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0 3 6 9 12 15
PBS (n=6-10)
DC alone (n =4)
DC/Nec-F/T (n=11-13)
DC/Nec-H/S (n=11-12)
MRL/lpr (n=10,16wks old)W/T mice
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PBS (n=6-10)
DC alone (n =4)
DC/Nec-F/T (n=11-13)
DC/Nec-H/S (n=11-12)
MRL/lpr (16wks old)
IL-10-/- mice
AgedMRL/lpr
Weeks post-injection
Pro
tein
uri
a (m
g/d
l)
Suppl. Fig. 1: Detection of proteinuria. Groups of W/T and IL-10-/- mice (8-wks of age, female) were injected, for three times and at bi-week intervals, with 1x106 syngenic DCs that had been preloaded with necrotic cells prepared either by the freeze-thaw (DC/necF/T) or heat-shock (DC/necH/S) procedures (see Methods), or with DC alone (DC) or PBS alone (PBS). Urine samples were collected bi-weekly and tested for the levels of proteinuria in the different treatment groups of both strains. For comparison, solid bars represent proteinuria levels detected in a group of aged lupus-prone MRL/lpr mice (16-wks old, n = 10). Data shown were the average levels (Mean + SE) calculated from mice of the respective groups, and the number of mice in each group indicated above (n).
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An
ti-S
M//
nR
NP
(I
gG
, AE
U/m
l)
Weeks post-injection
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ti-s
sD
NA
(I
gG
, AE
U/m
l)A
nti
-ch
rom
ati
n
(Ig
G, A
EU
/ml)
An
ti-h
isto
ne
(Ig
G, A
EU
/ml)
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ti-d
sD
NA
(I
gG
, AE
U/m
l)
W/T IL-10-/-
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DC alone
DC/Nec-F/T
DC/Nec-H/S
PBS
Suppl. Fig. 2: Serum autoantibody levels. Serum samples were taken from the mice described in Methods, at the indicated time points. Levels of 5 classical lupus-associated autoantibodies, i.e. anti-dsDNA, anti-ssDNA, anti-chromatin, anti-histone and anti-SM/nRNP, were quantified by ELISAs. Autoantibody levels against dsDNA and ssDNA were measured in all of the 4 treatment groups. Those against chromatin, histone and SM/RNP were measured selectively in the 2 main treatment groups (DC/necF/T, DC/necF/T) in comparison to those of DC alone control groups. Data shown are mean ± SEM calculated from individual mice of each group (n = 4-5). Arrows indicate the time of 3 injections given.
Suppl. Fig. 3: Isotypic analysis of serum anti-dsDNA antibodies induced by DC/nec treatments. Serum samples taken 8-weeks after DC/nec treatments were assayed for the subclasses of anti-dsDNA antibodies in the circulation. Data shown are mean±SEM calculated from individual mice of each group (n = 4-5).N.D = not detected. Serum samples from the PBS-treated groups contained no or very low level of anti-DNA antibodies (total IgG), hence were not included in the isotypic analysis.
IgG1
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60 DC/W
DC/K
DC DC/necF/T DC/necH/S
IgG2b
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60DC/W
DC/K
DC DC/necF/T DC/necH/S
IgG2c
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60
DC DC/necF/T DC/necH/S
N.D N.D N.D
IgG2b to IgG1 ratio
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0.5
1.0
1.5W/T
IL-10-/-
DC DC/necF/TDC/necH/S
ratio
Se
rum
an
ti-d
sD
NA
an
tib
od
ies
of
dif
fere
nt
iso
typ
es
IgG
1 (
AE
U/m
l)Ig
G2
b (
AE
U/m
l)Ig
G2
c/I
gG
2a
b (A
EU
/ml)
Suppl. Fig. 4: Characterization of the renal reactive autoantibodies in blood circulation. Frozen kidney sections prepared from normal C57BL/6 mice were used as the source of target autoantigens in this immunoreactivity assay. Serum samples taken from IL-10-/- mice treated with DC/necF/T (A), or DC/necH/S (B), and WT mice-treated with DC/necH/S (C) (8wks post 1st DC injection) were applied and incubated, followed by the FITC-conjugated goat anti-mouse IgG secondary antibody. Photomicrographs shown are results representative of 3 independent experiments. (D) Negative control: No serum (secondary antibody only).
A B
C D
