Igg g18 145

In order to assess HA- directed B cell binding and cross-reactivity, peripheral blood mononuclear cells (PBMC’s) from samples with different stem binding based on ELISA assay (n = 18) were further analyzed by flow cytometry. Three groups were selected for the analysis: negative for stem antibodies (n = 5), reactive to one stem only (n = 4), and samples with cross-reactive stem binding (n = 9). Cryopreserved PBMC’s were thawed in R-10 media containing 50 U/mL of Universal Nuclease (ThermoFisher). Cells were washed and resuspended in PBS for staining with UV-Blue viability dye (ThermoFisher) for 20 minutes at room temperature. After washing, surface staining was performed using antibodies against IgM (G20-127, BD Biosciences), IgG (G18-145, BD Biosciences), CD8 (RPA-T8, BioLegend), CD3 (OKT3, BioLegend), CD56 (HCD56, BioLegend), CD14 (M5E2, BioLegend), CD19 (J3-119, Beckman Coulter), CD27 (O323, BioLegend), CD38 (HIT2, BioLegend), and HA probes^{27 –29 (link)}. H1 NC 99 and H5 INDO 05 HAs were expressed and biotinylated followed by fluorochrome labeling as previously described^{29 (link)}. Stained cells were run on a BD LSRFortessa X-50 and data analysis was performed using FlowJo (TreeStar). The gating strategy is demonstrated in supplemental Fig. 2. Statistical significance at 95% confidence interval was done using two-tailed t-test.

Before staining with antibodies and RBD tetramers, B cells were enriched from PBMCs using a Pan B cell Isolation Kit (Miltenyi Biotech, #130-101-638) and LS column (#130-042-401); 1 × 10⁶ B cells were incubated with BD Horizon™ Fixable Viability Stain 510 for 15 min at room temperature in the dark. After washing, the cells were stained at 4 °C, protected from light for 30 min, using a panel of fluorochrome-conjugated primary antibodies and RBD tetramers in the optimized concentrations. The antibodies in the panel, mouse anti-human CD3 (SK7), CD56 (NCAM16.2), CD14 (M5E2), CD19 (HIB19), CD20 (2H7), CD27 (M-T271), IgD (IA6-2), CD38 (HB-7, BioLegend), CD10 (HI10a, BioLegend), CD21 (B-ly4, BD Biosciences), CD38 (HIT2), CD138 (MI15), IgM (UCH-B1), and IgG (G18-145), were purchased from BD Biosciences. Fluorescent-conjugated Omicron RBD-APC and -PE proteins were prepared by mixing biotinylated RBD protein (Acro Biosystem, SPD-C82E4) and Streptavidin-APC or -PE in a 4:1 molar ratio and incubated for 15 min at room temperature. Flow cytometry was performed on a BD LSRII (BD Biosciences), and data were acquired with BD FACSDiva Software (8.0.2) and analyzed using FlowJo software (version 10).

The following fluorochrome conjugated monoclonal antibodies reactive with macaque cells were used for flow cytometry studies: CD3 (SP34-2, BD Biosciences [BD]), CD4 (L200, BD), CD8 (SK1, BD), CD95 (DX2, BD), CD28 (CD28.2, BioLegend), CCR5 (3A9, BD), CXCR5 (MU5UBEE, Invitrogen), PD-1 (EH12.2H7, BioLegend), ICOS (C398.4A, BioLegend), CCR7 (150503, BD), α4β7 (A4B7, NHP Reagent Resource), LAG3 (3DS223H, eBioscience), Tim-3 (F38-2E2, BioLegend), TIGIT (MBSA43, Invitrogen), CD20 (2H7, BioLegend), CD19 (J3-119, Beckman Coulter), HLA-DR (L243, BioLegend), CD10 (HI10A, BioLegend), CD21 (B-ly4, BD), CD27 (O323, BioLegend), IgD (IADB6, Southern Biotech), IgG (G18-145, BD), IL-21R (2G1-K12, BioLegend), Ki-67 (B56, BD), BCL6 (IG191E/A8, BioLegend), CD80 (2D10, BioLegend), CD56 (B159, BD), CD45 (D058-1283, BD), CD14 (MoP9, BD), CD16 (3G8, BD), CCR2 (48607, R&D Systems), CD11b (ICRF44, BioLegend), CD11c (3.9, Invitrogen), PD-L1 (29E.2A3, BioLegend), PD-L2 (24F.10C12, BioLegend), CD80 (2D10, BioLegend), CD86 (FUN-1, BD), CD141 (1A4, BD), CD163 (GHI/61, BioLegend), and CD123 (7G3, BD).