Cd3 clone sk7

A total of 2 to 4 × 10⁶ human PBMCs expanded with specific Immuno-STAT or peptide were pretreated with brefeldin A (BFA) and monensin (ThermoFisher), plated in a 24-well plate, and stimulated at a 1:1 ratio with T2 cells (ATCC) that had been loaded with CMV pp65_495–503 (NLVPMVATV) or Mart1_26–35 (ELAGIGILTV) or HIV-1 p17 Gag_77–85 (SLYNTVATL; SL9) peptide for 2 h and washed twice. Cells were stimulated for 5 h, washed, stained with Fixed Viability Stain 780 (BD Biosciences), antibodies against CD3 (clone SK7, BioLegend), CD8 (clone SK1, BD Biosciences) and CD107a (clone H4A3, BD Biosciences), and fixed using IC fixation buffer (ThermoFisher). Cells were next washed in permeabilization buffer (eBioscience), stained with antibodies against TNF-α (clone MAb11, BD Biosciences), IFN-γ (clone 4S.B3, BioLegend), and granzyme B (clone GB11, BD Biosciences) for 30 min at room temperature, washed, and analyzed. For representative FACS plots and pairwise marker quantitation, PBMC were stimulated as described above using T2 cells loaded with 100 nM peptide. For peptide titration studies, PBMC were stimulated as described above using T2 cells loaded with 1 × 10^–13, 1 × 10^–12, 1 × 10^–11, 1 × 10^–10, 1 × 10^–9, 1 × 10^–8, 1 × 10^–7, 1 × 10^–6, or 1 × 10^–5 g/ml peptide.

The human CD14 positive selection kit (StemCell Technologies) was used to isolate or deplete CD14⁺ monocytes from PBMC according to the instructions of the manufacturer. CD14-depleted PBMC were cultured with 2.5% S. aureus-CFS in the presence or absence of LGG-CFS for 24 h with brefeldin A present during the last 4 h of incubation. Whole PBMC served as control. Isolated CD14⁺ monocytes were cultured at a concentration 0.5 × 10⁶/ml with 2.5% L. reuteri-CFS, 2.5% S. aureus-CFS, 100 ng/ml ultrapure LPS, or 10 μg/ml Pam3Cys (both Invivogen) for 14 h before culture supernatants were collected. Purity was assessed for all donors by staining with CD3 (clone: SK7) (Biolegend) and CD14 (clone: B159) (BD Biosciences). The mean percentage of CD14⁺ cells was 0.49 ± 0.28% in the monocyte-depleted PBMC and 97.4 ± 1.67% after purification.