Efluor 450 proliferation dye

Single cell suspensions of homogenized spleens and LNs (inguinal, cervical, axillary, mediastinal, and mesenteric) were prepared from vaccinated retrogenic mice (8–12 weeks after the last vaccination) or age-matched unvaccinated mice. CD8⁺ T cells were purified by negative selection using the CD8⁺ T cell isolation kit II (Miltenyi Biotec) or the EasySep mouse CD8 T cell enrichment kit (StemCell Technologies, Vancouver, BC, Canada) followed by magnetic separation. After purification, cells were stained with eFluor 450 proliferation dye (eBiosciences), antibody-stained and sorted by flow cytometry to achieve uniform populations of naïve or memory CD8⁺ T cells. For TB10Rg3 naïve/memory co-transfer experiments, 1x10⁴ cells of each population were mixed at a 1:1 ratio (confirmed by flow cytometry) and were transferred IV into congenic recipients (CD90.1 or CD45.1), which had been infected 0–7 d earlier with Mtb. TB10Rg3 CD8⁺ T cells used for the memory group were generated on the Thy1.1⁺, CD45.1⁺, and Thy1.2⁺CD45.2⁺ backgrounds to ensure that none of the observed effects were specific to congenic backgrounds of the mice. For protection experiments, 1x10⁵ TB10Rg3 Thy1.2⁺CD45.2⁺ memory or naïve cells were transferred into TCRα^-/- mice or sub-lethally irradiated (600 Rads) C57BL/6 mice, and challenged with Mtb the same day.

PBMCs were labelled with eFluor450 proliferation dye (20 μM, eBioscience) before the cells were incubated for 3 days in the absence or presence of the extracts and stimulated with phytohaemagglutinin-L (5 μg/mL, Roche). At the end of the stimulation period the cells were washed twice with PBS and stained with mouse anti-human CD3 antibody PE conjugated (BD Biosciences) for 20 minutes at 4°C. The cells were again washed and resuspended in 0.2% BSA in PBS, before assay by flow cytometry.

OT-I spleen cells (1–3x10⁵) were co-cultured with EG7 cells at a 1:1 ratio for 1 hour at 37°C. EL4 cells were used as a control target. The cells were harvested, washed twice with FACS buffer and stained with biotinylated-anti Ova_257–264/H-2Kb (clone 25-D1.16, eBioscience). Surface pMHC on CD8⁺ T cells was detected by flow cytometry following concomitant staining with anti-CD8-FITC and streptavidin-APC (both from eBioscience).
To measure effector CTL proliferation, OT-I spleen cells were co-cultured with EG7 or EL4 cells, as described above. CD8⁺ T cells (T-APC) were purified using BD IMag anti-mouse CD8 particles (BD Biosciences) and labeled with eFluor 450 proliferation dye according to the manufacturer’s instructions (eBioscience). eFluor 450-labeled T-APC were co-cultured at a 1:1 ratio for 3 days with naïve OT-I CD8⁺ T cells (effector CTL) labeled with carboxyfluorescein succinimidyl ester (CFSE, Life Technologies). The cells were harvested, washed and stained with anti-mouse CD8-allophycocyanin (eBioscience) followed by flow cytometry analysis of CFSE dilution in effector CTL (CD8⁺eFluor450^-). Proliferation of OT-I CD8⁺ T cells, treated with 2.5 μg/ml Concanavalin A (ConA) was used as a positive control.