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