An Introduction to NKT cells
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An Introduction to NKT cells. CD1d. Endogenous ligand: Isoglobotrihexosylceramide (iGb3) Foreign ligand: Microbial a-glycuranosylceramides Artificial ligand: a-galactosylceramide ( a -GalCer). mu: V a 14-J a 18 hu: V a 24-J a 18. mu: V b 8.2/V b 7 hu: V b 11. CDR3 b diverse. ( CD4).

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V a 14iNKT

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V a 14inkt

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

( CD4)

Va14iNKT

CD44high

NK1.1

Autoimmune diseases

Infectious diseases

Tumors

CD69high

Ly49

( DX5)

IFN-g

IL-4


V a 14inkt

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


V a 14inkt

Developmental pathway of Va14i NKT cells

CD4- CD8-

TCR

TCR

NK1.1

Thymus

NKT

NKT

CD1d

CD4+ CD8+

MHC I

MHC II

T

CD4+

T

CD8+

TCR

TCR

NK1.1

T

CD4+

T

CD8+

NKT

NKT

?

Periphery

MacDonald H.R.,Science, 2002


Development and selection of va14i nkt cells

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 dimers

Specific Identification of Va14i NKT cellsTetramers 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


V a 14inkt

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

53 ± 4

58 ± 2

80 ± 7

84 ± 4

Counts

Counts

Vb8.2

Vb8.2

Counts

16 ± 2

12 ± 2

6 ± 5

4 ± 1

Counts

Vb7

Vb7


Targeted expression of human cd1d in transgenic mice

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)


V a 14inkt

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+/-


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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+/-


Conclusions from human cd1d transgenic mice

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


V a 14inkt

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


V a 14inkt

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


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Frequency of thymic Vb7+ and Vb8.2+ Va14i NKT cellsreflects Vb rearrangement frequency in CD4+ CD8+ precursors

Va14i

NKT

DP

mouse dimer

CD8a

Vb8.2 (ic)

Vb8.2

Vb7 (ic)

Vb7

NK1.1

T

% Vb8.2 or Vb7 (ic)

% Vb8.2 or Vb7

CD4

TCR-b

mature

Va14i NKT

DP

50 ± 1

9.4 ± 0.5

Vbb/b

Ja18+/+

Vbb/b

Ja18+/+

Vbb/b

Ja18+/-

Vba/b

Ja18+/+

Vb8.2

Vb8.2 (ic)

mature

Va14i NKT

DP

DP

thymocytes

mature

Va14i NKT cells

2.7 ± 0.3

14 ± 3

Vb7 (ic)

Vb7


Preferential selection of vb7 nkt cells at limiting cd1d endogenous ligand concentration in vivo

Preferential Selection of Vb7 NKT Cells at Limiting CD1d:endogenous ligand Concentration in vivo

*

Vb8.2

Vb8.2 (ic)

Vb7 (ic)

Vb7

% Vb8.2

Vbb/b

% Vb7

DP

CD8a

*

Vb7 (ic)

Vb7

CD4

DP thymocytes

% Vb7

CD1d+/-

CD1d+/+

Vba/a

MFI,

122 ± 15

MFI,

59 ± 7

CD1d

CD1d+/+

CD1d+/-

CD1d+/+

CD1d+/-

DP

thymocytes

mature

Va14i NKT cells


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Preferential Selection of Vb7 NKT Cells in Va24 Transgenic Mice expressing a low avidity invariant TCRa chain

non-tg

huVa24-tg

Vb8.2

Vb8.2 (ic)

NKT

NKT

NK1.1

Vb7

Vb7 (ic)

non-NKT

non-NKT

% Vb8.2 or Vb7

TCR-b

non-

NKT

TCR-b

21 ± 10

88 ± 5

non-

NKT

NKT

NKT

DP

DP

DN

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


V a 14inkt

Vb7+ NKT cells are preferentially selected by endogenous ligands or exogenous self-ligand iGb3 in thymic culture

cell expansion (%)

cell expansion (%)


Role of vb domain in selection of va14i nkt cells by cd1d binding glycolipids

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


V a 14inkt

Diverse functions of the proto-oncogene c-Myc

From: Murphy MJ, Wilson A, Trumpp A. Trends Cell Biol 2005;15:128-137


V a 14inkt

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


Reduced numbers of va14i nkt cells in c myc haploinsufficient mice

57%

28%

29%

15%

Reduced numbers of Va14i NKT cells in c-myc haploinsufficient mice

Dimer positive cell number:

Thymus

c-Myc +/+c-Myc +/-

x10e3

Dimer

TCRb

Liver Thymus

Dimer positive cell number:

Liver

x10e3

Dimer

TCRb


V a 14inkt

44%

6%

38%

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 +/+4myc +/-4myc -/-

Dimer

TCRb

Dimer positive T cell number:

Thymus

Dimer negative T cell number:

Thymus

x10e3

x10e5

Thymus


Requirement for il 15 in va14i nkt cell development

Requirement for IL-15 in Va14i NKT cell development

Dimer positive cell number:

Thymus

IL15+/+IL15+/-IL15-/-

x10e3

5%

51%

30%

Dimer

TCRb

Liver Thymus

Dimer positive cell number:

Liver

x10e3

33%

18%

5%

Dimer

TCRb


Synergistic reduction in va14i nkt cells in combined c myc and il 15 haploinsufficiency

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


Preliminary conclusions c myc

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


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