Post on 28-Mar-2015
V14iNKT
mu: V14-J18hu: V24-J18
mu: V8.2/V7hu: V11
NK1.1
CD1d
Endogenous ligand: Isoglobotrihexosylceramide (iGb3)Foreign ligand: Microbial a-glycuranosylceramidesArtificial ligand: a-galactosylceramide (-GalCer)
IFN- IL-4
Autoimmune diseasesInfectious diseasesTumors
An Introduction to NKT cells
CD44high
CD69high
Ly49
( DX5)
( CD4)
CDR3 diverse
CD1 molecules present glycolipids
*Moody DB, Zajonc DM, Wilson IA. Nat Rev Immunol. 2005;5:387-399
CD1 family represents 5 MHC class I like molecules: CD1a, b, c, d, e
CD1 grooves provide “shoe-like” cavity serving to anchor the lipid antigens and to shield them from the aqueous environment
In contrast to MHC class I and II genes, allelic variation of CD1 genes is extremely limited
In mice only CD1d molecule is present
Developmental pathway of Va14i NKT cells
Thymus
Periphery
CD4- CD8-
TCD8+
CD4+ CD8+
TCD4+
TCD8+
TCD4+
MHC I MHC II
NKTCD1d
NKT
NKT NKT?
MacDonald H.R., Science, 2002
TCRNK1.1
TCR
TCR TCRNK1.1
DEVELOPMENT AND SELECTION OF Va14i NKT CELLS
• Cellular requirements for positive and negative selection
• Role of Vb domain in selection by endogenous glycolipids
• Role of c-myc in Va14i NKT cell development
V14iNKT
V14-J18 V8.2/V7
NK1.1
GalCermuCD1d
BiotinStreptavidin-fluorochrome
V14iNKT
V14-J18 V8.2/V7
NK1.1
GalCer
muCD1d/huCD1d
Specific Identification of Va14i NKT cellsTetramers Dimers
M1 M153 ± 4 58 ± 2
M1 M116 ± 2 12 ± 2
Co
un
tsC
ou
nts
R3
28
Thymus
mo
use
dim
ers R3
17
Liver
TCR-
R3 R3
12 9
M1 M180 ± 7 84 ± 4
M16 ± 5 M14 ± 1
V8.2
V7
Co
un
tsC
ou
nts
Thymus
hu
man
dim
ers
Liver
TCR-
V8.2
V7
Human CD1d:GalCer dimers bind preferentially to V14i NKT cells expressing V
Targeted expression of human CD1d in transgenic mice
• CD1d expression in CD4+CD8+ (DP) thymocytes driven by lck proximal promoter
• CD1d expression in thymic dendritic cells driven by CD11c promoter
• Monitor human CD1d-reactive ( Vb8.2+) Va14i NKT cells on CD1d-/- background (positive selection) or CD1d+/- background (negative selection)
muCD1d-/-
% V
8.2 % V
7
0
20
40
60
80 V?.V?7
0
5
10
15
0
5
30
. .n d. .n d
non-tgmuCD1d+/-
pLck-huCD1d-tgmuCD1d-/-
CD11c-huCD1d-tgmuCD1d-/-
DP thymocytes but not DC expressing huCD1dpositively select V8.2+ V14i NKT cells
muCD1d+/-
0
20
40
60
80V?.V?7
0
5
10
15
0
5
30
non-tgmuCD1d+/-
pLck-huCD1d-tgmuCD1d+/-
CD11c-huCD1d-tgmuCD1d+/-
% V
8.2
% V
7
Both DP thymocytes and DC expressing huCD1dnegatively select V8.2+ V14i NKT cells
CONCLUSIONS FROM Human CD1d TRANSGENIC MICE
• Human CD1d bound to mouse endogenous glycolipid ligands selects preferentially Vb8.2 Va14i NKT cells (like human CD1d bound to aGalCer),implying that residues on Vb8.2 interact preferentially with human CD1d
• DP thymocytes expressing human CD1d are sufficient to induce both positive and negative selection of developing Va14i NKT cells
• Thymic DC expressing human CD1d are sufficient to induce negative but not positive selection of developing Va14i NKT cells
• Thymic DC induce negative selection of developing Va14i NKT cells more efficiently than DP thymocytes
-Galactosylceramide (GalCer) serves as a model CD1d antigen
GalCer is a glycosphingolipid found in marine sponge and has no known physiological function in mammalian immunity
Isoglobotrihexosylceramide (iGb3) has been demonstrated as an endogenous agonist for CD1d restricted T cells
Role of Vb domain in selection of Va14i NKT cells by CD1d-binding endogenous glycolipids
R3
R4R5
R6
R2
R3
R4R5
R6
R2
mo
use
dim
ers
Thymus Liver
30
40
50
60
70
R6 R5 R4 R3
R2
Gate
30
40
50
60
70
R6 R5 R4 R3
R2
Gate
% V
8.2
+
0
10
20
30
40
R6 R5 R4 R3
R2
0
10
20
30
40
R6 R5 R4 R3
R2
% V
7+
Gate
Higher avidity binding of mouse CD1d:GalCer dimersby V14i NKT cells expressing V
TCR-
R2
R3
M1
T
V14i NKT
M1
TCR-
mo
use
dim
er
V8.2
matureV14i NKT 50 ± 1
M1
V7
14 ± 3
matureV14i NKT
R2
DP
CD4
CD
8
M1
V8.2 (ic)
9.4 ± 0.5
DP
M12.7 ± 0.3DP
NK1.1
0
2
4
6
8
10
12
0
20
40
60
% V
8.2
or
V7
(ic
)
% V
8.2 or V
7
DP thymocytes
mature V14i NKT cells
V8.2V7
Vb/b
J18+/-
Vb/b
J18+/+
Va/b
J18+/+
Vb/b
J18+/+
V8.2 (ic)V7 (ic)
V7 (ic)
Frequency of thymic V7+ and V8.2+ V14i NKT cellsreflects V rearrangement frequency in CD4+ CD8+ precursors
CD1d
CD4
R3
CD
8
DP thymocytes
DP
MFI,122 ± 15
MFI,59 ± 7
CD1d+/- CD1d+/+
0
20
40
60
80
100
0
5
10
15
20
25
*
% V
7
% V
8.2
Vb/b
V8.2V7
V8.2 (ic)V7 (ic)
% V
7
*
0
10
20
30
40
50
60
CD1d+/+
DP thymocytes
CD1d+/- CD1d+/+
matureV14i NKT cells
CD1d+/-
V7 (ic) V7
Va/a
Preferential Selection of Vb7 NKT Cells at Limiting CD1d:endogenous ligand Concentration in vivo
TCR-
mouse dimer
mouse tetramer
TCR-
NK
1.1
non-tg huV24-tg
R2
R3
NKT
non-NKT
R2
R3
NKT
non-NKT
M1
NKT
non-NKT
88 ± 5 M1non-NKT
NKT
21 ± 10
M1
NKT
non-NKT
81 ± 7 M1
NKT
non-NKT
7 ± 3
0
10
20
30
40
50
60
% V
8.2
or
V7
te
tram
er+
non-tg
dimer
+
dimer
+
DP DN DP
huV24-tg non-tg huV24-tg
V8.2V7
V8.2 (ic)V7 (ic)
NKTNKT
Preferential Selection of Vb7 NKT Cells in Va24 Transgenic Mice expressing a low avidity invariant TCRa chain
CD1d+/+
0
100
200
300
d0 d7 unstim d7 iGb310000 ng/ml
%Dimer+ Tcells
%Vb8.2+Dimer+
%Vb7+Dimer+
CD1d-/-
0
100
200
300
d0 d7 unstim d7 iGb310000 ng/ml
%Dimer+ Tcells
%Vb8.2+Dimer+
%Vb7+Dimer+
Vb7+ NKT cells are preferentially selected by endogenous ligands or exogenous self-ligand iGb3 in thymic culture
cell
exp
ansi
on
(%
)
cell
exp
ansi
on
(%
)
Role of Vb domain in selection of Va14i NKT cells by CD1d-binding glycolipids
• Vb8.2 binds the artificial agonist ligand aGalCer better than Vb7
• Vb7 binds endogenous ligands (including iGb3) better than Vb8.2
• Vb DOMAIN CONTRIBUTES TO GLYCOLIPID BINDING
From: Murphy MJ, Wilson A, Trumpp A. Trends Cell Biol 2005;15:128-137
Diverse functions of the proto-oncogene c-Myc
c-Myc deficiency in vivo
Conventional c-Myc deficiency is embryonic lethal*Trumpp A, Refaeli Y, Oskarsson T, Gasser S, Murphy M, Martin GR, Bishop JM. 2001. Nature 2001; 414: 768-773
*Baudino TA, McKay C, Pendeville-Samain H, Nilsson JA, Maclean KH, White EL, Davis AC, Ihle JN, Cleveland JL. Genes & Dev. 2002; 16:2530-2543
Conditional elimination of c-Myc in bone marrow (Mx cre;c-Myc flox/flox mice) results in failure to initiate normal stem cell differentiation*Wilson A, Murphy MJ, Oskarsson T, Kaloulis K, Bettess MD, Oser GM, Pasche AC, Knabenhans C, Macdonald HR, Trumpp A. Genes Dev. 2004;18:2747-2763
Haploinsufficiency of c-Myc leads to a significant decrease in the CD8 memory T cell population*Bianchi T, Gasser S, Trumpp A, Macdonald HR. Blood. 2006
c-Myc +/+ c-Myc +/-
Liv
er
Th
ymu
s 57% 28%
15%29%
0
150
300
c-Myc+/+ c-Myc+/-
x10
e3
Dimer positive cell number:Thymus
Dimer positive cell number:Liver
0
200
400
c-Myc+/+ c-Myc+/-
x10
e3
TCRb
Dim
er
TCRb
Dim
er
Reduced numbers of Va14i NKT cells in c-myc
haploinsufficient mice
CD4cre- CD4cre+ CD4cre+c-Myc fl/fl c-Myc fl/wt c-Myc fl/fl4myc +/+ 4myc +/- 4myc -/-
T
hym
us
38% 6%44%
0
250
500
4myc+/+ 4myc+/- 4myc-/-
0
150
300
4myc+/+ 4myc+/- 4myc-/-
Dimer positive T cell number:Thymus
Dimer negative T cell number:Thymus
x10e
3
x10e
5TCRb
Dim
er
T-cell specific conditional deletion of c-myc leads to a
dramatic and selective reduction in thymic Va14i NKT cells
IL15+/+ IL15+/- IL15-/-
Liv
er
T
hym
us
30% 5%
33% 18% 5%
0
200
400
IL15+/+ IL15+/- IL15-/-
Dimer positive cell number:Thymus
x10
e30
400
800
IL15+/+ IL15+/- IL15-/-x1
0e3
Dimer positive cell number:Liver
51%
TCRb
Dim
er
TCRb
Dim
er
Requirement for IL-15 in Va14i NKT cell development
IL15+/+ IL15+/+ IL15+/- IL15+/-c-Myc+/+ c-Myc+/- c-Myc+/+ c-Myc+/-
Th
ymu
s
56% 41% 7%43%
0
150
300
c-Myc+/+ c-Myc+/- IL15+/- c-Myc+/-IL15+/-
Dimer positive cell number:Thymus
x10
e3
TCRb
Dim
er
Synergistic reduction in Va14i NKT cells in combined c-myc and IL-15 haploinsufficiency
Preliminary conclusions (c-myc)
• C-myc plays a crucial cell-autonomous role in Va14i NKT cell development
• C-myc may be involved in IL-15 responsiveness of developing Va14i NKT cells
• Va14i NKT cells share properties with CD8 memory T cells