B7 h3 antibody clone 7 517

We performed flow evaluations using antibodies specific to human CD3, CD4, CD8, CD19, CD27, CD45, CD45RA, CD62L, CCR7, B7-H3, PD-1, PD-L1, and TIM3 and murine CD3, CD4, CD8, CD11b, CD11c, PD-1, and Ly-6G (from BD Biosciences and BioLegend) conjugated with BV421, BV510, BV605, BV711, fluorescein isothiocyanate (FITC), AF488, peridinin chlorophyll protein (PerCP)–cy5.5, phycoerythrin (PE), PE-cy7, allophycocyanin (APC), and APC-cy7 fluorochromes. Expression of human B7-H3 by tumor cell lines was assessed with the 376.96 monoclonal antibody and confirmed with the commercial B7-H3 antibody clone 7-517 (BD Biosciences) (16 (link)). Expression of B7-H3.CAR was evaluated using the fusion protein 2Ig-B7-H3-GFP. Samples were acquired with BD FACSCanto II or BD LSRFortessa using the BD FACSDiva software (BD Biosciences). Quantification and characterization of T cells within the tumor were obtained by generating single-cell suspension of the tissues using the Human Tumor Dissociation Kit (Miltenyi Biotec) as per the manufacturer’s instructions. For each sample, a minimum of 10,000 events was acquired, and data were analyzed using FlowJo version 10.

A FACSCanto II (BD Biosciences) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10 (BD Biosciences). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare). For all experiments, matched isotypes or known negatives (e.g., NT T cells) served as gating controls. CAR detection was performed using F(ab′)₂ fragment=specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA, USA). T cells were stained with fluorochrome-conjugated antibodies using combinations of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone G043H7, BioLegend, San Diego, CA, USA), CD45RO (clone UCHL1, BD Biosciences), and 41BBL (clone 5F4, BioLegend). Tumor cell lines were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7-517, BD Biosciences, or clone FM276, Miltenyi). To determine apoptosis, T cells were labeled with annexin V (BD Biosciences) and DAPI (BD Biosciences). The percentages of apoptotic cells were determined by the percent annexin V positive.