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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
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