Anti cd8a

Female C57BL/6j mice (6–8 weeks old) were anesthetized with a mixture of medical oxygen and isoflurane (induction: 5%; maintenance: 2%) and intracranially injected with 2 × 10⁴ viable GL261 glioma cells. The injection was performed using a stereotactic device 2 mm lateral and 2 mm posterior to the bregma and 3 mm below the dura mater according to a previously described protocol [14 (link)]. The skin was sutured, and meloxicam was administered subcutaneously (1 mg/kg, 2 mg/mL) to manage post-operative pain. To prepare the DC vaccine, cell death was induced in GL261 cells in vitro by PS-PDT or MTX as described above. The mice were injected intraperitoneally with a suspension containing 1 × 10⁶ of the prepared DCs (as described above) on days 2, 6, 10, and 17 after intracranial injection of viable GL261 cells. Local (inguinal and axillary) draining lymph nodes were collected on day 37 after intracranial injection with GL261 cells. The immune cells in the draining lymph nodes were stained by anti-CD8a (eBioscience, 12-0081-81), anti-CD45 (Biolegend, 103125), mouse Fc block (eBioscience, 16-0161-85), anti-CD11b (Invitrogen, 12-0112-83) and anti-CD11c (BD Pharmingen, 561022) and analyzed on a BD FACS Canto II flow cytometer.

Blood was collected by retro-orbital bleeding using heparinized capillaries, and erythrocytes were removed using ammonium chloride and washed twice. Subsequently, cells were stained with fluorescently labeled anti-CD8a, anti-CD4, anti-Thy1.2, anti-CD11a, and anti-CD49d antibodies (EBioscience, San Diego, CA) for 20 min. Flow cytometry was performed using a BD Fortezza cell analyzer.

Thymocytes and splenocytes from wild type and DJ-1-deficient mice were characterised by using surface and intracellular staining with relevant antibodies. In brief, thymocytes and splenocytes were collected and used for surface staining for anti-CD4, anti-CD8a, anti-CD25, anti-CD62L, anti-CD44 (eBioscience, Germany) or other antibodies depending on the experiment and washed with PBS. Cells were fixed with Foxp3 fixation/permeabilization buffer (eBioscience, Germany) for intracellular staining and incubated for 30 minutes. After incubation, cells were washed with 1x permeabilization buffer, exposed to added intracellular monoclonal antibodies for Foxp3 and incubated for additional 30 minutes. Cells were washed again with permeabilization buffer and PBS was added to acquire the cells on a flow cytometer (FACS-calibur from Becton Dickinson; Heidelberg, Germany).

For T cell activation, total PBMC samples from healthy donors (ALLCELLS, Alameda, CA) were enriched for CD3+ T cell populations using CD3 MicroBeads (Miltenyi Biotec, Auburn, CA, cat # 130-050-101) according to manufacturer’s instructions. Cells were then maintained in OpTmizer CTS T Cell expansion SFM plus T cell expansion supplement (Gibco, Grand Island, NY, cat # A1048501) complete with 5% CTS Immune Cell SR (Gibco, cat # A2596101), 2 mM Glutamine (Gibco, cat # 35050-061) and P/S solution. T cells were activated using Dynabeads Human T-Activator CD3/CD28 beads (Gibco, cat # 11131D). Surface FACS using anti-CD69 (Biolegend, London, UK, cat # 310904) staining was used to detect and confirm the activated state of T cells. TGFβ neutralizing antibody was added to cells within 24 h, and after 72 h incubation, cells were washed and stained with anti-CD4 (BD Biosciences, cat # 560768) and anti-CD8a (eBioscience, Grand Island, NY, cat # 56-0086-82) antibodies and subsequently analyzed with flow cytometry.