15h. 24h. Blander & Medzhitov supplementary Figure 1. Apoptotic cells. Apoptotic LPS blasts 30% 32% 32% + Exogenous LPS 0.1% 31% 21% 20% 48% 60% 53%

Transcription

1 a None Apoptotic cells Apoptotic LPS blasts 30% 32% 32% Apoptotic cells + Exogenous LPS 6h 0.1% 31% 21% 20% 48% 60% 53% 15h 0% 7% 5% 4% 30% 32% 32% 24h CD11c 0% 8% 2% 2% CFSE Blander & Medzhitov supplementary Figure 1

2 b Fig. 1. Kinetics of phagocytosis of apoptotic cells by DCs over time. A20 B cells or day 4 A20 LPS B cell blasts were labelled with CFSE, and induced to undergo apoptosis by UV irradiation. Apoptotic A20 B cells, A20 LPS B cell blasts, or A20 B cells with exogenous 10 ng/ml LPS were added to day 5 GM-CSF bone marrow cultures at a ratio of 2:1 apoptotic cells to non-adherent cultured cells. Apoptotic cells were briefly centrifuged onto cultures and incubated at 37 o C for 6, 15, and 24 h. At the end of each time point, non-adherent cells were harvested and resuspended in FACS buffer containing 1 m g/ml CD16/CD32 (Fc block) for 1 h at 4 o C. Cells were stained with APC-conjugated anti-cd11c and analyzed by flow cytometry. a. Contour plots showing log fluorescence intensity of CFSE on the X-axis and CD11c on the Y-axis, the percent CD11c + CFSE + representing DCs that have phagocytosed apoptotic cells, and the percent CD11c - CFSE + cells representing remaining apoptotic A20 cells that were not phagocytosed. b. Bar graphs showing the decrease in the percent of apoptotic A20 B cells that were not phagocytosed over time (left), the increase in the percent of DCs that have phagocytosed apoptotic cells over time (top right), and the decrease in the mean fluorescence intensity (MFI) of CFSE over time (bottom right) as DCs phagocytose and degrade apoptotic cells. Blander & Medzhitov supplementary Figure 1

3 * N * Fig. 2. Electron micrograph showing a DC phagocytosing apoptotic cells. Apoptotic A20 B cells were added at a ratio of 2:1 to bone marrow derived GM-CSF cultures. At 4 h, non-adherent cells were harvested, washed once in PBS and fixed. Cells were processed for electron microscopy as previously described in reference 28. Arrows indicate apoptotic cells surrounded by dendrites. Note the loss of plasma membrane integrity in these cells. Asterisks indicate internalized apoptotic cells. Bar scale, 5 m m. Blander & Medzhitov supplementary Figure 2

4 a Surface MHC Class II CTB Apoptotic Cells CTB Total MHC Class II Apoptotic Cells Apoptotic cells 1Apoptotic cells b CTB S. cerevisiae CTB S. cerevisiae S. cerevisiae S. cerevisiae Fig. 3. Confocal micrographs examining the distribution of at 6 h following phagocytosis of different types of cargo as indicated. Day 5 GM-CSF cultures were given different cargo at a ratio of 2:1 (apoptotic A20 cells:non-adherent cultured cells), 10:1(unlabelled heat-inactivated E. coli:non-adherent cells),10 m g/ml unlabelled S. cerevisiae (zymosan), or 10 ng/ml LPS. Cultures were incubated at 37 o C for 6 h. Non-adherent cells were harvested, and resuspended in endotoxin free PBS containing 1% heat-inactivated fetal bovine serum and CD11c Microbeads (Miltenyi) 20 min at 4 o C. Cells were washed and CD11c + DCs were isolated using AutoMACS (Miltenyi). 2x10 5 DCs were resuspended in 1 ml of complete RPMI medium, centrifuged onto Alcian blue glass coverslips, and incubated 20 min at 37 o C. Coverslips were washed with PBS and fixed in 0.5% paraformaldehyde 20 min followed by quenching with serum-free RPMI containing 0.2 M glycine for another 20 min. The cell surface was stained with Alexa-647-conjugated cholera toxin B subunit (CTB), followed by surface staining for (surface class II, left panels) or permeabilized in 0.2% saponin in complete RPMI, followed by intracellular staining for (total class II,right panels). was stained using anti-i-a b (KH74) that does not cross react with I-A d on apoptotic cells, followed by Alexa-594-anti-mouse Ab. S. cerevisiae were stained after cell permeabilization with zymosan A opsonizing reagent followed by Alexa-488-conjugated anti-rabbit Ab. Confocal Z-images acquired, and all Zs projected onto one plane using LSM image browser (Zeiss). Arrows indicate CD11c + containing phagocytosed cargo. Scale bars, 5 m m. Blander & Medzhitov supplementary Figure 3

5 Surface MHC Class II c CTB Apoptotic cells & E. coli Total MHC Class II CTB Apoptotic cells & E. coli Apoptotic cells Apoptotic cells d CTB Apoptotic cells & CTB Apoptotic cells & exogenous LPS exogenous LPS Apoptotic cells Apoptotic cells e CTB Apoptotic LPS blasts CTB Apoptotic cells Apoptotic cells Apoptotic LPS blasts Blander & Medzhitov supplementary Figure 3

6 WT DC TLR4 -/- DC LPS E. coli Apoptotic B cells Apoptotic LPS B cell blasts Apoptotic anti-ig B cell blasts Apoptotic B cells + exogenous LPS Apoptotic B cells + E. coli PBS CD40 Class II PBS CD40 Class II Fig. 4. Flow cytometry analyses of surface CD40 and by CD11c + bone marrow derived DCs. DCs were derived from either C57BL/6J mice, WT (left panels), or TLR4 -/- mice (right panels). DCs were stimulated on day 5 of culture as indicated on the left, and harvested at 12 h. Cells were centrifuged and resuspended in FACS buffer containing 1 m g/ml CD16/CD32 (Fc block) for 1 h at 4 o C. Cells were double stained with FITC-conjugated anti-cd11c and PE-conjugated anti-cd40 (CD40), or PE-conjugated anti-i-a b (AF ) (Class II), or single stained with FITC-conjugated anti-cd11c (PBS). Red histograms are unstimulated DCs for each group, and gray tinted histograms are stimulated DCs. Data were analyzed by gating on CD11c + cells, and plotting histograms to show the log fluorescence intensity of PE along the X-axes. The Y-axes represent cell numbers. All data were acquired using Cell Quest software (BD Biosciences), and analyses were performed using FlowJo software (Tree Star, Inc.). Blander & Medzhitov supplementary Figure 4

7 WT Dendritic Cells TLR4 -/- Dendritic Cells 6h 15h 24h 6h 15h 24h LPS Apoptotic cells Apoptotic cells +exogenous LPS Apoptotic LPS IA b B cell blasts CD40 CD11c + CFSE + Fig. 5. Flow cytometry analyses of bone marrow DCs derived from C57BL/6J (WT) or TLR4 -/- mice at different times following the phagocytosis of apoptotic cells as indicated. A20 B cells and day 4 A20 LPS B cell blasts were labelled with CFSE, and induced to undergo apoptosis by UV irradiation. Apoptotic B cells, apoptotic LPS B cell blasts and apoptotic B cells with 10 ng/ml exogenous LPS were centrifuged onto GM-CSF bone marrow cultures on day 5, and cultures were incubated at 37 o C for 6, 15, and 24 h. DCs were also stimulated with 10 ng/ml LPS for a comparison control. At the end of each time point, non-adherent cells were harvested, centrifuged, and resuspended in 1 m g/ml CD16/CD32 at 4 o C for 1 h. Cells were stained with APC-conjugated anti-cd11c, biotin-conjugated anti-i-a b (AF ) followed by PE-Cy5-conjugated streptavidin, and either PE-conjugated CD40 (3/23), or CD80 and CD86 (not shown). Events were acquired using Cell Quest software (BD biosciences), and data were analyzed using FlowJo software (Tree Star, Inc.). Cells were gated onto CD11c + CFSE + cells, and dot plots were generated depicting log fluorescence of CD40 PE on the X-axis and I-A b PE-Cy5 on the Y-axis. Red dots are unstimulated DCs (either WT or TLR4 -/- ) at each time point. Blue or black dots are stimulated WT or TLR4 -/- DCs, respectively, at each time point. Blander and Medzhitov supplementary Figure 5

8 Apoptotic cells Apoptotic cells +CD22 E. coli Apoptotic cells +EAP E. coli 57% Streptavidin/YAe CFSE + CD11c + Fig 6. Assessment of the simultaneous phagocytosis of apoptotic cells and E. coli by DCs. GM-CSF cultures of bone marrow cells were given different cargo as indicated on day 5. Apoptotic cells were labelled with CFSE. Cultures were incubated at 37 o C for 16 h. Non-adherent cells were harvested, centrifuged and stained with biotin-conjugated YAe followed by PE-conjugated streptavidin, and PE-Cy5-conjugated CD11c. Events were acquired by CellQuest software (BD Biosciences), and data were analyzed by FlowJo (Tree Star, Inc.) gating on CD11c + CFSE + DCs. FACS analyses show YAe expression by CD11c + CFSE + cells after simultaneous phagocytosis of CFSE apoptotic A20 cells and EAP E. coli. Blander and Medzhitov supplementary Figure 6

9 a Post sort CD8 + CD11c + 9% CD8 - CD11c + CD8 14% b CD11c Splenic CD8 - CD11c + Splenic CD8 + CD11c + * * * Fig. 7. presentation of particulate antigen derived from phagocytosed apoptotic cells by splenic CD8 + and CD8 - CD11c + DCs. C57BL/6J mice were injected subcutaneously with FLT3 L expressing B16 melanoma cells. Spleens were harvested 2 weeks later, and single cell suspensions were stained with FITC-conjugated anti-cd11c and PE-conjugated anti-cd8a in complete sterile RPMI medium. Stained cells were resuspended in sterile endotoxin-free Dulbecco s PBS (Gibco), and sorted by flow cytometry. a. Gates were set on CD11c + cells that were either CD8 + or CD8 - as shown in the dot plot of CD11c log fluorescence intensity on the X-axis, and CD8 log fluorescence intensity on the Y-axis. Enrichment is shown post sorting in the contour plots on the right. b. Sorted CD11c + CD8 + or CD11c + CD8 - DCs were seeded in 96-well plates, and apoptotic cells with different combinations as indicated on the X-axes were added. 18h later, CD4 + T cells were isolated from 1H3.1 TCR transgenic mice, and added to these DCs. All wells were pulsed with 3 [H]-thymidine at 72 h for a period of 16-18h. Wells were harvested and counted using a b -scintillation counter (Wallac). Asterisks denote not detected. Blander & Medzhitov supplementary Figure 7

10 b none/uv BALB/c B cells none/uv B6 B cells a UVBALB/c B cells/uvbalb/c LPS blasts UV A20/UV A20LPS blasts UVB6 LPS blasts/uvbalb/c LPS blasts c streptavidin YAe I-A b CD22 E. coli / EAP E. coli CD22 E. coli / UVBALB/c+CD22 E. coli CD22 E. coli/uv BALB/c B cells+cd22 E. coli streptavidin YAe I-A b CD22 E. coli/uv B6 B cells+cd22 E. coli Blander & Medzhitov supplementary Figure 8 streptavidin YAe I-A b Fig. 8. Expression of the YAe epitope, formed by presentation of EAP (52-68) peptide within I-A b, on the surface of DCs following their phagocytosis of different types of cargo as indicated. GM-CSF cultures of bone marrow cells were given different cargo on day 5, and incubated at 37 o C for 12 h. Non-adherent cells were harvested, centrifuged and resuspended in 1 m g/ml CD16/CD32 (Fc block) at 4 o C for 1 h. a, b. Cells were stained with FITC-conjugated anti-cd11c and either biotin-conjugated YAe or biotinconjugated anti-i-a b (AF ) followed by PE-conjugated streptavidin. c. Apoptotic cells (UV) were labelled with CFSE, and non-adherent cells were stained with PE-conjugated anti-cd11c and either biotin-conjugated YAe or biotin-conjugated anti-i-a b (AF ) followed by PE-Cy5-conjugated streptavidin. Events were acquired by CellQuest software (BD Biosciences), and data analyzed by FlowJo (Tree Star, Inc.) gating on CD11c + or CD11c + CFSE + DCs for panels a/b and c, respectively.

11 Ex-vivo/LPS stimulated Ex-vivo/Anti-Ig stimulated 24h 72h 120h PBS I-E d PBS I-E d Fig. 9. Kinetics of surface expression of I-E d on B cells prior to inducing their apoptosis. CD19 + B cells were isolated from the spleens of BALB/c mice by staining single cell suspensions with CD19 Microbeads (Miltenyi), and further enrichment by AutoMACS (Miltenyi). B cells were cultured in the presence of 25 m g/ml LPS or 10 m g/ml anti-mouse immunoglobulin (H+L) (Southern Biotech) for 24, 72, and 120 h as indicated on the left. All B cells were stained ex-vivo or at the indicated times in FACS buffer with PE-Cy5-conjugated anti-b220 and PE-conjugated anti-i-a/i-e (M5/ ). Data were acquired using Cell Quest software (BD Biosciences), and analyzed by gating on B220 + cells using FlowJo software (Tree Star, Inc.). Blander & Medzhitov supplementary Figure 9

12 E. coli UV B6 Ea - CD19 + UVBALB/c Ea + CD19 + Fig. 10. The lack of IL-2 production by T cells in response to apoptotic cell derived antigen is not due to the failure of apoptotic cells to induce co-stimulatory molecule expression on DCs. Here, we show that the co-stimulation independent T cell hybridoma 1H3.1, which has specificity for EAP, also failed to produce IL-2 when stimulated by DCs that have phagocytosed apoptotic cells. When these cells were stimulated by DCs that have phagocytosed apoptotic LPS B cell blasts, then IL-2 production was detected. This result demonstrated that the ligand for the TCR was not formed on DCs unless the antigen was derived from apoptotic cells carrying a TLR ligand. IL-2 was measured in culture supernatants at 24h post the addition of T cells to DCs. IL-2 levels were measured by ELISA. Blander & Medzhitov supplementary Figure 10

13 a Unstimulated/HEL microspheres Unstimulated/HEL/LPS microspheres 71% 86% CD86 CD40 Fig. 11. Only HEL/LPS microspheres engaged TLR signalling as assessed by DC maturation and IL-6 production. Bone marrow cells derived from CBA/J mice were cultured in GM-CSF for 5 days. 2x10 5 HEL or HEL/LPS conjugated microspheres were added per 1x10 6 non-adherent cultured cells. Microspheres were centrifuged onto cultured cells, and cells were incubated at 37 o C for 6, 15, and 24 h. At the end of each time point, both unstimulated and microsphere stimulated cells were harvested, centrifuged, and resuspended in FACS buffer containing 1 m g/ml CD16/CD32 (Fc block) at 4 o C for 1 h. The culture supernatants were collected for measurements of IL-6 levels by ELISA. Cells were stained with APC-conjugated anti-cd11c, FITC-conjugated anti-i-a k b (10-3.6), and PE-conjugated CD86, CD40 and anti-i-a k a (11-5.2). Data were acquired using Cell Quest software (BD Biosciences) and analyzed using FlowJo software (TreeStar, Inc.) gating on CD11c + I-A k+ b DCs. a. FACS analyses of DC maturation at 6h, b. IL-6 production at 24h, and c. (next page) Kinetics of maturation of DCs at 6, 15 and 24h following phagocytosis of HEL or HEL/LPS microspheres. Blander & Medzhitov supplementary Figure 11 (parts a and b)

14 c 6 h Unstimulated / HEL microspheres 15 h Unstimulated / HEL microspheres Unstimulated / HEL/LPS microspheres Unstimulated / HEL/LPS microspheres CD86 CD40 I-A a k 24 h Unstimulated / HEL microspheres CD86 CD40 I-A a k Unstimulated/HEL/LPS microspheres CD86 CD40 I-A a k Blander & Medzhitov supplementary Figure 11 (part c)

15 HEL microspheres C4H3 Phalloidin HEL/LPS microspheres C4H3 Phalloidin Fig. 12. Only HEL derived from HEL/LPS microspheres results in the formation of the C4H3 epitope specific for HEL peptide (48-62) within I-A k. Bone marrow cells derived from CBA/J mice were cultured in GM-CSF for 5 days. 2x10 5 HEL or HEL/LPS conjugated 10 m m Fluoresbrite BB fluorescent carboxylate microspheres (excitation 350, emission 407 nm) (Polysciences, Inc.) were added per 1x10 6 non-adherent cultured cells. Microspheres were centrifuged onto cultured cells, and cells were incubated at 37 o C for 6h. Cells were harvested, seeded onto Alcian blue treated coverslips, and fixed. Cells were stained with Alexa-Fluor 597-conjugated Phalloidin (Molecular Probes). For surface expression of C4H3, cells were stained with C4H3 followed by biotinylated anti-rat IgG2b (G15-337) (Pharmingen), and Alexa-488 streptavidin (Molecular Probes). Micrographs represent deconvoluted immunofluorescence Z-images that were compiled onto one image using the extended focus function/axiovision software (Zeiss). Scale bars = 5 m m. Blander & Medzhitov supplementary Figure 12

16 a Pre-enrichment fractions from discontinuous sucrose density gradients LAMP-2 Top Bottom b Post-enrichment Post-enrichment Blander & Medzhitov supplementary Figure 13 (part a and b)

17 c Blander & Medzhitov supplementary Figure 13 (part c)

18 e HEL HEL/LPS F10 F10 2 m g 2 m g Iip41 Iip31 Iip10 LAMP-2 Fig. 13. Biochemical characterization of phagosome enriched fractions. Day 5 bone marrow GM-CSF cultures were given microspheres in various combinations as described in the text. 12 h later, non-adherent cells were harvested, centrifuged, and dounce homogenized as described in supplementary methods. When present, magnetic microspheres denoted as (m), were removed as described. Post nuclear supernatants (PNS) were loaded onto discontinuous sucrose density gradients as described, and following ultracentrifugation 1 ml fractions were collected from top to bottom of the gradients starting with fraction F1 and ending with F13. a. Typical enzymatic activities for the lysosomal enzyme b -hexosaminidase and the golgi enzyme a -Mannosidase II as percent of activities in PNS, and LAMP-2 immunoblot for gradient fractions (F) b. b -hexosaminidase and a -Mannosidase II raw fluorescence numbers, and percent recoveries in fractions F10-11 following enrichment compared to activities in PNS. c. Enzymatic activities for b -hexosaminidase and a -Mannosidase II were determined per m g protein over 30 min as described in supplementary methods. These fluorescence values were plotted on the Y-axis in the top panel, and they showed that the highest activities were present in fractions F Similarly, these raw numbers were plotted on one graph together with the enzymatic activity for b -hexosaminidase (middle panel), and a -Mannosidase II (bottom panel) present in 1 m g of PNS over 30 min. The percent of total PNS activity that was present in the fractions is shown in b for both enzymes. d. Total protein yield in the PNS prepared after phagocytosis of both plain (pl) HEL microspheres and magnetic (m) HEL/LPS microspheres, or vice versa, shown in mg of protein (left graph). Total protein yield in the F10 HEL or F10 HEL/LPS fractions shown in m g of protein (middle graph). Percent protein in F10 compared to total pre-enrichment PNS protein (right graph). e. Western blot analyses for invariant chain (Ii) using anti-ii (CD74) (In-1) Ab (top) or LAMP-2 (bottom). Here, phagosome fractions were prepared following phagocytosis of either HEL microspheres alone, or HEL/LPS microspheres alone. The pattern of Ii chain degradation in the absence of LPS after phagocytosis of HEL microspheres is shown compared to that when LPS containing cargo, HEL/LPS microspheres, are phagocytosed. LAMP-2 levels were equivalent in the two fractions. Blander & Medzhitov supplementary Figure 13 (parts d and e)

19 a * * * * c 2 x 10 6 microsphere supernatants b * * * * Fig. 14. presentation of soluble HEL. a and b. Dose response curve for presentation of soluble HEL by CBA/J DCs to HEL specific 3A9 TCR Tg CD4 + T cells. The readout was IL-2 release at 24 h by CD4 + T cells after recognition of cognate peptide: complexes. a. At concentrations of 1 m g/ml or lower, the addition of 10 ng/ml LPS to these preparations markedly increases the efficiency of presentation. No presentation was detected at the lowest doses of HEL in the absence of LPS. However, when HEL was added at doses higher than 5 m g/ml, we detected equivalent presentation in the presence or absence of LPS. b. IL-6 release by CBA/J DCs in response to soluble HEL versus soluble HEL to which LPS was added. No IL-6 was produced in response to HEL showing that this preparation was free of endotoxin contamination. c. IL-2 by 3A9 (left) or OT-II (right) in response to F1 (CBA/JxC57BL/6J) DCs given 100 m l supernatants from 2x10 6 microspheres as indicated. This particular preparation of microspheres was used in the experiment shown in main text Fig. 4c. Asterisks denote not detected. Blander & Medzhitov supplementary Figure 14