H 2 db sslenfrayv pe

CD8+ T cells were isolated from PBMCs using a negative-selection microbeads kit (Miltenyi Biotec) according to the manufacturer’s protocol. Next, CD8+ T cells were labeled at room temperature for 20 min with H-2 Db ASNENMETM-APC and H-2 Db SSLENFRAYV-PE (Immudex, Virum, Denmark). Next, surface staining was performed using the following mAbs: CD3(17A2)FITC, CD4(GK1.5)- BrilliantViolet510, and CD8(53.6.7)-BrilliantViolet786 (all BD). CD3+CD4−CD8+dextramer+ cells were then sorted directly into RNAlater (Ambion Inc. Applied Biosystems) using a FACS Melody (BD) and subsequently stored at −80 °C for TCRβ clonotype analysis.

Approximately 2 million splenocytes, 2 million lung-derived lymphocytes, 4 million BM cells, and lysed whole blood were used for dextramer staining. Cells were stained using the commercial H-2 Db ASNENMETM-APC and H-2 Db SSLENFRAYV-PE (Immudex, Virum, Denmark) for 20 min at RT in the dark. Surface staining was added containing the following antibodies: CD44(IM7)-BrilliantBlue515, CD103(2E7)-BrilliantBlue700, CD4(RM4-5)-AF700, Fixable Viability Stain 780, CD8a(53-6.7)-BrilliantViolet510, CD62L(MEL-14)-BrilliantViolet650, CD49a(Ha31/8)-BrilliantViolet786, CD69(H1.2F3)-PE/Cy7, KLRG-1(2F1)-PE-CF594, CD3e(145-2C11)-Brilliant UV395 (all BD Biosciences), and CD127-A7R34-BV711 (Biolegend). These were incubated for 30 min at 4 degrees. After washing twice, cells were resuspended in FACS buffer. Acquisition was performed on an LSR Fortessa X-20 (BD), and data analyses were performed using FlowJo v10.6.2 (BD).