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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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6 7

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.


8 9

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 *


10 11

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



Tet

ram

er-P

E

Tet

ram

er-P

E

Tet

ram

er-P

E

Tet

ram

er-P

E

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





Tet

ram

er-P

E

Tet

ram

er-P

E

Tet

ram

er-P

E

Tet

ram

er-P

E

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


12 13


12 13

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.



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.


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.



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.


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


14 15

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

ram

er

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

ram

er

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


16 17


16 17

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

ram

er-P

E

Negative

0.00 %

0.00 %0.01 %

0.01 %0.06 %

EBV mix

0.25 %

0.17 %

Donor 4 Donor 8Donor 6


A*02:07A*02:06A*02:01

0.00 %6.64 % 3.33 %

0.00 %0.01 %0.17 %

CD8-FITC

Tet

ram

er-P

E


18 19


18 19

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



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

ram

er-P

E

CD8-FITC

20.3 %

0.00 % 1.37 %

31.9 %20.3 % 31.9 %

Tet

ram

er-P

E

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


0.00 %0.02 % 0.01 %

Tet

ram

er-P

E

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:


20 21


20 21

Tet

ram

er-P

E

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


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

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



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


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)


22 23


22 23

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


24 25

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


26 27

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)


28 29

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)


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 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%


DAY0

DAY7

Figure 2

CD8-FITC




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%


DAY0

DAY7


30 31


30 31

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


NegativeTetramer


32 33

CD8-FITC

Tet

ram

er-P

E


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)


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%


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)


NegativeTetramer


NegativeTetramer


♦Bacterial antigen

♦Cancer antigen

♦Minor antigen♦Model antigen


34 35


34 35

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


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


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.


36 37


36 37

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



38 39


38 39

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


CD8 (KT15)-FITC

Tet

ram

er-P

E

CD8 (53.6.7)-FITC

Tet

ram

er-P

E

AntibodyVolume

AntibodyVolume


10 μL

2.5 μL

0.63 μL

10 μL

2.5 μL

0.31 μL


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


40 41


40 41

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.


42 43


42 43

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


44 45


44 45


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 :


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



48 49


48 49

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


0.42 % 2.33 %

0.05 %0.00 %


CD3-FITC

Hu

man

CD

1d T

etra

mer

-PE

CD4 (GK1.5)-FITC

Mo

use

CD

1d T

etra

mer

-PE


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


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)


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)


Takahara M, et al., J. Leukoc. Biol. 83: 742−754 (2008)

Weishaupt C, et al., Clin Cancer Res 13: 2549-2556 (2007)


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)


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)


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)


Influenza Tetramer



He XS, et al., J Infect Dis 197: 803-811 (2008)


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

mblintl.com

Please refer to our website for the most updated FAQs


52 53

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)




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)


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)






Jalili A, et al., Blood 106: 3538−3545 (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


54

Tetramer References

Reagent Reference

Class II Custom Tetramer

Suemori K, et al., J. Gen. Virol. 90: 1806−1811 (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)






Jalili A, et al., Blood 106: 3538−3545 (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)


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 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 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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MEDICAL & BIOLOGICAL LABORATORIES CO.LTDKDX Nagoya Sakae Bldg. 10F, 4-5-3 Sakae, Naka-Ku, Nagoya, Aichi, 460-0008 Japan Tel: +81-52-238-1901Fax: +81-52-238-1440 www.mbl.co.jp

MBL International 15A Constitution Way Woburn, MA 01801 USATel: 1-800-200-5459mblintl.com





Documents

Tetramer Staining Guide - MoBiTec · The Tetramer Staining Guide is intended to highlight key and featured products. Please refer to ... H-2Kb OVA Tetramer 36 H-2Kb OVA Tetramer 36