Animals, cell lines and reagents

6- to 10-week-old female CB17-SCID, NOD SCID and BALB/c mice were purchased from Charles River Laboratories (Saint-Germain-Nuelles, France). Animal experiments were approved by the animal research committee of Geneva canton and experiments performed in accordance with the Swiss Federal Veterinary Office guidelines. CL-4 transgenic BALB/c mice with Hemagglutinin(HA)-specific TCR expressed by CD8+ T cells [25] were kindly provided by Dr. Roland Liblau (Research Center Toulouse Purpan, CPTP-INSERM).

The Burkitt’s lymphoma Raji cell line (CCL-86) was purchased from ATCC and cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS, Invitrogen) and 2 mM l-glutamine (Sigma-Aldrich). The Raji GFPhi cell line was generated by transfecting Raji cells with the randomly inserted GFP transgene (UniprotKB-C5MKY7) to allow the analysis of tumor cell uptake by phagocytes. The Raji HA-GFP cell line was developed by electroporating hemagglutinin (HA) gene from influenza virus strain A/Puerto Rico/8/1934 H1N1+ (UniProtKB-P03452) and GFP sequence integrated in an IRES-containing bicistronic vector. The transgene was inserted by targeting specifically the human Rosa26 locus using the CRISPR-Cas9 system. Stably expressing pool was enriched by successive flow cytometry cell sorting of GFP positive cells (Beckman Coulter MoFlo Astrios) and clones were generated by single cell sorting.

The generation and characterization of NI-1701, a fully human IgG1 anti-CD47xCD19 bsAb with unbalanced affinity towards CD19 (KD = 0.6 nM) and CD47 (KD = 500 nM), was previously described [24]. Human IgG1 isotype-matched control mAb was produced and purified at Light Chain Bioscience/Novimmune from Chinese Hamster Ovary (CHO) cell culture supernatants.

Raji Burkitt lymphoma xenograft model

NOD SCID mice were injected subcutaneously at the flank with 5 × 106 Raji GFPhi cells. Tumors were measured three times a week using a digital caliper and tumor volume determined using the formula (width × length × height) × π/6. NI-1701 or human IgG1 isotype-matched control mAb were administered by intravenous (i.v.) (lateral tail vein) or intraperitoneal (i.p.) injection on a weekly basis, at a dose of 20 mg/kg, when tumor volume reached about 100 mm3.

Tumor and spleen dissociation protocol

Mice were euthanized by CO2 inhalation and tumors or spleens directly excised and preserved in RPMI. Tumors were cut into small pieces using surgical scissors and further enzymatically and mechanically dissociated using the Gentle MACS dissociator (Miltenyi Biotec), following the recommendations provided by the human Tumor Dissociation Kit (Miltenyi Biotec). Spleens were incubated in collagenase IV (Gibco) and DNase I (Sigma-Aldrich) and mechanically dissociated using the Gentle MACS dissociator. After a washing step, red blood cells were lysed using ACK (Ammonium–Chloride–Potassium) buffer and cellular suspensions were filtered through a 70 µm cell strainer, washed and suspended in FACS buffer (PBS, BSA 1%, EDTA 2 mM) to obtain a homogeneous single-cell suspension for flow cytometry analysis.

Flow cytometry, imaging flow cytometry and cell sorting

The following anti-mouse Abs or viability dyes were used for staining: anti-Ly6C (HK1.4), anti-CD11c (N418) and anti-CD206 (C068C2) purchased from Biolegend; anti-CD45 (30F11), anti-Ly6G (1A8), anti-CD11b (M1/70), anti-CD8a (53-6.7), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-MHC Class I H-2Kd (SF1-1.1), anti-MHC Class II I-A/I-E (M5/114.15.2) and Fixable Viability Stain 620 purchased from BD Biosciences; anti-F4/80 (BM8), anti-NKp46 (29A1.4), anti-MHC II I-Ad (AMS-32.1), anti-SIRPα (P84) and LIVE/DEAD Fixable Violet Dead Cell Stain purchased from Thermo Fischer Scientific.

Cellular suspensions were first stained with viability dye (to exclude dead cells) for 20 min at room temperature following the manufacturer’s protocol. After 2 washes in FACS buffer, cells were incubated with purified rat anti-mouse CD16/32 Ab (Mouse BD Fc Block) for 5 min at 4 °C to block non-specific labeling. Then samples were directly processed for antibody staining for 20 min at 4 °C, washed 2 times and fixed in BD CellFIX (BD Biosciences). Data were acquired using a CytoFLEX S flow cytometer (Beckman Coulter, Indianapolis, IN, USA) and analysed with FlowJo software (FlowJo, LLC).

In vivo phagocytosis was determined by flow cytometry after tumor dissociation by detection of GFP in F4/80+ tumor-associated macrophage or F4/80CD11c+MHCII+ dendritic cell (DC) populations and validated using FlowSight imaging flow cytometer (Luminex Corp.) (see more details in Additional file 2).

For isolation of tumor-infiltrating mouse leukocytes, single-cell suspensions obtained from excised tumors were submitted to a double-step sorting. First, mouse CD45+ cells were enriched using CD45 (TIL) MicroBeads (Miltenyi biotec) following the manufacturer’s protocol. Then, after a 5 min staining with TOPRO-3 reagent to exclude dead cells (TOPRO-3+), the viable tumor-infiltrating mouse leukocytes (CD45+, TOPRO-3) were sorted using a S3e cell sorter (Bio-Rad).

HA-specific TCR CD8+ T cells were isolated from spleens of CL-4 transgenic mice. Briefly, spleens were dissociated and digested as described above and the single cell suspension was processed using the EasySep™ Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies) following the manufacturer’s instructions.

Nanostring nCounter targeted transcriptomic analysis of tumor-infiltrating mouse leukocytes

Total RNA was isolated from purified tumor-infiltrating mouse leukocytes using ReliaPrep™ RNA Cell Miniprep System (Promega) followed by DNAse treatment with DNA-free DNA Removal kit (Thermo Fischer Scientific). Purified total RNA was quantified by Qubit (Thermo Fischer Scientific) and checked for quality by the Bioanalyzer RNA 6000 Nano assay (Agilent Technologies). Gene expression was quantified with the NanoString nCounter platform using 100 ng of total RNA in the nCounter® Mouse Myeloid Innate Immunity panel v2 (NanoString Technologies). Briefly, the code set was hybridized with the purified RNA overnight at 65 °C. RNA transcripts were immobilized and counted using the NanoString nCounter Digital Analyzer. Normalized raw expression data were analysed when two SDs above the geometric mean of the code-set-internal negative control probes were reached. The 557 remaining genes after background filtering were normalized to the geometric mean of 18 housekeeping genes included in the panel and were log2-transformed for further analysis.

Macrophage and NK cell depletion in Raji Burkitt lymphoma xenograft model

For macrophage depletion experiment, CB17-SCID mice were injected s.c. into the flank with 5 × 106 Raji cells and concomitantly treated with a loading dose (i.v., lateral tail vein) of 100 μL of clodronate or control (PBS) liposomes (Liposoma B.V., The Netherlands), followed by maintenance doses of 50 μL, every 3 or 4 other days, until the end of the experiment.

For NK cell depletion, NOD SCID mice were injected s.c. into the flank with 5 × 106 Raji GFPhi cells and one week later treatments were initiated with 50 µg of anti-asialo GM1 Ab (Thermo Fischer Scientific) or PBS, with a weekly injection, until the end of the experiment.

Antibody dependent cellular phagocytosis assay with bone-marrow derived dendritic cells (BMDCs)

Single-cell suspensions of bone marrow cells were obtained from femur and tibia of BALB/c mice. Red blood cells were lysed using ACK buffer and then cell suspension was filtered with a 70 µm cell strainer. 10 million of bone-marrow cells were plated in 10-cm Petri dishes (day 0) and cultured in RPMI 1640 medium containing 10% heat-inactivated FCS, 2 mM l-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 50 μM 2-mercaptoethanol and 25 μg/mL gentamicin (Sigma-Aldrich) supplemented with 20 ng/mL of GM-CSF (Peprotech). At day 4 or 5, non-adherent cells were collected and pooled with adherent cell fraction detached with Trypsin–EDTA. Cells were resuspended in fresh medium with 20 ng/mL of GM-CSF and 10 million of cells were plated per Petri dishes. At day 10, BMDCs (non-adherent cell fraction) were harvested (whereas adherent cells were discarded) and characterized by flow cytometry.

Phagocytosis experiments were performed in ultra-low attachment plates (Corning/Sigma-Aldrich) by mixing Raji GFPhi cells with BMDCs (effector to target cells ratio of 1:1) in presence of 10 μg/mL of NI-1701 or hIgG1 control Ab. After 2.5 or 24 h of incubation at 37 °C, cells were harvested, suspended and stained with viability marker and anti-CD11c antibody and analysed by flow cytometry. CD11c+GFP+ double-positive events were identified as phagocytosis events, as confirmed by imaging flow cytometry (FlowSight, Luminex Corp.).

Antigen cross-presentation assay

First, an overnight phagocytosis step was performed by co-culturing 5 × 104 BMDCs with Raji HA-GFP cells (E:T ratio 1:1) in presence of 10 μg/mL of NI-1701 or hIgG1 control Ab. Then, purified HA-specific CD8+ T cells were stained with CellTrace Violet (Thermo Fisher Scientific) and 2.5 × 105 lymphocytes (5:1 ratio with BMDCs) were co-incubated for 48 h at 37 °C with the pre-mixed BMDCs and Raji HA-GFP tumor cells after the phagocytosis step. Cell suspensions were subsequently stained with a viability marker, anti-CD11c and anti-CD8a and T-cell proliferation assessed by flow cytometry.

Statistical analysis

GraphPad Prism was used for all statistical analysis. The unpaired t test or One-way ANOVA test were used for statistical comparison. Data were expressed as mean ± SEM or mean ± SD, as indicated. p-value of < 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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