Mice
Athymic nude mice were all purchased from The Jackson Laboratory. All mice were maintained in pathogen-free facilities in the UAB Brain Tumor Animal Models (BTAM) Facility. This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the ARRIVE guidelines. The protocol was approved by the Animal Care and Use Committee at the University of Alabama at Birmingham (Birmingham, AL). (APN20339). All surgery was performed under ketamine/xylazine anesthesia, and all efforts were made to minimize pain.
Patients and apheresis donors
Patient-derived tumor xenografts (PDX) were a gift of David James to the UAB Brain Tumor Tissue Facility (BTTF) headed by G. Yancey Gillespie, PhD and included the primary classical lines JX12 and JX14, their TMZ-resistant pairs JX12T and JX14T, and the primary mesenchymal subtype pair JX22P and JX59 and TMZ-resistant JX22T and JX59T. Expanded and activated γδ T cells were manufactured from deidentified apheresis products obtained from healthy volunteers via Hemacare, Inc., a commercial blood products vendor and as such met the exemption criteria for the human subjects review committee at the University of Alabama at Birmingham.
Intracranial injections
Intracranial gliomas were generated using 5 × 105 human glioma xenolines suspended in 5% methylcellulose in serum-free medium using a previously reported method18. The cells were drawn into a 250 μl Hamilton gas-tight syringe mounted in a Chaney repeating dispenser and fitted with a 30G ½-inch needle with a calibrated depth of 2.5 mm from the middle of the bevel opening. Under an operating microscope, the fascia on the skull of the anesthetized mouse were scraped off and a 0.5 mm burr hole made 2 mm to the right of the midline suture and 1 mm caudal to the coronal suture. The syringe was inserted into a Kopf stereotactic electrode clamp mounting bracket attached to an electrode manipulator (David Kopf Instruments; Tujinga, CA) mounted on a Kopf stereotactic frame electrode A-P zeroing bar (#1450). Each mouse was positioned on the stereotactic frame and the needle inserted to the depth marker into the right cerebral hemisphere. Approximately 90–120 s after injection of 5 ml, the needle was slowly withdrawn over the next minute. The burr hole was plugged with sterile bone wax and skin is closed with Tissu-Mend surgical adhesive (Stryker Orthopedics; Kalamazoo, MI). Tumor engraftment was confirmed by assessment of luminescence. Control mice received tumor only, tumor + DRI cells only, or tumor + TMZ only. The major endpoint in this study was animal survival; moribund animals that became unresponsive to mild external stimuli were euthanized and this date was used as an estimate of the date of death.
Treatment protocol
Tumor-bearing mice (n = 10/group) were treated twice weekly with intraperitoneal (IP) 60 mg/kg TMZ and intracranial (IC) of 1 × 106 MGMT-modified γδ T cells within 4 h of the TMZ injections. Mice receiving cell therapy received stereotactic intracranial injections of γδ T cells approximating the original tumor placement at a dose of 1 × 106 per injection. Chemotherapy-treated mice received 60 mg/kg Temozolomide intraperitoneally (IP). Tumor-bearing control mice were divided into three groups in which the first received IC MGMT-modified γδ T cells on days + 6, + 8, + 13, and + 15, the second received IP TMZ on days + 6, + 8, + 13, and + 15, and the third received no anti-tumor therapy. Survival was assessed using Kaplan–Meier analysis.
Histopathology and immunohistochemistry
Sections of mouse brain with tumor were prepared and fixed in neutral buffered formalin in the Brain Tumor Tissue Facility of the UAB Comprehensive Cancer Center. There formalin-fixed paraffin-embedded (FFPE) sections were sectioned and stained with hematoxylin and eosin and examined for size, infiltration, and histologic grade. The level of expression of checkpoint and stress-induced markers in whole mouse brain and tumor were assessed by immunohistochemistry. As previously described25, deparaffinized sections were post fixed in 4% neutral buffered formalin followed by antigen retrieval with Citra Plus (Biogenex Laboratories, Freemont CA). Sections were blocked sequentially with avidin, biotin (Biogenex Laboratories, Freemont CA) and FC receptor blocker (Innovex Biosciences, Richmond CA) for 20 min at RT. Primary antibody to PD-L1 was applied at 5 μg/ml overnight at 4 °C. Multilink secondary antibody (Biogenex Laboratories, Freemont CA) was applied for 30 min at RT, followed by Streptavidin-labeled peroxidase (Biogenex laboratories, Fremont CA) for 30 min. The immunostaining was developed with Turbo DAB chromogen (Innovex Biosciences, Richmond CA) for 2 min or until signal appeared.
Culture and activation of γδ T cells
Preparation and testing expanded/activated γδ T cells from healthy volunteers was performed using a modified version of a previously described method13. Up to 50 mL of peripheral apheresis products was obtained from healthy volunteers following informed consent and purchased from Hemacare (Van Nuys, CA). Mononuclear cells (MNC) were isolated using Ficoll and resuspended at a concentration of 1 × 106 cells/mL in a 50/50 mixture of RPMI-1640 (Life Technologies; Carlsbad, CA) and Clicks EHAA (Irvine Scientific; Santa Ana, CA) media supplemented with 15% pooled human AB serum, 5 mM Zoledronic Acid (Zoledronate; Novartis; Basel, Switzerland), and 50U/mL human rIL-12 (Miltenyi Biotech; Auburn, CA). Cultures were transduced with a P140k-MGMT expressing lentivector (Miltenyi Lentigen, Gaithersburg, MD) at a multiplicity of infection (MOI) of 10 at days 5 and 6 ± 1 and maintained for 14 ± 2 days with the addition of fresh complete media as needed to maintain a total cell concentration of 1 × 106/mL. Vector copy number (VCN) was determined by QRT-PCR.
Flow cytometry
Immunophenotyping of cultures was performed on a FACS Canto II flow cytometer or LSR Fortessa SE flow cytometer (BD Biosciences; San Jose, CA) using BD Lymphocyte Immunophenotyping System and Beckman-Coulter (Miami, FL) Duraclone tubes for TCR analysis and determination of effector/memory status. List mode data were analyzed using DiVa and Cell Quest Pro software (BD Biosciences; San Jose, CA). Characterization and enumeration of lymphocytes pre- and post- expansion culture and the final infused cell product was performed using single-platform flow cytometry via surface labeling using directly conjugated monoclonal antibodies (mAbs) against CD45 (2D1), CD3 (SK7), CD4 (SK3), CD8 (SK1), CD16/56 (B73.1/NCAM 16.2), CD19 (SJ25C1), CD27 (M-T271), CD45RA (L48), TCR-γδ (11F2), Vδ1 (TS 8.2), and Vδ2 (B6). Expression of known stress-associated molecules was measured in glioma xenolines by flow cytometry after staining with antibodies that bind cell stress-induced markers (NKG2D ligands).
Cytotoxicity assays
Cytotoxicity of expanded γδ T cells to xenolines was determined in vitro by flow cytometry using a commercial kit (Immunochemistry Technologies; Bloomington, MN) incorporating dye that labels target cells (CSFE) and a dye that labels dead cells (7-AAD). The standardized K562 erythroleukemia cell line was included in the assay as positive cytotoxicity control. Briefly, target cells were labeled with CSFE according to manufacturer’s protocol. Expanded/activated γδ T cells were then added to the tubes at ratios of 0:1 (Background), 5:1, 10:1, 20:1 effectors/targets, incubated for four hours at 37 °C and 5% CO2, washed once and resuspended in 1 ml HBSS. 7-AAD solution (20ul) was added prior to acquisition on the flow cytometer.
Percent specific release was calculated as follows:
$$% {text{ Specific }};{text{Release}} = frac{{{text{fluorescence}} _{{({text{experiment}})}} – {text{ fluorescence }}_{{({text{spontaneous }};{text{release}})}} }}{{{text{fluorescence}} _{{({text{maximum}})}} – {text{ fluorescence}} _{{({text{spontaneous}};{text{ release}})}} }} times 100$$
