Pe magnetic beads

Direct analysis of T-cell frequency was accomplished using our previously published enrichment approach (12 (link)). Briefly, 30–50 × 10⁶ PBMCs were resuspended in 200 µL of T-cell media, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 μg/mL phycoerythrin (PE)-labeled tetramer at room temperature for 100 min, followed by antibody staining with CD4-APC (eBioscience), CD45RO-FITC (eBioscience), and a combination of CD14-PerCP and CD19-PerCP (BD Biosciences) for 15 min at 4°C. Cells were washed, incubated with PE magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and enriched with a magnetic column, retaining 1% of the cells as a nonenriched sample. The PE-enriched and precolumn samples were labeled with Via-Probe (BD Biosciences) and analyzed on a FACSCalibur (BD Biosciences), gating on CD4⁺CD14⁻CD19⁻Via-Probe⁻ and plotting tetramer versus CD45RO. Frequencies were calculated as previously described (12 (link)). Statistical analysis was performed using unpaired t tests with Welch correction with Prism software (version 5.03, GraphPad Software Inc.).

Analysis of T-cell frequency was accomplished using our previously published approach (26 (link)). Briefly, 40–60 × 10⁶ PBMCs were resuspended in a total of 400–600 µL media, divided into two or three independent tubes of 20 × 10⁶ cells (200 µL) each, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 µg/mL PE-labeled, PE-CF594–labeled, or PE-Cy5–labeled HIP tetramers at room temperature for 120 min (three tetramers per tube for a total of six hybrid peptide tetramers plus an nonimmunogenic control tetramer if adequate cells were available for a third staining tube). Cells were washed, incubated with PE-magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and magnetically enriched; 1% of the cells were retained as a nonenriched sample. Enriched (bound) and nonenriched (precolumn) samples were stained with CD4 V500, CD14 PerCP-Cy5.5, and CD19 PerCP-Cy5.5 (eBioscience) and CD45RA AF700 (BD), CXCR3 FITC, CCR6 BV421, and CCR4 BV605 (BioLegend) for 15 min at 4°C. After washing, cells were labeled with ViaProbe (BD Biosciences) and analyzed on a FACSCanto (BD Biosciences), gating on CD4⁺CD14⁻CD19⁻ViaProbe⁻ cells and plotting tetramer versus CD45RA. Frequencies were calculated as previously described (26 (link)).