Cellular lipids and immunity: characterisation of

glycosphingolipids binding the antigen presenting molecule CD1

Karen MuindiOGBI seminar30th May 2007

CD1: an introduction

• MHC class 1- like glycoprotein

• Associates with 2m

• 5 CD1 genes (CD1a-e) expressed in humans

• 4 of these (CD1a-d) present self and foreign lipid antigens to T cells

Brigl and Brenner. Annual Reviews in Immunology, 2004

CD1a intracellular trafficking

Adapted from Hava et al. Curr Opin Immunol. 2005

CD1b,c and d intracellular trafficking

Hava et al. Curr Opin Immunol. 2005

CD1 antigens

• CD1 presentation of self GSLs implicated in etiology of several autoimmune diseases

• MS patients have elevated numbers of T cells responding to a variety of GSLs including sulfatide and GM1

• sera from Guillain-Barré patients has elevated levels of Abs reactive to GM1, GA1 and GD1b

• The most potent activators of CD1d-restricted T cells (-galactosylceramide and iGb3) are GSLs

Endogenous glycosphingolipids

• Wt mCD1d or mCD1d-tail deleted (TD) expressing cells assayed for their ability to activate CD1d restricted NKT cells

• CD1d-TD presentation greatly reduced.

• Suggests that lysosomal processing is required for activation of NKT cells

CD1 lysosomal trafficking and T cell activation

Chiu et al. JEM. 1999, 103-10.

2 construct system to resolve CD1 pre- and post- lysosomal GSL load

TEV protease

TEV protease

Secretorypathway only

Secretory and endocytic pathways

Adapted from Brigl and Brenner. Annual Reviews in Immunology, 2004

B2m Extracellular Domainlinker Hinge CH3CH2

IgG2a Fc

B2m Extracellular Domainlinker TM CYTEV

hCD1b-Fc

hCD1b-TEV

hCD1b-Fc is expressed and maintains native conformation

0 250 500 750 1000 1250 15000.00

0.25

0.50

0.75

1.00

1.25

hCD1b-Fc (ng/ml)

OD

@ 4

05n

m0 250 500 750 1000 1250 1500

0.00

0.25

0.50

0.75

1.00

1.25

hCD1b-Fc (ng/ml)

OD

@ 4

05n

m

markers hCD1b-Fc-

175

85

62

47.5

32.5

25

16.5

6.5

hCD1b-Fc (72.7)

KDamarkers hCD1b-Fc-

175

85

62

47.5

32.5

25

16.5

6.5

hCD1b-Fc (72.7)

KDa

TEV protease cleaves hCD1b-TEV: cleaved protein maintains

native conformation

100 101 102 103 104FL1-H

0

20

40

60

80

100

% o

f M

ax

100 101 102 103 104FL1-H

100 101 102 103 104100 101 102 103 104FL1-H

0

20

40

60

80

100

% o

f M

ax

0

20

40

60

80

100

0

20

40

60

80

100

% o

f M

ax

100 101 102 103 104FL1-H

100 101 102 103 104100 101 102 103 104FL1-H

0

20

40

60

80

100

% o

f M

ax

0

20

40

60

80

100

0

20

40

60

80

100

% o

f M

ax

100 101 102 103 104100 101 102 103 104FL1-H

100 101 102 103 104100 101 102 103 104FL1-H

0

20

40

60

80

100

0

20

40

60

80

100

% o

f M

ax

0

20

40

60

80

100

0

20

40

60

80

100

% o

f M

ax

mock wt hCD1b hCD1b-TEV (3:2-2)0

100

200

300

400

500

600

700untreated+ TEV protease

HeLa transfectants

hC

D1b

(n

g/m

l)

Lysosomal trafficking of hCD1b-TEV

wt hCD1b I I L Y W R N P T S I G S I V L A

hCD1b-TEV (2b4) I I L Y W A S G S E N L Y F Q G G S G S R N P T S I G S I V L A

hCD1b-TEV (151-150) I I L Y W G S E N L Y F Q G R N P T S I G S I V L A

hCD1b-TEV (05-150) I I L Y W E N L Y F Q G R N P T S I G S I V L A

hCD1b-tev (3:2-2) I I L Y W E N L Y F Q G S I V L A

hCD1b-a-tev-a (3a:2-2) I I L Y W A E N L Y F Q G A S I V L A

B2m Extracellular Domain linker TM CYTEV

hCD1b-TEV steady state localisation

hCD1b-TEV (151-150)hCD1b-TEV (2b4)

wt hCD1b

Lysosomal trafficking of recycling surface cleavable hCD1b constructs

wt hCD1b I I L Y W R N P T S I G S I V L A

hCD1b-TEV (2b4) I I L Y W A S G S E N L Y F Q G G S G S R N P T S I G S I V L A

hCD1b-TEV (151-150) I I L Y W G S E N L Y F Q G R N P T S I G S I V L A

hCD1b-TEV (05-150) I I L Y W E N L Y F Q G R N P T S I G S I V L A

hCD1b-tev (3:2-2) I I L Y W E N L Y F Q G S I V L A

hCD1b-a-tev-a (3a:2-2) I I L Y W A E N L Y F Q G A S I V L A

B2m Extracellular Domain linker TM CYTEV

Lysosomal trafficking of recycling surface cleavable hCD1b constructs

wt hCD1b I I L Y W R N P T S I G S I V L A

hCD1b-TEV (2b4) I I L Y W A S G S E N L Y F Q G G S G S R N P T S I G S I V L A

hCD1b-TEV (151-150) I I L Y W G S E N L Y F Q G R N P T S I G S I V L A

hCD1b-TEV (05-150) I I L Y W E N L Y F Q G R N P T S I G S I V L A

hCD1b-tev (3:2-2) I I L Y W E N L Y F Q G S I V L A

hCD1b-a-tev-a (3a:2-2) I I L Y W A E N L Y F Q G A S I V L A

B2m Extracellular Domain linker TM CYTEV

hCD1b-TEV (3:2-2) traffics to the lysosome …

wt CD1b hCD1b-TEV (3:2-2)

…and recycles back to the cell surface

10 100 1000 10000 no Ag0

10

20

30

40

50

GMM (nM)

IFN

- (

ng

/ml)

GMM C80

GMM C32

10 100 1000 10000 no Ag

5000

15000

25000

GMM (nM)

GM

-CS

F (

pg

/ml)

GMM C80

GMM C32

•Presentation of glucose monomycolate (GMM) with long alkyl chains (C80) to T cells requires lysosomal loading of lipid to CD1b (Moody et al. Nat Imm 2002)

•Not required for GMM with short alkyl chains (C32)

hCD1b-bound lipid extraction and characterisation

hCD1b-Fc harvested from cell culture

supernatant by passing it over protein A

beads

hCD1b-TEV recovered from cell surface using TEV

protease and purified over -CD1b coated beads

lipids extracted from CD1b

glycan headgroups of GSLs were cleaved off using ceramide glycanase

glycans were labelled with 2-aminobenzamide (2-AB), or anthranilic acid (2-AA), and analysed by HPLC.

Species structure was confirmed by exoglycosidase digests

Cleavage of GM1 by ceramide glycanase

Adapted from Expert Reviews in Molecular Medicine © 2002 Cambridge University Press

hCD1b-Fc expressed in CHO cells binds lactosylceramide and GM3.

Fluo

resc

ence

42 3 5Glucose units

LacCer

GM3

hCD1b-Fc

MHC1-Fc

hCD1b-TEV expressed in CHO cells binds lactosylceramide and GM3.

LacCer GM3CHO

CHO

hCD1b-TEV

Fluore

scenc

e (Mv

)

0.0

125

220

250

450LacCer GM3

Transfer of the 2 construct system to other cellular models

Flu

ores

cenc

e

CHO cells

RAW cells

U937 cells

LacCer

LacCerGM3

GM3

GM3

GD1aGM1a

LacCer

2 3 4 5 6Glucose units

GA2 GM2 GA1

paraglobosidesialosylparagloboside

Lysosomal processing of GSLs bound to mCD1d expressed in RAW

cellsmCD1d-Fc

MHC1-Fc

mCD1d-TEV

control

GM1a GD1a

GD1a

Flu

ore

scen

ce

A

B

53 4 6Glucose units

GM1a

Neu5Ac2-3Gal1-3GalNAc 1-4Gal 1-4GlcCer 3

Neu5Ac 2

Gal1-3GalNAc 1-4Gal 1-4GlcCer 3

Neu5Ac 2

GD1a GM1a

Conclusions

• We have successfully designed a 2 construct system to study lysosomal processing of CD1 GSL antigens

• Studies of mCD1d in RAW cells suggest that trimming of GSL headgroups from the non reducing end occurs in the lysosome (GD1a to GM1a)

Future Studies

• Using the U937 system, determine whether exchange or processing of hCD1b-bound GSLs occurs in the lysosomes

• Determine which of pre- and post- lysosomal GSL antigens is more antigenic

• Determine whether the GSL tails are modified in the lysosome

• Determine whether activation of APCs results in a change in GSL profile and CD1 GSL antigen load.

Acknowledgements

HMS/BWH (Boston)• Manuela Cernadas• Gerald Watts• Duarte Barral• Michael Brenner

UCD (Ireland)• Louise Royle• Pauline Rudd

OGBI• David Neville• Terry Butters• Raymond Dwek

Work supported by; • NIH grants to Michael

Brenner• OGBI studentship • DPhil scholarship awards

from ORS and Clarendon Fund schemes