Xgvhd Model with Immune Population Depletion

Supplementary Material xGVHD model with immune population depletion

3-4 week old Rag2-/-IL2Rγnull mice were injected i.p. with 1x107 human fresh PBMC or PBMC after

CD8+ or CD25+ or CD4+ or CD56+ or CD14+ cells depletion, on day 0 of the experiment. Treatment with Combo (nivolumab + urelumab, 200 μg per injection each) was administered by intravenous

(i.v.) injection on days 4, 7 and 10. A control group was injected i.p. with human fresh PBMC on day

0 and i.v. with IgG4, on same days as Combo. Plasma samples were collected as depicted before.

Animals that developed clinical symptoms of xGVHD (>15% weight loss, hunched posture, reduced mobility, fur loss, tachypnea) were sacrificed and an end point of survival was recorded. Depletion of immune cells was performed using CD8 (REF 130-045-201), CD4 (REF 130-045-101), CD25 (REF

130-091-301), CD14 (REF 130-050-201) or CD56 (REF 130-050-401) MicroBeads from Miltenyi

Biotec following manufacturer's instruction. An Automacs pro Separator (Miltenyi Biotec) was used to perform cell immunomagnetic sorting using the program "depletes". The efficacy of cell depletion was verified by flow cytometry.

Mouse syngeneic tumor model

A total of 0.5 × 106 MC38 murine colon cancer cells were injected subcutaneously into the flank of

C57/BL6 wild-type mice (5 to 6 weeks old) in a volume of 100 μL PBS on day 0. Mice were treated i.p. with anti-hCD137 (1D8, 200μg per injection) and anti-hPD-1 (RMP1-14, 200μg per injection) or

Control (Rat IgG, 200ug per injection) on days 12, 14 and 16. For depletion of immune cells, mice were injected i.p. with anti-CD4 (GK1.5, 100ug per injection), anti-CD8 (2.43, 100 ug per injection), anti-NK1.1 (PK136, 100ug per injection) or clodronate liposomes (200ul (containing 5mg clodronate per ml suspension) per injection). anti-CD4, anti-CD8 and anti-NK1.1 were administered on days 10, 14, 19 and 27 (anti-Nk1.1 was administered additionally on day 12) and clodronate was administered every single day starting on day 11 and finishing on day 22. Tumor size was monitored and mice were sacrificed when tumor size reached 1,000 mm3. Immunohistochemistry

Tissues were recovered from mice at necropsy, formalin-fixed and embedded in paraffin. Formalin-fixed, paraffin-embedded (FFPE) specimens were cut into 3 to 4 µm sections and mounted on glass slides. IHC was performed using the following monoclonal antibodies against

CD3 (clone SP7), CD4 (clone SP35) and CD8 (clone SP6). After deparaffinization and rehydration, the sections were washed in Tris-buffered saline (TBS) 0.55M. Sections were submerged in

Tris/EDTA (pH=9.0) for 30 minutes at 95°C in a Pascal pressure chamber (Dako). After blocking, the sections were incubated with the primary antibody overnight at 4°C. After washing with TBS

0.55M, the sections were incubated with EnVisionFLEX/HRP (Dako) for 30 minutes at room temperature. The sections were stained using the Liquid DAB + Substrate Chromogen System kit

(Dako), for 10 minutes, and were contrasted with Harris Haematoxylin for 5 minutes.

Multiplexed Immunofluorescence

Multiplexed quantitative immunofluorescence (QIF) was performed as recently reported (33) using monoclonal antibodies against hCD137 (clone GW2.4), hPD-1 (clone 5C3) and hPD-L1 (clone E1L3N).

The specificity of the antibodies was evaluated by the staining of control human tissues, Mel624 and

HEK293 cell line transfectants and experimentally activated human lymphocytes obtained from

PBMCs. Briefly, fresh full-face, whole tissue sections from the tumor samples were deparaffinized and subjected to antigen retrieval in citrate buffer pH=6.0 and boiling for 20 min at 102°C in a pressure-boiling container (PT module, Lab Vision). Slides were then blocked with 0.3% BSA in TBS for 30 min at room temperature followed by overnight incubation at 4oC with a solution containing the primary mouse antibody for CD137 (1:100) and a rabbit polyclonal pancytokeratin antibody

(1:50, Z0622; Dako Corp); or a solution containing primary rabbit anti PD-1 (1:20) or hPD-L1 (1:1600) antibodies and a mouse monoclonal anti-human pancytokeratin antibody, clone AE1/AE3, M3515;

Dako Corp.). Sections were incubated for 1 h at room temperature with Alexa 546-conjugated goat anti-mouse (A11003; Molecular Probes) or anti-rabbit (A11010; Molecular Probes) secondary antibody diluted 1:100 in mouse or rabbit EnVision amplification reagent, respectively (K4003,

Dako). Cyanine 5 (Cy5) directly conjugated to tyramide (FP1117; Perkin-Elmer) at a 1:50 dilution was used for target antibody detection. Prolong mounting medium (ProLong Gold, P36931; Molecular

Probes) with 40,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei in the preparations.

Additional QIF studies included simultaneous detection of hCK, hCD3, hCD8 and hCD20 as recently described (Schalper et al., 2015, JNCI). Briefly, fresh tumor cuts were deparaffinized and subjected to antigen retrieval using EDTA buffer (Sigma-Aldrich, St Louis, MO) pH=8.0 and boiled for 20 min at

97°C in a pressure-boiling container (PT module, Lab Vision). Slides were then incubated with dual endogenous peroxidase block (DAKO #S2003, Carpinteria, CA) for 10 min at room temperature and subsequently with a blocking solution containing 0.3% bovine serum albumin in 0.05% Tween for 30 minutes. Staining for targets was performed using a sequential multiplexed immunofluorescence protocol with isotype-specific primary antibodies to detect epithelial tumor cells (cytokeratin, clone

M3515, DAKO), T lymphocytes (CD3 IgG, clone E272, Novus biologicals, CO), cytotoxic T cells (CD8

IgG1, clone C8/144B, DAKO) and B-lymphocytes (CD20 IgG2a, clone L26, DAKO). Nuclei were highlighted using DAPI. Secondary antibodies and fluorescent reagents used were goat anti-rabbit

Alexa546 (Invitrogen), anti-rabbit Envision (K4009, DAKO) with biotynilated tyramide/Streptavidine-

Alexa750 conjugate (Perkin-Elmer); anti-mouse IgG1 antibody (eBioscience, CA) with fluorescein- tyramide (Perkin-Elmer), anti-mouse IgG2a antibody (Abcam, MA) with Cy5-tyramide (Perkin-Elmer).

Residual horseradish peroxidase activity between incubations with secondary antibodies was eliminated by exposing the slides to a solution containing benzoic hydrazide (0.136 mg) and hydrogen peroxide (50 µl). Objective signal quantification, definition of tumor/stroma compartments and phenotyping of cells in the stained preparations was performed using multispectral fluorescence analysis and spectral unmixing with the Mantra Quantitative Pathology

Workstation and the InForm software (PerkinElmer, Hopkinton, MA).

Antibodies and flow cytometry Lung, liver and tumor tissue was processed for flow cytometry analysis. To obtain unicellular cell suspensions, tissues were incubated in collagenase and DNase (Roche) for 15 min at 37°C, mechanically disrupted and passed through a 70-μm cell strainer (BD Falcon, BD Bioscience) by pressing with a plunger. Cells dissociated from tissues were centrifuged with Percoll © (GE

Healthcare) at 35% (500 g, 10 min, 20°C) and a gradient made in order to eliminate parenchymal cells. Erythrocytes were lysed with ACK buffer (Gibco). Single-cell suspension was treated with FcR-

Block and beriglobine in a PBS-based buffer containing 10% of fetal calf serum to avoid unspecific staining. Fluorochrome-conjugated mAbs to the following human antigens were used: CD3

(UCHT1), CD4 (OKT4), CD8 (RPAT8), CD45 (HI30), FoxP3 (150D), Ki-67 (B56), CD279/PD-1

(EH12.2H7), TIM-3 (F38-2E2) and PD-L1/B7-H1 (29E.2A3) from Biolegend, CD25 (BC96), Eomes

(WD1928) and LAG-3 (3DS223H) from eBioscience, CD137 (5D1) (35), Perforin (deltaG9) from BD

Pharmigen. Cytofix/Cytoperm (BD Biosciences) was used for perforin intracellular staining and anti- mouse/rat FoxP3 Staining Set for Eomes, Ki-67 and FoxP3 intranuclear staining (Biolegend, London,

Blood cells were centrifuged and the supernatant was discarded. Erytrocytes were lysed with RBC

Lysis buffer 1X (10 minutes, 37°C) from eBioscience. Thereafter, cells were stained as above.

FACS-Canto II and FACSCalibur (BD-Biosciences) were used for cell acquisition and data analysis was carried out using FlowJo software (Tree Star Inc).