Cd20 clone 2h7

Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail (1:100 dilution) of CD3 (clone SP34-2, BD Biosciences), CD4 (clone OKT4, Biolegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled biotinylated SIVmac239 SOSIP.664 Env at room temperature for 20 min in the dark. SIVmac239 Env trimer probes were labeled with two different fluorophores, from which SIVmac239 Env dual positive memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20⁺IgM⁻IgG⁺SIVmac239 SOSIP²⁺) were analyzed with BD FACSMelody or BD FACSFusion, and single-cell sorted, cultured, expanded in 384-well plates as described previously^{28 (link),29 (link)}. Briefly, sorted B cells were cultured with Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× MycoZap Plus-PR (Lonza), 100 U/mL human IL-2 (Roche), 50 ng/mL human IL-21 (Invitrogen), 50 ng/mL human IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H + L) (BioRad), and irradiated 3T3msCD40L feeder cells. Flow cytometric data were subsequently analyzed using FlowJo (v10.7.1).

Surface expression of CD157 was assessed by flow cytometry (Navios, Beckman Coulter, Krefeld, Germany) using a fluorochrome-conjugated monoclonal antibody (SY11B5; e-Bioscience, San Diego, California). CD157 expression on CD34⁺/CD38⁻ cells from BM of AML patients and HDs was analyzed using the following antibodies: CD45 (J33), CD34 (581), CD38 (LS198.4.3) and CD33 (D3HL60.251). Corresponding isotype controls were used. All antibodies were purchased from Beckman Coulter (Krefeld, Germany). A CD45^DIM/SSC^LOW gate was used to limit the analysis to myeloid progenitor cells.
Surface expression analysis of CD157 on AML cell lines and CD20 (clone 2H7, BioLegend, San Diego, California) on RAJI cells was performed on a BD LSR II (Becton Dickinson, Heidelberg, Germany). Staining was performed according to the manufacturer's instructions.
MFI values were determined using FlowJo (Version 9.4.11) (Tree Star Inc., Ashland, Oregon), and surface expression intensity (MFI ratio) was determined as previously described [20 (link)].

Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail of CD3 (clone SP34-2, BD Biosciences), CD4(clone OKT4, Biolegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (clone G20-127, BD Biosciences or clone MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled BG505 Env at room temperature for 20 min in the dark. BG505 SOSIP.664 was labeled with two fluorophores separately, from which dual positive Env-specific (CD3^-CD4^-CD8^-CD14^-CD20⁺IgM^-IgG⁺BG505 SOSIP²⁺) and/or 398F1 SOSIP⁺ memory B cells were analyzed with a FACSFusion sorter and then single-cell sorted, cultured, and expanded as previous described (Huang et al., 2013 (link)). In brief, cells were sorted into Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1X MycoZap Plus-PR (Lonza), 100 U/mL IL-2 (Roche), 50 ng/mL IL-21 (Invitrogen), 50 ng/mL IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H+L) (BioRad) and irradiated 3T3msCD40L feeder cells.

PBMCs were isolated from buffy coats as described earlier and pan-DCs (including myeloid and plasmacytoid DCs) were further enriched by negative selection using EasySep^TM human pan-DC pre-enrichment kit (STEMCELL Technologies) according to the manufacturer´s instructions. Cell purity of enriched live (fixable viability stain 780 ^– from BD Biosciences), lineage negative cells (CD14^– (clone M5E2, BD Biosciences), CD16^– (clone 3G8, BD Biosciences), CD3^– (clone UCHT1 BD Biosciences), CD20^– (clone 2H7, Biolegend) or CD19^– (clone HIB19, Biolegend), CD56^– (clone MEM-188, Biolegend), but HLA DR⁺ (clone L243, Biolegend)) was confirmed by flow cytometry. Isolated DCs were seeded at 1 × 10⁶ cells/ml in 96-well plates with 200 μl/well complete culture medium. Following 1–2 h rest, DCs were primed with 10% L. reuteri-CFS or were kept in RPMI as the control. Following 24 h incubation, cells were collected and washed twice with warm PBS and fresh medium with RA (1,15 μg/ml) or without RA was added. On day 4, the medium was replaced and on day 6 cells were re-activated with 10 μg/ml of Pam3SCK4 for 24 h. Supernatants were collected following 24 h priming with the first stimuli on day 1 and following 24 h stimulation with the second stimulus on day 7. Supernatants were stored at −20°C until further analysis.