Cytokine Complex Expanded Natural Killer Cells Improve Allogeneic Lung. Transplant Function via Depletion of Donor Dendritic Cells

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1 Cytokine Complex Expanded Natural Killer Cells Improve Allogeneic Lung Transplant Function via Depletion of Donor Dendritic Cells Wolfgang Jungraithmayr, Laura Codarri, Gregory Bouchaud,Carsten Krieg, Onur Boyman, Gabor Gyülvészi, Burkhard Becher, Walter Weder, and Christian Münz ONLINE DATA SUPPLEMENT

2 Jungraithmayr et al. 1 SUPPLEMENTARY METHODS Interventions in themouse model of orthotopic single lung transplantation Animals were approved by the institutional animal care committee (licence No. 1/21). For the IL-15 stimulation experiments, recipients were subcutaneously injected prior to lung transplantation with three consecutive doses of IL-15 complexes dissolved in 2 µl PBS, consisting of 1.5 µg of IL-15 and 3 µg of modified soluble IL-15R which corresponds to a 1:1 molar ratio. For CD11-DTR experiments, 1 ng of diphtheria toxin was given to each CD11c-DTR donor mouse 24 hours before implantation. Histology and immunohistochemistry At days 1, 3, 5, 7, 1 and 14 transplanted lungs were excised. After fixation in 4% phosphate-buffered formalin, the lungs were cut transversally into 2 mm thick slices and embedded in paraffin. 4 μm thick sections were cut and mounted on positively charged glass slides. Standard histological procedures (hematoxylin and eosin) and immunostainings were performed. For immunostaining, the following primary antibodies were used: CD3 (rat antimouse, BD Pharmingen), B22 (goat anti-mouse, Santa Cruz), F4/8 (rat anti-mouse, BD Pharmingen), and NKp46 (rat anti-mouse, BD Pharmingen). DAB was used as additional chromogen (Dako). Digital photographs were taken at a magnification of 1-4, and the nuclei of twenty random high-power fields (HPFs) from each lung sample were analyzed at 1 magnification. Cell isolation and mixed leucocytes reactions (MLRs)

3 Jungraithmayr et al. 2 Lungs were digested in 2 mg/ml collagenase and 1 mg/ml DNase (Roche) for 3 min, and lung cells were purified with a 7-μm cell strainer (BD Falcon) to obtain a single cell suspension. Cells were either directly stained with blocking monoclonal antibodies against Fc receptors (CD16 and CD32) or following a 4 h restimulation withphorbol 12- myristate 13-acetate (PMA) (5 ng/ml) and ionomycin (5 ng/ml) in the presence of GolgiPlug (BD Pharmingen). The cells were then stained for 3 min with a panel of antibodies for surface markers and fixed or permeabilized for cytokine staining. Where indicated lung derived cells were stimulated with syngeneic (C57BL/6) or allogeneic (BALB/c) T cell depleted irradiated (4Gy) splenocytes at an E:T ratio of 1 to 2 for 3 days and for the last 4-6 h cytokine release was induced by PMA and ionomycin while protein transport blocked with Golgistop treatment. Then antibody staining was performed as described above. Cells were acquired at the flow cytometer LSRII Fortessa (BD) and sorted purified with Aria III (BD).Analyses were performed with Flow Jo software (Tree Star). CD17a degranulation analysis and NK cell adoptive transfer Single cell suspensions from transplanted wild type or IL-15 complex treated naïve or transplanted mice lungs were stained for CD45 Pacific Blue (Biolegend), NKp46 Alexa 647 (ebioscience), and CD8 SRPD (BioLabs) before being FACS sorted. Sorted cells were cultured with 5 ng/ml IL-2 for 48 hours before co-culture with dendritic cells. Single cell suspensions of syngeneic (C57BL/6) or allogeneic (BALB/c) dendritic cells from lungs were MACS sorted using CD11c + magnetic beads (Miltenyi Biotech). Sorted NK were restained with CD45 (Biolegend), NKp46 and NK1.1 PE (BD Pharmingen) before being co-cultured with dendritic cells for 4 hours at 37 C in a ratio of 4:1 (DC:NK).

4 Jungraithmayr et al. 3 CD17a FITC (ebioscience) was added to the coculture right before the incubation time followed one hour later by GolgiStop (BD Pharmingen). At the end of the incubation time cells were immediately acquired at the flow cytometer. In adoptive transfer experiments sorted NK cells were cultured over night with IL- 2 (5 ng/ml) before being injected at 1 6 /mouse intra-venous into IL-15Ra -/- mice at the day of the transplant. Magnetic resonance imaging All measurements were performed in a Bruker 4.7T Biospec 47/4 with a gradient strength of 6 mt/m and a slew rate of 11 T/m/s equipped with a circular polarized 1H mouse whole body RF coil. The animal bed was equipped with a pad with continuous warm water supply to prevent temperature loss of the mouse. The imaging protocol consisted after gradient-echo (GRE) localizers in 3 spatial directions of a 3D T1w spoiled GRE sequence (TR/TE 15ms/4.7ms; matrix 192 x 192 x 32; FoV 4 x 4 x 45 mm; averages 3) and a 2D T2w fast spin-echo (FSE) sequence (TR/TE 25ms/11 ms; effective TE 33 ms; echo train length 8; matrix 256 x 256; FoV 4 x 4 mm; slice thickness 1 mm; averages 3). For relaxation measurement, a 3D ultra-fast echo-time (UTE) sequence was applied with 8 subsequent acquisitions with the echo-times TE = 5 s. Further protocol parameters of the UTE sequence were repetition time TR = 8 ms, matrix 128 x 128 x 128, FoV 45 x 45 x 45 mm; spatial resolution.35 x.35 x.35 mm, flip angle 18.9 ; averages 1, acquisition time 6m5s. Shorter TE than 5 s resulted in signal attenuation, which may be caused by the necessary time for the protection diodes to fire.

5 Jungraithmayr et al. 4 Image quality was assessed by computation of signal-to-noise and contrast-to-noise ratios of the UTE sequence with echo-time TE = 5 s. Signal-to-noise ratio (SNR) was defined as SNR S SD parenchyma background, wheres parenchyma is the mean signal of the lung parenchyma and SD background is the standard deviation of the background signal. SNR was evaluated by region-of-interest (RoI) analysis for normal lung parenchyma in the dorsal area (best ventilated) and in the lung transplant (Supplementary Methods Figure). Subpleural areas were chosen for RoI definition to avoid signal contamination with macroscopic vessels. Contrast-to-noise ratio (CNR) was defined as CNR S transplant SD S background lung withs transplant meaning the mean signal intensity of the transplanted lung, S lung the mean signal intensity in the dorsal area of the normal lung, and SD background the standard deviation of the background signal. Four different RoI positions were evaluated: the normal lung on the left side in the ventral, the dorsal and the apical part as well as the transplanted lung on the right side (typical RoI definition is shown in Figure 1). In the respective RoIs, spin density (normalized to the dorsal normal lung) and T2 values were measured.

6 Jungraithmayr et al. 5 Supplementary Methods Figure.Upper row: four different regions of interest (ROI) positions were evaluated: the normal lung on the left side in the ventral, the dorsal and the apical part as well as the transplanted lung on the right side (typical ROI is shown in right, naive Lung and left, transplanted Lung). In the respective ROIs, spin density (normalized to the dorsal normal lung) and T2 values were measured. Typical T2 decay curves with measurement points between 5μs up to 5ms are shown (lower row). SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1Leucocyte infiltrates in MHC class I and II mismatched mouse lung allografts after orthotopical lung transplantation. Nuclei scoring in histology after hematoxylin& eosin staining, and after immunostainings with antibodies against F4/8 + macrophages, CD3 + T cells, B22 + B cells, and NKp46 + NK cells (4 independent experiments per group are shown, means ± s.e.m.). Supplementary Figure 2 The expansion of NK cells by IL-15 complexes diminishes late allograft T cell infiltrates and favors improved transplantation acceptanceas determined by (a) kinetics of allograft infiltrating cells from single cells suspensions gated on CD45 + cells.ifn- (b)and TNF- (c) producing CD4 + T cells in transplanted lungs of mock- and IL-15 complex treated recipients ex vivo. Cumulative data of 3 experiments(means ± s.e.m.,

7 Jungraithmayr et al. 6 P <.5).(d)representative dot blot of IFN- and TNF- producing CD4 + T cells in untreated and IL-15 complex treated allo-graft recipients. (e)tnf- production by CD4 + (left panel) and IFN- secretion by CD8 + (right panel) T cells from transplanted allogeneic lungs of untreated or IL-15 complex treated mice after expansion with syngeneic (syn SPL) or allogeneic (allo SPL) splenocytes. Cumulative data of 3 experiments(means ± s.e.m., P <.5). (f)representative dot blots for the analysis in (e). Supplementary Figure 3Leucocyte infiltrates and graft function of lung allografts in IL- 15Rα-/- mice that lack NK cells (a) kineticsby flow cytometryof graft infiltrating cells from single cells suspensions gated on CD45 + cells and (b) oxygenation measurements from blood samples taken from the left pulmonary vein, (c) nuclei scoring from histology with hematoxylin& eosin staining (3 independent experiments per group are shown, means ± s.e.m., Mann-Whitney, NS= no statistical significance). Supplementary Figure 4NK cell mediated reduction of transplant donor DCs diminishes recipient derived T cell infiltration 3 days after transplantation(3 independent experiments per group are shown, means ± s.e.m., Mann-Whitney, P <.1).

8 Supplementary Figure 1 nuclei/2 fields (.1 mm 2 ) H&E CD3 B22 F4/8 NKp46

9 Supplementary Figure 2 a CD4 + T cells (x1 6 ) 1. +IL-15 cplx CD8 + T cells (x1 6 ) 1. +IL-15 cplx NKp46 + cells (x1 6 ) 8 +IL-15 cplx CD11c + cells (x1 6 ) IL-15 cplx... b Total IFN-γ + CD4 + T cell number (x1 3 ) d % TNF-α + CD4 + T cells IL-15 cplx IL-15 cplx Syn SPL Allo SPL c Total TNF-α + CD4 + T cell number (x1 3 ) e % IFN-γ + CD8 + T cells IL-15 cplx + IL-15 cplx Syn SPL Allo SPL IFN-γ IFN-γ TNF-α TNF-α IL-15 cplx IL-15 cplx CD4 + T cells CD4 + T cells CD8 + T cells

10 Supplementary Figure 3 a.8 IL-15Rα -/-.8 IL-15Rα -/- CD4 + cells (x1 6 ) CD8 + cells (x1 6 ) CD11c + cells (x1 6 ) e IL-15Rα -/- b nuclei/2 fields (.1 mm 2 ) H&E c PaO 2 (mmhg) Naive Lung IL-15Rα -/- NS

11 Supplementary Figure 4 % CD4 + T cells CD CD % CD8 + T cells CD CD IL-15 cplx +IL-15 cplx