Aurora spectral cytometer

For mouse lymphocyte staining, single-cell suspensions from iLNs were prepared by mechanical disruption of the tissues through a 70-µm mesh in 2% FBS in PBS. For stromal cell staining, single-cell suspensions from iLNs were prepared by enzymatic digestion with 0.2 mg ml⁻¹ Collagenase P (no. 11213865001, Sigma), 0.8 mg ml⁻¹ Dispase II (no. 4942078001, Sigma) and 0.1 mg ml⁻¹ DNase I (no. 10104159001, Sigma) in plain RPMI medium (no. 11875093 Gibco). The cell number and viability of samples were acquired using a CASY TT Cell Counter (Roche). For both mouse and human work, cells were stained with surface antibody stains (Supplementary Tables 1 and 2) for 30 min to 2 h at 4 °C in Brilliant stain buffer (no. 563794, BD Biosciences), then washed with 2% FBS in PBS and fixed using the Foxp3/Transcription Factor Staining Buffer Set (no. 00-5323-00, eBioscience). For intracellular staining, cells were incubated for 1 h at 4 °C with the appropriate antibodies. Samples were acquired on an LSR Fortessa (BD Biosciences) using BD FACSDiva software v.9.0 or on a Cytek Aurora Spectral Cytometer (Cytek) using SpectroFlo Software v.3.0, and analysis was done using FlowJo v.10 software (Tree Star).

PBMCs from healthy donors’ fresh blood were cultured in RPMI 1640 medium (Gibco) containing 10% of FBS and stimulated 50% (v/v) with supernatant from macrophages treated with 1 mM of SB and PBA. Stimulation was performed for 6 h at 37 °C 5% CO₂ in the presence of Golgi-Plug containing Brefeldin A (BD Biosciences, San Jose, CA, USA) and Golgi-Stop containing Monensin (BD Biosciences) added after 1 h of the stimulation according to the manufacturer’s instructions. After that, PBMCs were washed and stained with the Live/Dead fixable Blue (Invitrogen) and surface markers (listed in Supplementary Table S1) for 30 min at room temperature, twice washed, fixed and permeabilized using the eBioscience™ Transcription Factor Fixation/Permeabilization (Invitrogen) according to the manufacturer’s instructions. Subsequently, the fixed and permeabilized PBMCs were staining using fluorochrome-conjugated antibodies against intracellular makers listed in Supplementary Table S1. Labeled cells were acquired on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Fremont, CA, USA). Data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) v10.6.2 software.

Thawed PBMC were washed in RPMI culture medium containing 50U/ml benzonase nuclease and then washed by PBS. Cells were incubated with LIVE/DEAD Fixable Blue Dye (Life Technologies, Carlsbad, CA), which was used to exclude dead cells from analysis. Cells were incubated with fluorochrome-conjugated HAs for influenza B (B/Washington/02/2019 and B/Phuket/3073/2013 combined on the same fluorochrome), and Influenza A H1 (A/Hawaii/70/2019) and H3 (A/Hongkong/2671/2019) and fluorochrome-conjugated antibodies against IgM, IgA, CD21, CD85J, FCRL5, CD20, IgG, CD38, CD14, CD56, CD3, CD27, CD71, CD19, IgD for 30 min at 4 C in the dark. The dyes and detailed information of antibodies in the panel (Sarah Andrews, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health) are summarized in Table 1. After incubation with antibodies for 30 minutes, cells were washed two times with FACS buffer (0.1%BSA/PBS (pH7.4)) and fixed in 1% paraformaldehyde. Five million cells were acquired on Cytek Aurora spectral cytometer (Cytek Biosciences, Fremont, CA). Data were analyzed with FlowJo software version 10 (BD Biosciences).

PBMC were resuspended in DPBS, stained with Zombie NIR for 15 min at room temperature. The cells were washed, blocked, and stained using a 28-antibody cocktail (Supplementary Table 2). Cells were washed, fixed and samples were then acquired on a 4-laser Cytek Aurora spectral cytometer (Cytek, Fremont, CA). Single color controls were stained on UltraComp eBeads™ Compensation Beads (ThermoFisher Scientific, Cat# 01-2222-42), fixed, and used for spectral unmixing. Events were acquired using the gating strategy described in Supplementary Figure 1. Debris was removed using the SSC-B/SSC plot and autofluorescence was removed by selecting it as a fluorescence tag during unmixing. The unmixed data were analyzed in FCS Express 7 (De Novo Software, Pasadena, CA). A clean-up gate on HLA-DR/CCR7 plot was used to gate out very high fluorescence events due to antibody aggregation.

Livers perfused with 1X PBS (Thermo Fisher Scientific, Waltham, MA, USA) were digested with 2 mg/mL type IV collagenase (Worthington, NY, USA) to obtain single-cell suspensions and the lymphocytes were enriched by Percoll gradient centrifugation as described previously [11 (link)]. Enriched lymphocytes were stained with fluorochrome-conjugated antibodies and fixable viability stain (BD Biosciences, San Jose, CA, USA), and samples were acquired on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Bethesda, MD, USA) equipped with five lasers. Data were analyzed using FlowJo software v X.10 (Tree Star, Inc., Ashland, OR, USA) as described previously [11 (link)]. Total numbers were computed by multiplying a proportion of specific populations by the total number of enriched lymphocytes/liver. fluorochrome-conjugated antibodies against CD45 (30-F11), CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), CD44 (IM7), CD69 (H1.2F3), Tbet (4B10), α₄β₇ (LPAM-1; DATK32) and Ly6C (AL-21) were purchased from BD Biosciences (San Jose, CA, USA). fluorochrome-conjugated antibodies against Foxp3 (FJK-16s), Ki67 (SolA15), NK1.1 (PK136), F4/80 (BM8), CD11b (M1/70), Ly6G (RB6-8C5) and Live/Dead Aqua were purchased form ThermoFisher Scientific (Waltham, MA, USA).