Cd8 negative selection kit

Donor splenocytes were obtained from tandem dimer tomato (tdT) red fluorescent protein-transgenic mice expressing ovalbumin-specific OT-I TCR-transgenes (tdT-Tg/OT-I TCR-Tg mice), and CD8⁺ T cells were isolated using a CD8 negative selection kit (Miltenyi Biotech, San Diego, CA). After isolation, CD8⁺ T cells were labeled with 10 µM CFSE (Molecular Probes, Eugene, OR). Recipient human CD1d knock-in mice received 5×10⁶ labeled cells via the lateral tail vein, and were then vaccinated s.c. with 5×10⁶ CFU of glycolipid-modified or unmodified rBCG-OVA as indicated, or received PBS plus 0.05% tyloxapol as a negative control. Splenocytes were harvested 6 days later, stained with anti-B220-APC and anti-CD8-PerCP, and analyzed using a LSR-II-yellow flow cytometer (BD Biosciences). CFSE fluorescence of the transferred population was determined by gating on the B220^negCD8⁺ lymphocytes expressing tdTomato.

We prepared a single-cell suspension of LN cells that were harvested from female OT-I, P14, or gBT-I mice by mechanically passing LN tissue through a 70 to 100 μm strainer. To enrich transgenic T cells, we used MACS with a CD8 Negative Selection Kit (Miltenyi Biotec).
For OT-I memory mice, we adoptively transferred 1 × 10⁴ OT-I T cells in sterile 1 × PBS i.v. per C57BL/6J recipient and subsequently infected recipients i.v. with 1 to 2 × 10⁷ PFU VSV-OVA or 4 × 10³ CFU OVA-expressing L. monocytogenes (L. monocytogenes–OVA). For gBT-I memory mice, we adoptively transferred 5 × 10⁴ gBT-I T cells i.v. and subsequently infected recipient mice i.v. with or 4 × 10³ CFU HSV2 gB–expressing L. monocytogenes (L. monocytogenes–gB). We allowed 60 days or longer to pass after initial VSV or L. monocytogenes infections before assaying tissues.
For P14 memory mice, we adoptively transferred 2 × 10³ WT P14 T cells i.v. and subsequently infected recipient mice i.v. with 2 × 10⁵ PFU LCMV Armstrong clone (LCMV Arm.) or 2 × 10⁶ PFU LCMV Docile clone (LCMV Doc.). For Tox^–/– P14 memory mice, we adoptively transferred 2 × 10³Tox^–/– P14 memory mice and subsequently infected with 2 × 10⁵ PFU LCMV Arm.; we allowed 28 days to pass after initial LCMV infection before assaying tissues.

CD8⁺ T cells were isolated using the CD8 negative selection kit (Miltenyi Biotech) according to the manufacturer’s instructions via an AutoMACS pro seperator. The purity of the isolated cells was verified by flow cytometry and was consistently between 75% and 90%.

The generation of monocyte-derived mDC and antigen-specific T cells was performed^{57 (link)}. Briefly, DC were generated from monocyte cultures in GM-CSF (100 ng/ml, Berlex) and IL-4 (20 ng/ml, Miltenyi Biotec) and matured using CD40L-expressing K562 cells, IFN-γ (100 u/ml, CellGenix), poly I:C (Invivogen, Inc), and R848 (Invivogen, Inc.) for 16 h to generate mDC. CD8⁺ T cells were isolated from peripheral blood mononuclear cells, using a CD8 negative selection kit (Miltenyi Biotech, Auburn, CA). Purified CD8⁺ T cells (5 × 10⁶ cells/ml) were cultured at a 20:1 ratio with irradiated (2500 Rads) autologous mDC pulsed with peptide (40 μg per 1 × 10⁶ DC/ml) in 24-well trays in Optimizer CST media (Gibco) supplemented with 5% pooled human sera. Human IL-7 (10 ng/ml), IL-15 (5 ng/ml), and IL-12 (10 ng/ml) were added on day 0. Fresh media supplemented with IL-7 (10 ng/ml) and IL-15 (5 ng/ml) was added on day 7. Fourteen days after primary mDC stimulation, T cell cultures were harvested and re-stimulated with irradiated (2500 Rads) peptide-pulsed mDC. Cell culture media was supplemented with 50 U/ml IL-2 (Chiron, Emeryville, CA) starting day 2 then every 48 h following secondary stimulation. On days 10–14 of secondary mDC stimulation, antigen-specific T cell responses were identified by IFN-γ ELISPOT assay and flow cytometry by p-HLA multimer staining.