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