PBS nlg anti-CD8-nlg anti-CD4-nlg
CD4
CD
8
17.3 ± 4.3
27.1 ± 5.8
16.8 ± 4.6
27.8 ± 5.7
17.4 ± 4.2
28.9 ± 5.3
16.7 ±3.8
29.1 ± 5.2
Supplemental Figure 1. Empty nlg targeting to either CD4 or CD8 T cells does not affect T cell cellularity in vivo.
Age and sex matched MRL/lpr mice were treated i.v. with either anti-CD4 antibody or anti-CD8 antibody coated-empty-nlg (equivalent to 50 mg antibody
per mouse) for 48 hrs. PBS or empty-nlg were applied to two control groups separately. (a) Flow cytometry analysis of splenic T cells. Data represent
the mean ± SEM (n = 4 mice per group). (b) Dot plot graph shows quantitation of the absolute cell numbers of CD4 and CD8 T cells from the spleens of
mice subjected to the indicated treatment.
Cellu
larity
(10^6
)
a b
0
1.2
2.4
3.6
PBS Empty-nlg anti-CD8-nlg anti-CD4-nlg
CD4 T cells CD8 T cells
Control anti-CD4 anti-CD8
IgM CD3 ATTO590
200
300
400
500
CD4 T cells CD8 T cells DN T cells
Control anti-CD4 anti-CD8
AT
T0
590 M
FI
Supplemental Figure 2. Targeted delivery of ATTO590 by antibody-coated nlg.
12 weeks old MRL/lpr mice were treated i.v. with either anti-CD4 antibody or anti-CD8 antibody coated-nlg-ATTO590 (a fluorescent dye derived from
Rhodamine), and isotype control antibody coated-nlg-ATTO590 was used as control. Mice were euthanized 30 min after nlg administration for analysis,
n = 4 mice per group. (a) Confocal microscopic images show the distribution of ATTO590+ cells in representative splenic follicles. Original
magnification, ×20; Boxed areas in the upper panel are digitally magnified and shown in the bottom panels (IgM : blue, CD3 : green and ATT0590 :
red). (b) Dot plot graph shows quantitation of ATTO590 MFI in different T cell subsets from spleens of mice subjected to the indicated treatment. **P <
0.01, ***P < 0.005 vs. control, a 2-tailed Student’s t test.
**
*** ***
b a
0
0.5
1
1.5
2
2.5
3
Supplemental Figure 3. Empty nlg targeting to either CD4 or CD8 T cells does not alter production of auto-antibodies in MRL/lpr mice.
Age matched MRL/lpr mice were treated i.v. with either anti-CD4 antibody or anti-CD8 antibody coated empty-nlg (15 ml nlg-5-Aza/mouse) weekly for 4
weeks starting at 10 weeks of age. Empty-nlg was used in control group. Dot plot graph shows the ELISA analysis of IgG autoantibodies in the serum
from mice subjected to the indicated treatment. n = 4 mice per group.
Ab
so
rba
nce
(O
D4
50
)
Empty-nlg anti-CD4 anti-CD8
anti-Bip anti-dsDNA RF anti-Histone
a b
FSC
SS
C
Th
y1.2
Empty-nlg 5-Aza anti-CD4-nlg anti-CD8-nlg
3.2±0.9 4.1±1.1 1.8±0.4** 2.0±0.5**
TCRb
5.3±1.4 9.7±2.1 2.3±0.4** 1.1±0.4**
Cellu
larity
(10^6
)
**
***
**
** *
**
***
***
Supplemental Figure 4. Nlg-5-Aza targeting to either CD4 or CD8 T cells dramatically reduces intrarenal CD4, CD8 and DN T cells.
MRL/lpr mice were treated i.v. with either anti-CD4 antibody coated-nlg-5-Aza (15ml nlg-5-Aza/mouse) or anti-CD8 antibody coated−nlg-5-Aza (15ml nlg-
5-Aza/mouse) every ten days for 60 days starting at 12 weeks of age. Free-5-Aza (5mg/mouse) or empty-nlg were applied to two control groups
separately. (a) Flow cytometry analysis of intrarenal T cells. Data represent the mean ± SEM. (b) Dot plot graph shows quantitation of the absolute cell numbers of CD4, CD8 and DN T cells from the kidneys of mice subjected to the indicated treatment. *P < 0.05, **P < 0.01, ***P < 0.005 vs. control, a 2-tailed Student’s t test. n = 5-6 mice per group for 2 independent experiments.
0
1.2
2.4
*
Empty-nlg 5-Aza anti-CD4-nlg anti-CD8-nlg
CD4 T cells CD8 T cells DN T cells
IL-17
IFN
g
Empty-nlg 5-Aza anti-CD4-nlg anti-CD8-nlg
2.3±0.6 4.7±1.0 0.8±0.3** 0.5±0.2**
1.8±0.3 2.1±0.5 0.7±0.3** 0.6±0.2**
Thy1.2+CD4+ gated
Supplemental Figure 5. Nlg-5-Aza targeting to either CD4 or CD8 T cells dramatically reduces both inflammatory Th1 and Th17 cells in
the cervical lymph nodes.
MRL/lpr mice were treated i.v. with either anti-CD4 antibody coated-nlg-5-Aza (15ml nlg-5-Aza/mouse) or anti-CD8 antibody coated−nlg-5-Aza (15μl
nlg-5-Aza/mouse) every ten days for 60 days starting at 12 weeks of age. Free-5-Aza (5μg/mouse) or empty-nlg were applied to two control groups
separately. Flow cytometry analysis of IL-17 and IFNg expression in CD4+ T cells of spleens from mice subjected to the indicated treatment (gated in
CD3+CD4+TCRb+CD49b-). Data represents the mean ± SEM (**P < 0.01 vs. Control, a 2-tailed Student’s t test. n = 5-6 mice per group for 2
independent experiments).
Foxp3
CD
4
5.3±1.1 4.7±0.8 5.7±1.3 10.2±2.1***
Empty-nlg 5-Aza anti-CD8-nlg anti-CD4-nlg
Cellu
larity
(1
0^6
)
a b ***
Thy1.2+CD4+ gated
Supplemental Figure 6. Specific targeting of nlg-5-Aza in CD4+ T cells promotes expansion of Tregs in cervical lymph nodes.
MRL/lpr mice were treated i.v. with either anti-CD4 antibody coated-nlg-5-Aza (15μl nlg-5-Aza/mouse) or anti-CD8 antibody coated−nlg-5-Aza
(15μl nlg-5-Aza/mouse) every ten days for 60 days starting at 12 weeks of age. Free-5-Aza (5μg/mouse) or empty-nlg were applied to two control
group separately. (a) Flow cytometry quantization of the percentage of Foxp3+CD4 T cells (Thy1.2+CD4+) in cervical lymph nodes from mice
subjected to the indicated treatment. Data represent the mean ± SEM. (b) Dot plot graph shows quantitation of Foxp3+CD4 T cells (Thy1.2+CD4+) in cervical lymph nodes from mice subjected to the indicated treatment. ***P < 0.005 vs. control, a 2-tailed Student’s t test. n = 5-6 mice per group
for 2 independent experiments.
0
0.8
1.6
Empty-nlg 5-Aza anti-CD8-nlg anti-CD4-nlg
M
eth
yla
tio
n in
dex o
f on
in
dic
ate
d g
ene p
rom
ote
rs (
%)
Supplemental Figure 7. Nlg-5-Aza targeting to CD4 T cells but not free 5-Aza dramatically reduces Foxp3-associated DNA methylation in CD4 T
cells.
MRL/lpr mice were treated i.v. with either anti-CD4 antibody or anti-CD8 antibody coated-nlg-5-Aza (15μl nlg-5-Aza/mouse) for 10 hrs, free-5-Aza
(5μg/mouse) or Empty-nlg were applied to two control groups separately. n = 4 mice per group. Dot plot graph shows quantitation of DNA methylation on
indicated gene promoters or enhancers in CD4 T cells sorted from mouse spleens 10 hrs after indicated treatment. *P < 0.05, ***P < 0.005 vs. control, a 2-tailed Student’s t test.
0
0.5
1
1.5
2
2.5
Empty-nlg 5-Aza anti-CD4-nlg anti-CD8-nlg
***
***
*
***
*
Foxp3
3.2 ± 0.9 14.8 ± 3.2 20.3 ± 4.2 13.8 ± 3.9 19.8 ± 3.4
Cell polarization: - + + + +
5-Aza : - - + - -
Empty nlg : - - - + -
nlg-5-Aza : - - - - +
Supplemental Figure 8. 5-Aza or nlg-5-Aza promotes expansion of Tregs under polarization conditions in vitro.
Naive CD4 T-cells from healthy donors were polarized under Treg inducing conditions for 7 days, with Aza (1 mM), empty nlg or 5-Aza-nlg (equivalent
to 1mM) added 12 hrs before collection. (a) Flow cytometry analysis shows the induction of Foxp3 in CD4 T cells polarized in vitro. Data represent the
mean ± SEM. (b) Dot plot graph shows Mean fluorescence intensity (MFI) of Foxp3 expression in CD4 T cells polarized in vitro. n= 4 per group. **P < 0.01, ***P < 0.05 vs. control, a 2-tailed Student’s t test.
2000
2500
3000
3500
Cell polarization: - + + + +
5-Aza : - - + - -
Empty nlg : - - - + -
nlg-5-Aza : - - - - +
a
b *** **
MF
I of
Fo
xp3
Foxp3
CD
8
0.2±0.1 0.1±0.1 0.1±0.1 0.1±0.1
Empty-nlg 5-Aza anti-CD8-nlg anti-CD4-nlg
Thy1.2+CD8+ gated
Supplemental Figure 9. Specific targeting of nlg-5-Aza to CD8+ T cells did not induce Foxp3 expression in CD8 T cells.
MRL/lpr mice were treated i.v. with either anti-CD4 antibody coated-nlg-5-Aza (15ml nlg-5-Aza/mouse) or anti-CD8 antibody coated−nlg-5-Aza
(15ml nlg-5-Aza/mouse) every ten days for 60 days starting at 12 weeks of age. Free-5-Aza (5mg/mouse) or empty-nlg were applied to two
control group separately. Flow cytometry quantization of the percentage of Foxp3+ CD8 T cells (Thy1.2+CD8+) in spleens from mice subjected to
the indicated treatment. Data represent the mean ± SEM (n = 5-6 mice per group for 2 independent experiments).
54.6±6.2 63.4±7.3** 22.1±3.4***
CD4
CD
8
Empty-nlg 5-Aza anti-CD8-nlg
Cellu
larity
(10^6
)
a b
Empty-nlg 5-Aza CD8-nlg
***
**
Thy1.2+ TCRgd-TCRb+CD49b- gated
Supplemental Figure 10. Specific targeting of nlg-5-Aza to CD8+ T cells significantly reduces DN T cells in cervical lymph nodes.
MRL/lpr mice were treated i.v. with anti-CD8 antibody coated-nlg-5-Aza (15ml nlg-5-Aza/mouse) every ten days for total 60 days total starting at 12
weeks of age. Free-5-Aza (5mg/mouse) or empty-nlg were applied to two control group separately. (a) Flow cytometry quantization of the
percentage of Thy1.2+TCRb+TCRgd-CD49b-CD4-CD8-T cells in cervical lymph nodes from mice subjected to the indicated treatment. Data
represent the mean ± SEM. (b) Dot plots show quantitation of the absolute cell numbers of Thy1.2+TCRb+TCRgd-CD49b-CD4-CD8-T cells in cervical lymph nodes from mice subjected to the indicated treatment. **P < 0.01, ***P < 0.005 vs. control, a 2-tailed Student’s t test. n = 5-6 mice per
group for 2 independent experiments.
0
6
12
18
M
eth
yla
tio
n in
dex o
f on
in
dic
ate
d g
ene p
rom
ote
rs (
%)
Supplemental Figure 11. Nlg-5-Aza targeting to CD8 T cells but not free 5-Aza dramatically reduces cytolytic activity associated DNA
methylation in CD8 T cells.
MRL/lpr mice were treated i.v. with either anti-CD4 antibody or anti-CD8 antibody coated-nlg-5-Aza (15ml nlg-5-Aza/mouse) for 10 hrs, free-5-Aza
(5mg/mouse) or Empty-nlg were applied to two control groups separately. n = 4 mice per group. Dot plots show quantitation of DNA methylation on
indicated gene promoters or enhancers in CD8 T cells sorted from mouse spleens 10 hrs after indicated treatment. *P < 0.05, **P < 0.01, ***P < 0.005 vs.
control, a 2-tailed Student’s t test.
0
0.5
1
1.5
2
2.5
Empty-nlg 5-Aza anti-CD4-nlg anti-CD8-nlg
*** **
***
***
* ***
*
Meth
yla
tio
n in
dex o
n
indic
ate
d g
ene p
rom
ote
rs (
%)
Cd80
Il-1b
Cd86
H2-Ab
Tnfa
Cd69
Il-6
Ifng
Mφ B cells
Supplemental Figure 12. Free-5-Aza but not Nlg-5-Aza targeting to either CD4 or CD8 T cells dramatically reduces Inflammation-associated DNA
methylation in macrophages and B cells.
Age matched MRL/lpr mice were treated i.v. with either anti-CD4 antibody or anti-CD8 antibody coated-nlg-5-Aza (15ml nlg-5-Aza/mouse) for 10 hrs, free-
5-Aza (5mg/mouse) or Empty-nlg were applied to two control groups separately. n = 4 mice per group. Dot plot graph shows show quantitation of DNA
methylation on indicated gene promoters or enhancers in macrophages (Mφ,CD11b+) or B cells (CD19+) sorted from MRL/lpr mouse spleens 10 hrs after
indicated treatment. *P < 0.05, **P < 0.01, ***P < 0.005 vs. control, a 2-tailed Student’s t test.
0
1.6
3.2
4.8
0
0.8
1.6
2.4
0
3.2
6.4
9.6
0
0.3
0.6
0.9
0
2.4
4.8
7.2
0
0.3
0.6
0.9
0
0.8
1.6
2.4
0
3
6
9
Empty-nlg 5-Aza anti-CD4-nlg anti-CD8-nlg
**
***
*
*** **
*** ***
***
*
*
Mφ B cells Mφ B cells Mφ B cells
Mφ B cells Mφ B cells Mφ B cells Mφ B cells
OVA pulsed APCs: - - + + 5-Aza : - + - +
0.2±0.1 0.3±0.1 2.9±0.5* 4.8±0.7***
Supplemental Figure 13. 5-Aza did not induce perforin expression in naïve CD8 T cells but could enhance perforin expression in
activated CD8 T cells .
Flow cytometry analysis of intracellular perforin expression in CD45.1+OT-I TCR Tg T cells from CD45.1 OT-I TCR Tg Rag1-/- B6 mice
(CD45.1+TCRb+ gated) co-cultured with or without OVA257-264 loaded antigen-presenting cells (APC) for 12 hrs in the absence or presence of 5-Aza
(1mM). Data represent the mean ± SEM (n = 3-4 per group. *P < 0.05, ***P < 0.005 vs. control, a 2-tailed Student’s t test).
Perforin
CD8a
2.0 ± 0.8 21.3± 3.4 15.6 ± 3.1* 22.1 ± 3.9 15.2 ± 3.2**
Cell polarization: - + + + +
5-Aza : - - + - -
Empty nlg : - - - + -
nlg-5-Aza : - - - - +
Supplemental Figure 14. 5-Aza or nlg-5-Aza reduced activation induced CD8 downregulation in vitro.
MACS enriched peripheral CD8 T cells from healthy donors were cultured with anti-CD3/anti-CD28 stimulus for 12 hrs with 5-Aza (1mM), empty-
nlg or nlg-5-Azd (equivalent to 1mM). n = 4 per group. (a) Flow cytometry analysis shows the activation induced CD8 downregulation on cell
surface. Data represent the mean ± SEM. (b) Dot plots graph shows MFI of CD8 expression after stimulation. *P < 0.05, **P < 0.01 vs. indicated control, a 2-tailed Student’s t test.
AntiCD3+antiCD28 : - + + + +
5-Aza : - - + - -
Empty nlg : - - - + -
nlg-5-Aza : - - - - +
a
b
2000
3250
4500
5750
* **
MF
I of
CD
8