Dynabeads untouched mouse cd8 t cell negative isolation kit

ELISPOTS were performed 14–17 days post-vaccination. Splenic CD8⁺ T cells were isolated from individual mice using the Dynabeads Untouched Mouse CD8⁺ T Cell Negative Isolation Kit (Life Technologies). RNEU_420–429 (PDSLRDLSVF) and NP_118–126 (RPQASGVYM) peptides were synthesized at 95% purity by the Oncology Peptide Synthesis Facility (Johns Hopkins, Baltimore, MD). 10⁵ CD8⁺ T cells were incubated in triplicate with 10⁴ peptide-loaded T2D^q, NT2.5B7.1, or 3T3neuB7.1 target cells. NT2.5B7.1 and 3T3neuB7.1 cells were stimulated with IFNγ for 2 days prior to co-culture. T cell/T2D^q cells were co-cultured for 16 h and T cell/NT2.5B7.1 or T cell/3T3neuB7.1 cells were co-cultured for 24 h on pre-coated IFN-γ ELISPOT Multiscreen-HA plates (Millipore) according to the manufacturer's protocols (Ebioscience). ELISPOT plates were developed using an AEC staining kit (Sigma) according to the manufacturer's instructions. IFNγ-secreting CD8⁺ T cells were enumerated using the Immunospot counter (Cellular Technology, Ltd.). The average number of spots in control wells was subtracted from the average number of spots in each well containing both CD8⁺ T cells and targets.

Adoptive transfer of CD8⁺ T cells was carried out as described previously [20 (link)]. Briefly, FVB/N mice received subcutaneous injections of 5 × 10⁶ NT2.5 cells into the right upper mammary fat pad. One week post-tumor challenge, mice were vaccinated with 3 × 10⁶ irradiated 3T3neuGM cells or 3T3GM cells. Sorafenib (30 mg/kg) or vehicle was given by daily gavage starting on the day of vaccination. CD8⁺ T cells were isolated from Clone 100 TCR transgenic mice by CD8 negative selection using Dynabeads Untouched Mouse CD8⁺ T cell negative isolation kit (Life Technologies). 5 × 10⁶ CD8⁺ T cells per mouse were adoptively transferred via tail vein injection one day following the initiation of treatment administration. Spleens, lymph nodes, and tumors from adoptively transferred mice were harvested five days after adoptive transfer. Spleens and lymph nodes were collected and mashed through a 70 μM cell strainer. Tumors were minced and digested with Liberase TM (Roche) for 15 min and mashed through 70 μM cell strainers. Isolated single cell suspensions were analyzed for Thy1.2⁺ CD8⁺ cells on a Gallios (BD Coulter) or LSRII Flow Cytometer (BD Biosciences). Data were analyzed with FlowJo software (Treestar, Inc.).