Cd38 pe

After treatment with the various compounds, apoptotic cells were identified by labeling with Annexin V-APC/7-AAD (both Biolegend Inc., San Diego, CA, USA) for 15 min at RT. Cells were then collected and analyzed by flow cytometry (Attune NxT flow cytometer, Thermo Fisher Inc., Waltham, MA, USA). Dead cells were calculated by determining Annexin V⁺/7AAD⁺ cells in treated samples relative to a solvent control or untreated control (bone marrow samples). In multiple-staining experiments, cells were incubated on ice with CD19-FITC (Beckman Coulter, Brea, CA, USA, #A07768), CD38-PE (Beckman Coulter, Brea, CA, USA, #A07779), CD138-PE/Cy7 (Biolegend Inc., San Diego, CA, USA, #352318), CD45-AF700 (Beckman Coulter, Brea, CA, USA, #A79390), CD56-APC/Fire750 (Biolegend Inc., San Diego, CA, USA, #362554) and CD269-BV421 (Biolegend Inc., San Diego, CA, USA, #357520) for 20 min prior to Annexin V-APC/7-AAD staining. Multiple myeloma cells were identified as CD38⁺, CD138⁺, CD19⁻, CD56⁺, CD269⁺, CD45^+/−.

Blood samples were collected at W0, W8, W24 and W48 for immunophenotyping by flow cytometry in ethylenediaminetetraacetic acid (8.55 mg/tube). Peripheral blood samples (100 µL) were incubated with specific monoclonal antibodies (Beckman Coulter, Wycombe, UK) at room temperature (RT) for 15 min in the dark; afterward, the red cells were lysed, at RT in the dark, in a VersaLyse lysing solution (A09777, Beckman Coulter, Wycombe, UK) for 15 min and then analysed. LS were identified by the recognition of surface molecules belonging to the CD family. The samples successively underwent gating analysis (total lymphocytes and CD45+ vs. side scatter) (CD45-FITC, A07782, Beckman Coulter, Wycombe, UK), and the desired lymphocyte subpopulations were gated excluding doublets. The LS identification was based on monoclonal antibodies against Bmem CD19+/CD27+, Br1 CD19+/CD38+, Br2 CD19+/CD25+, Treg CD4+/CD25+, Th CD3+/CD4+ and Tc CD3+/CD8+ (CD19-PC7, IM3628; CD27-PE, IM2578; CD38-PE, AO7779; CD25-PE, A07774; CD4-FITC, A07750; CD3-PC5, A07749; CD8-PE, A07757; Beckman Coulter, UK). The samples were tested using a CytoFLEX flow cytometer and the CytExpert software (Beckman Coulter, Wycombe, UK).

Two panels were designed; Immune Activation Panel and Immune Exhaustion Panel. The Immune Activation Panel consisted of the following fluorochrome-coupled monoclonal antibodies: CD3-FITC; CD4-ECD; CD8-PC-7; CD45-KO; CD38-PE and; HLA-DR-APC (Beckman Coulter, MI). The Immune Exhaustion Panel was composed of CD3-FITC; CD4-ECD; CD8-PC7; CD45-KO; CTLA-4-PE and; PD-1-APC. Flow cytometry data acquisition was performed using a Navios flow cytometer (Beckman Coulter). Optimal volatges for acquisition were determined, compensation and fluorescence minus one analyses were performed before testing of study samples. The gating strategy is shown in Additional file 1: Figure S1. Data analysis was performed using Beckman Coulter Kaluza software.