Anti cd19 apc cy7

The ex vivo Ag presentation assays were performed using a protocol adapted from Langlet et al. OVA-specific CD8⁺ T and CD4⁺ T cells were isolated from OTI and OTII transgenic mice, respectively, and labeled with 10 µM CFSE. 10⁵ OT-I T cells or 10⁵ OT-II T cells were subsequently co-cultured for 48 hours with respectively 10⁴ sorted Ly6C^hi monocytes, MHCII^hi DCs or MHCII^int DCs obtained from the DLN of mice previously (24 h) immunized with 20 µl of an OVA (100 µg/ml)/CpG (100 µg/ml) mixture. OT-I and OT-II proliferation was determined by the loss of CFSE fluorescence by flow cytometry. Sorts were performed using a FACSAria sorter (BD Biosciences).
In vivo antigen presentation assays were performed through adoptive transfer of 2 × 10⁵ CFSE labeled OT-I or OT-II cells to respectively WT C57BL/6 J mice, CCR2^−/− mice, CCR7^−/− mice and Flt3L^−/− mice 48 hours prior to OVA/CpG immunization. Four days post immunization, DLN were dissected, stained with anti-CD3-V450, anti-CD4-PerCP, anti-CD8-PerCP, anti-Vα2 TCR-PE, anti-CD19-APC-Cy7 (all BD Biosciences), PE-labeled SIINFEKL specific dextramers (Immudex) and measured on a LSRII flow cytometer (BD Biosciences).

On day-1 prior to transplantation, recipient congenic B6 Pepboy mice (CD45.1) received a total of 11 Gy irradiation, divided into two doses of 5.5 Gy given 3 hours apart [39 (link)]. On day 0, mice were injected i.v. with 5 million BM cells from WT B6 donors (CD45.2) or IL-12p40 KO donors (CD45.2). Chimerism was confirmed a minimum of 50 days post-transplant using flow cytometric analysis of blood stained with anti-CD3-PE-Cy7, anti-CD19-APC-Cy7, anti-CD45.1-PE, anti-CD45.2-FITC, anti-CD90.1-PerCP, and anti-CD90.2-APC (BD Biosciences, San Diego, CA).

B- and T-cell subsets were enumerated in freshly thawed cryopreserved PBMCs. After washing and counting viable cells, PBMCs were surface-stained with the following conjugated mAbs: anti-CD3-AF488 (Biolegend; clone HIT3a), anti CD8-APC/Cy7 (Biolegend; SK1), anti-CD19-APC/Cy7 (BD Biosciences; SJ25C1), anti-CD19-PerCP/Cy5.5 (Biolegend; HiB19) anti-CD25-PE/Cy7 (Biolegend; BC96), anti-CD21-PE/Cy7 (BD Biosciences; B-ly4), anti-CD27-PerCP/Cy5.5 (BD Biosciences; M-T271), anti-CD38-PE/Cy7 (Biolegend; HIT2), anti-HLA-DR-PerCP/Cy5.5 (Biolegend; L243); anti-CD10-FITC (BD Biosciences; W8E7), anti-IL21R-PE (BD Biosciences; 17A12), and anti-CD39-APC (Biolegend; A1). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with anti-IL-10-PE (R & D Systems; 127107), anti-FOXP3-PE (Biolegend; 206D) and anti- TGFβ-PE (Biolegend; TW4-2F8), and analyzed with Guava easyCyte 8HT and FlowJo (Treestar). Subsets were expressed as percentages of the parent CD4+, CD8+ and CD19+ cell populations. The gating strategy is presented in S2 Fig.

A549 cells were seeded at 10⁴ cells per well in a 96-well plate in complete F-12K medium. PR8 infection was performed as described above. CYNK cells were added 24 hours post infection at an effector-to-target (E:T) ratio of 10:1 with anti-CD107a – BV786 (clone H4A3, BD) in assay buffer (RPMI (Gibco) with 10% FBS). After 1 hour, 2 μM monensin (Biolegend) and 3μg/ml Brefeldin A (BD) were added and incubated for 3 hours. Cells were stained with Live/Dead Fixable Aqua Stain (Thermo Fisher Scientific) and labelled with anti-CD56 (NCAM1) – Pe-Cy7 (clone: 5.1H11, Biolegend), anti-CD3 – APC-Cy7 (clone: sk7, BD), anti-CD14 – APC-Cy7 (clone: mΦp9, BD) and anti-CD19 – APC-Cy7 (clone: SJ25C1, BD). For intracellular cytokine staining, surface-stained cells were permeabilized using Cytofix/Cytoperm (BD) and cells were stained with anti-TNF-α - PE (clone: MAb11, BD) and anti-IFN-γ - APC (clone: B27, BD) antibodies. Cells were analyzed using Cytek Aurora (Cytek) and the data were analyzed using FlowJo Software (BD).