1  

Supplementary Information

Synergy between Rapamycin and FLT3 Ligand enhances Plasmacytoid

Dendritic Cell-dependent Induction of CD4+CD25+FoxP3+ Treg

Moanaro Biswas1, Debalina Sarkar1, Sandeep RP Kumar1, Sushrusha Nayak1,

Geoffrey L. Rogers1, David M. Markusic1, Gongxian Liao2, Cox Terhorst2,

and Roland W. Herzog1

1 Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA

2 Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA 02115. USA.

  2  

Supplementary Methods:

Drug treatments. DO11.10-tg x Rag-2-/- mice were repeatedly i.p. injected (3xweek for 4

weeks) with a drug combination of 4 mg/kg rapamycin (LC laboratories, Woburn, MA)

encapsulated in 0.02% carboxymethyl cellulose and 0.25% Tween-80 and 100 µg

OVA323-339 peptide antigen (Anaspec, Fremont, CA). To this combination, a third

compound was added: either Flt3L (80 µg/kg, Celldex Therapeutics, Needham, MA), IL-

2 (50 ng/kg, Miltenyi Biotec, Auburn, CA), IL-10 (50 ng/kg, Sigma Aldrich, St. Louis,

MO), Fc-glucocorticoid-induced tumor necrosis factor receptor-ligand (FcGITRL) (8

mg/kg) or a combination of Flt3L and Fc-GITR. In separate experiments, OVA323-339,

Flt3L, Flt3L/OVA323-339, Flt3L/irrelevant peptide (human F.IX peptide) or Flt3L/OVA323-

339/rapamycin were injected 3x/week for 3 weeks. Increased rapamycin doses of 8 mg/kg

(2x rapa) and 16 mg/kg (3x rapa) were also tested. Percentages of CD4+ T cells,

CD4+CD25+FoxP3+ Treg, cDC and pDC were determined by flow cytometry.

Gene therapy experiments. C57BL/6 mice were treated with Flt3L/OVA323-339, OVA323-

339/rapamycin or a combination of Flt3L/OVA323-339/rapamycin by i.p. injection, 3x/week

for 3 weeks. Control mice received no drugs. Subsequently, mice were intramuscularly

(i.m.) injected with 1x1011 vector genomes (vg)/mouse of scAAV1-CMV-OVA as

described.1 OVA-specific CD8+ T cell responses in peripheral blood were quantified by

surface labeling with the iTag tetramer/PE-H-2kb OVA(SIINFEKL) from MBL

International (Woburn, MA).

  3  

Protein/factor VIII replacement therapy. A combination therapy of Flt3L/rapamycin/0.3

IU FVIII (B domain-deleted recombinant human FVIII, Wyeth Pharmaceuticals,

Philadelphia, PA), FVIII/rapamycin, FVIII/Flt3L or Flt3L/rapamycin was i.v. injected

into hemophilia A mice (BALB/c F8e16-/-) 3x/week for 4 weeks. This was followed by 4

weekly i.v. injections (on weeks 5, 6, 7 and 8) of 1 IU FVIII. Plasma samples were

collected by tail bleed into 0.38% sodium citrate buffer as described.2 Antibodies to

FVIII in plasma were measured by Bethesda assay (for inhibitor titers) and by ELISA

(for anti-FVIII IgG) as published.3,4

Global immune profiles. WT BALB/c mice (n=4) received repeated injections of Flt3L/

OVA323-339/rapamycin 3x/week for 4 weeks. Splenocytes were stained with antibodies for

enumeration of immune cell types: CD4+ and CD8+ T cells, CD4+CD25+FoxP3+ Treg,

CD19+ B cells, CD11c+ and CD11c+PDCA-1+ DC, CD11b+Ly6Ghi neutrophils,

CD11b+Ly6Glo monocytes/macrophages and CD49b+ NK cells. Complete automated and

manual blood counts were also carried out. Naïve mice that did not receive drug

treatment were used as controls. 1 month after the treatment regimen had been

discontinued, immune profiles were tested on the same mice. Mice that received Flt3L/

OVA323-339/rapamycin were injected 1 month after drug treatment with 100 µg of keyhole

limpet hemocyanin (KLH) antigen by the i.p. route. Plasma was collected 3 weeks

following KLH injection, and the formation of IgG1 antibody against KLH was tested by

ELISA.

  4  

References:

1. Rogers GL, Suzuki M, Zolotukhin I, et al. Unique Roles of TLR9- and MyD88-

Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-

Mediated Gene Transfer. J Innate Immun. 2015.

2. Cao O, Hoffman BE, Moghimi B, et al. Impact of the underlying mutation and the

route of vector administration on immune responses to factor IX in gene therapy for

hemophilia B. Mol Ther. 2009;17(10):1733-1742.

3. Moghimi B, Sack BK, Nayak S, Markusic DM, Mah CS, Herzog RW. Induction

of tolerance to factor VIII by transient co-administration with rapamycin. J Thromb

Haemost. 2011;9(8):1524-1533.

4. Sack BK, Merchant S, Markusic DM, et al. Transient B cell depletion or

improved transgene expression by codon optimization promote tolerance to factor VIII in

gene therapy. PLoS One. 2012;7(5):e37671.

  5  

Supplementary Figures:

Spleen Naive Gate: Lymph

CD4 V450-A

FSC

-H

-102 102 103 104 1050

65536

131072

196608

262144

CD436.85%

Spleen FOR Gate: Lymph

CD4 V450-A

FSC

-H

-102 102 103 104 1050

65536

131072

196608

262144

CD422.07%

Spleen Rapa Gate: Lymph

CD4 V450-A

FSC

-H

-102 103 104 1050

65536

131072

196608

262144

CD429.85%

!"#$%& '"("& )*+,-./$".'"("&

Fig. S1. Flt3L enhances activation induced cell death of CD4+ T effector cells.

Representative dot plots showing depletion of CD4+ T cells in spleens of DO11.10-tg x

Rag-2-/- BALB/c mice injected 3X week for 3 weeks by the intraperitoneal route with

rapamycin (4mg/Kg) or a combination of Flt3L (80µg/Kg)/ Ova323-339 (100µg)/ rapamycin

(4mg/Kg) (n = 4/group).

  6  

Fig. S2. Rapamycin is required for Treg induction (replicate experiment). A.

Induction of OVA-specific CD4+CD25+FoxP3+ Treg in DO11.10-tg x Rag-2-/-BALB/c

mice treated with Flt3L/Ova323-339/rapamycin combination is rapamycin dependent. Mice

treated with Ova323-339, Flt3L, Flt3L/Ova323-339, or rapamycin only failed to generate Treg.

Data are average ±SD (n=5/group). Statistical differences were determined by 1-way

ANOVA with Bonferonni’s multiple comparison post-test analysis. All groups tested

were significantly different from the Flt3L/OVA/Rapa treatment group (p<0.0001).

Experiments were performed in duplicate and similar Treg induction percentages were

observed from both independent experiments. B. Expression of CTLA-4, Helios, CD62L

and GITR Treg associated markers in Treg induced by with Flt3L/Ova323-339/rapamycin

combination treatment. Data are average ±SD (n=5/group). Percent CTLA-4 expressing

Treg is likely underestimated in this experiment because of non-optimized staining.

!"#$%

& #$'

(&)*+,-&"./

001"

!!!"

!"#$%

& #$'

(&)*+,-&.#$%

& "/"01

223"A B

  7  

Fig. S3. Depletion of pDC in different mouse models and effect on CD4+ T cell

numbers. A. DO11.10-tg x Rag2-/- mice were treated with 2 weekly i.v. injections of

PDCA-1 antibody (clone 927) and continued to receive Flt3L/Ova323-339/rapamycin

combination for 3.5 weeks. Control mice received Flt3L/Ova323-339/rapamycin treatment

without pDC depletion. pDC were identified as CD11c+B220+ cells. Percentage of pDC

depletion compared to control is indicated below each plot. Percent CD4 effector T cells

of total splenocytes in pDC depleted and control mice are indicated in the graphs on the

right. Anti-PDCA-1 treatment resulted in ~40% depletion in pDC numbers as observed

by flow cytometry. A significant decrease in CD4+ T cell numbers was observed in pDC

depleted mice. Data are average ±SD (n=6/group). Statistical differences were calculated

  8  

by the Student’s t-test. B. BALB/c mice were infused with 1x107 CD4+CD25- effector T

cells from DO11.10-tg x Rag2-/- mice before receiving 5 i.v. injections of PDCA-1

antibody (clone 120G8) over a 3 week period. Mice simultaneously received

Flt3L/Ova323-339/rapamycin combination for 3 weeks. Control mice received

Flt3L/Ova323-339/rapamycin treatment without pDC depletion. pDC were identified as

CD11c+B220+SiglecH+ cells. Percentage of pDC depletion and of ova-specific CD4

effector T cells in pDC depleted and control mice are indicated. Anti-PDCA-1 treatment

resulted in ~76% depletion in pDC numbers as observed by flow cytometry. No

significant differences in total Ova323-339 specific (KJ1-26+) CD4+ T cell numbers were

observed between pDC depleted and control mice. Data are average ±SD (n=8/group). C.

BDCA-2-DTR mice were infused with 1x107 CD4+CD25- effector T cells from

C57BL/6-tg OTII mice before receiving i.v. injections of Diphtheria toxin (3x week for 3

weeks). Mice continued to receive Flt3L/Ova323-339/rapamycin combination for 3 weeks.

Control mice received Flt3L/Ova323-339/rapamycin treatment without pDC depletion. pDC

were identified as CD11c+PDCA-1+ cells. Diphtheria toxin treatment resulted in ~97%

depletion in pDC numbers as observed by flow cytometry. pDC depletion resulted in

significantly reduced endogenous Ova323-339-specific (MR9-4+) CD4+ T cell numbers in

recipient mice (analysis of transferred OT II cells is shown in Fig. S5). Data are average

±SD (n=4/group). Statistical differences were calculated by the Student’s t-test.

  9  

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'()'

!"#$%&*'+,-.'

)/01.-/123'+,-.'

Fig. S4. Gating scheme for enumerating i) induced ova-specific Treg in recipients of

adoptively transferred KJ1-26+ CD4+ T cells and ii) endogenous BALB/c CD4+ Treg

populations. Induced ova-specific Treg were identified as CD4+KJ1-26+CD25+FoxP3+

cells, whereas endogenous Treg were identified as CD4+KJ1-26-CD25+FoxP3+ cells.

  10  

  11  

Fig. S5. Adoptive transfer of OTII CD4+ T cells into BDCA-2-DTR mice. A.

Experimental timeline depicting adoptive transfer of OTII CD4+ T cells into BDCA-2-

DTR mice. BDCA-2-DTR mice received i.v. injections of Diphtheria toxin (3x week for

3 weeks) to deplete pDC. One day after the first pDC depletion treatment, mice were

infused with 1x107 CTV-labeled CD4+CD25- effector T cells from C57BL/6-Tg OTII

mice. Mice continued to receive Flt3L/Ova323-339/rapamycin combination during the

course of pDC depletion. Control mice received Flt3L/Ova323-339/rapamycin treatment

without pDC depletion. B. Gating scheme for detection of Treg induction from

adoptively transferred CTV+CD4+ T effector and of endogenous MR9-4+ T effector

populations. Transplanted Treg were identified as CTV+MR9-4+CD25+FoxP3+ cells,

whereas Ova323-339 specific endogenous Treg were identified as CTV-MR9-

4+CD25+FoxP3+ cells. C. No significant differences in the induction of Ova-specific

(CTV+MR9-4+) Treg from transplanted donor (OTII) cells were observed between pDC-

depleted and control mice after Flt3L/OVA323-339/rapamycin treatment. The percentage of

CD4+ T effector cells from transplanted cells (CTV+MR9-4+CD4+) also remained

unchanged between pDC depleted and control groups after Flt3L/OVA323-339/rapamycin

treatment. Data are average ±SD (n=4/group). Statistical differences were determined by

the student’s t-test.

  12  

cDC S

pleen

pDC Splee

n

cDC B

one Mar

row

pDC Bone M

arro

w0

20

40

60

80

100

**

% F

LT3+

cells

Fig. S6. Increased expression of Flt3 (receptor for Flt3L) in pDC. Increased

expression of Flt3 in spleen and bone marrow pDC of DO11.10-tg x Rag-2-/- BALB/c

mice. Cells were surface labeled with CD135 (Flt3)-PE antibody and simultaneously

stained with CD11c and PDCA-1 antibodies to determine relative percentage of Flt3

staining on DC subsets.

  13  

Spleen Unst Gate: PDC

pmTOR PE-A

Coun

t

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: PDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: PDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: CDC

pmTOR PE-A

Coun

t

-101 102 103 104 1050

12

24

35

47

Spleen Unst Gate: CDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

12

24

35

47

Spleen Unst Gate: CDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

12

24

35

47

pDC cDC

FLT3L

Rapa

Flt3L+Rapa

Spleen Unst Gate: PDC

pmTOR PE-A

Coun

t

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: PDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: PDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: CDC

pmTOR PE-A

Coun

t

-101 102 103 104 1050

12

24

35

47

Spleen Unst Gate: CDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

12

24

35

47

Spleen Unst Gate: CDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

12

24

35

47

pDC cDC

FLT3L

Rapa

Flt3L+Rapa

Spleen Unst Gate: PDC

pmTOR PE-A

Coun

t

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: PDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: PDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

14

28

41

55

Spleen Unst Gate: CDC

pmTOR PE-A

Coun

t

-101 102 103 104 1050

12

24

35

47

Spleen Unst Gate: CDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

12

24

35

47

Spleen Unst Gate: CDC

pmTOR PE-A

Cou

nt

-101 102 103 104 1050

12

24

35

47

pDC cDC

FLT3L

Rapa

Flt3L+Rapa

!"#$ %"#$

&'('

$)*+,*$

)*+,*-&'

('$

!./0!./12+3&$(4$

Ctrl%Flt3L%

Ctrl%Flt3L%

Ctrl%Rapa%

Ctrl%Rapa%

Ctrl%Flt3L%/Rapa%

Ctrl%Flt3L%/Rapa%

Fig. S7. Expanded histogram overlays showing differential p-mTORSer2448 expression in

splenic pDC and cDC of naïve DO11.10-tg x Rag-2-/- BALB/c mice after in vitro

incubation for 60 min with Flt3L, rapamycin or a combination of FLt3L/rapamycin.

  14  

CD11c+ CD11c+PDCA+0

1

2

3

ControlFlt3L+Ova+RapaFlt3L+Ova+Rapa 1month

2.5x

6x

*

***

% S

plen

ocyt

es

Naive

FOR

FOR 1month

0

10

20

30

Perc

ent F

oxP3

+ / C

D4+

T ce

lls

*

CD4+ CD8+ CD19+0

20

40

60

80

ControlFlt3L+Ova+RapaFlt3L+Ova+Rapa 1month

% S

plen

ocyt

es

CD11b+Ly6G+ CD11b+Ly6Glo CD49b+0

5

10

15

ControlFlt3L+Ova+RapaFlt3L+Ova+Rapa 1month

% S

plen

ocyt

es

!" #"

$"

%" &"

'

Neutrophils

Lymphocytes

Monocytes

Eosinophils

0

20

40

60

80

ControlFlt3L+Ova+RapaFlt3L+Ova+Rapa 1month

Perc

ent C

ells

/ Bl

ood

*** ***

Flt3L+Ova+Rapa Control0

20000

40000

60000

80000

100000!

-KLH

IgG

1!"#$%&'()*

&+,(-./0&

Fig. S8. Transient changes to the immune system induced by a 1-month

Flt3L/Ova323-339/rapamycin (FOR) regimen (mice recover complete immune function

within 1 month after completion of the regimen). A. Percentage of CD4+, CD8+ T cells

and B cells (CD19+) of total splenocytes in control, FOR treated or 1-month post FOR

treated wt BALB/c mice (n=4). B. FoxP3+ Treg as a percentage of CD4+ T cells in

control, FOR treated or 1-month post FOR treated wt BALB/c mice. C. Transient

upregulation of cDC (CD11chi) and pDC (CD11c+PDCA-1+) in FOR treated mice. D.

  15  

Percentage of neutrophils (CD11b+Ly6Ghi), monocytes/macrophages (CD11b+Ly6Glo)

and NK cells (CD49b+) of total splenocytes in control, FOR treated or 1 month post FOR

treated wt BALB/c mice. E. Complete automated and manual blood counts of control,

FOR treated or 1-month post FOR treated wt BALB/c mice. F. Anti-KLH IgG1 titers in

wt BALB/c mice that previously received FOR treatment or naïve controls. Mice were

injected i.p. with 100µg of KLH antigen, and the formation of IgG1 antibody against

KLH was tested by ELISA.

  16  

Naive 1 Gate: lymph

CD4 V450-A

CD

8 V

500-

A

-10310

010

310

410

5

-102

103

104

105

CD422.67%

CD810.60%

Naive 1 Gate: lymph

CD19 APC-Cy7-A

Ly6G

Per

CP

-A

-102

103

104

105

-102

101

103

104

105

CD1952.05%

Naive 1 Gate: lymph

CD11b PE-Cy7-A

Ly6G

Per

CP

-A

-103

-102

103

104

105

-102

101

103

104

105

Neutrophil4.79%

Mono/MF1.68%

Naive 1 Gate: lymph

CD11c QDot 605-A

PD

CA

-1 P

E-A

-102

103

104

105

-102

102

103

104

105

cDC0.60%

pDC0.52% Naive 1

Gate: lymph

CD11c QDot 605-A

CD

8 V

500-

A

-102

103

104

105

-102

103

104

105 10.32% 0.11%

88.99%0.59%

Naive 1 Gate: CD4

CD4 V450-A

FoxP

3 A

PC

-A

101

102

103

104

105

-102

102

103

104

105

85.91%0.00%

14.09%0.00%

Naive 1 Gate: Non CD11b

CD49b FITC-A

FoxP

3 A

PC

-A

-101

102

103

104

105

-102

102

103

104

105

NK1.26%

Naive 1 Gate: lymph

CD11b PE-Cy7-A

Ly6G

Per

CP

-A

-103

-102

103

104

105

-102

101

103

104

105

Neutrophil4.79%

Mono/MF1.68%

Non CD11b84.26%

Fig. S9. Gating scheme of immune cell types from spleens of wt BALB/c mice.

  17  

Naive Flt3L Rapamycin FOR0

2000

4000

6000

8000

10000TG

F-β

pg/m

l

Fig. S10. Treg expansion in Flt3L/OVA323-339/rapamycin treated mice is

accompanied by an increase in TGF-β production in vivo. Activated TGF-β levels

from plasma of BALB/c mice that were treated 3x/week for 3 weeks i.p. with Flt3L,

rapamycin or Flt3L/ Ova323-339/ rapamycin (n=4). Increases in TGF-β production in

experimental groups over control were observed to be non-significant (One-way ANOVA

with Tukey’s post-test comparison).