for research use only mhc class ii tetramer · antibody recognizing mouse i-ad / ova 323-339...
TRANSCRIPT
B cell
IL-4IL-5IL-10IL-13
Antibody production
CD4+ T
Th2
Th17
Th1 Th1
Treg
CD4
TGF-βIL-12
IL-4
IL-17
IL-6+
TGF-β
Lysosome
MHC class II
MHC class II tetramers can be used for direct detection of antigen-speci�c CD4+ T cells.
Biotinylated MHC/peptide complexes are tetramerized with fluorescently-labeled streptavidin, increasing the avidity of MHC/peptide complexes binding to TCR and allowing for direct detection of antigen-specific T cells.
I-Ab MOG35-55 tetramer-PEExperimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis induced by immunization with MOG35-55 peptide, is a widely used experimental model of autoimmune disease. It has been reported that regulatory T (Treg) cells and Th17 cells are involved in the onset of disease, suggesting that the immune balance of CD4+ T cells is important in EAE pathogenesis.
C57BL/6 mice were immunized intraperitoneally twice with 100 nmol of I-Ab-restricted MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK, MBL code no. TS-M704-P) and 10 μg cholera toxin (MBL code no. RK-01-511) in complete Freund's adjuvant. Eleven days later, splenocytes were isolated and stained with I-Ab MOG35-55 tetramer-PE (MBL code no. TS-M704-1; Day 0). An aliquot of splenocytes was stimulated with 10μg/mL MOG35-55 peptide for 6 days in vitro and then stained with MHC class II tetramer (Day 6). In vitro stimulation of with I-Ab MOG35-55 resulted in increased tetramer-positive CD4+ T cells from immunized (mouse 1 and 2), but not naïve (mouse 3), mice.
Mouse MHC class II Tetramer
Dendritic cell
MHC Tetramer
mouse 1
mouse 3
mouse 2
CD4 (GK1.5)-FITC CD4 (GK1.5)-FITC
Non-immunized splenocytes
MOG35-55 immunized splenocytes
In vitro MOG35-55
peptide stimulation
MOG35-55 immunized splenocytesDay 0
I-Ab M
OG
35-5
5 Tet
ram
er-P
E
0.17 % 0.22 %
0.05 %
mouse 1
mouse 3
mouse 2
Non-immunized splenocytes
Day 6
I-Ab M
OG
35-5
5 Tet
ram
er-P
E
13.4 % 9.43 %
0.05 %
Code no.
TS-M704-1
TS-M704-P
Product
T-Select I-Ab MOG35-55 Tetramer-PE
I-Ab MOG35-55 Peptide
Size
20 tests
100 μL (10 mg/mL)
Code no.
D341-4
Product
Anti-CD4 (Mouse) mAb-FITC
Size
1 mL (100 tests)
Clone
GK1.5
Isotype
Rat IgG2bκ
MHC
Peptide
Fluorochrome
TCRAntigenspecificT cells
β-chain
α-chain
For Research Use Only
http://ruo.mbl.co.jp/
I-Ab OVA323-339 tetramer-PEOVA 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 1-1). In vitro stimulation with specific peptide significantly increased the number of tetramer positive cells (Figure 1-2).
■ Figure 2: I-Ab OVA323-339 tetramer staining of splenocytes from peptide-immunized mice
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. 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.
OVA323-339 peptide immunized mouse splenocytes
Day 0 Day 8
I-Ab O
VA32
3-33
9
Tetra
mer
-PE
0.5 % 1.4 %
I-Ab M
OG
35-5
5 Te
tram
er-P
E
0.2 % 0.2 %
CD4 (GK1.5)-FITC
Code no.
TS-M710-1
TS-M703-P
Product
T-Select I-Ab OVA323-339 Tetramer-PE
I-Ad OVA helper peptide
Size
20 tests
100 μL (10 mg/mL)
Tetra
mer
-PE
I-Ab MOG35-55 Tetramer
1.2 %
I-Ab OVA323-339 Tetramer
1.9 %
OVA323-339 peptide
concentration
0 μg /mL
1 μg /mL
1.5 % 79.2 %
CD4 (GK1.5)-FITC
I-Ab O
VA32
3-33
9 Te
tram
er-P
E
mouse 16.4 %
mouse 275.2 %
CD4 (GK1.5)-FITC
I-Ab M
OG
35-5
5 Te
tram
er-P
E
0.5 %
CD4 (GK1.5)-FITC
< mouse 1 >
■ Figure 1-1: I-Ab OVA323-339 tetramer staining of freshly isolated OT-II splenocytes
■ Figure 1-2:I-Ab OVA323-339 tetramer staining of peptide-stimulated OT-II splenocytes (Day 6)
Antibody recognizing Mouse I-Ad / OVA323-339 specific TCR 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 (ISQAVHAA-HAEINEAGR, MBL code no. TS-M703-P) is often used as a helper peptide for induction of antigen-specific CTL in I-Ad-expressing mice.
■ Flow cytometry with Anti-mouse TCR DO11.10 mAb
Clear Back blocking reagent for tetramer stainingWhen 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 peripheral blood mononuclear cells (PBMC) with HLA-A*24:02 EBV BRLF1 tetramer in the tetramer-positive CD8-negative region (Figure 2).
■ Effect of Clear Back on PBMCs
0.3 %0.0 %
FL2
coun
ts
isotypic controlAnti-mouse TCRDO11.10
Anti-
mou
se T
CR
D
O11
.10-
PE
CD4 (GK1.5)-FITCCD4 (GK1.5)-FITC
Isot
ypic
con
trol-P
E
Sample: DO11.10 T cell hybridoma Sample: OVA323-339 peptide immunized BALB/c mouse splenocytes
I-A alleles of mouse strains:I-A allele
Mouse strains
I-Ak
C3H/He
I-Ab
C57BL/-,BXSB/Mp,
129/-
I-Ad
BALB/c,DBA/2
I-As
SJL/J,B10.S
Code no.
K0221-5
Product
Anti-TCR DO11.10 (Mouse) mAb–PE
Isotype
Mouse IgG2aκ
Clone
KJ1.26
Size
1 mL (50 tests)
Code no.
MTG-001
Product
Clear Back (Human Fc receptor blocking reagent)
Size
1 mL (50 tests)x2
SSC
-Hco
unts
coun
ts
FSC-H
FL1-H FITC FL1-H FITC
R1R2
PBMCs were stained with mouse IgG2a-FITC(5 μg/mL)
Red : Clear Back treatmentGreen : No Clear Back treatment
Lymphocytes (R1) Monocytes (R2)
Non-specific binding of antibody was not detected on lymphocyte (R1). Clear Back treatment reduced unwanted Fc-specific staining of monocyte (R2) by a mouse IgG2a.
Tetra
mer
-PE
CD8 (T8)-FITC
Clear Back without Clear Back
Clear Back treatment reduced non-specific binding of a tetramer reagent to tetramer-positive CD8-negative cell population.
Figure 1 Figure 2
Antigen
MOG35-55
FMLV
Eα52-68
ESAT-6
OVA
MHC
I-Ab
I-Ab
I-Ab
I-Ab
I-Ab
Sequence
MEVGWYRSPFSRVVHLYRNGK
EPLTSLTPRCNTAWNRLKL
ASFEAQGALANIAVDKA
MTEQQWNFAGIEAAASAIQG
ISQAVHAAHAEINEAGR
Location(aa)
35-55
123-141
52-68
1-20
323-339
Peptide100 μL (10 mg/mL)
TS-M704-P
—
—
TS-M707-P
TS-M703-P
PE-labeled20 tests
TS-M704-1
TS-M705-1
TS-M706-1
TS-M707-1
TS-M710-1
APC-labeled20 tests
■ Mouse MHC class II Tetramer
■ Mouse class II epitope peptide
T-Select Mouse MHC class II Tetramers & related products
Contact us
Antigen
OVA
OVA E1
OVA G4
OVA Q4H7
MHC
H-2Kb
H-2Kb
H-2Kb
H-2Kb
Sequence
SIINFEKL
EIINFEKL
SIIGFEKL
SIIQFEHL
Location(aa)
257-264
257-264
257-264
257-264
Peptide100 μL (10 mg/mL)
TS-5001-P
—
—
—
PE-labeled50 tests
TS-5001-1C
TS-M541-1
TS-M542-1
TS-M543-1
APC-labeled50 tests
TS-5001-2C
TS-M541-2
TS-M542-2
TS-M543-2
■ Mouse MHC class I OVA Tetramer
Product
T-Select Mouse CD1d Tetramer
PE-labeled50 tests
TS-MCD-1
PE-labeled10 tests
TS-MCD-1S
APC-labeled50 tests
TS-MCD-2
■ Mouse CD1d Tetramer
Origin
HBc
TT p30
OVAMOG
ESAT-6
HEL
MHC
I-Ab
I-Ad
I-Ab, I-Ad
I-Ab
I-Ab
I-Ak
Size
1 mg
1 mg
1 mg
1 mg
1 mg
1 mg
Sequence
TPPAYRPPNAPIL
FNNFTVSFWLRVPKVSASHLE
ISQAVHAAHAEINEAGR
MEVGWYRSPFSRVVHLYRNGK
MTEQQWNFAGIEAAASAIQG
DGSTDYGILQINSRW
Location(aa)
128-140
947-967
323-339
35-55
1-20
48-62
Code no.
TS-M701-P
TS-M702-P
TS-M703-P
TS-M704-P
TS-M707-P
TS-M708-P
■ AntibodyClone
GK1.5
KJ1.26
KJ1.26
KJ12.98
3G8
6H3
6H3
Size
1 mL (100 tests)
100 μg
1 mL (50 tests)
100 μg
50 μg/1 mL
100 μg/100 μL
10 μg/1 mL
Product
Anti-CD4 (Mouse) mAb-FITC
Anti-TCR DO11.10 (Mouse) mAb
Anti-TCR DO11.10 (Mouse) mAb–PE
Anti-TCR 3DT-52.5 (Mouse) mAb
Rat IgG2b (isotype control)-FITC
Mouse IgG2a (isotype control)
Mouse IgG2a (isotype control)-PE
Isotype
Rat IgG2bκ
Mouse IgG2aκ
Mouse IgG2aκ
Mouse IgG2aκ
Rat IgG2bκ
Mouse IgG2aκ
Mouse IgG2aκ
Code no.
D341-4
K0221-3
K0221-5
K0222-3
M090-4
M076-3
M076-5
■ ReagentSize
150 tests
1 mL (50 tests)x2
250 tests
200 tests
Product
7-AAD Viability Dye
Clear Back (Human Fc receptor blocking reagent)
OptiLyse B
OptiLyse C
Code no.
A07704
MTG-001
IM-1400
A11895
■ KitSize
50 tests
Product
IMMUNOCYTO Cytotoxity Detection Kit
Code no.
AM-1005
146064-140512014.05
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