powerpoint presentation student’s t test. n = 5-6 mice per group for 2 independent experiments. 0...

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

*


CD4 T cells CD8 T cells DN T cells

IL-17

IFN

g


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***


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


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


***

***

*

***

*

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


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


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


*** **

***

***

* ***

*

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


**

***

*

*** **

*** ***

***

*

*

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**


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

powerpoint presentation student’s t test. n = 5-6 mice per group for 2 independent experiments. 0...

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