Supplemental Figure 1 CD8 CD4
4 104 10 70 12 46 28 3 103 10 w.t. 2 102 10
1 101 10 11 6 6 20 100 100 100 101 102 103 104 100 101 102 103 104
104 104
CD62L 67 14 41 23
103 103
MFG-E8-/- 102 102
101 101 10 26 12 8 100 100 100 101 102 103 104 100 101 102 103 104
CD44
Supplemental Figure 1: Young w.t and MFG-E8-/- mice have similar splenic T cell activation. Splenic CD4 and CD8 T cells from 2 month old w.t or MFG-E8-/- mice were analyzed for the expression of CD44 and CD62L by flow cytometry. The results are representative of 5 mice from each group. Supplemental Figure 2 W.T. MFG-E8-/-
Α 104 104 Β W.T. MFG-E8-/- 0.4 0.37 103 103 CD169/ Apo
102 102
101 101
100 100 100 101 102 103 104 100 101 102 103 104 10x Apo 104 104
103 33 103 34 Apo+ 102 102
101 101 25 25 22 25
100 100 100 101 102 103 104 100 101 102 103 104 CD11c
104 104 40x 3.8 2.7 103 103 α
Apo+ CD8 102 102
101 101
100 100 100 101 102 103 104 100 101 102 103 104 CD11c Supplemental Figure 2: Similar distribution of apoptotic cells among splenic CD11c+ and CD11b+ cells between w.t and MFG-E8-/- mice. A: 20x 106 CM-Dil (red) labeled apoptotic cells were injected into either w.t. or MFG-E8-/- mice (n=3 in each group). CD11c+ and CD11b+ cells were isolated by positive magnetic sorting using a cocktail containing antibodies to CD11 and CD11b at 20 hour post injection. Apo+ (FL2) cells were analyzed for their expression of CD11c, CD11b and CD8α by flow cytometry. B: Spleen sections obtained from recipients in A were stained for CD169 (a marker for inner marginal zone macrophage, green). The distribution of apoptotic cells (red) were visualized by immunofluorescence microscopy. The samples are representative of 3 animals of each group. Supplemental Figure 3
No Ag W.T MFG-E8-/-
104 104 104 30 103 0.2 103 0.8 103 0.7 ) 4
102 102 102 25
101 101 101 CD45.1 20 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 I cells T (x10 CD8α - 15
4 4 4 10 10 10 + OT γ 2 17 15 - 3 103 103 10 10 γ -
2 102 102 10 IFN 5 1 101 101 10
0 of Number IFN 100 100 10 0 0 1 2 3 4 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10
CD8α W.T. n =4 MFG-E8-/- n =4
Supplemental Figure 3: OT-I T cells responded similarly to soluble OVA in either w.t or MFG-E8-/- mice. 5 x 106 CD45.1 /CD 45.2 OT-I T cells were transferred into 4 month old w.t or MFG-E8- /- mice. The recipients were challenged i.v. with 200 µg LPS depleted soluble OVA. At day 9 after challenge, spleen cells were recovered and restimulated with OVA peptide in vitro for 6 hours. The expression of IFN-γ by OT-I T cells was evaluated by flow cytometry. The total numbers of IFN-γ producing OT-I T cells are shown on the right. Supplemental Figure 4
W.T. MFG-E8-/-
104 104
103 13 103 14 LN 102 102
101 101
100 100 Foxp3 100 101 102 103 104 100 101 102 103 104
104 104
103 14 103 14 Spleen 102 102
101 101
100 100 100 101 102 103 104 100 101 102 103 104
104 104
3 3 10 3.3 10 3.6 Thymus
102 102
101 101
100 100 100 101 102 103 104 100 101 102 103 104 CD4
Supplemental Figure 4: The numbers of Foxp3+ regulatory T cells are similar between w.t and MFG-E8-/- mice. Spleen, lymph node and thymus from either 4 month old w.t or age matched MFG-E8-/- mice were analyzed for Foxp3 expression by flow cytometry. Similar results were obtained from 1 year old mice. The dot plots are representative of 5 mice examined. Supplemental Figure 5
MFG-E8-/- MFG-E8 -/- w.t. 4 (mild dermatitis) (severe dermatitis ) 10 104 104
3 3 10 2.9 103 2.7 10 2
DEC 205 2 2 10 102 10
1 1 10 101 10
0 0 10 100 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 10 10 10 10 10 CD11c
104 104 104
10 10 103 103 103 3.5 DEC 205 102 102 102 CD11c+ 51 53 47 2 101 101 101 6.4 5.5
100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD8α
104 104 104
103 103 103 9 2 8.4 2 2 2 DEC 205 10 10 10 3.8 7.6 CD11c+ 7.4 1 101 101 10 49 51 47 0 100 100 10 0 1 2 3 4 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10 CD103 CD86 CD80 PDL1 PDL2 ICOSL Isotype
100 100 100 100 100 100
80 80 80 80 80 80
60 60 60 60 60 60 DEC205+ 40 40 40 40 40 40
20 20 20 20 20 20
0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
100 100 100 100 100 100
80 80 80 80 80 80
60 60 60 60 60 60 CD8α+
40 40 40 40 40 40
20 20 20 20 20 20
0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
100 100 100 100 100 100
80 80 80 80 80 80 DEC205- 60 60 60 60 60 60 CD8α-
40 40 40 40 40 40
20 20 20 20 20 20
0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
Supplemental Figure 5: Similar expressions of co-stimulatory molecules on skin LNs APCs between w.t and MFG-E8-/- mice. A: Skin draining LNs from either 1 year old w.t or aged matched MFG-E8-/- with mild dermatitis or MFG-E8-/- mice (females) with severe dermatitis were collected. The percentages of various CD11c+ DC subsets were analyzed by flow cytometry. A significant reduction of DEC205+ Langerhans cells, CD103+ migrating DCs and CD8α + resident DCs was observed in MFG-E8-/- mice with severe dermatitis, presumably due to the death of DCs after continuous activation. B: The expressions of co-stimulatory molecules on different DCs subsets were compared between 1 year old w.t and aged matched MFG-E8-/- mice with mild dermatitis by flow cytometry. ( red---w.t., blue--- MFG-E8-/-) The results are representative of at least 5 mice analyzed. Supplemental Figure 6
W.T. CD11c hi MHCII + MFG-E8-/- CD11c hi MHCII +
104 104 104 104 α α
103 1.6 103 13 103 1.3 103 17 CD8 CD8 CD11c CD11c 102 102 102 102 85 80
101 101 101 101
100 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 MHCII CD86 MHCII CD86
CD86 CD80 PDL-1 PDL-2 ICOSL Isotype
100 100 100 100 100 100
80 80 80 80 80 80
60 60 60 60 60 60 % of Max % of Max % of Max % of % of Max % of Max % of % of Max % of CD8α+ 40 40 40 40 40 40
20 20 20 20 20 20
0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 PE-A PE-A PE-A PE-A PE-A PE-A
100 100 100 100 100 100
80 80 80 80 80 80
60 60 60 60 60 60 % of Max % of Max % of Max % of Max % of Max % of % of Max % of CD8α- 40 40 40 40 40 40
20 20 20 20 20 20
0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 PE-A PE-A PE-A PE-A PE-A PE-A
Supplemental Figure 6: Similar expressions of costimulatory molecules on splenic APCs between w.t and MFG-E8-/- mice. A: Spleens from either 1 year old w.t or aged matched MFG-E8-/- were collected. The percentages of splenic CD8α + and CD8α-DC subsets were analyzed by flow cytometry. B: The expressions of co-stimulatory molecules on CD8α+ and CD8α- splenic DCs subsets were compared between 1 year old w.t and aged matched MFG-E8-/- mice by flow cytometry. ( red---w.t., blue--- MFG-E8-/-) The results are representative of at least 5 mice analyzed. Supplemental Figure 7
IL10 IL12RB2 IL15RA IL17RA IL18 IL1B IL1RAP IL1RN IL20RB IL23A IL2RA IL4RA IL10RA IL13 IL15RA IL17RB IL18 IL1F10 IL1RAP IL1RN IL20RB IL23R IL2RB IL5 IL10RB IL13RA1 IL16 IL17RC IL18BP IL1F5 IL1RAP IL1RN IL20RB IL24 IL2RG IL5RA IL10RB IL13RA2 IL16 IL17RC IL18R1 IL1F6 IL1RAP IL1RN IL21 IL25 IL2RG IL6 IL11 IL15 IL16 IL17RC IL18R1 IL1F8 IL1RAPL1 IL2 IL21R IL27 IL3 IL6RA IL11RA1 IL15 IL17A IL17RD IL18R1 IL1F9 IL1RAPL2 IL20 IL21R IL27RA IL31 IL6RA IL11RA1 IL15 IL17B IL17RD IL18RAP IL1R1 IL1RAPL2 IL20RA IL21R IL27RA IL31RA IL6RA IL12A IL15 IL17C IL17RE IL18RAP IL1R1 IL1RL1 IL20RA IL22 IL28B IL33 IL6ST IL12B IL15 IL17D IL17RE IL19 IL1R2 IL1RL1 IL20RA IL22RA1 IL28RA IL3RA IL6ST IL12RB1 IL15RA IL17D IL17RE IL1A IL1RAP IL1RL1L IL20RB IL22RA2 IL28RA IL4 IL7 IL12RB1 IL15RA IL17F IL17RE IL1A IL1RAP IL1RL2 IL20RB IL23A IL28RA IL4I1 IL7
IL7 IFNA1 IFNA4 IFNAB IFNGR2 TGFB2 TGFBR2 Cd86 Icos IL7R IFNA1 IFNA5 IFNAR1 IFNGR2 TGFB3 TGFBR3 Cd86 Icosl IL7R IFNA1 IFNA5 IFNAR2 IFNK TGFBI TGFBR3 Cd86 Icosl IL7R IFNA11 IFNA6 IFNAR2 IFNZ TGFBI LTA Cd80 Cd28 IL7R IFNA11 IFNA6 IFNAR2 IFNZ TGFBR1 LTA Cd80 PD-L1 IL7R IFNA12 IFNA6 IFNB1 TNF TGFBR1 LTB Cd40 PD-1 IL8RA IFNA13 IFNA6 IFNE1 TNF TGFBR1 Tnfrsf1a Cd40 PD-L2 IL8RB IFNA14 IFNA7 IFNG TGFA TGFBR1 Tnfrsf1a Cd40 OX40 IL8RB IFNA14 IFNA9 IFNG TGFB1 TGFBR2 Tnfrsf1a IL9 IFNA2 IFNA9 IFNGR1 TGFB1I1 TGFBR2 Tnfrsf1b IL9R IFNA2 IFNAB IFNGR2 TGFB1I1 TGFBR2 Tnfrsf1b
List of probes of cytokines and costimulatory molecules of the microarray data. (duplicates are multiple probes for the same gene) 3
2
1
0
-1 Folds of change
-2
-3
Supplemental Figure 7:
Similar expressions of co-stimulatory molecules and cytokines by spleen cells between w.t and MFG-E8-/- mice. Spleen cells from either 4 month old w.t or aged matched MFG-E8-/- mice (females n =4) were analyzed by microarray. The fold changes of gene expression of cytokines and co-stimulatory molecules were compiled. The expression levels by w.t. mice were set as 0. None of the cytokines nor co-stimulation molecules had >2 fold change in mRNA expression in MFG-E8-/- spleens. The duplicate reflect multiple probes for the same gene. Supplemental Figure 8
medium +Apo-OVA
100 100
80 80
60 60
40 40
20 20
0 0 100 101 102 103 104 100 101 102 103 104
W.T 36% W.T. 32% MFG-E8-/- 30% MFG-E8-/- 28% CD86
Supplemental Figure 8: Phagocytosis of OVA loaded apoptotic cells did not induce upregulation of CD86 on BMDCs. Day6 BMDCs from either w.t. or MFG-E8-/- mice were mixed with Apo-OVA at Apo/DC ratio 5:1, the expression of CD86 on DCs was examined after 24 hours. (blue – MFG-E8-/-, red—w.t.). The results are representative of five independent experiments. Supplemental Figure 9
w.t MFG-E8-/- A C DAPI Apo LAMP-1 merge
MFG-E8-/- 2hr
Apo Lyso merge DAPI EEA-1 Apo merge B * D * w.t. 2hr * DAPI Apo EEA-1 merge
w.t. 2hr
Supplemental Figure 9: A: Lysotacker labeling of unfixed BMDCs. LysoTracker was added to BMDCs for 2 hours. DCs were collected and examined without fixation under wide-field microscope. Two typical examples of LysoTracker labeling from either w.t or MFG-E8-/- BMDCs are shown. B: Acidification of apoptotic cell requires phagocytosis: LysoTracker labeled w.t BMDCs were incubated with PKH 67 (green) labeled apoptotic cells for 6 hours. The acidification of apoptotic cell bodies within DCs was examined under wide-field microscope without fixation. Intact apoptotic cells (asterisks) outside DCs remained LysoTracker negative. C: MFG-E8 is not required for phagosome-lysosome fusion. PKH 67 (green) labeled apoptotic cells were incubated with MFG-E8-/- DCs for 2 hour. DCs were fixed, permeabilized and stained for LAMP-1. The few intact apoptotic cells phagocytosed by MFG-E8 DCs became LAMP-1 positive within 2 hour. D: Apoptotic cell containing phagosomes at an early stage of phagocytosis were associated with EEA-1. Apoptotic cells were labeled with either PKH 27 (red- upper panel) or PKH 67 (green – lower panel) and were incubated with w.t. BMDCs for 2 hour. DCs were fixed, permeabilized and stained for EEA-1. Supplemental Figure 10 6 hr 24 hr ApoOVA ApoOVA ApoBSA
4 4 4 A 10 10 10
3 3 10 10 103
2 2 10 10 102 W.T. 0.7 0.3 0
1 1 10 10 101
0 0 10 10 100 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 100 101 102 103 104
104 104 CD11c
103 103
102 1.2 102 1.1 MFG-E8-/-
101 101
100 100 100 101 102 103 104 100 101 102 103 104 25D1.16 6 hr 24 hr ApoBSA
100 100 250
80 80 200
60 60 150
40 40 100
20 20 50
0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 MHCI W.T. sOVA MFG-E8-/- sBSA B 4 4 4 10 10 10
103 103 103
2 2 2 CD11c 10 2 10 2 10 0
101 101 101
100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 25D1.16 Supplemental Figure 10: A: Increased detection of the OVAp/MHCI complex in MFG-E8-/- derived BMDCs is not caused by the up-regulation of MHCI. Apo-OVA were incubated with either w.t or MFG-E8-/- BMDCs for either 6 or 24 hours. The levels of OVAp/MHCI complex (dot plot) and MHCI (histogram) were evaluated simultaneously by flow cytometry. (Histogram: blue – w.t, red – MFG-E8-/-). The results are representative of two experiments. B: MFG-E8 deficiency does not affect the formation of OVAp/MHCI complex derived from soluble OVA. 100 µg of LPS-depleted OVA was incubated with either w.t or MFG-E8-/- BMDCs for 24 hours. Expression of OVAp/MHCI complex was analyzed as in A. The results are representative of two experiments. Supplemental Figure 11
Apo OVA B Apo BSA A LPS LPS
6 hr 24 hr 6 hr 24 hr 6 hr 6 hr 104 104 104 104 104 104
103 103 103 103 103 103
102 W.T. 102 102 102 102 102
101 101 101 101 101 101 1.5 0.5 0.64 0.34 0.1 0.1 100 100 100 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
104 104 104 104 104 104
103 103 103 103 103 103
MFG-E8-/- 102 102 102 102 102 102
101 101 101 101 101 101 1.8 1.4 0.99 0.22 0.1 0.1
100 100 100 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
6 hr 24 hr
335 v.s.247 250 v.s. 200 C 100 100 100 w.t. 80 Act-mOVA 80 80
60 60 60 % of Max % of 40 40 40
20 20 20
0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 APC-A 25D1.16 Supplemental Figure 11: Increased 25D1.16 signal in MFG-E8-/- DCs was not further enhanced by LPS. A and B: OVA (A) or BSA (B) loaded apoptotic cells were incubated with either w.t or MFG-E8-/- DCs . At 6 and 24 hr after incubation, DCs were first stained for CD11c, fixed, permeabilized and stained for the OVAp/MHCI complex using the 25D1.16 antibody. In the control experiment, 100ng/ml LPS was added to DCs 2 hours after apoptotic cells. C: Thymocytes from either w.t. or Act-mOVA transgenic mice were stained for OVAp/MHCI complex using 25D1.16. (blue—isotype, red—w.t., black—Act-mOVA transgenic. Supplemental Figure 12
MFG-E8 -/- W.T
IgG2c
C3
Supplemental Figure 12: Deposition of IgG2c and C3 in the kidneys of MFG-E8-/- mice on the B6 genetic background. Kidneys from 1 year old w.t. or MFG-E8-/- mice were stained for IgG2c (upper panel) and C3 (lower panel) by indirect immunofluoprescence.