Breast Cancer Cell Lines and ODN Stimulation

The breast cancer cell lines, AU565 (MAM-A⁺/HLA-A2⁺) and MCF-7 (MAM-A⁻/HLA-A2⁺), and human monocyte-like cell line, THP-1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human CD8+T cells from HLA-A2⁺ healthy subjects were obtained from StemCell technologies (Cambridge, MA, USA). All cell cultures and incubations were performed as per provider’s recommendations and described by us before [19 (link),20 (link)]. Briefly, cells were cultured in RPMI-1640 medium at 37 °C in a 5% CO₂ incubator until they were 80% confluent. The presence of MamA and HLA-A2 expression in the breast cancer cell lines was confirmed by western blot analysis (data not shown). The ODNs—ODN2216, ODN2006, and ODN M362 (respectively controls represented by suffix ‘c’)—were at vaccigrade and obtained from InvivoGen (San Diego, CA, USA). For ODN stimulation, the breast cancer cells and the THP-1 cells were cultured in 24 well plates, 1 × 10⁵ per well pre-stimulated with PMA (phorbol myristate acetate, 10 ng/mL medium) for 16 h, washed once with RPMI-1640 media, and stimulated with ODNs (10 µM) for 5 h. These cells were later used for various experiments detailed below.