Cd3 clone hit3a

The phenotype of PBMCs cell subsets was evaluated by multiparameter flow cytometry. Cells were incubated in the dark for 20 min RT with the following panel of anti-human fluorescent labelled antibodies: CD3 (clone HIT3a, APC conjugated, BioLegend), CD4 (clone SK3, APC conjugated, eBiosciences), CD8 (clone SK1, APC conjugated, BD Biosciences), CD14 (clone 61D3, FITC conjugated, eBiosciences), CD25 (clone BC96, Alexa Fluor 488 conjugated, eBiosciences), CD69 (clone FN50, PE conjugated, eBiosciences), CD45 (clone L48, PerCyP conjugated, BD Biosciences), Foxp3 (clone PCH101, PE conjugatated, eBiosciences), IL-1β (clone CRM56, FITC conjugated, eBiosciences), IL-6 (clone MQ2-13A5, FITC conjugated, eBiosciences), IL-10 (clone JES3-9D7, Alexa Fluor 488 conjugated, eBiosciences), HLA-DR (clone L243, APC.Cy7 conjugated, BD Biosciences), CD33 (clone P67.6, PE conjugated, BD Biosciences), CD11b (clone D12, APC conjugated, BD Biosciences), CD14 (clone MɸP9, Brilliant Violet 421 conjugated, BD Biosciences) and Arginase-1 (ARG-1, FITC-conjugated, R&D Systems). Isotype controls were used for each experiment. After incubation, cells were again washed, resuspended in flow buffer and analyzed using FACSCalibur and FACSCanto II cytometers (BD, Biosciences). At least 5 × 10⁴ events were collected, and the data was analyzed using Summit software (Summit Group Software).

Cell surface expression of L1CAM (clone REA163, Miltenyi Biotec), GD2 (clone 14.G2a; BD), CD3 (clone Hit3a, BioLegend, San Diego, CA, USA) and CD8 (clone SK1; BioLegend) was detected by fluorophore-conjugated monoclonal antibodies on a Fortessa X-20 (BD Biosciences, Franklin Lakes, NJ, USA) 4-laser flow cytometer. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb, New York, NY, USA) and a phycoerythrin (PE)-conjugated streptavidin antibody (cat #12-4317-87, BioLegend). Activation was assessed by fluorophore-conjugated monoclonal antibodies detecting TNFRSF9 (formerly CD137, clone 4B4-1; BioLegend) and IL2RA (formerly CD25, clone BC96; BioLegend). The Annexin V/7-AAD detection kit (BioLegend) was used to assess apoptosis. Dead cells were excluded from analyses using the LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (cat#L23101, Life Technologies). Precision count beads (BioLegend) were used to quantify T cell infiltration according to the manufacturer’s instructions. Flow cytometry data was processed using FlowJo_V10 Software (Tree Star Inc., Ashland, OR, USA).

Frozen tumors were cut with a Leica CM1850 UV cryostat (Leica) into 10 µm thickness serial sections and subsequently collected onto SuperFrost Plus glass slides (Avantor). Prior to staining, tumor sections were fixed with 4% PFA for 10 minutes and blocked with PBS containing 0.5% BSA for 30 minutes to reduce non-specific staining. Sections were then stained for human cell populations with the following monoclonal antibodies: CD66b-BB515 (clone G10F5; BD Biosciences), purified CD3 (clone HIT3a; BioLegend) followed by secondary PE-conjugated anti-mouse antibody (Invitrogen), and Biotin-labeled CD19 (clone HIB9; eBioscience) followed by APC-conjugated streptavidin (Invitrogen). Hoechst 33342 Solution (Thermo Fisher Scientific) was used for nuclear staining. Incubations were performed in the dark at room temperature for 45 minutes, using Tris-Buffered Saline with 0.1% Tween-20 detergent for washes between incubation steps. Sections were subsequently mounted with 10% Mowiol supplemented with 2.5% DABCO and analyzed with the Nikon Ti2e microscope (Leica Microsystems). A Tilescan of the entire tumor was taken with Kinetix sCMOS camera (objective 10x; Photometrics). Files were first Denoised using the Algorithm provided by Nikon and subsequently processed with a rolling ball filter (14.86 µm). Crops were taken from the Tilescans.