1. 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
CDR3b diverse
Va14iNKT
( CD4)
CD44high
NK1.1
Autoimmune diseases
Infectious diseases
Tumors
CD69high
Ly49
( DX5)
IFN-g
IL-4

2. 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:

3. Developmental pathway of Va14i NKT cells
CD4- CD8-
TCR
TCR
Thymus
NK1.1
CD4+ CD8+
CD1d
NKT
NKT
MHC I
MHC II T
CD8+ T
CD4+
TCR
TCR
NK1.1 T
CD8+ T
CD4+
NKT ?
NKT
Periphery
MacDonald H.R., Science, 2002

4. 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

5. Specific Identification of Va14i NKT cells Tetramers Dimers
Streptavidin-
fluorochrome
Biotin
muCD1d/
huCD1d
muCD1d
aGalCer
aGalCer
Va14-Ja18
Vb8.2/Vb7
Va14-Ja18
Vb8.2/Vb7
Va14iNKT
Va14iNKT
NK1.1
NK1.1

6. to Va14i NKT cells expressing Vb8.2
Human CD1d:aGalCer dimers bind preferentially
to Va14i NKT cells expressing Vb8.2
Thymus
Liver
Thymus
Liver 28 17 12 9
mouse dimers
human dimers
TCR-b
TCR-b
Counts
53 ± 4
58 ± 2
80 ± 7
84 ± 4
Counts
Vb8.2
Vb8.2
Counts
16 ± 2
12 ± 2
6 ± 5
4 ± 1
Counts
Vb7
Vb7

7. 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)

8. DP thymocytes but not DC expressing huCD1d
positively select Vb8.2+ Va14i NKT cells
muCD1d-/-
% Vb8.2
% Vb7
pLck-
huCD1d-tg
muCD1d-/-
CD11c-
huCD1d-tg
muCD1d-/-
non-tg
muCD1d+/-

9. Both DP thymocytes and DC expressing huCD1d
negatively select Vb8.2+ Va14i NKT cells
muCD1d+/-
% Vb7
% Vb8.2
pLck-
huCD1d-tg
muCD1d+/-
CD11c-
huCD1d-tg
muCD1d+/-
non-tg
muCD1d+/-

10. 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

11. 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

12. Higher avidity binding of mouse CD1d:aGalCer dimers
by Va14i NKT cells expressing Vb8.2
Thymus
Liver
mouse dimers
TCR-b
% Vb8.2+
% Vb7+
Gate

13. Frequency of thymic Vb7+ and Vb8
Frequency of thymic Vb7+ and Vb8.2+ Va14i NKT cells reflects Vb rearrangement frequency in CD4+ CD8+ precursors
Va14i
NKT DP
CD8a
mouse dimer
Vb8.2 (ic)
Vb8.2
Vb7 (ic)
Vb7
NK1.1 T
% Vb8.2 or Vb7 (ic)
CD4
TCR-b
% Vb8.2 or Vb7 DP
mature
Va14i NKT
9.4 ± 0.5
50 ± 1
Vbb/b
Ja18+/+
Vbb/b
Ja18+/+
Vbb/b
Ja18+/-
Vba/b
Ja18+/+
Vb8.2 (ic)
Vb8.2 DP
mature
Va14i NKT DP
thymocytes
mature
Va14i NKT cells
2.7 ± 0.3
14 ± 3
Vb7 (ic)
Vb7

14. Preferential Selection of Vb7 NKT Cells at Limiting CD1d:endogenous ligand Concentration in vivo
Vb8.2 (ic)
Vb8.2 *
Vb7 (ic)
Vb7
% Vb8.2
Vbb/b
% Vb7 DP
CD8a *
Vb7 (ic)
Vb7
CD4
DP thymocytes
CD1d+/-
CD1d+/+
% Vb7
Vba/a
MFI,
59 ± 7
MFI,
122 ± 15
CD1d
CD1d+/+
CD1d+/-
CD1d+/+
CD1d+/- DP
thymocytes
mature
Va14i NKT cells

15. Preferential Selection of Vb7 NKT Cells in Va24 Transgenic Mice expressing a low avidity invariant TCRa chain
non-tg
huVa24-tg
Vb8.2 (ic)
Vb8.2
NKT
NKT
NK1.1
Vb7 (ic)
Vb7
non-NKT
non-NKT
% Vb8.2 or Vb7
TCR-b
non-
NKT
TCR-b
88 ± 5
21 ± 10
non-
NKT
NKT
NKT DP DN DP
NKT
NKT
dimer+
dimer+
tetramer+
mouse tetramer
non-tg
huVa24-tg
non-tg
huVa24-tg
81 ± 7
7 ± 3
non-
NKT
non-
NKT
NKT
NKT
mouse dimer

16. Vb7+ NKT cells are preferentially selected by endogenous ligands or exogenous self-ligand iGb3 in thymic culture
cell expansion (%)
cell expansion (%)

17. 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

18. Diverse functions of the proto-oncogene c-Myc
From: Murphy MJ, Wilson A, Trumpp A. Trends Cell Biol 2005;15:

19. 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 Nature 2001; 414:
*Baudino TA, McKay C, Pendeville-Samain H, Nilsson JA, Maclean KH, White EL, Davis AC, Ihle JN, Cleveland JL. Genes & Dev. 2002; 16:
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:
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

20. Reduced numbers of Va14i NKT cells in c-myc haploinsufficient mice
Dimer positive cell number:
Thymus
c-Myc +/+ c-Myc +/-
x10e3
57%
28%
Dimer
TCRb
Liver Thymus
Dimer positive cell number:
Liver
29%
15%
x10e3
Dimer
TCRb

21. c-Myc fl/fl c-Myc fl/wt c-Myc fl/fl 4myc +/+ 4myc +/- 4myc -/-
T-cell specific conditional deletion of c-myc leads to a dramatic and selective reduction in thymic Va14i NKT cells
CD4cre CD4cre+ CD4cre+
c-Myc fl/fl c-Myc fl/wt c-Myc fl/fl
4myc +/ myc +/- 4myc -/-
44%
38% 6%
Dimer
TCRb
Dimer positive T cell number:
Thymus
Dimer negative T cell number:
Thymus
Thymus
x10e3
x10e5

22. Requirement for IL-15 in Va14i NKT cell development
Dimer positive cell number:
Thymus
IL15+/ IL15+/ IL15-/-
x10e3
51%
30% 5%
Dimer
TCRb
Liver Thymus
Dimer positive cell number:
Liver
x10e3
33%
18% 5%
Dimer
TCRb

23. Synergistic reduction in Va14i NKT cells in combined c-myc and IL-15 haploinsufficiency
c-Myc+/+ c-Myc+/- c-Myc+/+ c-Myc+/-
IL15+/+ IL15+/+ IL15+/- IL15+/-
Thymus
56%
41%
43% 7%
Dimer
TCRb
Dimer positive cell number:
Thymus
x10e3

24. 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