Transcript Restricted distribution of Isoglobotrihexosylceramide (iGb3) Implications for natural endogenous NKT cell selecting and activating ligand(s) Annie Speak CD1d & NKT CELLS CD1d:         MHC class I like.

Restricted distribution of
Isoglobotrihexosylceramide (iGb3) Implications for natural endogenous
NKT cell selecting and activating ligand(s)
Annie Speak
CD1d & NKT CELLS
CD1d:
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



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
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:



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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+
CD4+
CD8+
V14 TCR
CD4+
CD8+
Epithelial
cell
MHC I/II
+ peptide
CD4+
CD1d
+ GSL
CD8+
Conventional T cells
CD4-
CD4CD8-
NKT cells
Cortical
thymocyte
Science December 2004
GSL synthesis
Ceramide
Ceramide glucosyltransferase
GlcCer
Glc1-1Cer
Lactosylceramide synthase
Lacto and neolacto
series
LacCer
Gal1-4Glc1-1Cer
iGb3 Synthase
Ganglio series
Gb3 Synthase
iGb3
Gal1-3Gal1-4Glc1-1Cer
Gb3
Gal1-4Gal1-4Glc1-1Cer
iGb4/Gb4 Synthase
iGb4
GalNAc1-3Gal1-3Gal1-4Glc1-1Cer
Isoglobo series
Gb4
GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
Globo series
GSL catabolism Enzymes affected by lysosomal storage disorders (LSDs)
GM1
iGb4
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
-galactosidase
GM1 Gangliosidosis
GM2
-hexosaminidase A
Tay Sachs
iGb3
Gb4
-hexosaminidase A
-hexosaminidase B
Sandhoff

-galactosidase
Gb3


GM3
LacCer
Fabry
Neuraminidase

-galactosidase
GlcCer

-glucocerobrosidase
Ceramide
Gaucher
GSL products stored in LSDs
Relevant 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)
Gb3
iGb3
GT1b
GD1b
GD1a
GM1a
GA1
GM2
Gb4
Gb3
GM3
GA2
Lac
iGb3 standard
Identification of iGb4 in Rat stomach
iGb4
Rat stomach neutrals
Rat stomach neutrals -hex
iGb3 standard
DRG and spinal cord
SH-/-
DRG
iGb4
iGb3
SH+/+
Spinal cord
SH-/-
GT1b
GD1b
GD1a
GM1a
GA1
GM2
Gb4
Gb3
GM3
GA2
Lac
SH+/+
GT1b
GD1b
GD1a
GM1a
GA1
GM2
Gb4
Gb3
GM3
GA2
Lac
Human thymus
GT1b
GD1b
GD1a
GM1a
GA1
GM2
Gb4
Gb3
GM3
GA2
Lac
Human thymus neutrals
iGb3
iGb4
Mouse thymus neutrals
SH-/-
SH+/+
Fab
C57
iGb3
GT1b
GD1b
GD1a
GM1a
GA1
GM2
Gb4
Gb3
GM3
GA2
Lac
iGb4
Locations iGb3/4 identified
iGb3
iGb4
Rat Stomach
()

Rat Thymus
()

Mouse DRG


Mouse Spinal Cord


Mouse Stomach


Mouse Eye


Mouse Intestine
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
Mouse Thymus
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
Mouse Spleen
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Human thymus
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
Possible candidate ligands conserved GSLs in human, rat and control mouse thymi
Human
C57
NPC+/+
GT1b
GD1b
GD1a
GM1a
GA1
GM2
Gb4
Gb3
GM3
GA2
Lac
Rat
Possible candidate ligands conserved neutral GSLs in human, rat and control mouse thymi
Human
C57
NPC+/+
GT1b
GD1b
GD1a
M1a
GA1
GM2
Gb4
Gb3
GM3
GA2
Lac
Rat
Future work
CD1d ligands
 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?
Acknowledgements
Dr Fran Platt
Prof Raymond Dwek
Dr Dave Neville
Dr Terry Butters
Dr David Priestman
Dave Smith
Gabi Reinkensmeier
Prof Vincenzo Cerundolo
Dr Mark Exley
Everyone else on the 1st floor