Fc receptor binding inhibitor antibody

Collected cells were washed and resuspended in staining buffer (PBS 1X +1% BSA) with Fc Receptor Binding Inhibitor Antibody (Invitrogen, # 14-9161-73, Carlsbad, CA, USA) for 15 min at 4 °C. After blocking, samples were incubated in the dark for 1 h at 4 °C with a saturating concentration of anti-human primary mouse monoclonal antibodies. After two washes in PBS 1X +1% BSA, cells were incubated with secondary antibodies: Alexa Fluor 488^® F(ab’)2 fragment of goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) and/or Alexa Fluor 594^® F(ab’)2 fragment of goat anti-mouse IgG (H + L) (Invitrogen). The samples were then washed with PBS 1X + 1% BSA and resuspended in 200 μL of the same buffer. The macrophages were electronically gated according to light scatter properties to exclude cell debris and contaminating lymphocytes. Fluorescence was measured using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OH, USA).

T-ALL cell lines, HEK293T EV, HEK293T/CD1a, and primary T-ALL cells were labeled with Far-Red (Thermo Fisher Scientific) viable marker, according to manufacturer instructions. Labeled cells were co-cultured with PBMCs at different E:T ratios, in the presence of increasing concentrations of CD1a x CD3ε or vehicle for 24–48 h at 37 °C and 5% CO₂, and then stained with 7-AAD (BD Biosciences). Cytotoxicity was detected by flow cytometry (Attune NxT Flow cytometer, Thermo Fisher Scientific). To identify dead T-ALL cells, the following gating strategy was used: 7-AAD−/Far Red+ cells were considered as alive T-ALL cells while 7-AAD+/Far Red+ were considered dead T-ALL cells. Moreover, 7-AAD−/Far Red− were considered as alive PBMC while 7-AAD+/Far Red− were considered dead PBMC. In the cytotoxicity experiment with T cell depletion, immunomagnetic cell sorting using CD4, CD8, or CD56 microbeads (MACS Miltenyi Biotec, Bergisch Gladbach, Germany) was performed. In the cytotoxicity experiment with Fc blocking, Fc receptor binding inhibitor antibody (Invitrogen, Waltham, MA, USA) was added to cell co-culture according to manufacturer instructions.

Healthy donors derived PBMCs were labelled with Cell-Trace Violet (Thermo Fisher Scientific) viable marker, according to manufacturer instructions. Labeled PBMCs were co-cultured with EOC cell line at different E:T ratio, in the presence of increasing concentrations of pAXLxCD3ε or vehicle for 48–72 h at 37 °C and 5% CO₂, and then stained with 7-AAD (BD Biosciences). Cytotoxicity was detected by flow cytometry (Attune NxT Flow cytometer, Thermo Fisher Scientific) as 7-AAD⁺/ Cell Trace Violet⁻ cells (%). In the cytotoxicity experiment with T cell depletion, immunomagnetic cell sorting using CD4, CD8 microbeads (MACS Miltenyi Biotec) was performed. In the cytotoxicity experiment with Fc blocking, Fc receptor binding inhibitor antibody (Invitrogen) was added to cell co-culture according to manufacturer instructions.
For 3D re-directed T cell cytotoxicity assay, after EOC spheroids formation 1 × 10⁶ Cell Trace Violet PBMCs were added to each well. Cells were treated with increasing concentration of pAXLxCD3ε or vehicle. After 72 h, spheroids were monitored by microscope scoring. After treatment spheroids were pooled and dissociated with trypsin–EDTA (0.05%). Cells were labelled with 7AAD viable marker for 15 min at room temperature. Finally, cells were analyzed by flow cytometer.