Cd38 pe

Viable frozen peripheral blood samples from 2 DS patients with DS-associated myeloid preleukemia were obtained from the biobank commission of the Princess Máxima Center for Pediatric Oncology. Mononuclear cells were stained with a cocktail of the following antibodies: CD3-BV650 (Biolegend, Clone UCHT1, 300467, 1:100), CD4-PerCP/Cy5.5 (Biolegend, Clone OKT4, 317427, 1:200), CD8-BV785 (Biolegend, Clone SK1, 344739, 1:100), CD19-BV421 (Biolegend Clone HCD14, 30224, 1:100) , CD14-AF700 (Biolegend, Clone HCD14, 325614, 1:100), CD56-BV711 (Biolegend, Clone HCD56, 318335, 1:50), CD34-APC (Biolegend, Clone 561, 343607, 1:50), CD38-PE (Biolegend, Clone HIT2, 303505, 1:50) , CD33-PE/Cy7 (Biolegend, Clone WM53, 303433, 1:100), CD117-PE-dazzle594 (Biolegend, Clone 104D2, 1:100), CD16-FITC (Biolegend, Clone 3G8, 302005, 1:100), CD20-FITC (Biolegend, Clone IVB201, 302303, 1:100). Bulk T-cells (CD3+/CD4+ and CD3+/CD8+) and preleukemic blast cells were sorted with the Astrios-EQ. Preleukemic blast cells were sorted according to the diagnostics flow data. Cell pellets were used for DNA isolation. Public data was used for the other 4 myeloid preleukemia samples^{11 (link)}.

Flow cytometry analysis was performed as previously described.^{52 (link)} PBMCs were isolated from blood of COVID-19 convalescent subjects using human lymphocyte separation medium. Cells were stained and mixed with fluorescent-labeled DNA-barcoded antigens and other fluorescent antibodies, and sorted by FACS with a MoFlo Astrios EQ Flow Cytometer (Beckman). The following antibodies were used to label the B cell subgroups: CD19-PB (BioLegend, Cat 302232), CD27-APC (BioLegend, Cat 302810), CD38-PE (BioLegend, Cat 303506). CD19⁺ CD27⁺ memory B cells and CD19⁺ CD27^high CD38^high plasma B cells were sorted for further scRNA-seq.

For phenotyping of B cells, CD9⁺ and CD9⁻ B cells, the antibodies used were as follows: CD27-PE-Cy7 (clone 0323, Biolegend), CD38-PE (clone HB7, Biolegend), CD19-BV510 (clone HIB19, Biolegend), CD24-APC (clone SN3 A5-2H1D, eBioscience, San Diego, CA, USA), HLA-DR-PERCP (Clone L243, Biolegend), CD40-PE (Clone 5C3, BD Biosciences), CD86-PE (Clone 2331, BD Biosciences), CD25-PE/Cy5 (Clone M-A251, BD Biosciences), CD69-APC (Clone FN50, Biolegend), IgD-APC-Cy7 (Clone, IA6-2, Biolegend), IgM-FITC (Clone, SA-DA4, eBioscience), CD5-FITC (Clone, L17F12, eBioscience), CD10-APC (Clone, LT10, ImmunoTools, Friesoythe, Germany), CD20-PE-Dy647 (Clone, LT20, ImmunoTools) and CD21-FITC (Clone, LT21, ImmunoTools). Viability was assessed by 7AAD staining (BD Biosciences). All flow cytometric acquisition was performed with FACS Canto II, BD Biosciences. The data analysis was performed using FlowJo V7 (TreeStar Inc., Ashland, OR, USA). The gating strategy for the Bregs and single markers can be found in Supplementary Figure S1.

The staining for cell surface proteins were performed following standard protocols by incubation of cells with pre-optimized concentrations of antibodies at 4°C for 20 min in FACS buffer (PBS with 2% FCS and 1 mM EDTA). Flow cytometry data were acquired on BD FACS Canto II cytometer and analyzed with FlowJo software version X (Tree Star). Unless otherwise stated all antibodies were supplied by BD Biosciences: CD25 APC (M-A251) or CD25 PECy7 (2A3); CD8 APC (SK1); CD4 V500 (RPA-T4), CD3 PerCPCy5.5 (SK7), CD38 PE (HIT2); CD80 PE (L307.4); CD83 FITC (HB15e); CD86 PECy7 (FUN-1), HLA-DR PerCP (L243); CCR7 PECy7 (G043H7; Biolegends). Appropriate isotypes were used as controls.

B-cell whole peripheral blood samples (200 μL) were stained with the following anti-human monoclonal antibodies: IgD-Alexa Fluor 488 (cat. 348216, BioLegend, Inc., San Diego, CA, USA), CD38-PE (cat. A07779, Beckman Coulter, Brea, CA, USA), CD5-ECD (cat. A33096, Beckman Coulter, Brea, CA, USA), CD27-PC7 (cat. A54823, Beckman Coulter, Brea, CA, USA), CD19-APC/Cy7 (cat. 302218, BioLegend Inc., San Diego, CA, USA), and CD45-Krome Orange (cat. A96416, Beckman Coulter, Brea, CA, USA). All antibodies were utilized at the dilutions that were recommended by the manufacturers. After incubation at room temperature in the dark for 15 min, red blood cells were lysed for 15 min by adding 2 mL of VersaLyse Lysing Solution (Beckman Coulter, Inc., Brea, CA, USA) supplied with 50 μL IOTest 3 Fixative Solution (Beckman Coulter, Inc., Brea, CA, USA). Next, cells were washed (7 min, 330 g) twice with a buffer (sterile phosphate-buffered saline (PBS) containing 2% of heat-inactivated fetal bovine serum, Sigma-Aldrich, St. Louis, MO, USA) and were resuspended in 0.5 mL of PBS containing 2% of neutral buffered formalin solution (Sigma-Aldrich, St. Louis, MO, USA). Sample acquisition was performed using a Navios flow cytometer (Beckman Coulter, Inc., Brea, CA, USA), equipped with 405, 488, and 638 nm lasers. At least 5000 CD19+ B-cells were collected for analysis from each sample.