peripheral immune system of glycosphingolipid storage disorder mouse models aruna jeans

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