restricted distribution of isoglobotrihexosylceramide (igb3) - implications for natural endogenous...

Restricted distribution ofIsoglobotrihexosylceramide (iGb3) -

Implications for natural endogenousNKT cell selecting and activating ligand(s)

Annie Speak

CD1d & NKT CELLS

CD1d: MHC class I like molecule Presents GSLs Conserved throughout mammalian evolution Recognised by NKT cells carrying invariant TCRs Human NKTs can recognise mouse CD1d and vice versa Lysosomal trafficking required for antigen loading Lysosomal proteases are required Lysosomal lipid transfer proteins (saposins) required

NKT cells: Either single positive (CD4+) or double negative (CD4- CD8-) Have some natural killer cell markers Selected in thymus via GSL/CD1d on double positive cortical thymocytes Have an immunoregulatory role Display a memory phenotype Produce large amounts of Th1 (IFN-) and Th2 (IL-4) following stimulation

NKT cell development -TCR instructive model

CD4+

CD8+Immature thymocyte

Random TCR rearrangement

CD4+

CD8+

MHC I/II+ peptide

CD4+ CD8+

Conventional T cells

Epithelialcell

CD4+

CD8+

CD4- CD4-

CD8-

NKT cells

V14 TCR

CD1d+ GSL

Corticalthymocyte

CD4+

CD8+

Science December 2004

Ganglio seriesLacto and neolacto series

iGb3Gal1-3Gal1-4Glc1-1Cer

iGb4GalNAc1-3Gal1-3Gal1-4Glc1-1Cer

iGb3 Synthase

Isoglobo series

iGb4/Gb4 Synthase

Gb3Gal1-4Gal1-4Glc1-1Cer

Gb4GalNAc1-3Gal1-4Gal1-4Glc1-1Cer

Gb3 Synthase

Globo series

Ceramide

GlcCerGlc1-1Cer

LacCerGal1-4Glc1-1Cer

Ceramide glucosyltransferase

Lactosylceramide synthase

GSL synthesis

GSL catabolism -Enzymes affected by lysosomal storage disorders (LSDs)

Ceramide

GlcCer

LacCer

iGb3 Gb3

iGb4-hexosaminidase A-hexosaminidase B

GM3

GM2

GM1

-galactosidase

Sandhoff

Neuraminidase -galactosidase

-glucocerobrosidase

Gaucher

-hexosaminidase A

Tay Sachs

GM1 Gangliosidosis

-galactosidase

Fabry

Gb4

GSL products stored in LSDsRelevant disorders for isoglobo series storage

Disorder Enzyme deficiency Storage GSLs

Fabry -galactosidase Gb3, (iGb3)

Sandhoff -hexosaminidase A-hexosaminidase B

Gb4, (iGb4), GA2, GM2, GD2

Fabry dorsal root ganglion (DRG)

iGb3 standard

iGb3

Gb3La

c

GA

2

Gb3

GM

3

GM

2G

b4

GA

1

GM

1a

GD

1a

GD

1b

GT

1b

iGb3 standard

Rat stomach neutrals

Rat stomach neutrals -hex

iGb4

Identification of iGb4 in Rat stomach

SH-/-

DRG

Spinal cord

Lac

GA

2

Gb3

GM

3

GM

2G

b4

GA

1

GM

1a

GD

1a

GD

1b

GT

1b

SH+/+

SH+/+

SH-/-

iGb3

iGb4

DRG and spinal cord

Human thymus

Lac

GA

2

Gb3

GM

3

GM

2G

b4

GA

1

GM

1a

GD

1a

GD

1b

GT

1b

Human thymus neutrals

iGb3

iGb4

Lac

GA

2

Gb3

GM

3

GM

2G

b4

GA

1

GM

1a

GD

1a

GD

1b

GT

1b

Mouse thymus neutrals

SH-/-

SH+/+

Fab

C57

iGb3

iGb4

Lac

GA

2

Gb3

GM

3

GM

2G

b4

GA

1

GM

1a

GD

1a

GD

1b

GT

1b

Locations iGb3/4 identified

iGb3 iGb4

Rat Stomach ()

Rat Thymus ()

Mouse DRG

Mouse Spinal Cord

Mouse Stomach

Mouse Eye

Mouse Intestine

Mouse Thymus

Mouse Spleen

Human thymus

Human

NPC+/+

Rat

C57

Possible candidate ligands -conserved GSLs in human, rat and control mouse thymi

Lac

GA

2

Gb3

GM

3

GM

2G

b4

GA

1

GM

1a

GD

1a

GD

1b

GT

1b

Possible candidate ligands -conserved neutral GSLs in human, rat and control mouse thymi

Human

C57

NPC+/+

Rat

Lac

GA

2

Gb3

GM

3

GM

2G

b4

GA

1

GM

1a

GD

1a

GD

1b

GT

1b

Future work

Characterise the GSL profile of human thymus

Characterise the GSL profile of mouse thymus

Development of acid strip protocol to elute CD1d ligands

Using -GalCer to out compete endogenous ligands

Use of cleavable CD1d

Changes in the storage models?

Explains loss of NKT cells?

CD1d ligands

Acknowledgements

Dr Fran PlattProf Raymond Dwek

Dr Dave NevilleDr Terry Butters

Dr David PriestmanDave Smith

Gabi Reinkensmeier

Prof Vincenzo CerundoloDr Mark Exley

Everyone else on the 1st floor