Cd20 2h7

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).

QVOAs were conducted on B cells isolated from PBMCs and spleens from SIV-infected ART-suppressed macaques. B cells were isolated using an anti-CD20 biotinylated antibody (clone 2H7; Biolegend), antibiotin antibody (EasySep), and EasySep magnetic nanoparticles. The CD20 selection was performed following the same method as the CD11b isolations previously described (3 (link)– (link)5 (link)). In brief, cells were thawed, counted, brought up in 50 μl of selection buffer (2% FBS, 1 mM EDTA in 1× PBS), and incubated with anti-CD20 antibody for 20 min at 4°C. The cells were then washed and brought up in 100 μl/10⁷ cells of selection buffer and incubated with the EasySep antibiotin antibody for 15 min at 4°C. EasySep magnetic particles were then added and incubated for 10 min at 4°C. Magnetically labeled cells were then removed using the EasySep EasyEights magnet. The cells were washed, counted, and saved for FACS purity analysis (CD3 SP34-2 [BioLegend]; CD20 2H7 [BioLegend]) and plated at limiting dilutions following the CD4 QVOA protocol as described above and previously (39 (link)).

Human and NHP lung tissues were placed in optimal cutting temperature compound (OCT, KMA-0100-00A, CellPath, UK) and snap frozen in liquid nitrogen. 10 µm sections were cut on to Super Frost microscope slides (12312148, Thermo Fisher) using a cryostat. Sections were air dried overnight at room temperature and then fixed in -20°C acetone for 10 minutes. Sections were air dried for <20 minutes prior to x2 washes with PBS. Multiplex immunofluorescence was performed on lung sections using a fully automated Bond-Rx Multiplex IHC Stainer (Leica Biosystems) and OPAL Multiplex IHC Detection Kit and counterstained with DAPI (Akoya Biosciences) according to a previously published protocol (27 (link)). T_RM were stained with purified, cross-reactive CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD69 (FN50, Biolegend) and CD103 (2G5.1, Bio-Rad, UK) antibodies. B_RM were stained with purified, cross-reactive CD20 (2H7, Biolegend), CD27 (M-T271, Biolegend) and CD69 (FN50, Biolegend) antibodies. Human lung (n=5) and NHP lung (n=3) sections were stained simultaneously using the same protocol and antibody concentrations. For full details, see online supplement.