Cd3 bv785

For detection of antigen-specific GC B cells, freshly isolated LN cell suspensions from individual mice were stained with Aqua Viability Dye (Thermo) and Fc blocked with an anti-CD16/32 antibody (clone 93; BioLegend). Cells were then surface stained with the relevant probes (APC or PE labelled stem, HEL, OVA or gp120 proteins for baiting antigen-specific GC B cells) [10 (link)] and the following antibodies: CD45 APC-Cy7 (30-F11; BD), CD3 BV785 (145-2C11; BioLegend), F4/80 BV785 (BM8; BioLegend), Streptavidin BV785 (BD), B220 BUV737 (RA3-6B2; BD), IgD BUV395 (11-26c.2a; BD), CD38 PE-Cy7 (90; BioLegend), GL7 AF488 (GL7; BioLegend). For detection of Tfh cells ex vivo, freshly isolated LN cell suspensions from individual mice were stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD Biosciences), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD Biosciences), CXCR5 BV421 (L138D7; BioLegend), PD-1 BV786 (29F.1A12; BioLegend). For Ag-specific Tfh identification [23 (link)], cells were cultured as described above and then stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD), CD25 BB515 (PC61; BD), OX40 PeCy7 (OX-86; BioLegend). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.

Pre-cultured PBMCs (5 × 10⁵) with or without C-Vx for 72 h, were stained with anti-human-CD107a-APC mAb (Biolegend, San Jose, CA, USA) and cultured alone or together with K562 cells (5 × 10⁴) at a 10:1 effector/target (E:T) ratio for 5 h at 37°C incubator. After the culture, PBMCs were labelled with anti-human-CD56-BV711, -CD16-BV570, -CD3-BV785, and -CD8-FITC (Biolegend, San Jose, CA, USA) mAbs. In order to determine the levels of intracellular cytotoxic granules; perforin and granzyme B, samples were fixed and permeabilized according to the manufacturer’s directions (Cytofix&Cytoperm Kit, BD Biosciences, San Jose, CA, USA), and stained with anti-human-Perforin-PerCp/Cy5.5 and -Granzyme B-Alexa Fluor 700 (Biolegend, San Jose, CA, USA) mAbs. Stained cells were measured and analyzed by flow cytometry.

Primary T cells were obtained by isolation of PBMCs from buffy coats of HLA-A2 negative donors by ficoll density gradient lymphoprep, followed by pan-T cell isolation according to manufacturer’s protocol (139-096-535, Miltenyi Biotec, Leiden, The Netherlands). T cells were lentivirally transduced with EV or TCR-E7 as described in (27 (link)). THP-1.EV, THP-1.SIRP-ß2 and THP-1.SIRP-ß2.K202L were inactivated for 1 h (37°C) by incubation with 6 µg/mL Mitomycin-C, followed by washing thrice in PBS. 100 µL of inactivated cells were plated at 1x10⁴ cells per well, followed by a 2 h incubation (37°C, 5% CO₂) with or without 10 µg/mL of E7 HPV peptide (E711-20, Peptides & Elephants, Berlin, Germany). Then, 100 µL of T cells (expressing EV or TCR-E7) were added to the THP-1 cells at a 5:1 E:T ratio (based on GFP positive T cells) and incubated for 48 h (37°C, 5% CO₂). Samples were collected, spun down (300 xG, 5 min) and stained for CD3-BV785 (317330, BioLegend, San Diego, USA), CD8-BV421 (344748, BioLegend, San Diego, USA), CD69-PerCP (310928, BioLegend, San Diego, USA) and CD25-APC (21810256, ImmunoTools, Friesoythe, Germany) and analyzed using flow cytometry. The supernatant was analyzed for IFN-y, using an IFN-y cartridge (SPCKB-PS-002574, Biotechne, Minneapolis, USA) with the Ella automated immunoassay system, following manufacturer’s protocol.

Spleens were mechanically disrupted using the cap of an Eppendorf tube to grind tissue against a piece of 100 μm nylon mesh that was placed in a petri dish. The disrupted spleen tissue was then incubated in red blood cell lysing buffer (Biolegend, San Diego, CA, United States) for 2 min and then resuspended in RPMI media. Mesenteric lymph nodes were mechanically disrupted in RPMI media alone. Cell pellets from blood samples were resuspended in red blood cell lysing buffer (Biolegend, San Diego, CA, United States), incubated for 2 min, centrifuged, then incubated in lysis buffer a second time. Cell suspensions from all tissues were filtered through 100 μm nylon mesh and aliquots containing 1 × 10⁶ – 5 × 10⁶ cells stained with the fluorescently conjugated antibodies: CD4 (Pacific. Blue), CD8 (PE), and CD3 (BV785) (Biolegend, San Diego, CA, United States). Samples were analyzed using a Beckman Cytoflex flow cytometer and analyzed using Flowjo flow cytometry analysis software (FlowJo LLC, OR).