H 2kd

Naïve B cells were purified from the spleens of the mice mentioned above (“Mice”) by 2-step negative sorting, first by an iMag system (BD Biosciences) using biotinylated MoAbs against CD43 (S7: BD Pharmingen), CD4, CD8α, CD11b, CD49b (DX5), Ter-119 (BioLegend), and streptavidin-particle-DM (BD Biosciences) and then by passing of the unbound cells through a MACS LS column (Miltenyi Biotec), yielding B cells of >97% purity. B cells strongly binding HEL were purified from naïve B cells of Hy10 mice prepared as above by sorting the cells brightly stained with biotinylated-HEL plus streptavidin-APC and with CD19-PE/Cy7 (BioLegend) with FACSAria II (BD Biosciences). HEL (Sigma) was conjugated with biotin using EZ-Link Biotinylation kit (Pierce). iGB cells were purified by removing the feeder cells, IgE⁺ cells and plasmablasts/plasma cells with an iMag system as described previously [17] (link) using primary MoAbs against H-2K^d (Biolegend), IgE (R35–72: BD Pharmingen), CD138 (281–2 : BD Pharmingen), and FasL (MFL3 : Biolegend) when removing feeder cells expressing FasL. Purified naïve B cells were cultured on a feeder layer of irradiated 40 LB cells with IL-4 and IL-21, sequentially, to generate iGB cells, as described previously [17] (link). The purified iGB cells were used for the adoptive transfer into non-irradiated recipient mice, as described below.

Single-cell suspensions of patient tumor tissue were stained using the following antibodies: phycoerythrin (PE)-conjugated anti-CD44 (550989 BD Pharmigen), PE-Cy7-conjugated anti-CD90 (561558 BD Pharmigen), APC-conjugated anti-CD49f (313616 BioLegend), a lineage mixture containing Pacific-blue conjugated anti-CD45 (304022 BioLegend), anti-CD31 (303114 BioLegend) and live/dead stain (L34964 LIVE/DEAD Fixable Violet Dead Cell Stain Kit, Life Technologies). Patient-derived xenograft tissue was stained using the above antibodies with the addition of Pacific-blue conjugated anti-CD45 (103126 BioLegend), anti-CD31 (102422 BioLegend), and H2K^d (116616 BioLegend). Flow cytometry was performed using the BD LSRFortessa and BD LSR-II and cells were sorted using BD FACSAriaIII under 20psi using a 100 μm nozzle.

For the ALDEFLUOR assay (StemCell), dissociated single cells were suspended in assay buffer contain ALDEFLUOR substrate and incubated with or without DEAB. Analysis of tumor cell suspensions from xenograft tumors were performed as previous report. Briefly, PE-conjugated anti-mouse lineage antibodies (CD45 (BD), CD31 (BD), CD140b (BD), CD235a (BD), and H2KD (Biolegend)) were used for gating out non-breast cancer cells. For cell cycle analysis, cells were fixed with 70% alcohol at 4 °C overnight and stained with propidium iodide (Sigma-Aldrich) in the presence of 1% RNAase A (Takara) at 37 °C for 30 min prior to analysis. For apoptosis analysis, cells were stained with propidium iodide (Sigma-Aldrich) and Annexin V (BD), according to the manufacturer’s instructions. DCFH-DA (Sigma-Aldrich) was added to the cells without FBS for detecting ROS production. CytoFLEX (Beckman Coulter) was used for detection and data acquisition and analysis were performed in CytoExpert software. A MoFlo Astrios instrument (Beckman Coulter) was used for sorting cells.