Murine il 2

Blood was donated at Massachusetts General Hospital blood, as approved by the institutional review boards of the Lemuel Shattuck Hospital and Partners Healthcare. Biotinylated CD1b monomers were obtained from the NIH tetramer core facility. The polyclonal T cell line named C58L and T cells clones named 11, 20, and 28 were derived after stimulation of peripheral blood mononuclear cells with autologous monocyte-derived dendritic cells and C80 MA mixture for 5 hours, followed by magnetic sorting of IFN-γ producing cells using the IFN-γ secretion assay and cell enrichment kit PE (Miltenyi). For cloning, cells were plated at 1 cell/well in round-bottom 96-well plates containing 2 × 10⁵ irradiated allogeneic PBMCs, 4 × 10⁴ irradiated Epstein Barr Virus-transformed B cells, 30 ng/ml anti-CD3 Ab OKT3, and 1 ng/ml IL-2, which was added on day 2 of the culture. Clones were validated by CD1b-MA tetramer staining and in the case of clone 20, a contaminating population of tetramer-negative cells was removed by cell sorting. Established T cell lines and clones were grown under the same conditions in 25 ml cultures. ELISPOT was performed using Mabtech antibodies according to the manufacturer’s instructions. Cytokine ELISAs were performed with antibodies from eBioscience (human IL-2), Thermo scientific (human IFN-γ), or BD Pharmingen (murine IL-2).