Pd 1 clone eh12

Tonsils were mechanically disrupted by first cutting the tissue samples with a scalpel into small pieces, and then dissociating into single cells using gentleMACS technology (C tube and gentleMACS; Miltenyi Biotec). Alternatively, cells were dissociated by forcing through a 40-µm cell strainer. Single-cell suspension was then enriched for light density cells by a Ficoll gradient centrifugation (Lymphoprep). Tonsil CD4⁺ T cells were enriched using CD4⁺ T cell isolation kit (Miltenyi Biotec) and used for intracellular flow cytometry or further purified by cell sorting. Naive CD4⁺ T cells, GC Tfh cells, and extra-follicular Tfh cells were sorted using antibodies against CD4 (clone RPA-T4; BioLegend), CD19 (clone HIB19; eBioscience), CD45RO (clone UCHL1; BD Biosciences), CXCR5 (clone RF8B2 from BD Biosciences or clone J252D4 from BioLegend), and PD-1 (clone EH12.1 or clone EH12.2H7 from BD Biosciences) on a FACS Aria instrument (BD Biosciences).

Fresh tumor tissues were obtained from 20 patients undergoing surgical resection of glioma in accordance with the institutional review board approval at Seoul National University Hospital. Histological diagnosis consisted of 10 glioblastomas, 4 anaplastic oligodendrogliomas, 3 anaplastic astrocytomas, 1 diffuse midline glioma, 1 anaplastic ganglioglioma, and 1 oligodendroglioma. Tumor specimens were collected in RPMI media at room temperature immediately after surgical resection. MACS brain tumor dissociation kit and gentleMACS™ Dissociators (Miltenyi Biotec) were used to dissociate tissue samples within 2 hours following collection, and debris were removed using MACS Debris Removal Solution (Miltenyi Biotec) according to the manufacturer protocol.
Immune cells were stained for surface markers using fluorescence-conjugated monoclonal antibodies including CD45 (clone HI30, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone SK1, BD Biosciences), PD1 (clone EH12.1, BD Biosciences), and TIGIT (clone 741182, R&D Systems). Flow cytometry was performed using FACS LSR Fortessa™ (BD Biosciences), and data was analyzed using FlowJo software (Treestar).