Negative magnetic selection

Effector memory CD4⁺ T cells were purified from cryopreserved PBMCs by magnetic negative selection (Miltenyi) and rested overnight in RPMI/10%FBS media. The following day, cells were stimulated with PMA (50ng/mL) and ionomycin (1μg/mL) for 6 hours. Brefeldin A and monensin (both 1:1000, eBioscience) were added for the last 5 hours. Cells were washed twice in cold PBS, incubated for 30 minutes with Fixable Viability Dye eFluor 780 (eBioscience), washed in PBS/1%BSA, and stained with anti-CD4-BV650 (RPA-T4), anti-CD27-BV510 (TB01), anti-HLADR-BV605 (G46–6), anti-CD20-APC-Cy7 (2H7), and anti-CD14-APC-Cy7 (M5E2). Cells were then washed and fixed and permeabilized using the eBioscience Transcription Factor Fix/Perm Buffer. Cells were then washed in PBS/1%BSA/0.3% saponin and incubated with anti-IFN-γ-FITC (B27), anti-TNF-PerCp/Cy5.5 (mAb11), anti-IL-10-PE (JES3–9D7) and anti-IL-2-PE/Cy7 (MQ1–17H12) or anti-granzyme A-AF647 (CB9) and anti-perforin-PE/Cy7 (B-D48) for 30 minutes, washed once, filtered, and data acquired on a BD Fortessa analyzer. Gates were drawn to identify singlet T cells by FSC/SSC characteristics, and dead cells and any contaminating monocytes and B cells were excluded by gating out eFluor 780-positive, CD20+, and CD14+ events.

Splenic neutrophils were purified by mechanical disaggregation of freshly isolated spleen without the use of erythrocyte lysis. Splenic neutrophils were isolated for RNAseq analysis pre-enrichment by magnetic negative selection (Miltenyi), followed by sorting of CD11b^hiLy6G^hiLy6b^hiLy6C^int cells with doublet and dead (DAPI+) cell exclusion to obtain a purity of ≥97% CD11b^hiLy6G^hi cells, as determined by post-sort analysis. Neutrophils were kept cold (at 4˚C) throughout the entire course of neutrophil isolation and handled gently to avoid mechanical activation. Antibodies used for FACS purification: anti-Ly6B (REA115), anti-Ly6C (HK1.4), anti-Ly6G (1A8), anti-CD11b (M1/70).

All procedures were performed with approval of the Ohio State University Institutional Review Board. Normal human pediatric tonsils were obtained following routine tonsillectomy from Nationwide Children’s Hospital (Columbus, OH). ILC3s and developmental precursors were isolated as previously described (20 (link)). Briefly, total mononuclear cells were depleted of CD19⁺ and/or CD3⁺ cells via magnetic negative selection (Miltenyi Biotec). For some experiments, B and/or T cell depleted mononuclear cells were used immediately for flow cytometric analysis. Alternatively, ILC3s were sorted directly from the depleted fraction by gating on CD3⁻CD14⁻ CD19⁻CD20⁻CD34⁻CD16⁻CD94⁻CD117⁺ events on a FACSAria II cell sorter (BD Biosciences). Purity analysis routinely revealed that sorted populations were ≥97% pure.

Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll density gradient centrifugation. As described before^{50 (link)}, CD4⁺ T cells were purified from PBMC using magnetic negative selection (Miltenyi Biotech), cultured in Xvivo15 supplemented with 10% inactivated human AB serum, and activated in vitro with Phytohemagglutinin (PHA) (2.5 μg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann-La Roche Inc)^{51 (link)} for 1 to 3 days prior to HIV-infection. Purity higher than 98% was obtained after CD4⁺ T cell magnetic isolation.