
MLPC protocol, MBL has successfully induced cancer antigen-specific CTL from
peripheral blood of healthy subjects.
1) Collect blood by venipuncture into a blood collection tube containing heparin sodium. Turn the blood collection tubes over repeatedly to mix. If the blood cannot be treated immediately, allow to stand at room temperature.
2) After low speed centrifugation (-400 xg), aliquot and store the separated plasma at -30°C. Do not repeatedly freeze and thaw.
3) Add back an equal volume of wash medium to blood and isolate PBMC using a Ficoll gradient. Wash cells once.
4) Prepare culture medium: 5% autologous plasma/50 U/mL IL-2/50 μM 2-ME/antibiotics/RPMI1640.
5) Resuspend PBMC at 1-3x106 cells/ml, add 10 μg/mL of an antigen peptide, and seed 100 μL/well into a 96-well U-bottom plate.
6) After 48 hours, add 100 μL/well IL-2 containing medium. After that, using the color of the medium as a guide, change half of the medium about once in the first week and every 2-3 days in the second week. The optimal concentrations of peptide and IL-2 added may be different for each antigen, depending on solubility of the peptide, epitope prediction score, etc.
Note: Induction of viral antigen-specific CTL can often be achieved by the MLPC method using tubes. In order to induce cancer antigen-specific CTL, the 96-well plate MLPC method is recommended because the frequency of cancer antigen-specific CTL is low.
CD8
Tet
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Centrifugation RT3,000 rpm
10 min.
Heparinizedblood
Separationof plasma
Separationof PBMCs
Stimulationby Antigen
Virusantigen
Several hours – 15 days
Cancerantigen
12– 15 days
Tetramer staining
96 well plate
DAY0
Add equal volumeof serum free
RPMI1640
PBMCs 1-3 x 105 cells/well5% autologous plasma + antigen peptide
IL-2
AB
CD
EF
GH
Day 0
Column1 Column3 Column11
Column10
Row G
Row E
Row F
Column2
Column12
0.04 0.02
64.6 35.4
CD8
Tet
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0.02 0.36
64.2 35.4
0.06 2.74
61.6 35.6
0.01 0.23
61.6 39.2
Separation of PBMCs
Stimulation by antigen
Analysis
: Tetramer-positive well
12– 15 days
DAY2
1 2 3 4 5 6 7 8 9 10 11 12
96-well plate
(1)Create pools by harvesting aliquots from each column (2)Deconvolute by staining aliquots
from wells in the pool
DAY0
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Identification of virus-specific T cells using MHC Tetramers
■EBV ■CMVHLA-A*24:02 EBV Tetramer HLA-A*02:01 CMV pp65 Tetramer
MBL offers HLA-A*24:02 Tetramers to five different EBV epitopes. HLA-A*24:02
EBV mix Tetramer (MBL code no. TS-M009-1, upper middle plot) is a pool of all
five Tetramers used to identify multiple types of EBV-specific CTL using a single
reagent. Because reactivity of epitopes varies from person to person, the mix
Tetramer allows for identification of EBV-specific CTL in HLA-A*24:02 positive
individuals. Samples from those testing positive for EBV reactivity can then be
selectively stained with the individual Tetramers to determine epitope specificity,
if desired. The value on the upper right of each plot indicates the percentage of
Tetramer-positive CD8 T cells.
PBMC were separated from peripheral blood of HLA-A2 positive healthy subjects
and immediately stained with HLA-A*02:01 CMV pp65 Tetramer (MBL code
no. T01009). Tetramer positive CTL could be detected in the blood of HLA-
A*02:01 and HLA-A*02:06, but not HLA-A*02:07-positive donors. This suggests the
CMV peptide is cross-presented by HLA-A*02:01 and HLA-A*02:06 to induce
CMV specific CTL and that HLA-A*02:01 CMV pp65 Tetramer can be used to
monitor patients of either haplotype. The value on the upper right of each plot
indicates the percentage of Tetramer-positive CD8 T cells.
EBV BRLF1 EBV EBNA3A EBV EBNA3B
EBV BMLF1 EBV LMP2
CD8-FITC
Tet
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Negative
0.00 %
0.00 %0.01 %
0.01 %0.06 %
EBV mix
0.25 %
0.17 %
Donor 4 Donor 8Donor 6
Donor 5 Donor 9Donor 7
A*02:07A*02:06A*02:01
0.00 %6.64 % 3.33 %
0.00 %0.01 %0.17 %
CD8-FITC
Tet
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PBMC were separated from peripheral blood of HLA-A*11:01 or HLA-B*15:01
positive healthy subjects and immediately stained with HLA-A*11:01 CMV pp65
Tetramer (MBL code no. TS-M012-1) and HLA-B*15:01 CMV pp65 Tetramer (MBL
code no. TS-M013-1), respectively (Figure 3). After two weeks of culture with the
MLPC method, significant populations of CMV pp65-specific CTL were detected
in samples that had only yielded of 0.01 and 0.06% Tetramer-positive T cells
immediately after blood collection. The value on the upper right of each plot
indicates the percentage of Tetramer-positive CD8 T cells.
Identification of virus-specific T cells using MHC Tetramers
HLA-A*11:01, HLA-B*15:01 CMV pp65 Tetramer
CMV-specific T cells could be detected by day 8 of culture from PBMC that
had no detectable Tetramer-positive T cells immediately after blood collection.
CMV-specific CTL were induced from peripheral blood of ten HLA-A*24:02 positive
healthy subjects studied using the MLPC method. The value on the upper right of
each plot indicates the percentage of Tetramer-positive CD8 T cells.
Day 0 Day 8
Day 11 Day 14
Tet
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CD8-FITC
20.3 %
0.00 % 1.37 %
31.9 %20.3 % 31.9 %
Tet
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CD8-FITC
0.01 % 12.3 %
0.06 % 52.5 %
12.3 %
52.5 %
HLA-A*11:01CMV pp65 Tetramer-PE
(TS-M012-1)
HLA-B*15:01CMV pp65 Tetramer-PE
(TS-M013-1)
Day 0 Day 14
Donor 1 Donor 3Donor 2
0.00 %0.02 % 0.01 %
Tet
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CD8-FITC
HLA-A*24:02 CMV pp65 Tetramer staining following MLPC induction
HLA-A*24:02 CMV pp65 Tetramer staining immediately after blood collection
PBMC were separated from peripheral blood of HLA-A*24:02 positive healthy
subjects and immediately stained with HLA-A*24:02 CMV pp65 Tetramer (MBL
code no. T01043) or were cultured to induce antigen-specific T cells using the
MLPC method. HLA-A*24:02 CMV pp65 specific CTL responses tend to be more
rare and variable than HLA-A*02:01 responses and cannot always be detected
immediately after blood collection (Figure 1). However, CMV-specific CTL can be
detected by induction using the MLPC method (Figure 2).
Figure 2 :
Figure 1 :
Figure 3:
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Tet
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Tax301-309 (TS-M018-1) Tax12-20 (TS-M020-1) Tax187-195 (TS-M021-1) Env11-19 (TS-M022-1)
CD8-FITC
HLA-A*24:02 HTLV-1a
Tax11-19 (TS-M017-1) Tax178-186 (TS-M019-1)
HLA-A*02:01 HTLV-1a
Tax88-96 (TS-M023-1) HTLV-1 Tax272-280 (TS-M024-1)
HLA-A*11:01 HTLV-1b
HLA-A*02:01 HLA-A*02:01 / HLA-A*02:06 HLA-A*02:06 HLA-A*02:07
HLA-A*02:01 HLA-A*02:01 / HLA-A*02:06 HLA-A*02:06 HLA-A*02:07
Tet
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ECD8-FITC
0.00 % 0.01 %0.01 %0.01 %
0.19 % 0.06 %0.43 %0.12 %
HLA-A*02:01 Influenza M1 Tetramer-PE
Negative Tetramer-PE
A*02:01A*02:01A*02:01A*02:01A*02:01A*02:01A*02:01A*02:01A*24:02A*24:02A*24:02A*24:02A*24:02A*24:02A*24:02A*24:02Tax CTL positivesEnv CTL positivesTotal CTL positives
Tax11-19Tax123-131Tax155-163Tax178-186Tax307-315Env175-183Env239-247Env442-450Tax12-20Tax187-195Tax289-297Tax301-309Tax311-319Env11-19Env21-29Env153-161
73%9%0%9%9%0%0%0%
11%11%0%
94%0%6%0%0%
22%1%
14%
(8/11)(1/11)(0/11)(1/11)(1/11)(0/11)(0/11)(0/11)(2/18)(2/18)(0/18)(17/18)(0/18)(1/18)(0/18)(0/18)(32/145)(1/87)(33/232)
(3/9)(0/9)(0/9)(0/9)(0/9)(0/9)(0/9)(0/9)(0/22)(0/22)(0/22)(12/22)(0/22)(0/22)(0/22)(0/22)(15/155)(0/93)(15/248)
(9/10)(2/10)(1/10)(2/10)(0/10)(0/10)(1/10)(0/10)(2/15)(1/15)(2/15)(13/15)(1/15)(6/15)(2/15)(2/15)(33/125)(11/75)(44/200)
(6/11)(5/11)(0/11)(2/11)(0/11)(1/11)(2/11)(2/11)(4/17)(3/17)(3/17)(13/17)(1/17)(8/17)(6/17)(4/17)(37/140)(23/84)(60/224)
33%0%0%0%0%0%0%0%0%0%0%
55%0%0%0%0%
10%0%6%
90%20%10%20%0%0%
10%0%
13%7%
13%87%7%
40%13%13%26%15%22%
55%45%0%
18%0%9%
18%18%24%18%18%76%6%
47%35%24%26%27%27%
allele Tetramers AC ATLL HAM/TSP AC with autoimmune diseases
HLA-A*02:01 HLA-A*02:01 / HLA-A*02:06 HLA-A*02:06 HLA-A*02:07
HLA-A*02:01 HLA-A*02:01 / HLA-A*02:06 HLA-A*02:06 HLA-A*02:07
Tet
ram
er-P
ECD8-FITC
0.00 % 0.01 %0.01 %0.01 %
0.19 % 0.06 %0.43 %0.12 %
HLA-A*02:01 Influenza M1 Tetramer-PE
Negative Tetramer-PE
Identification of virus-specific T cells using MHC Tetramers
Figure 2
Figure 1
Frequency of HTLV-1-specific CTL positivity in asymptomatic HTLV-1 carriers (AC), ATLL, HAM/TSP and carriers with autoimmune diseases
PBMC were separated from peripheral blood of HLA-A2
positive healthy subjects and immediately stained with
HLA-A*02:01 Influenza M1 Tetramer (MBL code no.
T01011). Tetramer positive CTL could be detected in
the blood of HLA-A*02:06 and, to a lesser extent, HLA-
A*02:07, as well as HLA-A*02:01 donors, suggesting this
Tetramer can be used to detect flu-specific responses in
subjects of all three haplotypes (Figure 1). The value on
the upper right of each plot indicates the percentage of
Tetramer-positive CD8 T cells.
HTLV-1 (Human T cell leukemia virus type 1) is known
to infect T cells and induce adult T cell leukemia (ATLL)
and HTLV-1-associated myelopathy (HAM/TSP). HTLV-1 is
one of the viruses receiving extra attention due to the high prevalence of HTLV-1
infected patients and seriousness of symptoms after onset of associated diseases.
It is believed that HTLV-1 infection and the corresponding host immune response
involving mainly CTL are closely linked to the onset of disease, and HTLV-1-specific
CTL epitopes have been described.1-3 Using sixteen different Tetramer reagents, a
group from Kagoshima University showed that the variety and number of HTLV-1-
specific CTL was lower in ATLL patients4-5 and higher in HAM/TSP patients,
compared with asymptomatic carries of HTLV-1 (Figure 2 and Table).6
■ Influenza M1HLA-A*02:01 Influenza M1 Tetramer
■HTLV-1HTLV-1 Tetramer
(The number of subjects with detectable CTL / Tetramers tested)
aData provided by Dr. Tomohiro Kozako, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Fukuoka University
bData provided by Dr. Mari Kannagi, Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University.
1) Kannagi M, et al., J. Virol. 66: 2928-2933 (1992)2) Yashiki S, et al., AIDS Res Hum Retroviruses 17: 1047-1061 (2001)3) Harashima N, et al., J. Virol. 79: 10088-10092 (2005)4) Kozako T, et al., J. Immunol. 177: 5718-5726 (2006) 5) Akimoto M, et al., J. Med. Virol. 79: 977-986 (2007)6) Kozako T, et al., J. Med. Virol. 83: 501-509 (2011)
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1 %
CD107a CD107a
Tet
ram
er X
Tet
ram
er Y
Stimulation
35 %
0 %100 %99 %
11 % 89 % 65 %
control peptide
cognatepeptide
2.5 5.0 10
Unstimulated
Stimulatedwith Peptide
(QYDPVAALF)
×104 cells/well
Measurement methods for antigen-specific CTLusing MBL Kits
*This kit does not include CFSE
Prior to the development of MHC Tetramers, identification and isolation of antigen-
specific T cell was dependent on cell function, based on cytokine production or
cytotoxic activity. MHC Tetramers can directly detect CTL based on TCR expression,
regardless of function, and are often run in parallel with traditional functional
assays such as ELISPOT, intracellular cytokine staining, and 51Cr release assay
to distinguish between anergic and functional T cells. Tetramer staining can be
combined with other flow cytometric assays to directly assess the function of
antigen-specific T cells. MBL offers kits for detecting activity of antigen-specific
CTL. When combined with Tetramer reagents, these kits enable detection of
antigen-specific CTL in an activated state.
IFN-γ, a cytokine involved in regulating the inflammatory
immune response, is secreted mainly by activated CD8+
T cells, CD4+ T cells, and NK cells. Measurement of IFN-γ is widely used for evaluation of immune response. The
ELISPOT method can detect and quantify the number of
IFN-γ-releasing cells as spots.
■ IMMUNOCYTO IFN-γ ELISPOT Kit
When activated by antigen stimulation, CTL release cytotoxic factors including
perforin and granzyme from intracellular granules. During this process, CD107a
(LAMP-1) in the inner membrane of intracellular granules is exposed on the cell
membrane. IMMUNOCYTO CD107a Detection Kit (MBL code no. 4844) enables
indirect measurement of CTL cytotoxicity by detecting CD107a mobilization.
While intracellular flow staining for IFN-γ has been found to be more sensitive
than CD107a mobilization, a recent study conducted by MBL identifying new CTL
epitopes showed the latter better correlated with Tetramer staining. In addition to
being more specific, the CD107a mobilization assay is more rapid than cytokine
flow cytometry.
■ IMMUNOCYTO CD107a Detection Kit
1) Immobilize anti-human IFN-γ mAb on membrane2) Add IFN-γ secreting cells on the membrane3) Incubate cells and capture IFN-γ on antibody4) Wash membrane
1
2
3
4
5
6
7
8
5) Add biotinylated anti-human IFN-γ mAb6) Add SA-PE7) Add substrate8) Detect signal
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Living cells Apoptotic cells
PS of living cells is not exposed on the cell surface and does not bind to Annexin V.
In apoptotic cells, the membrane structure is damaged, PS is exposed on the surface of the cells, and PS binds to Annexin V.
Annexin VKO
Annexin VCa2+
Ca2+
Ca2+PS
PS
Ca2+
Annexin V
KO
Annexin V
KO
KO
Cytotoxicity: 84.64%
E:T=0:1 E:T=1:4
CFS
EC
FSE
FSC-H
Annexin V-KOE: A*24:02 EBV BRLF1 CTL lineT: BRLF1 peptide-pulsed HLA-A24 LCL
7.79 % 85.84 %7.79 % 85.84 %
Cytotoxicity: 0.32%
E:T=0:1 E:T=1:4
CFS
EC
FSE
FSC-H
Annexin V-KOE: A*24:02 EBV BRLF1 CTL lineT: Unpulsed HLA-A2 LCL
8.43 % 8.72 %8.43 % 8.72 %
0102030405060708090
100(%)
1:4 1:2 1:1
peptide (+)peptide ( - )
E:T ratio E:T ratio
010203040506070
HLA-A*24:02 (CMV)HLA-A*24:02 (HIV)HLA-A*02:01 (CMV)
1:2 1:1 2:1
(%)
Effector cells: HLA-A*24:02 CMV pp65 CTL lineTarget cells: OKT3 stimulated HLA-A*24:02 PBMC line
Effector cells: HLA-A*24:02 CMV pp65 CTL lineTarget cells: Lymphoblastoid cell lines of each allele pulsed with peptide
Figure 3
Measurement methods for antigen-specific CTL using MBL Kits
Principle of apoptosis detection by Annexin V
Phosphatidylserine (PS) located on the inner side of the lipid bilayer is exposed
on the cell surface in apoptotic cells due to change in membrane structure.
Annexin V binds with high affinity to PS in a Ca2+-dependent manner, effectively
labeling apoptotic cells (Figure 1). By using fluorescently-labeled Annexin V,
the target cells damaged by effector cells can be detected by flow cytometry.
IMMUNOCYTO Cytotoxity Detection Kit contains fluorescent protein Kusabira-
Orange (KO; Excitation Max 548 nm/Emission Max 559 nm)-labeled Annexin V.
Graphs showing cytotoxic activity
Graphs showing cytotoxic activity by HLA-A*24:02 CMV pp65 CTL line against
various targets illustrate the specificity of CTL killing, as well as the effect of
different E:T ratios (Figure 3).
Data analysis by flow cytometry
Cytotoxic activity measured by IMMUNOCYTO Cytotoxity Detection Kit is shown
for specific and negative control targets. Effector cell (E) to target cell (T) ratios
are indicated in Figure 2. When killing takes place, CFSE-single positive target
cells become dual positive for CFSE and Annexin V. Cytotoxic activity can be
calculated from percentages of single and dual positive cells using the following
equation:
Cytotoxicity (%) = [(ET-T0)/(100-T0)]×100
ET: % CFSE+ Annexin V+ cells when effector cells and target cells are co-cultured.T0: % CFSE+ Annexin V+ cells when only target cells are cultured.
Figure 1
Figure 2
T cells and NK cells (effector cells) directly recognize virus-infected and tumor cells
(target cells) and can respond with cytotoxic activity to injure or kill target cells.
While the 51Cr release assay has been a reliable method to measure cytotoxic
activity, special training and facilities for radioisotopes are required. Therefore,
methods to measure cytotoxic activity without using radioisotopes have been
developed. The principle to detect cytotoxic activity of effector cells using
IMMUNOCYTO Cytotoxity Detection Kit (MBL code no. AM-1005) is to double
stain the target cells with both CFSE and Kusabira-Orange labeled Annexin V, and
measure cytotoxic activity by flow cytometry. Target cell death (as a consequence
of effector cell cytotoxicity) is measured based on the ratio of cells stained with
Annexin V to cells stained with CFSE, as described below. Because the effector
cells are not stained with CFSE, they are easily distinguished by flow cytometry
for analysis. This non-radioactive method is reported to have high correlation with
the 51Cr release assay making it a safer alternative.
■ IMMUNOCYTO Cytotoxity Detection Kit
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SS
C-H
cou
nts
cou
nts
FSC-H
FL1-H FITC FL1-H FITC
R1
R2
R1
R2
mouse IgG2a-FITC (5 μg/mL)
Red:Clear Back treatmentGreen:No Clear Back treatment
Lymphocyte (R1) Monocytes (R2)
Clear Back treatment reduced unwanted Fc-specific staining of isotype control by monocytes.
Tet
ram
er-P
E
CD8-FITC
Clear Back treatment reduced non-specific binding of CD8 negative Tetramer positive cells.
WithoutClear Back
Clear Back
MHC Tetramer related products
When a very rare cell population is detected by a Tetramer reagent, it is important
to reduce non-specific and Fc-specific staining. Clear Back (MBL code no. MTG-
001) is a reagent that blocks non-specific binding to both human and murine cells
in flow cytometry and fluorescence microscopy experiments. As shown in Figure 1,
Clear Back reduced Fc-specific staining of monocytes by a mouse IgG2a. Clear
Back also reduced background staining of PBMC with HLA-A*24:02 EBV BRLF1
Tetramer in the Tetramer-positive CD8-negative region (Figure 2).
MBL offers ready-to-use epitope peptides in liquid form for inducing CTL by the MLPC
method. These are the same peptides that are used to produce MHC Tetramers.
Peptides for inducing murine CTL and antigenic peptides that induce T-helper
immunity are also available. Please refer to the product list for product lineup.
■Clear Back blocking reagent for tetramer staining
■Peptides for antigen-specific CTL induction
Concentration Volume Solvent Purity
10 mg/mL 100 μL (1 mg) DMSO 95%
♦Effect of Clear Back on peripheral blood mononuclear cells
Figure 1 Figure 2
Day 7
DAY-10*antigenic peptide,
helper peptide,CFA, PBS
Immunization
Spleen Harvest Dissection
Tetramerstaining
PeptideStimulation
DAY0
Analyze
DAY7
Analyze
CD8-FITC
Tet
ram
er-P
E
mouse strain
Class I allele
H-2K H-2D H-2L
C57BL/- b b -
BXSB/Mp b b -
129/- b b -
BALB/c d d d
DBA/2 d d d
B10D2 d d d
C3H/He k k -
CBA/N k k -
Induction method for antigen-specific murine CTL
Mouse models are commonly used to study various in vivo immune responses,
and antigen-specific CTL can be detected using mouse MHC Tetramers.
Antigen-specific CTL can be induced rapidly and easily using the following
method. First, the antigen peptide of interest is mixed with a second "helper"
peptide designed to induce T helper activity. This mixture is emulsified
with adjuvant, and injected intraperitoneally (IP). Spleens are harvested
7-11 days after the final immunization, and splenocytes are stimulated by the
same peptides in vitro for 1 week. The times of immunization depend on the antigen
used. In a study conducted by MBL, antigen-specific CTL could be induced in
1-4 rounds of immunization. Immunization of two or more mice per antigen is
recommended due to individual differences.
A variety of influenza virus epitopes and variants are reported in mouse models
and used for various purposes including structural analysis, MHC binding studies,
vaccine development, and infectious disease research. Influenza virus epitopes and
variants are among the antigens suitable for CTL induction by peptide immunization.
■ Induction of antigen-specific murine CTL by in vitro stimulation (influenza virus)
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CD8-FITC
CD8-FITC
1 μg/mL peptideStimulation
Mouse A Mouse B
Tet
ram
er-P
ET
etra
mer
-PE
12.7%
0.2%0.3%
1.3%
0.0%0.0%
Influenza NP(TS-M502-1)
Influenza NP(TS-M502-1)
Negative(TS-M505-1)
Induction method for antigen-specific murine CTL
Figure 1 H-2Db Influenza NP Tetramer-PE (MBL code no. TS-M502-1)
The antigen peptide derived from H-2Db-restricted Influenza NP (ASNENMDTM,
MBL code no. TS-M502-P) was mixed with I-Ab HBc helper peptide (MBL code
no. TS-M701-P), an antigen peptide which induces T cell help. This mixture was
emulsified with adjuvant and IP immunization was performed on C57BL/6 mice.
The spleen was harvested 11 days later, and spleen cells were stimulated in
culture with the antigen peptide derived from Influenza NP (1 μg/mL) for 1
week. Aliquots were stained with the MHC Tetramer reagent on (days 0 and 7 of
culture), and live lymphocytes were assessed for Tetramer reactivity.
In mice immunized with the antigen peptide derived from influenza NP
(ASNENMDTM), expansion of influenza NP-specific CTL by in vitro stimulation
was observed (Figure 1). Negative staining with a Tetramer to an unrelated
epitope (H-2Db human gp100 Tetramer-KVPRNQDWL-PE, MBL code no. TS-
M505-1) demonstrated the specificity of the expanded CTL to NP. The value on
them upper right of each plot indicates the percentage of Tetramer-positive
CD8 T cells.
Figure 2 shows in vitro expansion of various Influenza-specific T cells from mice
immunized with the corresponding peptide.
*Please refer to the data sheet of each reagent for induction condition for each epitope.
CD8-FITC
H-2Db Influenza NP(TS-M527-1)
H-2Db Influenza NP(TS-M508-1)
H-2Db Influenza NP(TS-M502-1)
H-2Db Influenza PA(TS-M528-1)
H-2Kd Influenza HA(TS-M520-1)
3.5%
0.0% 0.0% 0.1% 0.2% 0.2%
12.7% 2.3% 10.6%2.6%
Specific Tetramer
Negative Tetramer
Tet
ram
er-P
E
Tet
ram
er-P
E
0.3%
Influenza NP(TS-M502-1)
DAY0
DAY7
Figure 2
CD8-FITC
H-2Db Influenza NP(TS-M527-1)
H-2Db Influenza NP(TS-M508-1)
H-2Db Influenza NP(TS-M502-1)
H-2Db Influenza PA(TS-M528-1)
H-2Kd Influenza HA(TS-M520-1)
3.5%
0.0% 0.0% 0.1% 0.2% 0.2%
12.7% 2.3% 10.6%2.6%
Specific Tetramer
Negative Tetramer
Tet
ram
er-P
E
Tet
ram
er-P
E
0.3%
Influenza NP(TS-M502-1)
DAY0
DAY7
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CD8-FITC CD8-FITC
Tet
ram
er-P
E
0.1%0.1% 0.1% 0.1% 0.2%0.39% 0.2%0.4%0.0%
17.8% 11.6% 1.6%10.8%3.1% 0.9% 2.8% 1.3%4.5%
H-2Kb HSV-1 gB(TS-M523-1)
H-2Kb SeV(TS-M509-1)
H-2Kb VSV NP(TS-M529-1)
H-2Dd HIV P8-I10(TS-M516-1)
H-2Dk polyomavirus MT(TS-M530-1)
H-2Dk HTLV-1(TS-M531-1)
H-2Kd RSV(TS-M506-1)
H-2Ld MCMV IE1(TS-M510-1)
H-2Ld HBsAg(TS-M522-1)
Specific Tetramerreagent
Negative Tetramer
CD8-FITC CD8-FITC
Tet
ram
er-P
E
0.1%0.1% 0.1% 0.1% 0.2%0.39% 0.2%0.4%0.0%
17.8% 11.6% 1.6%10.8%3.1% 0.9% 2.8% 1.3%4.5%
H-2Kb HSV-1 gB(TS-M523-1)
H-2Kb SeV(TS-M509-1)
H-2Kb VSV NP(TS-M529-1)
H-2Dd HIV P8-I10(TS-M516-1)
H-2Dk polyomavirus MT(TS-M530-1)
H-2Dk HTLV-1(TS-M531-1)
H-2Kd RSV(TS-M506-1)
H-2Ld MCMV IE1(TS-M510-1)
H-2Ld HBsAg(TS-M522-1)
Specific Tetramerreagent
Negative Tetramer
Staining with Mouse MHC class I Tetramer
MBL offers Tetramer reagents for murine
model antigens, tumor antigens, viral antigens,
etc. All the data shown below are results of
staining with a Tetramer reagent (upper column)
or a negative Tetramer (lower column) of each
antigen peptide-specific CTL expanded by in vitro
stimulation after peptide immunization. Negative
Tetramers were constructed using the matching
allele with a different (non-reactive) peptide
epitope sequence. Murine spleen cell samples
6 days after peptide stimulation are shown. The
value on the upper right of each plot indicates
the percentage of Tetramer-positive CD8 T cells.
♦LCMV ♦MuLV
♦Other Viral antigens
*Please refer to the data sheet of each reagent for induction condition for each epitope.
CD8-FITC CD8-FITCCD8-FITCT
etra
mer
-PE
Tet
ram
er-P
E
H-2Db LCMV gp33(T03001)
H-2Db LCMV gp33 (C9M)(TS-M512-1)
H-2Kb MuLV p15E(TS-M507-1)
H-2Db LCMV NP396(TS-M513-1)
H-2Ld LCMV NP118(TS-M514-1)
H-2Ld MuLV gp70(TS-M521-1)
15.3%3.3%
0.1%
0.7%
0.1%0.13% 0.2%0.4% 0.0%
2.3%4.3%7.9%
SpecificTetramerreagent
NegativeTetramer
SpecificTetramerreagent
NegativeTetramer
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CD8-FITC
Tet
ram
er-P
E
SpecificTetramerreagent
NegativeTetramer
H-2Kb
β-galactosidase(TS-M501-1)
H-2Kd
EGFP(TS-M525-1)
H-2Ld
β-galactosidase(TS-M511-1)
7.2% 3.7%
0.1%
2.0%
0.0% 0.0%
CD8-FITC
Tet
ram
er-P
E
8.3%
H-2Db HY Uty (TS-M524-1)
SpecificTetramerreagent
H-2Db HY Uty (TS-M524-1)
0.2%
NegativeTetramer
CD8-FITC
CD8-FITC
Tet
ram
er-P
E
H-2Kd
Listeria LLO91-99
(TS-M503-1)
H-2Dd
BCG MPT51(TS-M517-1)
H-2Kd
Malaria Pb9(TS-M515-1)
2.1% 4.4%
0.3%
1.4%
0.1% 0.2%
SpecificTetramerreagent
NegativeTetramer
CD8-FITC
Tet
ram
er-P
E
0.0%0.1%
2.2% 0.9%
H-2Ld P815(TS-M519-1)
H-2Kd HER2(TS-M526-1)
0.4%0.0%
0.6% 5.4%
H-2Db human gp100(TS-M505-1)
H-2Db CEA(TS-M518-1)
SpecificTetramerreagent
NegativeTetramer
SpecificTetramerreagent
NegativeTetramer
Staining with Mouse MHC class I Tetramer
♦Bacterial antigen
♦Cancer antigen
♦Minor antigen♦Model antigen
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Negative Tetramer Negative Tetramer Negative Tetramer
0.0% 0.0%
0.0%0.2%
0.5%
0.6%
WT1 Tetramer WT1 Tetramer WT1 Tetramer
0.0% 0.6%
0.2%0.1%
3.2%
4.9%
0.1 μg/mL peptide
stimulation
0.1 μg/mL peptide
stimulation
DAY0
DAY7
DAY14
Mouse1
Mouse2
CD8-FITC
Tet
ram
er-P
E
Negative Tetramer Negative Tetramer Negative Tetramer
0.0% 0.0%
0.0%0.2%
0.5%
0.6%
WT1 Tetramer WT1 Tetramer WT1 Tetramer
0.0% 0.6%
0.2%0.1%
3.2%
4.9%
0.1 μg/mL peptide
stimulation
0.1 μg/mL peptide
stimulation
DAY0
DAY7
DAY14
Mouse1
Mouse2
CD8-FITC
Tet
ram
er-P
E
Staining with Mouse MHC class I Tetramer
Induction of murine WT1 Tetramer-specific CTL
Wilms tumor factor WT1 is a transcription factor regulating cell growth,
differentiation, and apoptosis. The WT1 gene is highly expressed in leukemia and
various solid cancers and is known to contribute to their onset and progression. It
has been shown that WT1-specific CTL kill tumor cells with high WT1 expression,
but not normal cells. WT1 is receiving widespread attention as a target molecule
for cancer immunotherapy and clinical trials are in progress.
The Figure above shows an increase in H-2Db WT1 Tetramer positive cells
(using MBL code no. TS-M504-1) in splenocyte cultures from peptide immunized
mice stimulated with peptide in vitro. The value on the upper right of each plot
indicates the percentage of Tetramer-positive CD8 T cells.
It is sometimes difficult to obtain adequate amounts of antigen-specific CTL with
a single round of in vitro peptide stimulation. A second peptide stimulation may
amplify the specific CTL, but because the peptide is presented on MHC class
I molecules of the cells in culture, those cells can become targets of the MHC
peptide-specific CTL. This will often cause a significant decrease in CD8 negative
cells, as shown in the day 14 plots, or even a decrease in CTL themselves,
as they too can become targets of the antigen-specific CTL. To avoid these
complications, the addition of peptide-pulsed antigen-presenting cells (such as
dendritic cells), rather than soluble peptide, is recommended for the second
round of stimulation.
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Mouse 2 Mouse 2Mouse 1 Mouse 1
Tet
ram
er-P
E
0.04 % 0.01 % 0.42 %0.43 %
1.14 %7.68 %0.02 %0.03 %
NegativeTetramer
OVATetramer
1WEEK
CD8-FITC
Mouse 2 Mouse 2Mouse 1 Mouse 1
Tet
ram
er-P
E
0.04 % 0.01 % 0.42 %0.43 %
1.14 %7.68 %0.02 %0.03 %
NegativeTetramer
OVATetramer
1WEEK
CD8-FITC
H-2Kb OVA Tetramer
H-2Kb OVA Tetramer
H-2Kb OVA Tetramer staining
Ovalbumin (OVA), the major protein found in chicken egg whites, is a T cell-
dependent antigen commonly used as a model protein for studying antigen-
specific immune responses in mice. H-2Kb OVA Tetramer detects OVA-specific
T cells in C57BL/6 and other mouse strains expressing the class I allele, H-2Kb.
An antigenic peptide derived from H-2Kb-restricted OVA was mixed with a
T-helper peptide. This mixture was emulsified with adjuvant, and IP immunization
was performed on two C57BL/6 mice. Spleens were harvested 10 days later,
and spleen cells were prepared. Some cells were sampled and stained with the
OVA-specific or negative Tetramers (left figure). Remaining spleen cells were
incubated in vitro for 1 week with naïve splenocytes pulsed with OVA peptide,
and then stained with Tetramers (right figure). OVA-specific T cells were detected
one week following in vitro stimulation. The value on the upper right of each plot
indicates the percentage of Tetramer-positive CD8 T cells.
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CD8 (KT15)-FITC
Tet
ram
er-P
E
CD8 (53.6.7)-FITC
Tet
ram
er-P
E
AntibodyVolume
AntibodyVolume
Negative Tetramer OVA Tetramer
10 μL
2.5 μL
0.63 μL
10 μL
2.5 μL
0.31 μL
Negative Tetramer OVA Tetramer
CD8 (KT15)-FITC
Tet
ram
er-P
E
CD8 (53.6.7)-FITC
Tet
ram
er-P
E
AntibodyVolume
AntibodyVolume
Negative Tetramer OVA Tetramer
10 μL
2.5 μL
0.63 μL
10 μL
2.5 μL
0.31 μL
Negative Tetramer OVA Tetramer
Figure 2
H-2Kb OVA Tetramer
Effect of CD8 antibody clones on H-2Kb OVA Tetramer staining:
Cells from OT-I mice
Splenocytes from mice transgenic for OVA-specific T cells (OT-I) were used to
explore staining differences among mouse CD8 antibody clones in combination
with H-2Kb Tetramers. OT-I mouse spleen cells (1×106 cells/sample) were
stained with H-2Kb OVA Tetramer-PE (10 μL/sample) and serially diluted
anti-mouse CD8 (clone KT15 or clone 53.6.7) antibody in a final assay volume
of 100 μL. H-2Kb β-galactosidase (β-gal) Tetramer-PE (10 μL/sample) was used
as a negative Tetramer to assess non-specific binding. When anti-CD8 clone
53.6.7 was used, positive staining was observed on CD8 positive cells with
both OVA Tetramer and β-gal Tetramer (Figure 1). Even when 53.6.7 was diluted,
the cells were stained with both Tetramers similarly, suggesting the Tetramer
staining was not specific.
When anti-CD8 clone KT15 was used, however, CD8 positive cells among OT-I
mouse spleen cells were specifically stained only with OVA Tetramer at all
antibody concentrations tested (Figure 2). Cells stained with β-gal Tetramer
were negative. Titration of KT15 revealed an optimal signal to noise ratio at
0.63 μL/sample for OT-I splenocytes. In summary, when staining cells with
H-2Kb Tetramers it is important to use appropriately titrated anti-CD8 clone
KT15 and include a negative control Tetramer in the experiment.
The concentration used for this evaluation is only a guide for use. Antibody titrations should be performed to optimize concentration according to the experimental system used.
Staining with anti-mouse CD8 (Clone 53.6.7)-FITC/Tetramer -PE Staining with anti-mouse CD8 (Clone KT15)-FITC/Tetramer -PEFigure 1
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100100
101
102
103
104
101 102 103 104 100 101 102 103 104
8.6 %
Cells were incubated with Tetramer for 4 hr.
OV
A-T
etra
mer
CD8-FITC CD8-FITC
2 : 1 1 : 1E : T
0.5 : 1
10
101
102
103
104
0
OV
A-T
etra
mer
A. Wild type B. OT-I transgenic mice
Tetramer staining inhibits TCR-specific cytotoxic activity Cytotoxic activity is inhibited dependent on concentration of the Tetramer reagent.
About 90% of CD8+ T cells in splenocytes of OT-I mice are OVA Tetramer positive.
60
0
10
20
30
40
50
C D
Pre-treatedCyt
oto
xici
ty(%
)
Cyt
oto
xici
ty(%
)
60
0
10
20
30
40
50TetramerNon-treated
E : T = 4 : 1
vs E.G7 vs EL-4
(μg/mL)
0.9 % 90.3 %
98.5 %
90.3 %
98.5 %
OT-I mousesplenocyte
Cytotoxicityassay
Analysis
WithTetramer
WithoutTetramer
51Cr labeledtarget cells
(E.G7)
No-tet. No-tet.OVA-tet.0.025
OVA-tet.0.175
OVA-tet.3.3
HBV-tet.3.3
OT-I CTL vs E.G7 Targets
OT-I CTL vs EL-4 Controls OT-I CTL + Tetramer vs E.G7 Targets
CD8-FITC
clone KT15 clone 53.6.7
Tet
ram
er-P
E
1.31%
0.71%
32.7%
61.5%
H-2Kb β-galactosidaseTetramer-PE
(negative control)
H-2Kb OVATetramer-PE
H-2Kb OVA Tetramer
Effect of CD8 antibody clones on H-2Kb OVA Tetramer staining:
Cells from peptide-immunized mice
Mouse spleen cells prepared using peptide immunization for OVA-specific
CTL induction were stained with H-2Kb OVA Tetramer-PE (10 µL/sample) and
anti-mouse CD8 (clone KT15 or clone 53.6.7). H-2Kb β-galactosidase Tetramer-PE
(10 µL/sample) was used as a negative Tetramer to assess non-specific staining.
When KT15 was used, OVA-specific Tetramer positive cells were observed
compared with the negative Tetramer, while when 53.6.7 was used, marked non-
specific staining was observed in both H-2Kb β-galactosidase and OVA Tetramer
samples. Because clone 53.6.7 is known to have poor compatibility with OVA and
other H-2Kb Tetramers, anti-mouse CD8 clone KT15 is recommended.
Figure 1
Staining using cancer- related MHC Tetramers
1) Wakita D, et al., Int. Immunol. 18: 425−434 (2006) 3) Yokouchi H, et al., Cancer Sci. 97: 148−154 (2006) 2) Chamoto K, et al., Cancer Res. 66: 1809−1817 (2006) 4) Yokouchi H, et al., Clin Exp Metastasis 24: 533−540 (2007)
Data provided by Dr. Kenji Chamoto and Dr. Takashi Nishimura, Division of Immunoregulation, Institute for Genetic Medicine Hokkaido University.
The Tetramer blocking assay is an experimental system showing inhibition of
antigen specific cytotoxic activity by blocking TCR using MHC-Tetramer reagents.
The assay shows whether or not the cytotoxic activity observed in the 51Cr release
assay or the IMMUNOCYTO Cytotoxity Detection Kit is caused by Tetramer positive
CTL. Cytotoxic activity of the Tetramer-stained CTL is reported to recover about 24
hours after the blocking assay is completed.
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Mouse MHC class II Tetramer
OVA is a model antigen commonly used to study immune responses in mice. OT-II
transgenic mice express TCR specific for OVA323-339 epitope (ISQAVHAAHAEINEAGR)
in the context of I-Ab and serve as an important tool to study differentiation and
activation of CD4 T cells. I-Ab OVA323-339 Tetramer can be used to monitor antigen-
specific CD4 T cell responses in OT-II mice and in various experimental systems
using OT-II cells for adoptive transfer. Reactivity of I-Ab OVA323-339 Tetramer reagent
was assessed using freshly isolated spleen cells from OT-II mice. The majority of
CD8 T cells were Tetramer positive in mouse 2, but not in mouse 1 (Figure 1). In
vitro stimulation with specific peptide greatly increased the number of Tetramer
positive cells (Figure 2.)
OVA-specific CD4 T cells can also be induced in wild-type mice. C57BL/6 mice
were immunized twice IP with a mixture of 100 nmol of OVA323-339 peptide and
10 μg of cholera toxin emulsified with adjuvant. After 11 days, spleen cells were
harvested and cultured with a final concentration of 1 μg/mL OVA323-339 peptide for
8 days. Cells were tested on days 0 and 8 of culture for OVA Tetramer reactivity
(Figure 3). In mice immunized with OVA323-339 peptide, induction of specific T cells
by in vitro peptide stimulation was confirmed using I-Ab OVA323-339 Tetramer. I-Ab
MOG35-55 Tetramer, used as a negative control, showed no specific staining.
■ I-Ab OVA323-339 Tetramer-PEI-
Ab O
VA
323-
339
Tet
ram
er-P
E
Mouse 1
6.4 %
Mouse 2
75.2 %
CD4-FITC
I-A
b M
OG
35-5
5 T
etra
mer
-PE
0.5 %
CD4-FITC
Tet
ram
er-P
E
I-Ab MOG35-55
Tetramer
1.2 %
I-Ab OVA323-339 Tetramer
1.9 %
OVA323-339
peptide concentration
1.5 % 79.2 %
CD4-FITC
0 μg /mL
1 μg /mL
I-A
b O
VA
323-
339
Tet
ram
er-P
EMouse 1
6.4 %
Mouse 2
75.2 %
CD4-FITC
I-A
b M
OG
35-5
5 T
etra
mer
-PE
0.5 %
CD4-FITC
Tet
ram
er-P
E
I-Ab MOG35-55
Tetramer
1.2 %
I-Ab OVA323-339 Tetramer
1.9 %
OVA323-339
peptide concentration
1.5 % 79.2 %
CD4-FITC
0 μg /mL
1 μg /mL
OVA323-339 peptide immunized mouse splenocytes
Day 0 Day 8
I-A
b O
VA
323-
339
Tet
ram
er-P
E
0.5 % 1.4 %
I-A
b M
OG
35-5
5 T
etra
mer
-PE
0.2 % 0.2 %
CD4-FITC
Figure 1: I-Ab OVA323-339 Tetramer staining of OT-II splenocytes
Figure 2: I-Ab OVA323-339 Tetramer staining of peptide stimulated OT-II splenocytes (Day 6).
Figure 3: I-Ab OVA323-339 Tetramer staining of splenocytes from peptide-immunized mice
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Mouse MHC class II Tetramer
Human tubercle bacillus (Mycobacterium tuberculosis, Mtb) is well known as a
causative agent of pulmonary tuberculosis. ESAT-6 (Early Secreted Antigenic
Target 6) is a low molecular weight protein secreted by Mtb and possesses strong
antigenicity. M. bovis BCG strain used for tuberculosis vaccination lacks the ESAT-
6 gene and thus the T cell response specific to the antigen is used for diagnosis of
tuberculosis infection in BCG-vaccinated subjects. ESAT-61-20 is an immunodominant
I-Ab restricted epitope in the mouse and widely used for monitoring of immune
response in vivo for research and development of new vaccines. I-Ab ESAT-61-20
Tetramer is anticipated to become an important tool in various experimental systems
involving transgenic mice expressing I-Ab ESAT-61-20-specific TCR.
■ I-Ab ESAT-61-20 Tetramer-PE
FSC/SSC plot
Staining of ESAT-61-20 specific CD4+- T cells harvested from spleen lung
Staining of ESAT-61-20 specific CD4+ T cells harvested from spleen
Live CD4 positive lymphocytes were defined by a FSC/SSC lymphocyte region
(R1), CD4 positive cell region (R2), and 7-AAD negative cell region to analyze CD4
positive T cells for Tetramer reactivity.
Human tubercle bacillus H37Rv strain or BCG Pasteur strain was transmitted to
mice via the tail vein (5x105 CFU/animal). Cells harvested from lung and spleen
four weeks later were stained with the MHC Tetramer I-Ab ESAT-61-20. Tetramer
positive cells were detected in 7.73% of CD4 positive lymphocytes from lungs of
human tubercle bacillus-infected mice (Figure 1). I-Ab ESAT-61-20 Tetramer positive
cells were detected in spleen cells of the human tubercle bacillus-infected, but
not BCG-infected, mice, (Figure 2).
Data provided by Dr. Shunsuke Sakai and Dr. Masao Mitsuyama, Department of Microbiology, Kyoto University Graduate School of Medicine
FSC-H
SS
C-H
CD
4-FI
TC
7-A
AD
CD4-FITC
Non-infection After Mtb infection
I-A
b E
SA
T-6
1-20
Tet
ram
er-P
E
0.08 % 7.73 %
CD4-FITC
Tet
ram
er-P
E
I-Ab ESAT-61-20 Tetramer-PEI-Ab MOG35-55 Tetramer-PE
(Negative control)
1.19 %
0.02 %
0.03 %
0.03 %
0.02 %
1.19 %
0.02 %
0.01 %
0.02 %
Non-infection
After BCGinfection
After Mtbinfection
Figure 2 :
Figure 1 :
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46 47
CD4-FITC
Tet
ram
er-P
E
I-Ab MOG35-55
Tetramer-PE(Negative control)
I-Ab ESAT-61-20
Tetramer-PE
2.2 % 1.0 %
0.3 % 0.2 %
RT 1hour On ice 1hour
CD4(GK1.5)-FITC
day 0 day 8
Tet
ram
er-P
E
0.18 % 4.85 %
0.06 % 0.27 %
I-Ab MOG35-55
Tetramer-PE(Negative Tetramer)
I-Ab ESAT-61-20
Tetramer-PE
Spleen cells from the tubercule bacillus infected mice were stained at room
temperature or on ice. The percent positive and fluorescence intensity of I-Ab
ESAT-61-20 Tetramer was found to be higher when staining was performed at room
temperature for 1 hour, than staining on ice for 1 or 2 hours (Figure 1 and data not
shown).
Mice were immunized twice IP with a mixture of 100 nmol of I-Ab ESAT-61-20
peptide (MTEQQNFAGIEAAASAIQG, MBL code no. TS-M707-P) and 10 μg
of cholera toxin emulsified with adjuvant. After 11 days, spleen cells were
harvested and cultured with ESAT-61-20 peptide (0.1 μg/mL) for 8 days. Cells were
tested on days 0 and 8 of culture for antigen-specific T cells. Induction of specific
CD4+ T cells was confirmed using I-Ab ESAT-61-20 Tetramer. I-Ab MOG35-55
Tetramer, (MBL code no. TS-M704-1), used as a negative Tetramer, showed no
specific staining (Figure 2).
*In this data, live lymphocytes were defined by FSC/SSC plot, lymphocyte region and 7-AAD negative cell region to analyze CD4 positive T cells for Tetramer reactivity
Anti-Mouse TCR DO11.10
(clone KJ1.26, MBL code no.
K0221-5) is an antibody that
binds to the TCR specific for
OVA323-339 presented in the
context of I-Ad. This antibody
can be used to identify
OVA323-339-specific CD4 T
cells derived from I-Ad mice.
OVA323-339 peptide
(ISQAVHAAHAEINEAGR,
MBL code no. TS-M703-P)
is often used as a helper
peptide for induction of
antigen-specific CTL in
I-Ad-expressing mice.
Data provided by Dr. Shunsuke Sakai and Dr. Masao Mitsuyama, Department of Microbiology, Kyoto University Graduate School of Medicine.
■ I-Ab EAST-61-20 Tetramer-PE
■Reagents for studying OVA specificity in the context of I-Ad murine class II molecules
0.0 % 0.3 %
cou
nts
isotypic controlAnti-mouse TCRDO11.10
An
ti-m
ou
se T
CR
D
O11
.10-
PE
CD4-FITC CD4-FITCIs
oty
pic
co
ntr
ol-
PE
Sample: DO11.10 T cell hybridoma
Sample: OVA323-339 immunized mouse splenocyte
Figure 1 Figure 2
Anti-mouse TCR DO11.10
OVA323-339 peptide BALB/c mouse splenocyte
DO11.10 T cell hybridoma
Mouse MHC class II Tetramer
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CD1d Tetramer
CD1d is a membrane protein non-covalently
bonded to β2-microglobulin (β2m) and shows
high homology between human and mouse.
CD1d can present α-galactosylceramide
(α-GalCer), a glycolipid extracted and isolated
from the marine sponge, and this complex can
activate human and murine CD1d-restricted
NKT cells. CD1d Tetramer is a reagent
prepared by tetramerization of complexes of
CD1d and β2m by PE- or APC- labeled
streptavidin. Binding this reagent to α-GalCer
enables highly sensitive detection of
CD1d-restricted NKT cells and can be combined
with antibodies to study NKT cell function by
flow cytometry.
MHC
FluorochromeGlycolipid
CD1d Tetramer
TCR
IFN-γ
Perforin GranzymeIFN-γTNF-α
DC maturation
IL-4CD161Inhibitoryreceptor
NKT
Target cells
Cell death
Dendritic cells
Peptide
TCR
CD1d
NKT
β2m
NKT cellactivation
Cytokineproduction
1) Stephane S, et al., J. Immunol. Methods 268: 107−121 (2002)2) Matsuda JL, et al., J. Exp. Med. 192: 741−754 (2000)3) Benlagha K, et al., J. Exp. Med. 191: 1895−1903 (2000)
Dissolving and loading α-GalCer on CD1d Tetramer
*This product does NOT include α-GalCer
α-GalCerstock solution
(1 mg/mL)
Pyridine0.9% NaCl/0.5% Tween-20α-GalCer diluted solution
(200 µg/mL)
Add diluted α-GalCerinto CD1d-tetramer-PE
Store at 4˚C
Incubate for 12-18 hr
at RT (20-30˚C) or 37˚C
protected from light
*This product does NOT include α-GalCer
Product50 tests 10 tests
MBL code no. MBL code no.
Human CD1d Tetramer-PE TS-HCD-1 TS-HCD-1S
Human CD1d Tetramer-APC TS-HCD-2 -
Mouse CD1d Tetramer-PE TS-MCD-1 TS-HCD-1S
Mouse CD1d Tetramer-APC TS-MCD-2 -
Detection of NKT cells with CD1d Tetramer
Detection of human NKT cells with CD1d Tetramer
PBMC were separated from peripheral blood of healthy subjects and incubated
at room temperature for 5 minutes with 40 μL of Clear Back (Human Fc receptor
blocking reagent, MBL code no. MTG-001). CD3-FITC and human CD1d
Tetramer-PE (with or without binding of α-GalCer) were added and incubated for 30
minutes at 4°C protected from light, and cells were analyzed by flow cytometry.
Results showed total cells contained 0.05% NKT cells, as defined by CD1d/CD3
dual positivity.
α-GalCer (−) α-GalCer loaded
CD3-FITC
Hu
man
CD
1d T
etra
mer
-PE
CD4 (GK1.5)-FITC
Mo
use
CD
1d T
etra
mer
-PE
α-GalCer (−) α-GalCer loaded
0.42 % 2.33 %
0.05 %0.00 %
α-GalCer (−) α-GalCer loaded
CD3-FITC
Hu
man
CD
1d T
etra
mer
-PE
CD4 (GK1.5)-FITC
Mo
use
CD
1d T
etra
mer
-PE
α-GalCer (−) α-GalCer loaded
0.42 % 2.33 %
0.05 %0.00 %
C57BL/6 mouse splenocytes were stained with CD4-FITC and mouse CD1d
Tetramer-PE (with or without binding of α-GalCer) for 30 minutes at 4°C protected
from light, and analyzed by flow cytometry. Results showed splenocytes
contained 2.33% NKT cells, as defined by CD1d/CD4 dual positivity.
Detection of murine NKT cells with CD1d Tetramer
life.science.discovery.™ mblintl.com life.science.discovery.™mblintl.com
50 51
MBL has been developing, manufacturing, and selling Tetramer reagents in Japan
since 2002. In 2013 MBL acquired an exclusive sublicense from Beckman Coulter
USA for the entire portfolio of iTAg™ MHC Tetramer products. MBL has worked
with a number of physicians and researchers on various immunological studies
centering on our core technology, Tetramer reagents. We are committed to building
our knowledge base and experience so that we may continue to provide high
quality products and support to customers using Tetramer products.
MHC Tetramer and CD1d products are for Research Use Only and not for use in
diagnostic procedures.
iTAgTM MHC Tetramer and MBL
0.00 % 31.9 %31.9 %
day 0 day 14
CTL
Tetramer
Induction Expansion
Detection Functional Assay
Tetr
amer
CD107a
non-specificpeptide
specificpeptide
100 % 0 % 65 % 35 %
α1
α2
α3
β2m
Tetramer References
Reagent Reference
gp100 Tetramer
Walker EB, et al., Clin Cancer Res 15: 2541-2551 (2009)
Frleta D, et al., J Immunol 182: 2766-2776 (2009)
Walker EB, et al., Clin Cancer Res 14: 5270-5283 (2008)
Shi H, et al., J Immunol 176: 2134-2141 (2006)
Morgan RA, et al., J Immunol 15: 3287-3295 (2003)
Mart-1 Tetramer
Uzana R, et al., J Immunol 188: 632-640 (2012)
Favre D, et al., Blood 117: 2189-2199 (2011)
Frleta D, et al., J Immunol 182: 2766-2776 (2009)
Takahara M, et al., J. Leukoc. Biol. 83: 742−754 (2008)
Weishaupt C, et al., Clin Cancer Res 13: 2549-2556 (2007)
Shi H, et al., J Immunol 176: 2134-2141 (2006)
Akiyama Y, et al., J. Transl. Med. 3: 4−13 (2005)
Tomiyama M, et al., Anticancer Res. 24: 3327−3334 (2004)
Tyrosinase TetramerShi H, et al., J Immunol 176: 2134-2141 (2006)
Akiyama Y, et al., J. Transl. Med. 3: 4−13 (2005)
PR-1 TetramerMorita Y, et al., Int. J. Cancer 119: 1360−1367 (2006)
Burchert A, et al., Blood 101: 259-264 (2003)
CMV Tetramer
Gratama JW, et al., Blood 116: 1655-1662 (2010)
Brooimans RA, et al., Cytometry A 73: 992-1000 (2008)
Chan WK, et al., J Immunol 191: 1625-1636 (2013)
Favre D, et al., Blood 117: 2189-2199 (2011)
Wang X, et al., Blood 117: 1888-1898 (2011)
Walker EB, et al., Clin Cancer Res 14: 5270-5283 (2008)
Watanabe K, et al., Int J Hematol. 88: 311−320 (2008)
Sabouri AH, et al., Blood 112: 2411−2420 (2008)
Heijnen IA, et al., Cytometry B Clin Cytom 62: 1-13 (2004)
Mohty M, et al., J Immunol 171: 3385-3393 (2003)
Gratama JW, et al., Blood, 98: 1358-1364 (2001)
EBV Tetramer
Shultz LD, et al., PNAS 107: 13022−13027 (2010)
Tomiyama M, et al., Anticancer Res. 24: 3327−3334 (2004)
Sato K, et al., Blood 117: 5663−5673 (2011)
Watanabe K, et al., Int J Hematol. 88: 311−320 (2008)
Influenza Tetramer
Frleta D, et al., J Immunol 182: 2766-2776 (2009)
Walker EB, et al., Clin Cancer Res 14: 5270-5283 (2008)
He XS, et al., J Infect Dis 197: 803-811 (2008)
Akiyama Y, et al., J. Transl. Med. 3: 4−13 (2005)
HIV Tetramer Pahwa R, et al., J Infect Dis 193: 879-887 (2006)
HTLV-1 Tetramer
Kozako T, et al., Hum Immunol. 72: 1001−1006 (2011)
Kozako T, et al., J. Med. Virol. 83: 501−509 (2011)
Tanaka Y, et al., Cancer Res. 70: 6181−6192 (2010)
Sabouri AH, et al., Blood 112: 2411−2420 (2008)
Akimoto M, et al., J. Med Virol. 79: 977−986 (2007)
Kozako T, et al., J. Immunol. 177: 5718−5726 (2006)
Human
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Tetramer References
Reagent Reference
HLA-A*24:02 Negative Tetramer
Akiyama Y, et al., Anticancer Res. 29: 647−655 (2009)
Tsukahara T, et al., Cancer Sci. 99: 368−375 (2008)
Akimoto M, et al., J. Med Virol. 79: 977−86 (2007)
Kozako T, et al., J. Immunol. 177: 5718−5726 (2006)
Akiyama Y, et al., J. Transl. Med. 3: 4−13 (2005)
Reagent Reference
OVA Tetramer
Kurachi S, et al., J. Exp. Med. 208: 1605−1620 (2011)
Nierkens S, et al., Cancer Res 71: 6428-6437 (2011)
Yanai H, et al., PNAS 108: 11542−11547 (2011)
Crozat K, et al., J Exp Med 207: 1283-1292 (2010)
Takeshima T, et al., Cancer Res. 70: 2697−2706 (2010)
He D, et al., J Immunol 184: 2281-2288 (2010)
Asano J, et al., J. Immunol. 184: 736−745 (2010)
Willmon CL, et al., Cancer Res 69: 7713-7720 (2009)
Bowers EV, et al., J Leukoc Biol 86: 1259-1268 (2009)
Senju S, et al., Stem Cells 27: 1021−31 (2009)
Tang C, et al., J. Leukoc. Biol. 86: 187−194 (2009)
Kijima M, et al., J. Immunol. 182: 3566−3572 (2009)
Miyakoda M, et al., J. Immunol. 181: 1420−1428 (2008)
Wakabayashi A, et al., J. Immunol. 180: 4000−4010 (2008)
Lou Y, et al., Clin Cancer Res 14: 1494-1501 (2008)
Kumar H, et al., J. Immunol. 180: 683−687 (2008)
Sugiyama T, et al., Int. Immunol. 20: 1−9 (2008)
Hao S, et al., J Leukoc Biol 82: 829-838 (2007)
Li W, et al., J. Immunol. 178: 4482−4488 (2007)
Zhang Y, et al., Int. Immunol. 19: 151−161 (2007)
Hao S, et al., Immunology 120: 90-102 (2007)
Wallace A, et al., Cancer Res 67: 7011-7019 (2007)
Haas AR, et al., Clin Cancer Res 12: 214-222 (2006)
Bertholet S, et al., J Immunol 177: 3525-3533 (2006)
Hao S, et al., Cell Mol Immunol 3: 205-211 (2006)
Taneichi M, et al., J. Immunol. 177: 2324−2330 (2006)
Loeffler DI, et al., Infect Immun 74: 3946-3957 (2006)
Saito K, et al., J. Immunol. 176: 2496−2504 (2006)
Chamoto K, et al., Cancer Res. 66: 1809−1817 (2006)
Yokouchi H, et al., Cancer Sci. 97: 148−154 (2006)
Wakita D, et al., Int. Immunol. 18: 425−434 (2006)
Yajima T, et al., J. Immunol. 176: 507−515 (2006)
Yajima T, et al., J. Immunol. 174: 3590−3597 (2005)
Li W, et al., Infect. Immun. 72: 7005−7011 (2004)
Teramoto K, et al., Cancer Res. 63: 7920−7925 (2003)
Kelly J, et al., 170: 210-217 (2003)
TRP2 Tetramer
Kuwada E, et al., Anticancer Res. 31: 881−891 (2011)
Takeshima T, et al., Cancer Res. 70: 2697−2706 (2010)
Okano F, et al., J. Immunol. 174: 2645−2652 (2005)
Reagent Reference
Influenza Tetramer
Nakamura R, et al., J. Virol. 84: 5574−5582 (2010)
Koyama S, et al., J. Immunol. 179: 4711−4720 (2007)
Seo S−U, et al., J. Virol. 84: 12713−12722 (2010)
Kayamuro H, et al., J. Virol. 84: 12703−12712 (2010)
LLO TetramerOzawa Y, et al., Am. J. Respir. Cell Mol. Biol. 41: 440−448 (2009)
Hayashi T, et al., J. Immunol. 182: 6360−6368 (2009)
LCMV Tetramer
Takagi A, et al., Clin. Vaccine Immunol. 16: 1383−1392 (2009)
Jung A, et al., J. Virol. 82: 196–206 (2008)
Kawagoe T, et al., J. Exp. Med. 204: 1013−1024 (2007)
Gil MP, et al., Blood 107: 987−993 (2006)
H-2Kb Negative Tetramer
Asano J, et al., J. Immunol. 184: 736−745 (2010)
Chamoto K, et al., Cancer Res. 66: 1809−1817 (2006)
Wakita D, et al., Int. Immunol. 18: 425−434 (2006)
Mouse CD1d Tetramer Yoshiga Y, et al., Clin Exp Immunol. 164: 236–247 (2011)
Reagent Reference
Mamu-A*01 SIV TetramerCecchinato V, et al., J. Immunol. 180: 5439−5447 (2008)
Mao H, et al., J Virol 79: 14887-14898 (2005)
Reagent Reference
Class I Custom Tetramer
Hofer MJ, et al., J Virol 86: 6932-6946 (2012)
Luo M, et al., J Virol 86: 1166-1180 (2012)
Takatsuka N, et al., Int. Immunol. 21: 1089−1100 (2009)
Tsukamoto T, et al., J. Virol. 83: 9339−9346 (2009)
Yoshizaki M, et al., Blood 114: 1518−1527 (2009)
Suemori K, et al., J. Gen. Virol. 90: 1806−1811 (2009)
Akiyama Y, et al., Anticancer Res. 29: 647−655 (2009)
Loisel-Meyer S, et al., Mol Cancer Ther 8: 692-702 (2009)
Jeng RR, et al., Blood 113: 1574-1580 (2009)
Harao M, et al., Int J Cancer 123: 2616−2625 (2008)
Vasir B, et al., J Immunol 181: 808-821 (2008)
Wakabayashi A, et al., J. Immunol. 180: 4000−4010 (2008)
Tsukahara T, et al., Cancer Sci. 99: 368−375 (2008)
Akimoto M, et al., J. Med Virol. 79: 977−86 (2007)
Kozako T, et al., J. Immunol. 177: 5718−5726 (2006)
Shi H, et al., J Immunol 176: 2134-2141 (2006)
Jalili A, et al., Blood 106: 3538−3545 (2005)
Akiyama Y, et al., J. Transl. Med. 3: 4−13 (2005)
Class II Custom Tetramer
Hofer MJ, et al., J Virol 86: 6932-6946 (2012)
Luo M, et al., J Virol 86: 1166-1180 (2012)
Takatsuka N, et al., Int. Immunol. 21: 1089−1100 (2009)
Tsukamoto T, et al., J. Virol. 83: 9339−9346 (2009)
Yoshizaki M, et al., Blood 114: 1518−1527 (2009)
Mouse
Non-Human Primate
Custom
life.science.discovery.™ mblintl.com
54
Tetramer References
Reagent Reference
Class II Custom Tetramer
Suemori K, et al., J. Gen. Virol. 90: 1806−1811 (2009)
Akiyama Y, et al., Anticancer Res. 29: 647−655 (2009)
Loisel-Meyer S, et al., Mol Cancer Ther 8: 692-702 (2009)
Jeng RR, et al., Blood 113: 1574-1580 (2009)
Harao M, et al., Int J Cancer 123: 2616−2625 (2008)
Vasir B, et al., J Immunol 181: 808-821 (2008)
Wakabayashi A, et al., J. Immunol. 180: 4000−4010 (2008)
Tsukahara T, et al., Cancer Sci. 99: 368−375 (2008)
Akimoto M, et al., J. Med Virol. 79: 977−86 (2007)
Kozako T, et al., J. Immunol. 177: 5718−5726 (2006)
Shi H, et al., J Immunol 176: 2134-2141 (2006)
Jalili A, et al., Blood 106: 3538−3545 (2005)
Akiyama Y, et al., J. Transl. Med. 3: 4−13 (2005)
Vukmanovic-Stejic M, et al., J Immunol 190: 977-986 (2013)
Scriba TJ, et al., J Immunol 175: 6334-6343 (2005)
Wong R, et al., Clin Cancer Res 10: 5004-5013 (2004)
Ardern-Jones MR, et al., PNAS 104: 5557-5562 (2007)
Van Overtvelt L, et al., J Immunol 180: 4514-4522 (2008)
Muthumani K, et al., J Virol 82: 11536-11544 (2008)
Scriba TJ, et al., J Immunol 175: 6334-6343 (2005)
Jahn-Schmid B, et al., J Immunol 181: 3636-3642 (2008)
Mizote Y, et al., Vaccine 28: 5338−5346 (2010)
Watanabe K, et al., Int J Hematol. 88: 311−320 (2008)
Reagent ReferenceIMMUNOCYTO Cytotoxity Detection Kit Tanaka Y, et al., Cancer Res. 70: 6181−6192 (2010)
IMMUNOCYTO CD107a Detection Kit Sabouri AH, et al., Blood 112: 2411−2420 (2008)
Anti-mouse CD8-FITC (clone KT15)Asano J, et al., J. Immunol. 184: 736−745 (2010)
Kayamuro H, et al., J. Virol. 84: 12703−12712 (2010)
Please refer to our web site for the latest information on products including MHC
Tetramer reagents, peptides for CTL induction, and custom construction of MHC
Tetramers. The attached document of each product can be downloaded.
MHC and CD1d Tetramer regents are for Research Use Only and not for use in
diagnostic procedures.
Support
We do more than sell products. All of our commercial teams possess advanced
scientific degrees and extensive laboratory experience. Our goal is to serve
as advisors and consultants to assist you in project planning and product
recommendations. To have your questions answered or to receive a pricing quote,
please contact our team.
By email: [email protected]
By phone: 1-800-200-5459, Option 1
By fax: 847-544-5051
For international customers, please contact your local distributor.
Please visit mblintl.com for a complete listing.
■SALES SUPPORT
MBL International Corporation recruits only the best scientific talent to provide
answers to all of your questions regarding the use and applications of our products.
We offer a variety of convenient methods to contact our technical support team.
By email: [email protected]
By phone: 1-800-200-5459, Option 3
■TECHNICAL SUPPORT
From order to delivery and at every point in between, our world-class customer
service team is dedicated to ensure that your orders are processed efficiently and
accurately. Customer service representatives are available.
By email: [email protected]
By phone: 1-800-200-5459, Option 8
(8:30am - 6:00pm EST, Monday-Friday)
By fax: 847-544-5051
Contact your local distributor for assistance and your inquiries will be directed
appropriately.
■CUSTOMER SUPPORT
US Patent Number 5,635,363Inventors: Altman JD, et al., (Stanford University)"Compositions and methods for the detection, quantitation and purification of antigen-specific T cells."
French Application Number FR9911133Inventors: Lang F, et al., (INSERM)"Means for detecting and for the purifying CD8+ T-lymphocyte populationsspecific for peptides presented in the HLA context."
License
Accessory Reagents
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