Interferon γ regulates idiopathic pneumonia syndrome, a Th17 + CD4 + T-cell-mediated GvH disease Nora Mauermann, Julia Burian, Christophe von Garnier, Stefan Dirnhofer, Davide Germano, Christine Schuett, Michael Tamm, Roland Bingisser, Urs Eriksson, and Lukas Hunziker Online Data Supplement
Methods Flow cytometry Lung digestion was performed as previously described (1). Single-cell lung suspensions from diseased and control mice were Fc blocked with anti-mouse CD16/32 (ebioscience) and stained with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin direct labeled, or secondary antibodies to biotin-labeled antibodies using Streptavidin-allophycocyanin-Cy7,, streptavidin PerCP-Cy5.5 labeled antibodies against CD3e (145-2C11), CD4 (L3T4), CD8α (53-6.7), MHC-II (I-A/I-E, clone 2G9), CD11b (M1/70), CD11c (HL3), CD45.1 (A20), CD45.2 (104), and BrdU (all from BD Pharmingen). Intracellular stains were performed after stimulation with 50 ng/ml PMA and 500 ng/ml ionomycin, together with brefeldin A (10 ng/ml) for 6 hours. Cells were fixed with 2% paraformaldehyde and incubated with 0.1% saponin (all chemicals from Sigma) and PE-labeled antibody to IL-17A (clone TC11-18H10, Pharmingen). Propidium iodide (PI) exclusion was routinely used to differentiate living from dead cells, when applicable. BrdU staining procedures followed available protocols (BrdU flow kit, BD Pharmingen). Samples were analyzed on a FACSCalibur (BD Biosciences) or Cyan (DAKO) flow cytometer, using FlowJo (Treestar) software. Depending on the experiment, cell sorting was performed with FACSAria (BD Biosciences) to isolate at least 99% pure CD3 + CD4 + or CD3 + CD8 + T cells. References E1. von Garnier C, Filgueira L, Wikstrom M, Smith M, Thomas JA, Strickland DH, Holt PG, Stumbles PA. Anatomical location determines the distribution and function of dendritic cells and other apcs in the respiratory tract. J Immunol 2005;175:1609-1618.
Figure E1. Characterization of lung APC populations during IPS. (Left) Single cell suspension of naïve CB6F1 mouse lung was prepared and stained for CD11c and MHC-II (I-A/I-E) and analyzed by FACS to identify the different APC populations. The percentages of DC (MHC-II high CD11c high ), macrophages (MHC- II low CD11c high ), and B cells (MHC-II high CD11c neg ) of total cells are indicated. (Right) CB6F1 recipients reconstituted with BALB/c Rag2 -/- bone marrow together with either WT CD4+ (top right) or IFN-γ -/- CD4 + (bottom right) T cells. After 6, 10, and 13 days post reconstitution, single cell preparations of the lungs were
stained for CD11c and MHC-II. The percentage of each cell population is shown. Percentage of each population in naïve animals is also shown. Figure E2. Influence of donor T cells on BAL cell content. CB6F1 recipient mice reconstituted with BALB/c Rag2 -/- bone marrow together with WT CD4 + T cells (n=4), WT CD8 + T cells (n=4), IFN-γ -/- CD4 + T cells (n=4), or IFN-γ -/- CD8 + T cells (n=4) were sacrificed 13 days post reconstitution. Bronchio-alveolar lavage (BAL) cells were isolated, counted, spun onto glass slides and stained with Diff- Quik. The percentage of neutrophils, lymphocytes, and macrophages of total cells are plotted for each group of mice.
Figure E3. Anti-TNF-α treatment has beneficial effect on severe IPS. CB6F1 hosts were reconstituted with BALB/c Rag2 -/- bone marrow and WT or IFN-γ -/- CD4 + T cells together with i.v. 40 μg anti-tnf-α, on day 4, 8, and 11 post reconstitution, while control mice received vehicle alone (PBS). The mice were
sacrificed on day 13. BAL cells were isolated. (A) BAL cells of vehicle and anti- TNF-α treated mice were counted, spun onto glass slides, and stained with Diff- Quik. The count of neutrophils, lymphocytes, and macrophages are shown as a percentage of total cells. (B) Representative H&E stained sections of lung from mice belonging to each experimental group (n=4 for each group) is shown. Scale bar in the upper row represent 500μm and the lower row shows a magnified portion of the same image (scale bar: 100μm).