Anti cd3 pe cy7 clone sk 7

Frozen PBMCs from HLA-A2 patients (at least 30 × 10⁶ cells at day 0 and 6 days after vaccination) were thawed as previously described.⁵⁰ Cells were washed twice with ice-cold PBS and were further subjected to magnetic bead separation with human CD8⁺ T Cell Isolation Kit (Miltenyi Biotec cat. no. 130-096-495). The purity of separation was determined with FACS analysis of CD3 (anti-CD3-PE-Cy7; clone SK-7, BioLegend cat. no. 344816) and CD8 (anti-CD8a-FITC; clone RPA-T8, BioLegend cat.no. 301006) cell surface markers. Separated cells were resuspended in a total volume of 50 μl PBS. Cells were incubated with PE-labeled HLA-A0201 MHC dextramers loaded with AHDL1A1_88-96 (LLYKLADLI) or negative control peptides (Immudex cat.no. WB3794, cat. no. WB2666) in the dark at room temperature for 10 min, according to manufacturer’s instructions. Next, cells were labeled with recommended amounts of anti-CD3-PE-Cy7 (clone SK-7, BioLegend cat. no. 344816), anti-CD8a-FITC (clone RPA-T8, BioLegend cat.no. 301006), anti-CD45-APC (clone HI100, BD Biosciences cat. no. 304112), and anti-CCR7-PerCP/Cy5.5 (clone G043H7, BioLegend cat. no. 353220) monoclonal antibodies in the dark at 4°C for another 20 min. Labeled cells were washed twice with PBS, resuspended in 0.4 ml PBS, and immediately analyzed on a FACSCanto flow cytometer. Data acquisition was performed using BD FACSDiva v.6.1.2.

Freshly isolated PBMCs (2 million) were washed once with PBS containing 5% FBS and re-suspended in a total volume of 50 μl PBS with 5% FBS. Cells were incubated with PE-labeled HLA-A0201 MHC dextramers loaded with AHDL1A1_88-96 (LLYKLADLI) or negative control peptides (Immudex cat.no. WB3794, cat. no. WB2666) in the dark at room temp. for 10 min, according to the manufacturer’s instructions. Next, cells were labeled with recommended amounts of anti-CD3-PE-Cy7 (clone SK-7, BioLegend cat. no. 344816), anti-CD8-APC (clone RPA-T8, BioLegend cat. no. 301049) and anti-CD4-FITC (clone SK3, BD Biosciences cat. no. 347413) monoclonal antibodies in the dark at 4°C for another 20 min. Labeled cells were washed twice with PBS with 5% FBS, and dead cells were stained with 7-AAD (BD Biosciences cat. no. 559925) in the dark for 10 min. Cells were resuspended in 0.4 ml PBS and immediately analyzed on a FACSCanto flow cytometer. Data acquisition was performed using BD FACSDiva v.6.1.2. Dextramer positive cells were analyzed within CD3⁺CD8⁺ gate, after the exclusion of doublets, CD4⁺, and dead cells. Supplemental Figure 1 presents the gating scheme for the analysis of ALDH1A1_88-96-specific CD8⁺ T cells.