Transcript Document

An Introduction to NKT cells
CD1d
Endogenous ligand: Isoglobotrihexosylceramide (iGb3)
Foreign ligand: Microbial a-glycuranosylceramides
Artificial ligand: a-galactosylceramide (a-GalCer)
mu: Va14-Ja18
hu: Va24-Ja18
mu: Vb8.2/Vb7
hu: Vb11
( CD4)
CD44high
CD69high
NK1.1
Va14iNKT
Ly49
(
IFN-g
DX5)
IL-4
CDR3b diverse
Autoimmune diseases
Infectious diseases
Tumors
CD1 molecules present glycolipids
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
*Moody DB, Zajonc DM, Wilson IA. Nat Rev Immunol. 2005;5:387-399
Developmental pathway of Va14i NKT cells
CD4- CD8TCR
TCR
NK1.1
Thymus
CD4+ CD8+
MHC I
T
CD8+
Periphery
T
CD8+
CD1d
NKT
NKT
TCR
TCR
MHC II
T
CD4+
T
CD4+
NKT
?
MacDonald H.R., Science, 2002
NK1.1
NKT
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
Specific Identification of Va14i NKT cells
Tetramers
Streptavidinfluorochrome
Dimers
Biotin
muCD1d/
huCD1d
muCD1d
Va14-Ja18
aGalCer
Vb8.2/Vb7
aGalCer
Va14-Ja18
Vb8.2/Vb7
NK1.1
Va14iNKT
NK1.1
Va14iNKT
Human CD1d:aGalCer dimers bind preferentially
to Va14i NKT cells expressing Vb8.2
Thymus
R3
R3
28
17
human dimers
Liver
R3
12
M1
58
±2
M1
80
±7
12M1± 2
Vb7
M1
84
±4
Vb8.2
Vb8.2
M1
16
±2
9
TCR-b
Counts
M1
53
±4
Liver
R3
TCR-b
Counts
Counts
Counts
mouse dimers
Thymus
6M1± 5
4M1± 1
Vb7
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)
DP thymocytes but not DC expressing huCD1d
positively select Vb8.2+ Va14i NKT cells
muCD1d-/-
30
Vb8.2
Vb7
25
60
20
40
15
10
20
5
n.d.
0
non-tg
muCD1d+/-
n.d.
pLckCD11chuCD1d-tg huCD1d-tg
muCD1d-/- muCD1d-/-
0
% Vb7
% Vb8.2
80
Both DP thymocytes and DC expressing huCD1d
negatively select Vb8.2+ Va14i NKT cells
muCD1d+/80
Vb8.2
Vb7
30
60
20
40
15
10
20
5
0
non-tg
muCD1d+/-
pLckhuCD1d-tg
muCD1d+/-
CD11chuCD1d-tg
muCD1d+/-
0
% Vb7
% Vb8.2
25
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
Role of Vb domain in selection of Va14i NKT cells by
CD1d-binding endogenous glycolipids
a-Galactosylceramide (aGalCer) serves as a model
CD1d antigen
aGalCer 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
Higher avidity binding of mouse CD1d:aGalCer dimers
by Va14i NKT cells expressing Vb8.2
mouse dimers
Thymus
Liver
R2
R2
R3
R3
R4
R5
R4
R5
R6
R6
TCR-b
% Vb8.2+
70
70
60
60 R2
R2
50
50
40
40
30
R6
% Vb7+
40
R5
R4
30
R3
R6
40
Gate
R5
R4
R3
Gate
30
30
20
R2
20
R2
10
0
10
R6
R5
R4
R3
0
Gate
R6
R5
R4
R3
Frequency of thymic Vb7+ and Vb8.2+ Va14i NKT cells
reflects Vb rearrangement frequency in CD4+ CD8+ precursors
CD4
NK1.1
T
R3
TCR-b
DP
9.4 ± 0.5
mature
Va14i NKT
50 ± 1
% Vb8.2 or Vb7 (ic)
DP
12
10
Vb8.2
2.7 ± 0.3
Vb7 (ic)
DP
mature
Va14i NKT
14 ± 3
Vb7
Vb8.2
Vb7
8
60
40
6
4
20
2
0
Vb8.2 (ic)
Vb8.2 (ic)
Vb7 (ic)
Vb b/b
Ja18+/+
Vb b/b Vb b/b Vb a/b
Ja18+/+ Ja18+/- Ja18+/+
DP
thymocytes
mature
Va14i NKT cells
0
% Vb8.2 or Vb7
mouse dimer
R2
CD8a
Va14i
R2
NKT
Preferential Selection of Vb7 NKT Cells at Limiting CD1d:endogenous
ligand Concentration in vivo
100
CD8a
DP
60
CD1d+/+
MFI,
122 ± 15
MFI,
59 ± 7
% Vb7
CD1d+/-
40
10
20
5
0
0
Vb7 (ic)
Vb7
*
40
30
Vba/a
20
10
CD1d
25
15
Vbb/b
50
DP thymocytes
*
20
60
CD4
Vb8.2
Vb7
0
CD1d+/+ CD1d+/-
CD1d+/+ CD1d+/-
DP
thymocytes
mature
Va14i NKT cells
% Vb7
R3
% Vb8.2
80
Vb8.2 (ic)
Vb7 (ic)
Preferential Selection of Vb7 NKT Cells in Va24 Transgenic Mice
expressing a low avidity invariant TCRa chain
non-tg
huVa24-tg
R2
R3
R3
non-NKT
non-NKT
TCR-b
nonNKT
60
NKT
NKT
88 ± 5
nonNKT
TCR-b
21 ± 10
% Vb8.2 or Vb7
NK1.1
R2
Vb8.2
Vb7
Vb8.2 (ic)
Vb7 (ic)
50
40
30
20
10
0
NKT
NKT
mouse tetramer
81 ± 7
nonNKT
nonNKT
NKT
mouse dimer
7±3
NKT
non-tg huVa24-tg
non-tg
huVa24-tg
Vb7+ NKT cells are preferentially selected by endogenous ligands
or exogenous self-ligand iGb3 in thymic culture
300
CD1d-/-
%Dimer+ Tcells
cell expansion (%)
cell expansion (%)
CD1d+/+
%Vb8.2+Dimer+
%Vb7+Dimer+
200
100
%Dimer+ Tcells
300
%Vb8.2+Dimer+
%Vb7+Dimer+
200
100
0
0
d0
d7 unstim
d7 iGb3
10000 ng/ml
d0
d7 unstim
d7 iGb3
10000 ng/ml
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
Diverse functions of the proto-oncogene c-Myc
From: Murphy MJ, Wilson A, Trumpp A. Trends Cell Biol 2005;15:128-137
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;cMyc 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
Reduced numbers of Va14i NKT cells in c-myc
haploinsufficient mice
28%
x10e3
57%
c-Myc +/-
Dimer
Thymus
c-Myc +/+
Dimer positive
cell number:
Thymus
300
150
TCRb
0
c-Myc+/+
Dimer positive
cell number:
Liver
15%
x10e3
Dimer
Liver
400
29%
c-Myc+/-
200
TCRb
0
c-Myc+/+
c-Myc+/-
T-cell specific conditional deletion of c-myc leads to a
dramatic and selective reduction in thymic Va14i NKT cells
CD4crec-Myc fl/fl
4myc +/+
CD4cre+
c-Myc fl/wt
4myc +/38%
6%
Dimer
500
Dimer positive
T cell number:
Thymus
300
x10e5
TCRb
x10e3
Thymus
44%
CD4cre+
c-Myc fl/fl
4myc -/-
250
150
0
0
4myc+/+ 4myc+/- 4myc-/-
Dimer negative
T cell number:
Thymus
4myc+/+ 4myc+/-
4myc-/-
Requirement for IL-15 in Va14i NKT cell
development
51%
IL15+/30%
IL15-/-
x10e3
IL15+/+
5%
200
Dimer
Thymus
400
Dimer positive
cell number:
Thymus
TCRb
0
IL15+/+
x10e3
5%
400
Dimer
Liver
18%
IL15-/-
Dimer positive
cell number:
Liver
800
33%
IL15+/-
TCRb
0
IL15+/+
IL15+/-
IL15-/-
c-Myc+/+
IL15+/+
c-Myc+/IL15+/+
56%
c-Myc+/+
IL15+/-
41%
43%
Dimer
TCRb
Dimer positive
cell number:
Thymus
300
x10e3
Thymus
Synergistic reduction in Va14i NKT cells in
combined c-myc and IL-15 haploinsufficiency
150
0
c-Myc+/+
c-Myc+/-
IL15+/-
c-Myc+/-
c-Myc+/IL15+/7%
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