Supporting Information Lau-Kilby et al. 10.1073/pnas.1009738108 SI Materials and Methods Conditional STAT5KO (tie2-cre, STAT5 fl/fl ) Mice. Conditional STAT5KO (tie2-cre, STAT5 fl/fl ) mice [a gift from John O’Shea (National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD) and Lothar Hennighausen (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD)] were typed by genomic DNA (for cre) and hematocrit levels (1). Athough tie2, a tyrosine kinase TEK, is expressed specifically by hematopoietic stem cells, its expression can also be found on endothelial cells (EC) (2). There is some evidence that EC can impact DC development and maturation (3, 4). The re- sults obtained using these mice can therefore be an indirect effect of stat5 deletion on EC function. The deletion of STAT5 was confirmed by RT-PCR using RNA isolated from BM-derived cells. Cells from all stat5 KO mice show reduction of 10 cycles, i.e., >1,000-fold reduction in stat5a expression. Isolation and Culturing of FL-BM. The tibias and femurs were re- moved from mice and flushed to obtain bone marrow cells. The red blood cells were lysed and remaining bone marrow cells plated out (10 6 /mL) with complete RPMI-1640 (cRPMI) [10% FCS, 2 mM L-gluamine, 100 units/mL penicillin, 100 μg/mL strepto- mycin, 50 μM 2-mercaptoethanol (MCE), 100 μM nonessential amino acids (NEAA), 1 mM sodium pyruvate]. Where indicated, 25 μg/mL of LEAF-purified αIL2Rα/CD25 (PC61) (isotype control: purified Rat IgG1) or αIL2Rβ/CD122 (isotype control: purified Rag IgG2a) (Biolegend) was also added. For lineage depletion, biotin-conjugated antibodies against CD3 (145-2C11), CD11b (M1/70), Gr1 (RB6-8C5), TER119, NK1.1 (PK136), and CD19 (6D5) (Biolegend) were used. Cells were depleted of line- age by anti-biotin beads followed by negative selection using a magnetic column (Miltenyi Biotec). Flow Cytometry. The following antibodies were used: anti-mouse Siglec-H (440c), CD317/bone marrow stromal cell antigen 2 (BST2) (927), ckit/CD117 (2B8), and MCSF-R/CD115 (AFS98) (eBioscience); CD11c (N418), CD11b (M1/70), CD3 (145-2C11), NK1.1 (PK136), CD25 (PC61), and CD24 (M1/69) (Biolegend); and SIRPα (P84) and IA b (AF6120.1) (BD). T cells were defined as CD3 + NK1.1 − , NK cells as CD3 − NK1.1 + , and NKT cells as CD3 + NK1.1 + . Cells were analyzed on a FACS-CyAN machine (Beckman Coulter) or FACS-LSRII (BD). To calculate absolute numbers of cells per culture well/sample, a known number of Countbright beads (Invitrogen) was added to the sample im- mediately before flow analyses. The ratio of known vs. the de- tected number of beads was used to calculate the absolute number of cells from the detected number of cells. All data shown are representative of at least three separate experiments showing similar results (Figs. 1–7), and each dot represents one culture well (Figs. 1–7, S4, and S5) except where indicated. For all statistical analyses, a nonparametric Student’s t test was used. P values ≤0.05 were considered statistically significant. Isolation of DCs from FL-BM Cultures. To sort pDCs/cDCs from 7-d FL-BM cultures (starting cells: either total BM or sorted Lin − flt3 + CD11c − BM cells), cells were harvested and stained with anti-mouse PECy7-CD11c (N418), biotin-NK1.1 (PK136), PB-CD11b (M1/70), and Al488-CD317 (clone 120G8) (all from Biolegend except Al488-CD317, from Imgenex) and then SA-PO (Invitrogen) and sorted using FACS Aria (BD Biosciences) (Fig. S2C). Because the antibody to Siglec-H, 440c, has been shown to alter pDC function by modulating type 1 IFN production (5), we used anti-CD317/BST2 (clone 120G8) to sort pDCs. In a check for purity, >90% of these cells were found to express Siglec-H. Phenotype of DCs. To analyze the phenotype of DCs after 7-d FL- BM cultures, cells were harvested, washed, and stained with anti- mouse CD40 (3/23), CD86 (GL-1), CD80 (16-10A1), MHCII IAb (AF6120.1), and PD-L1 (M1H5) (all from Biolegend except PD- L1 from eBioscience and MHCII from BD). Cytokine Secretion. For analyses of cytokine secretion by sorted pDCs or cDCs after overnight TLR stimulation, sorted DCs were plated at 5 × 10 5 /mL and cDCs were stimulated with 100 ng/mL LPS (Sigma) or 1 μM CpG-1826 (Invivogen), whereas pDCs were stimulated with 1 μM CpG-2216 (Invivogen) for 18–22 h. Supernatants were harvested and analyzed using mouse IL-6, TNF-α, and IL-12p70 ELISAs (R&D systems). IFN-α secretion by pDCs was also analyzed using a sandwich ELISA coated with rat monoclonal anti-mouse IFN-α (RAMMA-1) followed by in- cubation with culture supernatants and then with rabbit poly- clonal anti-mouse IFN-α (both antibodies from PBL). The amount of IFN-α was then detected using goat anti-rabbit horseradish peroxidase (Promega) followed by TMB substrate (R&D Systems). For analyses of cytokine secretion in DC–T-cell cocultures, supernatents were harvested at the end of 3-d co- culture and analyzed using mouse IL-10 and IL-12 ELISAs (R&D Systems). DC–T-Cell Cocultures. T cells were isolated from the spleen and splenocytes were lysed of RBC and then incubated with biotin- conjugated anti-mouse CD11b (M1/70), B220, CD49b (DX5), Gr1 (RB6-8C5), CD8 (53-6.7), TER119, and CD25 (PC61) (Biolegend). CD4 + CD25 − splenic T cells were then purified by adding anti-biotin beads followed by negative selection via a magnetic column (Miltenyi Biotec). DC–T-cell cocultures were analyzed by staining for CD11c (N418), CD25 (PC61), and CD4 (GK1.5) (Biolegend). Where appropriate, cells were then stained with Annexin V and/or fixed and permeabilized for in- tracellular staining of Foxp3 (eBioscience). Blocking of IL-12 Activity. Where applicable, 500 ng/mL anti-mouse IL-12p40 (C17.8) was added to DC–T-cell cocultures to block IL- 12 activity. 1. Zhu BM, et al. (2008) Hematopoietic-specific Stat5-null mice display microcytic hypochromic anemia associated with reduced transferrin receptor gene expression. Blood 112:2071–2080. 2. Kisanuki YY, et al. (2001) Tie2-Cre transgenic mice: A new model for endothelial cell- lineage analysis in vivo. Dev Biol 230:230–242. 3. Despars G, O’Neill HC (2006) Splenic endothelial cell lines support development of dendritic cells from bone marrow. Stem Cells 24:1496–1504. 4. Tian F, et al. (2007) The endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces dendritic cell maturation. J Immunol 179: 3742–3751. 5. Blasius AL, Cella M, Maldonado J, Takai T, Colonna M (2006) Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12. Blood 107:2474–2476. Lau-Kilby et al. www.pnas.org/cgi/content/short/1009738108 1 of 6
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Supporting InformationLau-Kilby et al. 10.1073/pnas.1009738108SI Materials and MethodsConditional STAT5KO (tie2-cre, STAT5fl/fl) Mice. ConditionalSTAT5KO (tie2-cre, STAT5fl/fl) mice [a gift from John O’Shea(National Institute of Arthritis and Musculoskeletal and SkinDiseases, Bethesda, MD) and Lothar Hennighausen (NationalInstitute of Diabetes, Digestive and Kidney Diseases, Bethesda,MD)] were typed by genomic DNA (for cre) and hematocritlevels (1). Athough tie2, a tyrosine kinase TEK, is expressedspecifically by hematopoietic stem cells, its expression can also befound on endothelial cells (EC) (2). There is some evidence thatEC can impact DC development and maturation (3, 4). The re-sults obtained using these mice can therefore be an indirect effectof stat5 deletion on EC function. The deletion of STAT5 wasconfirmed by RT-PCR using RNA isolated from BM-derivedcells. Cells from all stat5 KO mice show reduction of 10 cycles,i.e., >1,000-fold reduction in stat5a expression.
Isolation and Culturing of FL-BM. The tibias and femurs were re-moved from mice and flushed to obtain bone marrow cells. Thered blood cells were lysed and remaining bonemarrow cells platedout (106/mL) with complete RPMI-1640 (cRPMI) [10% FCS,2 mM L-gluamine, 100 units/mL penicillin, 100 μg/mL strepto-mycin, 50 μM 2-mercaptoethanol (MCE), 100 μM nonessentialamino acids (NEAA), 1 mM sodium pyruvate]. Where indicated,25 μg/mL of LEAF-purified αIL2Rα/CD25 (PC61) (isotypecontrol: purified Rat IgG1) or αIL2Rβ/CD122 (isotype control:purified Rag IgG2a) (Biolegend) was also added. For lineagedepletion, biotin-conjugated antibodies against CD3 (145-2C11),CD11b (M1/70), Gr1 (RB6-8C5), TER119, NK1.1 (PK136), andCD19 (6D5) (Biolegend) were used. Cells were depleted of line-age by anti-biotin beads followed by negative selection using amagnetic column (Miltenyi Biotec).
Flow Cytometry. The following antibodies were used: anti-mouseSiglec-H (440c), CD317/bone marrow stromal cell antigen 2(BST2) (927), ckit/CD117 (2B8), and MCSF-R/CD115 (AFS98)(eBioscience); CD11c (N418), CD11b (M1/70), CD3 (145-2C11),NK1.1 (PK136), CD25 (PC61), and CD24 (M1/69) (Biolegend);and SIRPα (P84) and IAb (AF6120.1) (BD). T cells were definedas CD3+ NK1.1−, NK cells as CD3−NK1.1+, and NKT cells asCD3+NK1.1+. Cells were analyzed on a FACS-CyAN machine(Beckman Coulter) or FACS-LSRII (BD). To calculate absolutenumbers of cells per culture well/sample, a known number ofCountbright beads (Invitrogen) was added to the sample im-mediately before flow analyses. The ratio of known vs. the de-tected number of beads was used to calculate the absolutenumber of cells from the detected number of cells. All datashown are representative of at least three separate experimentsshowing similar results (Figs. 1–7), and each dot represents oneculture well (Figs. 1–7, S4, and S5) except where indicated. Forall statistical analyses, a nonparametric Student’s t test was used.P values ≤0.05 were considered statistically significant.
Isolation of DCs from FL-BM Cultures. To sort pDCs/cDCs from7-d FL-BM cultures (starting cells: either total BM or sortedLin−flt3+CD11c− BM cells), cells were harvested and stainedwith anti-mouse PECy7-CD11c (N418), biotin-NK1.1 (PK136),PB-CD11b (M1/70), and Al488-CD317 (clone 120G8) (all fromBiolegend except Al488-CD317, from Imgenex) and then SA-PO(Invitrogen) and sorted using FACS Aria (BD Biosciences) (Fig.S2C). Because the antibody to Siglec-H, 440c, has been shown toalter pDC function by modulating type 1 IFN production (5), weused anti-CD317/BST2 (clone 120G8) to sort pDCs. In a checkfor purity, >90% of these cells were found to express Siglec-H.
Phenotype of DCs. To analyze the phenotype of DCs after 7-d FL-BM cultures, cells were harvested, washed, and stained with anti-mouse CD40 (3/23), CD86 (GL-1), CD80 (16-10A1), MHCII IAb(AF6120.1), and PD-L1 (M1H5) (all from Biolegend except PD-L1 from eBioscience and MHCII from BD).
Cytokine Secretion. For analyses of cytokine secretion by sortedpDCs or cDCs after overnight TLR stimulation, sorted DCs wereplated at 5 × 105/mL and cDCs were stimulated with 100 ng/mLLPS (Sigma) or 1 μM CpG-1826 (Invivogen), whereas pDCswere stimulated with 1 μM CpG-2216 (Invivogen) for 18–22 h.Supernatants were harvested and analyzed using mouse IL-6,TNF-α, and IL-12p70 ELISAs (R&D systems). IFN-α secretionby pDCs was also analyzed using a sandwich ELISA coated withrat monoclonal anti-mouse IFN-α (RAMMA-1) followed by in-cubation with culture supernatants and then with rabbit poly-clonal anti-mouse IFN-α (both antibodies from PBL). Theamount of IFN-α was then detected using goat anti-rabbithorseradish peroxidase (Promega) followed by TMB substrate(R&D Systems). For analyses of cytokine secretion in DC–T-cellcocultures, supernatents were harvested at the end of 3-d co-culture and analyzed using mouse IL-10 and IL-12 ELISAs(R&D Systems).
DC–T-Cell Cocultures. T cells were isolated from the spleen andsplenocytes were lysed of RBC and then incubated with biotin-conjugated anti-mouse CD11b (M1/70), B220, CD49b (DX5),Gr1 (RB6-8C5), CD8 (53-6.7), TER119, and CD25 (PC61)(Biolegend). CD4+CD25− splenic T cells were then purified byadding anti-biotin beads followed by negative selection viaa magnetic column (Miltenyi Biotec). DC–T-cell cocultures wereanalyzed by staining for CD11c (N418), CD25 (PC61), and CD4(GK1.5) (Biolegend). Where appropriate, cells were thenstained with Annexin V and/or fixed and permeabilized for in-tracellular staining of Foxp3 (eBioscience).
Blocking of IL-12 Activity.Where applicable, 500 ng/mL anti-mouseIL-12p40 (C17.8) was added to DC–T-cell cocultures to block IL-12 activity.
1. Zhu BM, et al. (2008) Hematopoietic-specific Stat5-null mice display microcytichypochromic anemia associated with reduced transferrin receptor gene expression.Blood 112:2071–2080.
2. Kisanuki YY, et al. (2001) Tie2-Cre transgenic mice: A new model for endothelial cell-lineage analysis in vivo. Dev Biol 230:230–242.
3. Despars G, O’Neill HC (2006) Splenic endothelial cell lines support development ofdendritic cells from bone marrow. Stem Cells 24:1496–1504.
4. Tian F, et al. (2007) The endothelial cell-produced antiangiogenic cytokine vascularendothelial growth inhibitor induces dendritic cell maturation. J Immunol 179:3742–3751.
5. Blasius AL, Cella M, Maldonado J, Takai T, Colonna M (2006) Siglec-H is an IPC-specificreceptor that modulates type I IFN secretion through DAP12. Blood 107:2474–2476.
Lau-Kilby et al. www.pnas.org/cgi/content/short/1009738108 1 of 6
Bone marrow
Peripheral LNs
Spleen
B22
0
CD11cC
D31
7Siglec-H
1.9%
0.7%
1.4%
69.5%
8.4%
5.3%
7AAD- 7AAD-CD11c+B220+
Fig. S1. CD11c+B220+ cells are not all pDCs. Bone marrow, peripheral lymph nodes (inguinal, popliteal, and axillary) and spleen of C57BL/6 mice were col-lagenase treated and stained for flow analyses. Plots shown are first gated on live cells (Left) and then on CD11c+ B220+ cells (Right). Data shown are rep-resentative of at least two experiments showing similar results. Although both CD317 and Siglec-H have been described as markers for pDCs, CD317 expressioncan be found on activated cDCs and plasma cells (1, 5). In addition, not all CD11c+B220+ cells are pDCs (Right and ref. 2). For this reason, Siglec-H was used toidentify pDCs.
1. Blasius AL, et al. (2006) Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFNstimulation. J Immunol 177:3260–3265.
2. Segura E, Wong J, Villadangos JA (2009) Cutting edge: B220+CCR9- dendritic cells are not plasmacytoid dendritic cells but are precursors of conventional dendritic cells. J Immunol 183:1514–1517.
Lau-Kilby et al. www.pnas.org/cgi/content/short/1009738108 2 of 6
A
B
(x 1
,000
)
(x 1,000)FSC
7AA
D
flt3
Line
age
FSC
SSC
flt3
Line
age
CD11b
CD
317
CD11b
Sigl
ec-H
cDC
pDC
7AAD- 7AAD-CD11c+NK1.1-
NK1.1
CD
11c
C
Lin depleted BM cells
Total BM cells
NK1.1
CD
11c
Siglec-H
Cou
nt
98%
800
600
400
200
0
Fig. S2. Sorting strategy for Lin−flt3+ or Lin−
flt3− BM cells and pDCs/cDCs after 7-d BM cultures. (A) Schematic diagram showing the flow sorting of Lin−flt3+
and Lin−flt3− BM cells. On average, we were able to sort 105 Lin−
flt3+ cells per mouse. (B) The gate for Lin− was set by using lineage staining of total BM cellsbefore magnetic depletion. (C) Sorting strategy for cDCs (Upper) or pDCs (Lower) after 7-d culture of BM cells with flt3L ± IL-2. cDCs were sorted asCD11c+NK1.1−CD317−CD11b+SiglecH−. pDCs were sorted as CD11c+NK1.1−CD317+CD11b− and Siglec-H expression was measured. Data shown are represen-tative of at least two experiments showing similar results.
Lau-Kilby et al. www.pnas.org/cgi/content/short/1009738108 3 of 6
C
CD11c+ SiglecH-
CD11b- CD24-Pre-cDC
A
CD11c+ pre-DCs
fl +50U IL2 +100U IL20
10000
20000
30000
abso
lute
num
ber
B
MHCII
SIR
Pα
pDC CD11bhicDC
CD24hi
cDC
102 103101 101 102 103101 102 103101 102 1030
101
102
103
104102 103101 101 102 103101 102 103101 102 103
104
101
102
103
flt3L
flt3L +100U IL-2
104
104
0
0
101
102
103
104
32.8%
5 10 20 25
24.8%
0
101
102
103
104
87.6%
101 102 103 104
All live cells
FSC
ckit
CX3CR1
ckit
CX3CR1
CD
115
D
0
101
102
103
104
53.2%
101 102 103 104
46.0%
0
101
102
103
104
66.6%
101 102 103 104
0
101
102
103
104
70.6%
5 10 20 25
21.9%
IL2Rα gated
CX3CR1
ckit
CX3CR1M
CSF
-RFSC
ckit
0
101
102
103
104
4.96%
101 102 103 104
95.0%
CD
11c
Siglec-H
pDC
Flt3L only +IL-2 100U/ml
0
101
102
103
104
101 102 103 104 101 102 103 104
CD11b
CD11bhi
cDC
CD24hi cDC
Pre-cDC
0
101
102
103
104
101 102 103 104 101 102 103 104
CD
24
Fig. S3. Gating strategy for CD24hi cDCs and different stages of DC development. Sorted Lin−flt3+ BM cells were cultured with flt3L ± IL-2 and harvested at day
7 for flow cytometry analysis. (A) Live cells were first gated according to CD11c and Siglec-H expression. cDCs are CD11c+Siglec-H−. Within cDCs, CD24hi DCs aregated as CD24hi CD11blow/−, CD11bhi DCs are CD24low/− CD11bhi. The bar chart shows the absolute number of CD11c+Siglec-H−CD24−CD11b− cells from theindicated culture conditions. (B) Expression of SIRPα and MHCII on the indicated populations after culture with flt3L ± IL-2. (C) Schematic diagram showing thedifferent stages of DC development as previously defined. (D) Live cells were first gated for c-kit expression. c-kithi cells were then gated for CX3CR1/gfp−
myeloid progenitors (MPs) and CX3CR1/gfp+ monocyte and DC progenitors (MDPs). c-kitlo cells were gated for MCSF-R (CD115)hiCX3CR1/gfp+ common DCprogenitors (CDPs). This gating strategy was based on that previously published (1). All data shown are representative of at least three experiments showingsimilar results.
1. Liu K, et al. (2009) In vivo analysis of dendritic cell development and homeostasis. Science 324:392–397.
Lau-Kilby et al. www.pnas.org/cgi/content/short/1009738108 4 of 6
no stim +CpG-22160
2000
4000
6000
8000
10000
no stim +LPS0
100
200
300
400
500
pg/m
l
B
no stim +CpG-22160
100
200
300
400
500
pg/m
l
no stim +LPS0
2000
4000
6000
8000
10000
no stim +CpG-22160
200
400
600
800
1000
no stim +LPS0
50
100
150
200
250
A
ND ND ND ND
ND ND ND
pDC
cDC
ns
nsns
ns
**p<0.02
C
no stim +LPS +CpG-18260
50
100
150
200
ND ND
no stim +CpG-22160
50
100
150
200
ND ND
Lin-flt3+ Total BM
FL-pDC FL+IL2-pDC
IL6 IFNα TNFα IL12
IL6 IFNα TNFα IL12
FL-cDC FL+IL2-cDC
**p<0.01
Fig. S4. Cytokine secretion of FL+IL2-DCs. (A and B) cDCs (A) and pDCs (B) were sorted from 7-d FL-BM cultures (using total BM) and stimulated overnight witheither LPS (A) or CpG-2216 or -1826 (B). (C) Supernatants were taken from day 7 of FL-BM cultures. Total BM or sorted Lin−
flt3+ cells were cultured with flt3L ±IL-2. Supernatants were collected on day 7 of culture and secretion of IL-10 and IL-12 was analyzed by ELISAs (R&D Systems). All cytokine measurements weredone by ELISAs. ND, none detected. Data shown are representative of at least two experiments showing similar results.
A CD4 gated
CD25
CD11c gated CD4gated
Fl-cDCFL+IL2-cDC
020406080
020406080
% A
nnex
in V
hi
FL-DC FL+IL2-DC0
20
40
60
B
0.5μg/ml αCD3
0.1μg/ml αCD3
200ng/ml OVA323-339
Annexin V
Cou
nt
0 101 102 103 104
%m
ax
CFSE
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
CD25
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0.5μg/ml αCD3
0.1μg/ml αCD3
200ng/ml OVA323-339
40ng/ml OVA323-339
Fig. S5. T-cell stimulation of Lin−flt3+BM-derived FL+IL2-DCs. Sorted Lin−
flt3+-derived cDCs were cocultured with naive CD4+CD25− splenic T cells (fromnontransgenic or OTII mice) under αCD3 or OVA 323–339 peptide stimulation, respectively. (A) After 3 d of coculture, T-cell proliferation was analyzed by CFSEdilution (Left) and T-cell activation was measured by CD25 expression (Right). (B) Cells were harvested after 16–20 h of culture to compare DC apoptosis(Annexin V) and T-cell activation (CD25). Data shown are representative of at least two experiments showing similar results.
Lau-Kilby et al. www.pnas.org/cgi/content/short/1009738108 5 of 6
Foxp3C
ount
Fl-cDCFL+IL2-cDC
0
10
20
30
40
50
FL FL+IL2 FL FL+IL2
IL-1
0 pg
/ml
ND ND ND ND
pDC cDC
Before co-culture
CD4 gated
Crudesplenocytes
PurifiedCD4+CD25-
OT2 cells
PurifiedCD4+CD25-
NT cells
Foxp3
Cou
nt
A
0.5μg/mlCD3
0.1μg/mlCD3
200ng/mlOVA323-339
40ng/mlOVA323-339
CD4 gated
After 3 days of cocultureB
C
0 101 102 103 104
20
40
60
80
100
0 101 102 103 104
20
40
60
80
100
0 101 102 103 104
20
40
60
80
100 0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
0 101 102 103 104
Co-culture supernatant
0 101 102 103 104
D
0 101 102 103 104
0 101 102 103 104 0 101 102 103 104
CFSE
Cou
nt
Fl-cDCFL+IL2-cDC
0.5μg/mlCD3
0.1μg/mlCD3
+ IL-12p40
0
10
20
30
40
50
FL FL+IL2 FL FL+IL2
ND ND ND ND
pDC cDC
IL12
pg/
ml
10.51
0.29
0.41
Fig. S6. Decreased stimulatory capacity of FL+IL2-DCs is not due to regulatory pathways or differential IL-12 activity. (A) Purified CD4+CD25− naive T cells, fromnontransgenic or OTII mice, were checked for Foxp3 expression before coculturing with T cells. (B) After 3 d of DC–T-cell coculture, the indicated populationswere harvested and analyzed for Foxp3 expression. (C) Supernatants were collected after 3 d of DC–T-cell coculture stimulated with anti-CD3 (0.5 μg/mL), andIL-10 and IL-12 were measured by ELISA. ND, not detectable. (D) DC–T-cell cocultures were set up as described in Fig. 7 with the addition of anti-mouse IL12p70as indicated and T-cell proliferation was analyzed by CFSE dilution. Data shown are representative of at least two experiments showing similar results.
Lau-Kilby et al. www.pnas.org/cgi/content/short/1009738108 6 of 6
Related Documents
