www.sciencetranslationalmedicine.org/cgi/content/full/6/250/250ra113/DC1

Supplementary Materials for

Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis

Christine Happle, Nico Lachmann, Jelena Škuljec, Martin Wetzke, Mania Ackermann, Sebastian Brennig, Adele Mucci, Adan Chari Jirmo, Stephanie Groos, Anja Mirenska, Christina Hennig, Thomas Rodt, Jens P. Bankstahl, Nicolaus Schwerk, Thomas Moritz,

Gesine Hansen*

*Corresponding author. E-mail: hansen.gesine@mh-hannover.de

Published 20 August 2014, Sci. Transl. Med. 6, 250ra113 (2014) DOI: 10.1126/scitranslmed.3009750

The PDF file includes:

Fig. S1. In vitro differentiation of CD45.1 wild-type lin− cells toward macrophage progenitors. Fig. S2. Pulmonary engraftment and therapeutic effect after organotropic transplantation in the Csf2rb−/− model. Fig. S3. Function and phenotype of in vivo differentiated cells in the Csf2rb−/− model. Fig. S4. In vitro differentiation of human CD34+ cells toward macrophage progenitors. Fig. S5. Pulmonary engraftment after organotropic cell transplantation in the humanized herPAP model. Fig. S6. Therapeutic effect of intrapulmonary cell transplantation in the humanized model and function of donor-derived cells. Fig. S7. CCT gating strategy and correlation of CCT data with BALF proteinosis. Fig. S8. Gating and controls for CD11b upregulation assay and cellular engraftment in the murine herPAP model. Fig. S9. Gating and controls functional assays and cellular engraftment in the humanized herPAP model. Fig. S10. Gating and controls for murine macrophage progenitor characterization. Fig. S11. Gating and controls for human macrophage progenitor characterization and cellular engraftment in both models. Fig. S12. Full unedited blot for Fig. 4C. Table S1. List of antibodies used to assess surface marker expression in murine and human cells.

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Fig. S1: In vitro differentiation of CD45.1 WT lin− cells towards macrophage progenitors. (A) Four-fold increase of murine lineage negative cells after four days of in vitro differentiation in M-CSF containing medium. (B) Representative cytospins of day four differentiated macrophage progenitors (scale bar 20µm). (C) Surface marker expression in day four adherent cell fraction, flow cytometry. (D) Colony forming units in methylcellulose assay before (d0) and after 4 days (d4) of differentiation of lin- cells (2000, 5000 or 20.000 cells/ml methylcellulose; n.d.- not detected). All graphs display mean plus SEM from 3 experiments.

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Fig. S2: Pulmonary engraftment and therapeutic effect after organotropic transplantation in the Csf2rb-/- model. (A) BALF cells of treated Csf2rb-/- mice compared to nontreated Csf2rb-/- and CD45.1 WT mice (chipcytometry: CD45.1- yellow, DAPI - blue, 6 weeks post transplantation). (B) Chipcytometry of a lung cryosection nine months post PCT, CD45.1 - yellow, AF=autofluorescence - turquiose; far right: isotype staining for CD45.1 (red) and background fluorescence, scale bars 100µm (C) Flow cytometry of BALF cells in recipient Csf2rb-/- mice, 9 months post transplantation. (n=3 representative mice, 3 experiments. (D) CD45.1+ BALF cells 6 weeks and 9 months post PCT compared to nontransplanted Csf2rb-/- mice (n=6-11 mice per group, from 2 experiments per time point, **P = 0.0059). (E) Time-dependent BALF proteinosis in treated (black) and untreated (red) Csf2rb-/- mice (n=6-9 mice per group from 3 independent experiments per time point, *P = 0.0166 and **P = 0.0037). All graphs display means and SEM calculated by One-way ANOVA with Dunnett’s (D) or Tukey’s (E) post hoc testing, [n.s.] not significant.

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Fig. S3: Function and phenotype of in vivo differentiated cells in the Csf2rb−/− model. (A) Phagocytyosis rate and (B) antigen presentation in donor derived cells sorted from the lungs of transplanted Csf2rb−/− mice versus control cells (values from 2 experiments, ***P = 0.0002). (C) Maximum cellular diameters of donor-derived versus control cells (analysis of PAS stained semithin slices prepared for electron microscopy, one experiment *** P < 0.0001, *P = 0.0123). All graphs display mean plus SEM calculated by One-way ANOVA with Dunnett’s post hoc testing, [n.s.] not significant.

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Fig. S4: In vitro differentiation of human CD34+ cells towards macrophage progenitors. (A) Five-fold increase of numbers after four days of in vitro cultivation of human CD34+ cells in macrophage differentiation cocktail. (B) Representative cytospins of day four differentiated CD34+-derived cells (scale bar 20µm). (C) Expression of surface markers after four days of differentiation (flow cytometry). (D) Total number of colony forming units in methylcellulose assays: day four of in vitro differentiation. (E) Exemplary hematopoietic colonies derived from methylcellulose culture of day four differentiated cells (scale bars 500µm). All graphs display mean plus SEM, values from 3 experiments.

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Fig. S5: Pulmonary engraftment after organotropic cell transplantation in the humanized herPAP model. (A) Engraftment of hCD45+ donor derived cells in the lungs and not in other organs of recipient huPAP mice (6.5 months post PCT), compared to nontransplanted mice and primary human AM; representative flow cytometry; n≥6 mice per group from 3 independent experiments. (B) Donor derived cells that are found in BALF but not in liver, spleen or BM of transplanted mice (n=5 mice per group from 2 experiments, ***P = 0.0003 BALF compared to liver and ***P = 0.0003 BALF compared to spleen and BM). (C) Percentage of hCD45+ cells in the BALF of treated huPAP mice (n=10 untreated mice and n= 8, 2 and 10 mice for 2 months, 4 months and 6.5 months post PCT respectively, *P = 0.0208 and ***P = 0.0001. (D) Percentage of hCD45+ cells in the lungs of treated huPAP mice (n=10 untreated mice and n= 8, 2 and 10 mice for 2 months, 4 months and 6.5 months post PCT respectively, *P = 0.0287, and ***P = 0.0009). (E/F) Chipcytometry of lung cryosections from huPAP mice 2 months (E) and 6.5 months (F) post PCT: hCD45 - yellow, DNA - blue, AF=autofluorescence - turquiose; far right: isotype staining for hCD45 (red), scale bars 100µm, n= 2 experiments. All graphs display display mean plus SEM., One-way ANOVA with Dunnett’s post hoc testing.

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Fig. S6: Therapeutic effect of intrapulmonary cell transplantation in the humanized model and function of donor-derived cells. (A) Time-dependent BALF proteinosis in treated (black) and untreated (red) huPAP mice (n=6-11 mice per group for 0, 2 and 6.5 months from 3 independent experiments; n=2 mice from one experiment for 4 months, ****P <0.0001 and **P = 0.0025). (B) Phagocytosis of donor derived cells sorted from huPAP recipient lungs, 2 experiments. All graphs display mean and SEM calculated by One-way ANOVA with Tukey’s (A) and Dunnett’s (B) posthoc testing, [n.s.] not significant.

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Fig. S7: CCT gating strategy and correlation of CCT data with BALF proteinosis. (A) Representative Chest computed tomography (CCT) scans illustrating the gating for volumetric analyses and 3D-rendering of CCT-data (1-3): segmentation of lung volumes (blue) in micro-CCT. (4) 3D-Rendering of the segmented lung volume. (B) Correlation of BALF protein levels and CCT values (linear regression).

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Fig. S8: Gating and controls for CD11b upregulation assay and cellular engraftment in the murine herPAP model. (A)

Gating of human granulocytes for CD11b upregulation assay as in Fig. 1E (-1ctrl = minus one control). (B) Gating of murine

granulocytes for CD11b upregulation assay as in Fig. 1E. (C) Gating strategy for Csf2rb-/- lung cells and BALF cells as in Figure

2B with staining controls. (D) Gating for determination of extrapulmonary engraftment of CD45.1+ donor derived cells as in Fig.

2C. (E) Isotype controls for chipcytometry stainings as used in Fig. 2D.

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Fig. S9. Gating and controls functional assays and cellular engraftment in the humanized herPAP model. (A) Gating

strategy to determine phagocytic capacity for Fig. 3C. (B) Gating strategy to determine the proliferation rate of T responder cells

for Fig. 3C. (C) Gating strategy to determine engraftment of hCD45+ donor derived cells in the humanized model as in Fig. 4B.

(D) Staining and isotype controls for the used human hCD45 antibody in primary human granulocytes. (E) Gating strategy to

determine cellular phagocytic capacity for Fig. 5D.

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Fig. S10: Gating and controls for murine macrophage progenitor characterization.

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Fig. S11: Gating and controls for human macrophage progenitor characterization and cellular engraftment in both

models. A: Gating strategy for engraftment of CD45.1+ cells in the BALF of Csf2rb-/- recipients as in Fig. S2C. B: Gating

strategy and controls for murine macrophage progenitors as in Fig. S4C. C: Gating strategy and staining controls for engraftment

of hCD45+ cells in BALF and lung samples of huPAP recipients as in Fig. S5C and D.

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Fig. S12: Full unedited blot for Fig. 4C.

Specificity

Epitope

Conjugate

Company

Clone

Application

mouse B220 eFlour 450 eBioscience RA3-GBL flow cytometry mouse CD117 (c-Kit) APC eBioscience 2B8 flow cytometry mouse CD11b PE eBiosciense M1/70 chip/flow cytometry mouse CD11c PE BD HL3 chipcytometry mouse CD11c APC eBiosciense N418 flow cytometry mouse CD14 PE/FITC eBiosciense Sa2-8 chip/ flow cytometry mouse CD16/32 PE-Cy7 eBiosciense 93 chipcytometry mouse CD19 PE. APC BD 1D3 chipcytometry mouse CD3e PE/PE-Cy7 eBioscience 145-2C11 flow cytometry mouse CD4 PE BD RM4-5 chipcytometry mouse CD34 PE BD HM34 chipcytometry mouse F4-80 PE/APC eBioscience BM8 chip/ flow cytometry mouse Ly-6A/E (Sca-1) PerCP-Cy5.5 eBioscience D7 flow cytometry mouse Ly-6G (Gr-1) eFlour 450 eBioscience RB6-8C5 flow cytometry mouse MHC-II PE eBioscience M5/114.15.2 chipcytometry mouse Siglec-F PE BD E50/2440 chipcytometry mouse podoplanin FITC eBioscience 8.1.1. chipcytometry mouse CD45.1 APC/PE eBioscience A20 chip-/flow cytometry human CD11b PE BD KRF 44 chip/ flow cytometry human CD11c PE BD S-HCL-3 chipcytometry human CD14 PE BC RMO52 chipcytometry human CD15 PE BD HI98 chipcytometry human CD163 APC eBioscience eBioGHI/61 flow cytometry human CD19 PE eBioscience HIB19 chip-/ flow cytometry human CD193 PE eBioscience eBio5E8-G9-B4 flow cytometry human CD20 PE BD 2H7 chipcytometry human CD200R PE eBioscience OX108 flow cytometry human CD206 eFluor 450 eBioscience 19.2 flow cytometry human CD3 PE BD UCHT 1 chipcytometry human CD33 PE-Cy5 eBioscience HIM 3-4 flow cytometry human CD34 PE/eFluor 450 eBioscience 4H11 flow cytometry human CD38 APC eBioscience HIT2 flow cytometry human CD4 PE Biolegend OKT4 chipcytometry human CD45 APC eBioscience HI30 flow cytometry human CD64 PE eBiosciene 10.1 chipcytometry human CD71 PE Biolegend CY1G4 chipcytometry human CD8a PE BD RPA-78 chipcytometry human DAPI/Hoechst 3342 Invitrogen n.a. chipcytometry human MHC-II PE BD G46-6 chipcytometry

Table S1. List of antibodies used to assess surface marker expression in murine and human cells.