Cd27 v450 m t271

Multi-color flow cytometry was performed on PBMC by acquiring 106 formaldehyde-fixed cells at the Gallios (Beckman Coulter) flow cytometer, and data analysed with FlowJo 8.8.7 (Treestar Inc.). The following mouse antihuman fluorochrome-conjugated monoclonal antibodies were used: CD19-PER-CP-Cy5.5 (SJ25-C1), CD10-PE-Cy7 (Hl10a), CD38-APC (HIT2), CD21-PE (B-ly4), CD27-V450 (M-T271), IgD-APC-H7 (IA6-2) and IgM-FITC (G20-127) (BD Biosciences). Dead cells were excluded using the Live/Death Vivid detection kit labeled with a near-infrared dye (Invitrogen, Carlsbad, California, USA). Gating strategy was performed as follows: only singlets were acquired and live B cells were gated on Vivid-CD19+ lymphocytes. Only samples with viability above 85% were subjected to the B-cell analysis. Transitional (“TR”) B cells were identified as CD19+CD27-CD10+. Thereafter, the CD10- cell population was gated on CD27++CD38high+ to identify Plasma-cells. Whereas on the CD27+CD38high- gate the following populations were identified: naive (“NV” CD27-CD21high+), resting memory (“RM” CD27+CD21high+), activated memory (“AM” CD27+CD21-) and tissue-like memory (“TLM” CD27-CD21-). RM, AM and TLM were each further characterized on the basis of Ig-expression into switched (“SW”, IgM-IgD-), Marginal Zone-like (“MZL”, IgM+IgD+), IgM-expressing (“IgM+”, IgM+IgD-) and IgD-expressing (“IgD+”, IgM-IgD+).

Flow cytometry was performed on PBMCs from both the influenza vaccine immunized volunteers and those from the VRC 902 Study. There were 2 panels of antibodies; one for CXCR5⁺CD4⁺ T cell subsets, and the second panel for memory B cells. For the staining of T cells, the following antibodies were used: CXCR5-AF488 (RF8B2, BD Biosciences, San Diego CA), PD1-PE (eBioJ105, eBioscience, San Diego, CA), CD45RO-PE/CF594 (UCHL1, BD Biosciences), CCR6-PerCP/Cy5.5 (G034E3, Biolegend, San Diego, CA), ICOS-PE/Cy7 (C398.4A, Biolegend), CCR7-AF647 (3D12, BD Biosciences), CXCR3-AF700 (1C6/CXCR3, BD Biosciences), CD4-APC/eFluor 780 (SK3, eBioscience), CD27-V450 (M-T271, BD Biosciences), CD8-BV510 (SK1, BD Biosciences), and CD3-BV605 (SK7, BD Biosciences). For B cells, IgD-AF488 (IA6-2, Biolegend), CD19-PE (HIB19, Biolegend), CD38-PE/Cy7 (HB-7, Biolegend), and CD27-BV450 (M-T271, BD Biosciences) were used. To exclude dead cells, Zombie UV Fixable Viability Kit (Biolegend) was used. For compensation, UltraComp eBeads (eBioscience) were used on each day the experiment was performed.