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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