ImageStream cytometer analysis. Cells were cultured as described above in vented-cap

Transcription

1 ImageStream cytometer analysis. Cells were cultured as described above in vented-cap polypropylene tubes, stained with αcd66b-fitc, αm-dc8-pe and αcd56-pe-cy5.5 mabs, washed and fixed with 4 % (w/v) paraformaldehyde. Samples were acquired by the Amnis ImageStream cytometer (Amnis, Seattle, WA, USA) and data analysis performed using IDEAS (Amnis). In our experiments, cells were tested at a concentration of 1 * 10 6 cells/100 µl. IDEAS application builds a matrix of spectral compensation values using control files, which contain fluorescence values of cells stained with a single fluorochrome as well as unlabeled cells. Debris and cell aggregates were excluded, and doublets were identified by gating on size and aspect ratio. Doublets were confirmed by visual inspection of the fluorescence pattern. IL-12 and IFNγ immunostaining procedures. Frozen tissue blocks were retrieved from the archives of the Istituto Dermopatico dell Immacolata (IDI-IRCCS, Rome, Italy) or kindly provided by Dr. F. Annunziato (Department of Internal Medicine, Immunology and Cellular Therapies Unit, Florence, Italy). Five micron frozen tissue sections were stained with either αil-12 (rat, clone 20C2, dilution 1:50) or αifnγ (mouse IgG2a, clone 25718, R&D Systems, dilution 1:200) mabs. IL-12 was revealed using Rat HRP polymer (Biocare Medical, Concord, CA, USA) whereas IFNγ was revealed using Mach 4 AP polymer (Biocare Medical) and developed using DAB chromogen.

2 Table S1. List of agonists used Agonist Final Provider concentration Ultra-Pure E.coli LPS 100 ng/ml Alexis Biochemicals, San Diego, USA (0111:B4 strain) GM-CSF 50 ng/ml PeproTech, Rocky Hill, USA IL U/ml R&D Systems, Minneapolis, USA IFNγ 100 U/mL R&D Systems, Minneapolis, USA IL pg/ml PeproTech, Rocky Hill, USA IL ng/ml Immunotools, Friesoythe, Germany IL ng/ml MBL International, Nagoya, Japan

3 Table S2. List of antibodies used Antibody Clone Final Application Provider dilution CD3 SP7 dil. 1:100 IHC Thermo Scientific, Fremont, USA CD11c 5D µg/ml IHC Monosan, Uden, The Netherlands CD15 MMA 4 µg/ml IHC Thermo Scientific, Fremont, USA CD18 IB4 10 µg/ml 5 µg/ml B/N LCM Dr. C. Laudanna, Dept. Pathology and Diagnostics, Verona CD18 7E4 20 µg/ml B/N Thermo Scientific, Fremont, USA FC CD56 123C3.D5 6.7 µg/ml IHC Thermo Scientific, Fremont, USA CD66b G10F5 dil. 1:600 IHC BD Pharmingen, San Diego, USA GM-CSF polyclonal 10 µg/ml B/N R&D Systems, Minneapolis, USA HLA-DR CR3/ µg/ml IHC Dako, Glostrup, Denmark ICAM-1 BBIG-I1 2 µg/ml B/N R&D Systems, Minneapolis, USA 5 µg/ml FC LCM ICAM-2 CBRIC2/2 2 µg/ml B/N AbD Serotec, Oxford, UK FC ICAM-3 CBR-IC3/1 2 µg/ml B/N Bender MedSystems, Vienna, Austria 5 µg/ml FC LCM ICAM-3 Cal µg/ml CSA R&D Systems, Minneapolis, USA IFNγ B µg/ml B/N Dr. G. Trinchieri (NCI, Frederick, USA) IFNγ dil. 1:200 IHC R&D Systems, Minneapolis, USA IL-12p35 20C2 20 µg/ml B/N Dr. G. Trinchieri dil. 1:50 IHC IL-12p40 C µg/ml B/N Dr. G. Trinchieri 6-sulfo LacNAc DD1 FC Miltenyi Biotec, Bergisch Gladbach, Germany 6-sulfo DD2 dil. 1:50 IHC ref. 34 LacNAc 5 µg/ml LCM MPO polyclonal 1.2 µg/ml IHC Dako, Glostrup, Denmark TNFα B µg/ml B/N Dr. G Trinchieri Abbreviations: B/N: blocking/neutralizing; CSA: co-stimulation assay; dil: dilution; FC: flow citometry; IHC: immunohistochemistry; LCM: laser confocal microscopy; MPO: myeloperoxidase

4 Table S3. List of ELISA kits used Cytokine Detection limit Provider GM-CSF 2 pg/ml Biolegend, San Diego, USA IFNγ 2 pg/ml Immunotools, Friesoythe, Germany IL-1β 3.9 pg/ml ebioscience, San Diego, USA IL pg/ml Immunotools, Friesoythe, Germany IL-12p pg/ml ebioscience, San Diego, USA IL pg/ml MBL International, Nagoya, Japan IL pg/ml ebioscience, San Diego, USA TNFα 1.4 pg/ml Immunotools, Friesoythe, Germany

5 Table S4. Expression levels of CD18, ICAM-1, ICAM-2 and ICAM-3 by neutrophils, NK cells, and slandc as evaluated either immediately after isolation or upon co-culture neutrophils NK cells slandc t0 18 h t0 18 h t0 18 h LPS plus LPS plus LPS plus medium medium medium IL-15/IL-18 IL-15/IL-18 IL-15/IL-18 ICAM ICAM ICAM CD Neutrophils, NK cells and slandc were analyzed for ICAM-1, ICAM-2, ICAM-3 and CD18 expression by flow cytometry, immediately after isolation (t0) as well as after an 18 h-co-culture in the absence or the presence of LPS plus IL-15/IL-18. Numbers represent the mean fluorescence intensity (MFI) of each antigen after subtraction of the MFI values obtained from specific isotype controls. One representative experiment is shown out of 2 performed with similar results.

6 Figure S1. Neutrophils potentiate the release of IFNγ and IL-12p70 by stimulated NK cells and slandc, respectively. (A) NK cells were cultured with or without neutrophils and treated with LPS, alone or in combination with either IL-2 or IL-15/IL-18. (B) Neutrophils and slandc were cultured, separately or together, in the presence or absence of LPS and/or different concentrations of IFNγ. After 18 h, IFNγ (A) and IL-12p70 (B) were measured in culture supernatants by ELISA. In (A), asterisks stand for p < (***) by Student s t test using a two-tail distribution of paired

7 samples (n = 8), while a representative experiment out of 2 performed with similar results is shown in (B).

8 Figure S2. Neutrophils cooperate with slandc to potentiate the release of IFNγ by CD56bright and CD56dim NK cell subsets. (A, B) Purified NK cells were incubated with slandc, in presence or absence of neutrophils, and cultured either untreated or treated with LPS in combination with either IL-2 or IL-15/IL-18. After 18 h of culture, both the CD56bright (black

9 dots) and the CD56dim (gray dots) subsets were analyzed for IFNγ secretion by a specific cytokine secretion assay. In (A), the dot plots of the different experimental conditions are shown as representative of one experiment out of 3 performed with similar results. In (B), the corresponding fluorescence intensities are displayed in an overlay graph, and depicted in the absence (thin line) or the presence (thick line) of neutrophils.

10 Figure S3. The neutrophil-dependent increase of IL-12p70 production by slandc is responsible for the augmented NK-derived IFNγ in neutrophil/nk cell/slandc co-cultures. NK cell/slandc co-cultures were incubated in the presence or absence of neutrophils, and then treated with LPS plus either IL-2 (A) or IL-15/IL-18 (B). After 18 h, IFNγ, IL-12p70 and TNFα were measured in culture supernatants. The fold-increase in the release of IFNγ, IL-12p70 and TNFα was calculated as the ratio between the amount of each cytokine produced in the presence of neutrophils and the amount produced in their absence (n = 13). The values obtained for each experiment were plotted as fold increase of the release of IFNγ versus the corresponding increase in either IL-12p70 (upper panels) or TNFα (lower panels). The respective degrees of correlation (r) and p values were calculated and reported in each plot.

11 Figure S4. Neutrophils, NK cells and slandc form small aggregates upon their activation under co-culture conditions. Freshly isolated neutrophils (PMN) or neutrophil/nk cell/slandc co-cultures incubated with LPS plus IL-15/IL-18 for either 18 h (A), or 2 h (C-E), were collected

12 and cytocentrifuged. Smears were then stained either by May-Grunwald Giemsa for microscopy analysis (A-B), or by conjugated αcd66b (yellow), αm-dc8 (orange) and αcd56 (pink) Abs for ImageStream cytometer analysis (C-E). Panels A and B show representative images (n=3 for each condition), while panels C-E display representative images of doublets between neutrophils (yellow) and slandc (orange) (C), slandc (orange) and NK cells (pink) (D), neutrophils (yellow) and NK cells (pink) (E), from a representative experiment out of three performed with similar results.

13 Figure S5. CD18, ICAM-1, ICAM-3, and M-DC8 labeling in neutrophils, NK cells and slandc under co-culture conditions. Neutrophil/NK cell/slandc co-cultures were incubated in the absence of stimulation. After 2 h, cells were harvested, cytocentrifuged and co-stained with either αcd18 and αicam-1 (A) or αicam-3 (B) Abs, prior to confocal microscopy analysis. For (C), cytospins of neutrophil/nk cell/slandc co-cultures were labeled using both the αm-dc8 mabs, to identify slandc (violet cells, as indicated by the arrows), and DAPI, to visualize the nuclei (blue)

14 for the specific morphological recognition of neutrophils. NK cells are thus identified as M-DC8 negative, mononuclear cells. Representative images from 3 experiments are shown.

15 Figure S6. Identification of NK cells and staining of IL-12 and IFNγ by immunohistochemistry. Immunohistochemistry was performed using sections from biopsies of patients affected by psoriasis (A-C) or Crohn s disease (D-F). Frozen sections of lesional skin (A, B) or intestinal mucosa (D, E) from patients with psoriasis and Crohn s disease, respectively, are stained for either IFNγ (A, D) or IL-12p35 (B, E). IFNγ reactivity is observed on round cells (arrows), whereas IL-12 signal is observed on large monocytoid/dendritic cells (arrows). (C, F) Double-staining analysis of formalin-fixed paraffin-embedded sections revealed that CD56 + cells do not stain for CD3, thus representing NK cells. The images are representative of at least 3 independent experiments for each pathology.