Peripheral Immune System of Glycosphingolipid Storage

Disorder Mouse Models

Aruna Jeans

Plasma membrane

Golgi –GSL synthesis

Lysosome–GSL degradation

GlycosphingolipidStorage Disorders – GSL degradation is blocked

Cytosol

Glycosphingolipids

Sandhoff Mouse Model

• Mutation in hexB, loss of -hexosaminidase A and B

• Accumulation of GM2 and GA2• Neurodegenerative disease course with

symptom onset at 8 weeks (head tremor)• End stage 16-18 weeks (ataxic gate, hind limb

paralysis)• Characterised by CNS inflammation

(contributes to disease progression)

Peripheral Immune System

• GSLs accumulate in haematopoietic cells.• GSLs have a number of specific roles within the

peripheral immune system, making particular lymphocyte subsets vulnerable to GSL accumulation.

• Human disease course is highly variable – peripheral inflammation is a potential influential factor.

• Common neurodegenerative disorders characterised by CNS inflammation are exacerbated by peripheral inflammation.

Aims…

• Phenotype the peripheral immune system

• Functionally characterise the immune system

• Determine whether peripheral inflammation can exacerbate disease progression

Phenotype

Cell type  

CD4 T cells Reduction in splenic CD4 T cells from 6/8 weeks

CD8 T cells No detected change in numbers, but double positive thymocytes show a decrease in CD8 expression

NKT cells Loss of NKT cells (defective selection)

B cells Reduction in marginal zone B cells from 8 weeks. Elevation in peritoneal B1 cells. Reduction in splenic and circulating B cells at 16-18 weeks (end stage)

Monocytes/macrophages

Elevation in monocytes 3-6 weeks. Decrease in CD11b expression in a subset of splenic macrophages

Neutrophils Elevation in neutrophils at 16-18 weeks (end stage)

Immune response• Innate response – recognition by pre-formed

non-specific effectors, activation of adaptive response

• Adaptive response – generation of specific effectors and immunological memory

Antigen presenting cell – process antigens and display them on cell surface in conjunction with molecules required to activate lymphocytes

CD8+ killer T cells –kill infected cells (cytosolic pathogens)

CD4+ helper T cells – activate B cells in response to T-Dependent antigens

B cell – produces antibodies (inactivate toxins, label pathogens for complement recognition or opsonisation)

Killer T cells

Female mice primed and boosted with UTY peptide.

Percentage of UTY specific CD8+ T cells determined using UTY-tetramer

CD8+ CTL production against UTY

0

5

10

15

20

25

30

1

Mouse

Per

cen

tag

e o

f U

TY

+ C

D8+

T

cell

s Con

Sandhoff

Killer T cells – VITAL assay

Percentage of specific lysis

0

20

40

60

80

100

120

Male splenocytes med peptide hi peptide

Target cell

Per

cen

tag

e sp

ecif

ic l

ysis

(%

)

Het

Hom

•Mice injected with stained male splenocytes or female splenocytes loaded with UTY-peptide.

•After 24 hrs, mice bled and cell populations recorded – calculate percentage of specific lysis, relative to female non-pulsed

YX XX XX XX

Injected into mice (IV), bled at 24 hrs

FL1

FL2

10uM1uM

T-Dependent ResponseAntigen binds BCR, and is internalised and processed

Antigen taken up, processed and presented by APC

CD4+ T cells with specific TCR ‘recognises’ presented antigen. T cells are activated and divide. T cells secrete IL4 and IFN influencing the immune response

B cell is partly activated. Antigen-complexed with MHC II is presented at cell surface T cells recognise antigen

presented by partially activated B cells in lymph node. Delivers second activation signal.

Germinal Centre – Fully activated B cells undergo clonal expansion, affinity maturation and B cell maturation to produce plasma cells and memory cells

T-dependent

IL4

IFNIL4

IFN

T-Dependent Response in the Sandhoff Mouse

Mice were injected with 100ug TNP-KLH, precipitated with alum. Mice were bled on day 7 and serum Ig levels measured by ELISA.

Antibody production in response to TNL-KLH

0

5000

10000

15000

20000

25000

30000

35000

8 14

Age (weeks)

Ig t

itre

(n

g/m

l)

Con.

Sandhoff

Relative IgG and IgM production by 14 week Sandhoff mice

020406080

100120140

IgG IgM

Isotype

Rel

ativ

e an

tib

od

y p

rod

uct

ion

Con.

Sandhoff

CD4+ T cellsPercentage of Splenic CD4+ T Cells

02468

1012141618

3 6 8 12 14

Age (weeks)

Per

cen

tag

e (%

)

Con

Sandhoff

Antigen processing and presentation

IL4

IFNIL4

IFN

IFN-gamma producing cells

0

10

20

30

40

50

60

70

80

8 14

Age (weeks)

Nu

mb

er (

x10^

6)

Con.

Sandhoff

IL4 producing cells

0

2

4

6

8

10

12

14

16

8 14

Age (weeks)

No

. ce

lls

(x10

^6)

Con.

Sandhoff

Germinal Centre Formation

Percentage of Germinal Centre B cells

0

2

4

6

8

10

TNP-KLH SRBC

Antigen

Per

cen

tag

e (%

)

Con

Sandhoff

Mice were injected with TNP-KLH or Sheep red blood cells

On day 7, mice were culled and the percentage of germinal centre B cells (PNAhiCD19+) determined by flow cytometry.

Plasma Cell Formation

Percentage of Plasma Cells(Ig+CD138+)

MFI of CD138 expression

Control 0.9+/- 0.06 1141+/-49

Homozygote 0.6 +/- 0.14 876+/-33

Plasma cells – 0.87%

Serum anti-TNP titre- 37139 ng/ml

Plasma cells – 1.0%

Serum anti-TNP titre – 23670 ng/ml

Heterozygote Homozygote

Conclusion

• GSL accumulation effects all major groups of myeloid cells and lymphocytes, altering the response to immune stimulus.

• Sandhoff mice are able to generate functional killer T cells.

• Sandhoff mice have a reduced response to T-dependent antigens, due to a defect in plasma cell production and/or function .

• Defect in plasma cell formation could be due to errors in cell signalling and/or interaction with follicular dendritic cells

• Changes in the way human patients respond to infection, may make them vulnerable to secondary infection.

Future Work

• Look at germinal centre architecture

• Look at secondary response to T-dependent antigens

• Determine whether peripheral inflammation exacerbates disease progression

Acknowledgements

Fran PlattProf. Raymond Dwek

David SmithGabriele Reinkensmeier

All of the first floor

Jon Silk (IMM)Prof. Crundolo (IMM)

T-Independent Response

T-independent

Germinal centre?

Mice were injected with 25g TNP-Ficoll IP. On day 7, mice were bled and serum expressed. Anti-TNP antibodies were measured by Elisa.

Antibody production in response to TNP-Ficoll

010002000300040005000600070008000

8 14

Age (weeks)

Tota

l Ig

(n

g/m

l se

rum

)

Con.

Sandhoff

Macrophages

MIP2 production in response to immune stimulus

0

0.5

1

1.5

2

2.5

3

nothing 10ng/ml LPS 10ug PGN

Stimulant

MIP

2 n

g/m

l/u

g p

rtn

Con

Sandhoff

MIP-1 alpha production in response to stimulus

0

0.2

0.4

0.6

0.8

1

10ng/ml LPS 10ug/ml PGN 1ug/ml Zymosan

Stimulant

MIP

1-a

(ng

/ml/

ug

prt

n)

Con

Sandhoff

TNF-alpha production in response to immune stimulus

0

0.05

0.1

0.15

0.2

0.25

0.3

10ng/ml LPS 10ug/ml PGN 1ug/ml Zymosan

Stimulant

TN

F-a

(n

g/m

l/u

g p

rtn

)

Con.

Sandhoff