Easysep kits

BM cells were differentiated into cDC1-like cells and incubated for 4 h in 96-well U-bottom plates (5 × 10⁴ per well) in 200 µl of medium in the presence of 2 mg OVA protein (10 mg ml^–1; vac-stova; InvivoGen) and individual interleukins (IL-2, IL-12, IL-15, IL-18, IL-21, IL-23 or IL-27). The concentration of each interleukin was 2 ng ml^–1, 8 ng ml^–1 or 20 ng ml^–1 (in 200 µl). Meanwhile, OT-I CD8⁺ and OT-II CD4⁺ cells were isolated using EasySep kits (STEMCELL Technologies) and resuspended in T cell medium (complete RPMI medium with 50 μM beta-mercaptoethanol, minimal non-essential amino acids, 1 mM sodium pyruvate and 10 mM HEPES). OVA-loaded cDC1-like cells were then washed and co-cultured with OT-I or OT-II cells in T cell medium in the presence of the aforementioned interleukins. Co-culture of OVA-loaded cDC1-like cells with OT-I or OT-II cells was continued for 3 days and 5 days, respectively. T cell activation was measured by intracellular staining with antibodies against IFNγ (clone XMG1.1, BD Biosciences) using BD Golgi Stop kit (BD Biosciences).

Human memory CD4+CD45RO+ and memory CD8+CD45RO+ T cells were purified from PBMC of healthy buffy coat donors. PBMC were first isolated from buffy coats (Gulf Coast Regional Blood Center, Houston, TX) by using Ficoll-Paque PLUS (GE Healthcare). Memory CD4 and memory CD8 T cells were then negatively selected from PBMC with magnetic bead-based EasySep kits (Stemcell Technologies). Purities of memory CD4+CD45RO+ T cells were usually >90% and memory CD8+CD45RO+ T cells >80% as determined by flow cytometry. Natural killer cells were purified from PBMC with negative selection kits (Stemcell Technologies), and CD3-CD56+ purity was >90%. Cells were cultured at 37°C + 5%CO₂ in complete RPMI-1640 medium, supplemented with 10% FBS, 0.1 mM MEM nonessential amino acids, 2mM sodium pyruvate, 2mM HEPES, 1× antibiotic-antimycotic and 2mM L-glutamine.
Most experiments involved no stimulation (UT) or stimulation of 1-5×10⁵ memory CD4 and memory CD8 T cells, or NK cells (200 μl/well of 96-well flat-bottom plates, or 1 ml/well of 24-well plates, as indicated) for 24 hrs with 1 ml/ml coated CD3 (clone UCHT1, BD Biosciences) or soluble CD3 (clone CD3-2, Mabtech) + 1 μg/ml CD28 (clone CD28.2, BD Biosciences) mabs. For some experiments, cells were stimulated with 100 ng/ml recombinant IL2 (Biolegend) or soluble CD3 mab alone for 24 hrs with or without brefeldin (GolgiPlug, BD Biosciences).

CD45.1⁺ OT-I mice were used as cell donors for adoptive cell transfer into syngeneic CD45.2⁺ C57BL/6J recipient animals. Lymphocytes were isolated from spleen and OT-I CD8⁺ T cells purified using EasySep kits from StemCell Technology according to the manufacturer’s protocol. 1 × 10⁴ OT-I CD8⁺ T cells were injected intravenously via the tail vein in a 100-µl volume a day prior to i.n. immunization.

PBMC, MM and HS-5 cell lines, primary BMSCs from patients were isolated and cultured as previously described [31 (link), 33 (link), 43 (link)]. Malignant CD138⁺ PCs and healthy B cells were isolated with EasySep™ kits (STEMCELL Technologies, USA), after achieving informed consent according to the declaration of Helsinki. The internal Institutional Board approved the use of human material. SaMMi cell line was obtained from a 81 year old woman with a 20% PCs in the bone marrow. CD138⁺ SaMMi PCs were cultured in DMEM 20% FBS with the addition of IL-6 2.5 ng/ml. Co-cultures of HS-5 or BMSCs with INA-6 or patient derived CD138⁺ PCs were stabilized as described in [33 (link)]. The clinical features of the patients analyzed and the immunophenotype/karyotype of SaMMi are detailed in Tables 1 and 2.