PBS

01234567

CD11c+

CD83+

(R1)

CD11c+

CD83–

(R2)

+ PBS+ Clodro

Cel

l # (%

)

P = 0.12

P = 0.003

100

101

102

103

104

CD11C

B6/COLODR/SPL/11C/83/LAP/#2.006

100

101

102

103

104

CD11C

B6/PBS/SPL/11C/83/LAP/#1.003

CD

83

R1

R2

R1

R2

CD11c

01

23

4

56

+ PBS+ Clodro

CD11c+

CD86+hi

(R1)

CD11c+

CD86–/low

(R2)

P = 0.97

P = 0.02

Cel

l # (%

)

100

101

102

103

104

CD11C

B6/COLODR/SPL/11C/86/LAP/#2.016

100

101

102

103

104

CD11C

B6/PBS/SPL/11C/86/LAP/#1.013

CD

86

CD11c

R1

R2

R1

R2

0

1

2

3

4

5

6

CD11c+

CD80+hi

(R1)

CD11c+

CD80–/low

(R2)

+ PBS+ Clodro

Cel

l # (%

)

P = 0.27

P = 0.05

100

101

102

103

104

CD11C

B6/COLODR/SPL/11C/80/LAP/#2.011

100

101

102

103

104

CD11C

B6/PBS/SPL/11C/80/LAP/#1.008

CD

80

CD11c

R1

R2

R1

R2

a

b

c

Clodronate

Supplementary Fig. S1 Clodronate-loaded liposomes selectively deplete iDCs. C57BL/6 mice wereinjected i.v. with PBS- or clodronate-loaded liposomes. Spleen cells were then harvested 24 h later,stained with CD11c-FITC and CD83- (a), CD86- (b), or CD80-PE (c) antibodies and analyzed withFACScalibur. Data are shown as representative FACS profiles of CD11c vs. indicated surface markersin each group of mice (left panel). Data are shown as mean ± s.d. of indicated cell numbers ofindividual mice (n = 2-3 in each group, right panel). The experiment was repeated twice with similarresults.

CD3-specific antibody-induced immune tolerance and suppression of autoimmuneencephalomyelitis involves TGF-β production through phagocytes digesting apoptotic T cellsSylvain Perruche1,3, Pin Zhang1, Yongzhong Liu1, Jeffrey A. Bluestone2, and WanJun Chen1*

aF4/80+ MΦ:Red; Apo-T cells: Green.

F4/80+ MΦ: Red; Apo CD4+ T cells: Green.b c

0

100

200

300

400

MΦ+Med

TGF-β1

(pg/

ml)

MΦ+Apo

100

101

102

103

104

FL4-LAP-TGFb

M1

PBSanti-CD3

Rel

ativ

e ce

ll nu

mbe

r

LAP-TGF-β

d e

0

5

10

15

20

25

30

35P = 0.0002

LAP-

TGF-β+

cel

ls (%

)(w

ithin

F4/

80+ )

PBS

in vivo treatment

anti-CD3

Supplementary Fig. S2 Macrophages engulf and digest apoptotic T cells and produce TGF-β. (a) Macrophagesstained with PE-F4/80 antibody (red) were co-cultured with apoptotic thymocytes pre-labeled with CFSE for 12 hand examined under immuno-fluorescence microscopy. White arrows: apoptotic cells that are engulfed andingested by macrophages; yellow arrows: apoptotic contacting macrophages. (b) The supernatants frommacrophages cultured alone and plus apoptotic thymocytes were tested for total TGF-β1 by ELISA. The data areshown as mean ± s.d. of the duplicate wells and representative of three independent experiments. (c) Macrophagestake up and digest apoptotic CD4+ T cells in vivo. The experiment was done as described in Fig. 2e. Green color:apoptotic T cells, red color: F4/80+ macrophages in spleen. (d,e) Macrophages increase LAP-TGF-β after CD3-antibody treatment in vivo. C57BL/6 mice were injected with PBS or CD3-antibody. Spleen cells were stained forF4/80 and LAP-TGF-β 24 h later. (d) A representative FACS profile of LAP-TGF-β expression on macrophages.Dashed line indicates the histogram of isotype control antibody. (e) Data are shown as mean ± s.d. of LAP-TGF-β+

cells within F4/80+ macrophages in individual mice (n = 7 mice in each group).

PBSanti-CD3-day 1anti-CD3-day 4

0

5

10

15

20

25

CD

4+C

D25

+ Fox

p3+ T

regs

(%)

P = 0.04

P = 0.003

Blood

P = 0.000003

P = 0.0000002

PLN

P = 0.003

P = 0.02

MLN

P = 0.04

P = 0.30

Spleen

P = 0.005

nd

Lungs

Supplementary Fig. S3 CD3-antibody treatment induced increase in CD4+Foxp3+ Treg cells invivo. C57BL/6 mice were injected with PBS- or clodronate-loaded liposomes one day beforeCD3-antibody treatment. On days one and four post-antibody injection, T cells from blood,spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and lungs were isolatedand stained with CD4-, CD8-, CD25- and Foxp3-antibodies and analyzed by FACScalibur. CD4+

T cells were gated and the frequency of CD4+CD25+Foxp3+ Treg cells is shown. The data areshown as mean ± s.d. of individual mice in each group. (PBS vs. anti- CD3-day 4: blood: n = 7mice; spleen: n = 11 mice, PLN: n = 9-11 mice; MLN: n = 4 mice; lungs: n = 5 mice. anti-CD3-day 1: n = 2 mice per group). nd: not done.

a

b

05

1015202530354045

PBS

Tota

l # o

f DLN

cel

ls p

erm

ouse

(*10

6 )

+ Apo cell + Necro cell

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

3 H u

ptak

e (C

PM)

P = 0.03 P = 0.008

in vivo treatment

PBS + Apo cell + Necro cell

Supplementary Fig. S4 Necrotic T cells fail to induce immune tolerance in vivo. C57BL/6 micewere injected with apoptotic (+ Apo cell) or necrotic T cells (+ Necro cell) followed byimmunization with OVA peptide in CFA. (a) One week later, the draining lymph nodes wereharvested and pooled for each group (n = 3 mice) and total cell numbers were calculated. (b) Thecells were then cultured with OVA protein (50 µg/ml) for 3 d and antigen-specific T cellproliferation was determined by 3H thymidine incorporation assay. The data are shown as mean ±s.d. of the triplicate wells of the plate.

a

b

c

0

0.05

0.15

0.25

0.35

IL-1

7+ ce

lls in

sple

en (%

)

PBS anti-CD3

Clodro anti-CD3+ Clodro

0

0.02

0.04

0.06

0.08

IFN

-γ+

cells

inD

LNs

(%)

PBS anti-CD3

Clodro anti-CD3+ Clodro

TNF-α

in D

LNs

(ΔO

.D. v

alue

s)

0

0.02

0.04

0.06

0.08

0.10

0.12

0.14

PBS anti-CD3

Clodro anti-CD3+ Clodro

Supplementary Fig. S5 MOG-specific T cell tolerance in CD3-specific antibody treated mice isreversed by phagocyte depletion. IL-17 expressing cells (a) were detected in spleens and IFN-γexpressing cells in the draining lymph nodes (DLNs) (b) ex vivo 21 d after MOG-immunization inthe different groups: PBS, clodronate-loaded liposomes (Clodro), CD3-specific (anti-CD3) orclodronate in addition to CD3-specific antibody (anti-CD3 + Clodro). IL17 and IFN-γ expressingcell percentages are given in the CD4+ T cell population. (c) T cells from draining lymph nodes ofthe different groups were also cultured for 3 d with MOG-stimulation and the supernatants werequantified for TNF (O.D., mean ± s.e.m.). Representative results from 2 independent experiments.

0

1

2

3

4

5

0 3 6 9 12 15 18 21 27 30 33

Time (d) post immunization

PBSanti-CD3anti-CD3 + anti-TGF-β

*

ns

*

Mea

n cl

inic

al s

core

Supplementary Fig. S6 Injection of anti-TGF-β antibody reverses intact CD3-antibody-mediated therapeutic effects on relapsing-remitting EAE. SJL mice were immunized withPLP139–151 peptide and then randomly divided into the following 3 groups: untreated control(PBS, n = 12), treated with CD3-antibody (anti-CD3, n = 6), or with CD3-antibody plus anti-TGF-β1,2,3-antibody (anti-CD3 + anti-TGF-β, n = 6). As indicated (blue arrow), CD3-antibody(50 µg/per mouse) was injected i.p. on days 13-14. *P < 0.05; ns: no statistical significance(one-way ANOVA test). The data are shown as mean ± s.e.m. of clinical scores of individualmice in two independent experiments.

Mice

αCD3injection

activation/proliferation/

apoptosis

IL2,IFN-γ TNF

APC activation/phagocytosis

TGF-β

-

Tolerance

TNF

iDCsMØ

T cell

Foxp3+

Treg

T

Supplementary Fig. S7 A proposed model of T cell apoptosis/phagocyte mediated immunetolerance induced by intact CD3-specific antibody treatment in mice. Intact antibody inducestransient activation and release of T cell-inflammatory cytokines, followed by T cell deletion.Macrophages and iDCs then engulf and digest apoptotic T cells to produce TGF-β. TGF-β thenin turn not only downregulates consequent activation of immune cells and resolve theinflammation, but also establishes an immunosuppressive microenvironment under which newlygenerated antigen specific T cells are anergized and/or promoted to differentiate into Tregulatory cells. Immune tolerance ensues.

Supplementary Table 1 Depletion of phagocytes abrogates CD3-antibody treatment mediated increase in ratios of Foxp3+ to IL-17+ or IFN-γ+ in spinal cords of MOG-induced EAEa

Treatment Ratio of Foxp3+/IL-17+ Ratio of Foxp3+/IFN-γ+

PBS 20 2.0

Clodronate 5.1 0.9

Anti-CD3 + PBS 37.8 4.2

Anti-CD3 + Clodronate 3.8 0.6

a Four days before immunization with MOG35–55 peptide, C57BL/6 mice received PBS (n = 3), Clodronate (n = 4), anti-CD3 + PBS (n = 3) or anti-CD3 + Clodronate (n = 3). 21 days post immunization, spinal cords from each group were pooled together and cells were isolated for flow cytometry analysis. The ratios of CD4+Foxp3+ Treg cells to CD4+IL-17+ or IFN-γ+ cells were calculated as the following formula: the % of Foxp3+ Treg cells/ % of IL-17+ or IFN-γ+ cells.

Supplementary Table 2 Depletion of phagocytes abrogates CD3-antibody treatment-mediated increase in the ratios of Foxp3+ to IL-17+ or IFN-γ+ cells in relapsing-remitting EAEa

Treatment Tregs (%, SC)

Ratio (SC)

(Foxp3+/IL-17+)

Ratio (SC)

(Foxp3+/IFN-γ+) Treg / CD25+Foxp3– cells

(%, Spleen)

PBS 16.0 2.2 0.8 24/17

Anti-CD3 + PBS 34.0 4.1 2.3 32/15

Anti-CD3 + Clodronate 3.7 0.9 0.7 24/19

3-IgG3 + PBS 5.3 7.1 2.3 29/5.3

3-IgG3 + Clodronate 13.0 3.0 6.8 34/5.1

a SJL mice were immunized with PLP139-151 peptide in CFA. At the peak of the acute EAE (d 13), the mice were randomly divided into the five following groups: PBS (n = 4), anti-CD3 + PBS (n = 3), anti-CD3 + Clodronate (n = 3), 3-IgG3 + PBS (n = 3) and 3-IgG3 + Clodronate (n = 4). Mice from each group were pooled together. Cells were isolated and then divided into two parts, one for Foxp3+ Treg cell staining and the other to study IL-17 and IFN-γ production in CD4+ T cells, both by flow cytometry. The ratios of CD4+Foxp3+ Treg cells to CD4+IL-17+ or IFN-γ+ cells were calculated as the following formula: the % of Foxp3+ Treg cells/% of IL-17+ or IFN-γ+ cells. Foxp3+ Treg cells to CD25+Foxp3– effector T cells ratio was also assessed in the pooled spleens.

Supplementary methods.

Antibodies. The following antibodies were purchased form BD Biosciences: Purified

anti-CD16/CD32; FITC-conjugated-CD3-, CD4-, CD11c-, and IFN-γ- antibodies; PE-

conjugated CD8-, CD11c-, CD80-, CD83-, CD86-, MAC3-, TNF-, IL-4-, and IFN-γ-

antibodies ; PercP-conjugated CD4-, CD8- antibodies; APC-conjugated CD45LCA-antibody.

FITC-conjugated F4/80 antibody was purchased from Caltag (Invitrogen). APC-

conjugated IL-17 and PE-conjugated Foxp3 antibodies were purchased from eBioscience.

Isolation and culture of dendritic cells and macrophages. Briefly, spleens were injected

with 1 mg of collagenase D (Gibco, Invitrogen), cut into small pieces and incubated at

37°C for 30 minutes. Single cell suspension was then passed trough a 70 µm cell strainer,

washed and incubated with purified CD16/CD32-antibody to block non-specific FcR

binding. CD11c+ dendritic cells were then purified using CD11c-antibody coated

microbeads (Miltenyi Biotec) and MAC3+ or F4/80+ cells were isolated using PE-

conjugated MAC3- or F4/80- antibodies followed by incubation with anti-PE microbeads

(Miltenyi Biotec). For culture and phagocytosis assay, DCs or macrophages were plated

into 24 well plates (0.1 to 1 x 106 cells/well) and incubated in the absence or presence of

apoptotic cells (5:1 ratio) overnight in a serum-free milieu x-vivo 20. Supernatants were

then collected for cytokine quantification. Apoptotic cells were obtain from thymocytes

after γ-irradiation (1500 Rad) followed by a 3-8 h incubation in complete DMEM at

37°C. In some experiments, apoptotic cells were first labeled with carboxyfluoroscein

succinimidyl ester (CFSE, 2.5 µM) and then incubated with splenic DC or macrophages

overnight. Cells were then stained with PE-conjugated CD11c- or F4/80- antibodies and

then analyzed under immunofluorescence microscopy. For phagocytosis assay in vivo,

syngenic CD4+ T cells were labeled with CFSE and injected into Rag1-/- mice followed

by CD3-antibody treatment. 1-2 h later, spleens, lungs and liver were harvested and DCs

or macrophages were stained with purified CD11c- or F4/80- antibodies followed by

Cy5-goat anti-hamster IgG or Rhod-Red goat anti-rat IgG (Jackson Lab) secondary

staining respectively and examined under the confocal microscopy. For coculture

experiments DC (0.2 x 106 cells) plus apoptotic cells (106 cells) were placed in the upper

level of a transwell culture insert (0.4 µm pore size; BD Biosciences) and CD4+CD25-

naïve purified T cells (0.5 x 106, >98-99% purity, Miltenyi Biotec) were plated below the

transwell in CD3 antibody-coated wells (5 µg/ml) with soluble CD28-antibody (2 µg/ml)

for 3-4 d. In some wells, TGF-β1,2,3 antibody (50 µg/ml; clone: 1D11; R&D Systems)

was added. T cells were counted and prepared for Foxp3 mRNA quantification by real-

time PCR or staining with Foxp3-antibody45.

Phagocyte depletion. In some experiments, mice were scarified at 24 hours and DCs

were stained with FITC-CD11c- together with PE-CD80, -CD86,- or -CD83 antibodies to

examine the phenotype of the DC subsets. In other experiments, DO11.10 OVA

peptide3332-339 TCR transgenic mice were injected i.p. with PBS or OVA protein (2 mg

/mouse). On days 4-5, blood samples were collected and leukocytes were stained

immediately with CD4- and CD8- antibodies together with Annexin V and 7-AAD ( BD

Biosciences) to detect apoptosis. Some OVA-treated mice were then injected with

clodronate on day 5 to deplete phagocytes. On day 10, all mice were scarified and spleen

cells were isolated to determine the OVA-specific T cell proliferation and Foxp3+ Tregs.

In other experiments, C57BL/6 mice were injected with PBS- or clodronate-loaded

liposomes followed by CD3-antibody treatment one day later. T cells were isolated (day

4) and stained with CD4-, CD8-,CD25- and Foxp3- antibodies and analyzed by

FACScalibur.

T cell culture and proliferation assay. 106 T cells/ml of complete DMEM were

stimulated either by soluble CD3-specific antibody (5 µg/ml) or MOG peptide (0.01 to 1

µg/ml) or PLP peptide 139-151 ( 0.1 to 100 µg/ml). Cells were cultured at 37°C in 5% CO2

for 3 to 5 d and pulsed with 1 µCi [3H] thymidine for the last 6–16 h for proliferation

assay. Radioactivity incorporation was counted using a flatbed β-counter (Wallac). For

cytokine assay, cells were culture in complete DMEM in the same peptide- or TCR-

stimulations as proliferation assay for 3 or 5 d and supernatants were collected for TNF

and IFN-γ quantification. For IL-17 expressing cell detection, phorbol 12-myristate 13-

acetate (PMA, 5 ng/ml, Sigma), ionomycine (250 ng/ml, Sigma) and GolgiPlug (1 µl/ml,

BD Biosciences) were added for the last 5 h of culture. Then cells were prepared for

intracellular staining.

Flow cytometry analysis of LAP-TGF-β . For analysis of the latent form of TGF-β

(LAP-TGF-β) on macrophages and DCs, C57BL/6 mice were injected with PBS or

CD3-antibody (50 µg/per mouse). Spleen cells were isolated 24 h later and incubated

with CD16/32 FcR blocking antibody (BD Biosciences). Cells were then stained with

antibodies against F4/80 or CD11c together with biotinylated-LAP-TGF-β antibody (0.5

µg/ per 106 cells, R&D systems) followed by Streptavidin-APC staining (0.2 µg/ 106

cells).

EAE induction, scoring and analysis. For induction of EAE, C57BL/6 mice were

immunized subcutaneously on day 0 with 200 µg/mouse of MOG35-55 peptide (Research

Genetics), emulsified in CFA (IFA supplemented with 400 µg/ml heat-killed M.

tuberculosis) at two sites in the lower back as well as in each rear footpads (50 µg per

site). Mice received also 200 ng of Bordetella pertussis (List Biological Lab) i.p. on the

day of immunization and 2 d later. For induction of relapsing-remitting EAE, SJL mice (

Jackson Laboratory) were immunized with with PLP peptide139-151 ( 75 µg/mouse) in

CFA ( 200 µg/mouse) at three sites on the back followed by i.p. injection of Bordetella

pertussis toxin ( 100 ng per mouse) 2 d later. All the immunized mice developed the

acute disease on d 10-12. Since some mice died from the acute EAE, the surviving mice

were then randomly divided into the indicated groups to ensure that each group had

similar average score before the treatments. As indicated, PBS- or clodronate- loaded

liposomes were injected on day 13, intact CD3-antibody (50 µg) was injected once on

day 14 and CD3-IgG3 antibody ( 50 µg per mouse per day) was injected for 5 d ( d 14-

18). The mice were monitored and scored daily for 33 or 62 d . Whenever a mouse died

of score 5, the mean EAE score of the involved group did not include the dead mouse

during the following days. The basic scoring system used was 0: no disease; 1: limp tail;

2: weak/partially paralyzed hind legs; 3: completely paralyzed hind legs; 4: complete

hind and partial front leg paralysis and 5: complete paralysis/death. Animals were scored

twice a week or daily when the disease started to occur. On day 21 after MOG

immunization or days 33 or 62 after PLP peptide 139-151 immunization, spinal cords were

harvested and a part was fixed with neutral 10% formalin, extracted, embedded in

paraffin and cut in 5 µm sections for H&E staining whereas the other part was submitted

to collagenase D digestion (10 mg/ml) for 30 min at 37°C. Then the solution was passed

through a 70 µm cell strainer, washed once with PBS and resuspended in 38% Percoll

solution for 20 min at 2000 rpm. Then pellets of cells were resuspended in the culture

medium for further staining or culture.