Fernandez 102408
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Do murine T cells express MHC-II? McFarland BJ, et al. PNAS July 2001 peptide-MHC-II complex (pMHC-II) Luis P. Fernández October 24, 2008
Transcript of Fernandez 102408
Do murine T cells express MHC-II?
McFarland BJ, et al. PNAS July 2001
peptide-MHC-II complex(pMHC-II)
Luis P. FernándezOctober 24, 2008
Otten LA et al (Acha-Orbea), J Immunol 2006
Naïve CD4 T cells pooled from B6, Balb/c, CBA and purified 99.5%
+ anti-CD3
T lymphocytes from most mammals express (synthesize) surface MHC II following activation by MHC II/peptide complexes, or mitogens (reviewed in (1)).
- Among rodents, rat activated T cells also express MHC II (2).
1. T. M. Holling, E. Schooten, P. J. van Den Elsen, Hum Immunol 65, 282-90 (2004).2. B. Reizis, C. Schramm, I. R. Cohen, F. Mor, Eur J Immunol 24, 2796-802 (1994).
Class II transactivator (CIITA):Master regulator of MHC-II expression
• Importantly, MHC-II expression is not regulated at the level of MHC-II genes themselves, but by several promoters that control the CIITA gene.
• Cell type-specific pattern of expression
• Temporal expression
• -interferon responsiveness
• Knockout of CIITA
• Gain-of-function:•plasma cell (plasmacytoma) lines•Bare lymphocyte syndrome cell lines•mouse T cells•CIITA-Tg mice
Gourley et al (Chang, Flavell), Immunity 1999Thomas et al (Christine Graham), J Immunol 1996
Activated mouse T cells express MHC-II
shared or unshared MHC between T-regs and T effectors
class Ia class II class Ia
Syn comb. (13)
Stim Suppr.
Allo APCs Yes
B10; B6 Allo APCs Yes
class IbQa-2 TL Q1
BALB/c; B6 Allo APCs No
BALB/c; B6 Anti-CD3 Yes
BALB.B; B6 Allo APCs Yes
A.SW; B6 Allo APCs No
B10.D2; B6 Allo-APCs No
B10.A; B6 Allo-APCs No
B10.A; B10.BR Allo-APCs Yes
B10.A; B10.MBR Allo APCs Yes
A.TH; A.TL
Allo APCsB10; B10.MBR Allo APCs No
No
Suppression of Th proliferation to allogeneic APC appears MHCII-restricted
MHC-II on activated mouse T cells
Mouse T cells capture MHC-II from APCs
MHC-II
MHC-II
MHC-II
APCCD4 T cell
TCRpeptide
Nagy et al (Pernis), J Exp Med 1976Sharrow et al (Sachs), J Immunol 1980Sharrow et al (Singer), J Immunol 1981Lorber et al (Fitch), J Immunol 1982Patel et al (Mannie), J Immunol 1999Tsang et al (Lechler), Blood 2003
Mouse T cells make MHC-II
MHC-II
MHC-II
MHC-II
APCCD4 T cell
TCRpeptide
Thomas et al (Graham), J Immunol 1996Gourley et al (Chang, Flavell), Immunity 1999
Tsang et al (Lechler), Blood 2003
DO11.10 CD4 T cells cocultured overnight with BM-DCs
CD4+ gate
Aims
• Verify expression of endogenous MHC-II on activated T cells from mouse spleen.
• Furnish evidence that activated T cells acquire MHC-II by capture from APCs.
• Do mouse T cells express the same type of MHC-II that is expressed on APCs?
Aim 1
• Verify expression of endogenous MHC-II on activated T cells from mouse spleen.
– Metabolic labeling and immunoprecipitation with MHC-II-specific antibodies
• Furnish evidence that activated T cells acquire MHC-II by Furnish evidence that activated T cells acquire MHC-II by capture from APCs.capture from APCs.
• Do mouse T cells express the same type of MHC-II that is Do mouse T cells express the same type of MHC-II that is expressed on APCs?expressed on APCs?
Metabolic labeling and immunoprecipitation:MHC-II biosynthesis
Starve cells in cysteine- and methionine-free media
Immunoprecipitate
Resolve
Detect
Solubilize protein
Metabolically label cells with 35S cysteine and methionine
Activate with PMA and ionomycin
Lyse
Splenocytes from B10A.5R (I-Ab, I-Eb/k)
191 kDa
97
64
51
39
28
19
14
I-Ab I-Ab I-E Pig CD8
B10A.5R (I-Ab, I-Eb/k)splenocytes (P+I stimulated)
18 h labeling(4-12%)PAGE
MHC-II
Biosynthesis of MHC-II in activated splenocytes
IP:
Antibodies used:
Lane 1: Y3PLane 2: 25-9-17Lane 3: 40DLane 4: isotype control
1 2 3 4
Pig CD8
B10A.5R (I-Ab, I-Eb/k) splenocytes (P+I stimulated)
18 h labeling10% PAGE
97
64
51
39
28
191 kDa
Improved resolution in 10% acrylamide gel
IP:
Antibodies used:
Lane 1: Y3PLane 2: 25-9-17Lane 3: 40DLane 4: isotype control
I-Ab I-Ab I-E
MHC-II
1 2 3 4
Ii
Pig CD8 Y3P
191 kDa
97
64
51
39
28
19
14
B10A.5R (I-Ab, I-Eb/k) CD4+ splenocytes
21 h labeling(10%)PAGE
MHC-II in enriched CD4 T cells
IP:
MHC-II
I-Ab I-Ab I-E
Antibodies used:
Lane 1: Y3PLane 2: 25-9-17Lane 3: 40DLane 4: isotype control
B220
CD
4
100
101
102
103
104
100
101
102
103
104
12.1 0.5
61.825.6
100
101
102
103
104
100
101
102
103
104
3.38
92.1 1.6
2.9
B220
CD
4
B10A.5R Spleen
CD4 MACS
Metabolic labeling and activation
Enriched CD4+ T cells
1 2 3 4
10 0 101 10 2 10 3 104
TCR
10 0
10 1
10 2
10 3
10 4
I-E
1.3 0.6
93.34.9
10 0 101 10 2 10 3 10410 0
10 1
10 2
10 3
10 4
0.1 0.1
99.40.43
MACS-enrichedCD4 T cells
(negative selection)Sorted by TCR
100 101 102 103 104
TCR
100
101
102
103
104
CD
25
0.01
6.1
93.40.47
100 101 102 103 104
TCR100
101
102
103
104
CD
25
1.36
93.5
4.820.33
Unactivated
21h PMA & iono
100 101 102 103 104
TCR
100
101
102
103
1040.24
0.1
99.40.24
100 101 102 103 104
TCR
100
101
102
103
1040.16
0.1
98.41.34
Activated
No MHC-II on purified CD4 T cells after activation
CBA
TCR
FACS sort
I-E
I-E
I-E
191 kDa
97
64
51
39
28
19
14
I-E Pig CD8
TCR+ PMA + iono 21hrs.
IP:
CBA TCR+ splenocytes21 h labeling10% PAGE
No endogenous biosynthesis of MHC-II on activated CD4 T cells
Antibodies used:
Lane 1: 40DLane 2: isotype control
1 2
MHC-II on activated T cells
Mouse T cells capture MHC-II from APCs
MHC-II
MHC-II
MHC-II
APCCD4 T cell
TCRpeptide
Nagy et al (Pernis), J Exp Med 1976Sharrow et al (Sachs), J Immunol 1980Sharrow et al (Singer), J Immunol 1981Lorber et al (Fitch), J Immunol 1982Patel et al (Mannie), J Immunol 1999Tsang et al (Lechler), Blood 2003
Mouse T cells make MHC-II
MHC-II
MHC-II
MHC-II
APCCD4 T cell
TCRpeptide
Thomas et al (Graham), J Immunol 1996Gourley et al (Chang, Flavell), Immunity 1999
Aim 2• Verify expression of endogenous MHC-II on activated T cells Verify expression of endogenous MHC-II on activated T cells
from mouse spleen.from mouse spleen.
• Furnish evidence that activated T cells acquire MHC-II by capture from APCs.
– MHCII-GFP mice* / FACS & confocal microscopy*Boes M, Nature 2002
– B10A.5R / Yae (E55-72/I-Ab)
• Do mouse T cells express the same type of MHC-II that is Do mouse T cells express the same type of MHC-II that is expressed on APCs?expressed on APCs?
Activated mouse T cells display surface MHC-II
CD4+ T cells (~90% pure) from MHCII beta-GFP mouse
with or without PMA + Ionomycin
20 hrsFACS
F
SC
-W
0 1000 2000 3000 4000
0
1000
2000
3000
4000
79
0 102 103 104 105
<PE-A>: B220
0
102
103
104
105
769
SSC-A
act
activated
<A
PC
-A>
: C
D2
5
0 102 103 104 105
GFP
0
20
40
60
80
100
T cells
B220neg (CD4+ T cells)
0 102 103 104 105
GFP
0
20
40
60
80
100
TCR+CD25+
MHCII WT
MHC-GFP0 1000 2000 3000 4000
0
1000
2000
3000
4000
88
0 102 103 104 105
0
102
103
104
105
<A
PC
-A>
: C
D2
5
578
Gate on singlets
No act
B220neg or B220+ cellsinactivated
F
SC
-W
0 102 103 104 1050
20
40
60
80
100
% o
f M
ax
B cells
B220+ (B cells)
GFP
act B_GFP + anti-TCR (red)
20 µm
Activated mouse T cells display surface MHC-II
CD4+ T cells (93% pure)or
B220+ B cells (95% pure)
PMA + Ionomycin
18 hrs
cytospin slidesconfocal microscopyfrom
IAβ–GFP mouse
act T_GFP + anti-TCR (red)
Activated mouse T cells display surface MHC-II by capture from APC
CD4+ T cells (~90% pure) from B6 mice
PMA + Ionomycin
20 hrsFACS+/- APC from MHCII-GFP(B6)
no APC
<A
PC
-A>
: C
D2
5
Gate on singlets TCR+ CD25+
F
SC
-W
0 1000 2000 3000 4000
0
1000
2000
3000
4000
72
0 102 103 104 105
0
102
103
104
105
24
with APC
FS
C-W
SSC-A 0 1000 2000 3000 4000
0
1000
2000
3000
4000
69
<PE-A>: TCR
<A
PC
-A>
: C
D2
5
0 102 103 104 105
0
102
103
104
105
15
0 102
103
104
105
GFP
0
20
40
60
80
100
% o
f M
ax
TCR+ CD25+
Activated T cells capture and re-expose surface MHC class-II/peptide complexes
CD4+ T cells (90% pure) from B10A.5R (IAb, IEb/k)
IAb & IEα pept 55-72
PMA + Ionomycin
21 hrsFACS
Ya-e
100
101
102
103
104
FL1-H: TCR FITC
100
101
102
103
104
FL4
-H: C
D25
AP
C
anti- Pig CD8 (76-2-11)anti- IAb (25-9-17)pan anti-IE (ISCR3)IAb + IEα peptide (Ya-e)
100 101 102 103 104
FL2-H: PEA
0
20
40
60
80
100
% o
f Max
TCRneg CD25+
ContYae IAb
IE
100 101 102 103 104
FL2-H: PE
0
20
40
60
80
100
% o
f Max
TCR+ CD25hi
Yae IAb
100 101 102 103 104
FL2-H: PE
0
20
40
60
80
100
% o
f Max
TCR+ CD25neg
Aim 3
• Verify expression of endogenous MHC-II on activated T Verify expression of endogenous MHC-II on activated T cells from mouse spleen.cells from mouse spleen.
• Furnish evidence that activated T cells acquire MHC-II by Furnish evidence that activated T cells acquire MHC-II by capture from APCs.capture from APCs.
• Do mouse T cells express the same type of MHC-II that is expressed on APCs?
– Use monoclonal antibody that recognizes MHC-II dimer for both FACS and IP
Do mouse T cells express the same type of MHC-II that is expressed on APCs?
100 101 102 103 104100
101
102
103
104
Streptavidin-PE
Biotinylate, IP, SDS-PAGE
100 101 102 103 104100
101
102
103
104
0
99.1
B2
20
TCR
Sort T cells to >99% purity
% o
f Max
100 101 10 2 103 1040
20
40
60
80
100
B220-TCR+
MHC-II (I-Ab)
MHC-II FACS(25-9-17 Ab)
Stimulate T cells and APCs with PMA+iono
100 101 102 103 104
I-Ab
100
101
102
103
104
TC
R
83.9
3.5
100 101 102 103 104
I-Ab
100
101
102
103
104
TC
R
84.5
Enriched CD4 T cells
Enriched APCs
B6 spleen
100 101 102 103 104100
101
102
103
104
13.6
61.5
T cells
100 101 102 103 1040
20
40
60
80
100
% o
f M
ax
B cells
IP:E = I-Ab
C = Pig CD8
100 101 102 103 104100
101
102
103
104
57
Pre-FACS sort Post-FACS sort
10% PAGE
MHC-II (I-Ab)
B2
20
B2
20
TCR TCR TCR
B2
20
100 101 102 103 104100
101
102
103
104
98.5
100 101 102 103 1040
20
40
60
80
100
TcR+
E
APC
C
105# cells per lane:
C E
APC
E
105 5 x 106
% o
f M
ax
MHC-II
Sort for TCR+B220-
100 101 102 103 1040
20
40
60
80
100
% o
f Max
B220-TCR+
100 101 102 103 104100
101
102
103
104
0
99.1
E C E C
Sorted CD4+ APC
TCR
B2
20
100 101 102 103 104100
101
102
103
104
93.2
100 101 102 103 1040
50
100
150
# C
ells
B220+TCR-
B2
20
MHC-II (I-Ab)
Post-FACS sort B cells
Sort for TCR+MHCII-/lo
IP:E = I-Ab
C = Pig CD8MHC-II
TCR
MH
C-I
I (I
-Ab)
Pre-FACS sort
Biotinylated MHC-II cannot be detected on activated CD4 T cells
191 kDa
97
64
51
39
28
19
14
Unsorted splenocytes MHC-II KO
IP:
TCR+ P+I 16h
APC P+I 16h
B6
12% Bis-Tris gel
2-m(12 kDa)
MHC-I HC(45 kDa)
Antibodies used:
20-8-4 = H-2Kb. Db
25-9-17 = I-Ab
Isotype control = Pig CD8
I-AbPigCD8
PigCD8
PigCD8K/Db K/Db K/Db
Biotinylated MHC-I can be detected on activated CD T cells
Conclusion
• Verify expression of endogenous MHC-II on activated T cells from mouse spleen.– No endogenous expression of MHC-II
• Furnish evidence that activated T cells acquire MHC-II by capture from APCs.– Activated mouse T cells express or acquire MHC-II
exclusively by capture as pMHC complexes
• Do mouse T cells express the same type of MHC-II that is expressed on APCs?– Yes, but MHC-II on T cells is less stable than on APCs
Acknowledgments
Sharon GermanaChristian LeGuernKaela Goldstein
Marianne BoesDavid Dumkoswki
Taroko GorgeTaiwanMay 2008
0
20
Cel
l nu
mb
er (
x 10
6 )
After MACS
After o/n P+I activation
TCR+ cells collected from sorting
TCR+ cells available for biotinylation
TCR+ cells after biotinylation
Diminishing returns:CD4 T cells from 12 B6 mice
40
60
80
100
120
140
