Cd8 buv496

The AIM assay for epitope identification was performed mirroring the previously described protocol (Tarke et al., 2021). Cryopreserved PBMCs were thawed in RPMI 1640 media supplemented with 5% human AB serum (Gemini Bioproducts) in the presence of benzonase [20 μl/10 ml]. Cells were stimulated for 24 hours in the presence of CCC specific MPs or mesopools at 1 μg/ml and then deconvoluted with 15-mer peptides [10 μg/ml] to reach the epitope level. Stimulation was carried out in 96-wells U bottom plates with 1×10⁶ PBMC per well. An equimolar amount of DMSO was used as negative control in triplicates, while stimulation with phytohemagglutinin (PHA, Roche, 1 μg/ml) was included as the positive control. The cells were stained with CD3 AF700 (2:100; Life Technologies Cat# 56–0038-42), CD4 BV605 (1:100; BD Biosciences Cat# 562658), CD8 BUV496 (2:100; Biolegend Cat#612942), CD14 V500 (2:100; BD Biosciences Cat# 561391), CD19 V500 (2:100; BD Biosciences Cat#561121), and Live/Dead eFluor 506 (25:1000; eBioscience Cat# 65–0866-18). Activation was measured by the following markers: CD137 APC (4:100; Biolegend Cat# 309810) and OX40 PE-Cy7 (2:100; Biolegend Cat#350012). All samples were acquired on ZE5 cell analyzer (Bio-rad laboratories) and analyzed with FlowJo software (Tree Star).

The AIM assay for epitope identification was performed mirroring the previously described protocol (Tarke et al., 2021). Cryopreserved PBMCs were thawed in RPMI 1640 media supplemented with 5% human AB serum (Gemini Bioproducts) in the presence of benzonase [20 μL/10 mL]. Cells were stimulated for 24 h in the presence of CCC specific MPs or mesopools at 1 μg/mL and then deconvoluted with 15-mer peptides [10 μg/mL] to reach the epitope level. Stimulation was carried out in 96-wells U bottom plates with 1 × 10⁶ PBMC per well. An equimolar amount of DMSO was used as negative control in triplicates, while stimulation with phytohemagglutinin (PHA, Roche, 1 μg/mL) was included as the positive control. The cells were stained with CD3 AF700 (2:100; Life Technologies Cat# 56-0038-42), CD4 BV605 (1:100; BD Biosciences Cat# 562658), CD8 BUV496 (2:100; Biolegend Cat#612942), CD14 V500 (2:100; BD Biosciences Cat# 561391), CD19 V500 (2:100; BD Biosciences Cat#561121), and Live/Dead eFluor 506 (25:1000; eBioscience Cat# 65-0866-18). Activation was measured by the following markers: CD137 APC (4:100; Biolegend Cat# 309810) and OX40 PE-Cy7 (2:100; Biolegend Cat#350012). All samples were acquired on ZE5 cell analyzer (Bio-rad laboratories) and analyzed with FlowJo software (Tree Star).