Apc sa

For selected neopeptides, pMHC tetramers were generated for staining of neoepitope-specific T cells. Neopeptides were selected based on the observed NART responses from the DNA barcode-labelled multimer screening. Following the observed increase in NART responses at 3 weeks post-treatment, NART responses in 3-week post-treatment PBMC samples were interrogated wherever sufficient patient material remained, otherwise 9-week post-treatment samples were analyzed. Single-fluorochrome pMHC specificity tetramers using were generated as described in detail previously^{81 (link),82 (link)}, using a library of streptavidin (SA)-conjugated flourochromes consisting of PE-SA (BioLegend, cat. #405204), APC-SA (BioLegend, cat. #405207), BV421-SA (BD, cat. #563259), PE-Cy7-SA (BD, cat. #557598), BV605-SA (BD, cat. #563260), PE-CF594-SA (BD, cat. #562284), BV650-SA (BD, cat. #563855), BUV395-SA (BD, cat. #564176). Up to eight patient-specific pMHC tetramers per sample were investigated. PBMC samples were stained with respective library of pMHC tetramers and with an antibody mix consisting of CD8-BV480, dump channel antibodies and a dead cell marker, as above. Tetramer-specific T cells analyzed as lymphocytes, single, live, CD8⁺, FITC^‒ and tetramer⁺ cells. Due to staining strategy, tetramer⁺ cells were gated by being CD8⁺.

Secondary antibodies: rat anti-mouse IgG2a-bio (BioLegend), rat anti-mouse Igλ-bio (BD Biosciences), rat anti-mouse Igκ-bio (BD Biosciences), and rat anti-mouse Igκ-FITC (BD Biosciences). Streptavidin (SA)-conjugated detection proteins: PE-SA (eBioscience) and APC-SA (BioLegend).
E0771-GFP cells or SK-BR-3 cells (a Her2-positive cell line) were routinely cultured. Cells were then blocked in FACS buffer containing human IgG (1 μL/50 μL FACS buffer). Mouse serum, obtained from tumor-bearing or control animals, was added to the cells at dilutions of 1/10 and 1/100, and the cells were incubated on ice for 15 min. Cells were washed and biotinylated secondary anti-mouse-Ig antibodies were added and incubated for 15 min on ice. Again, cells were washed, and SA-conjugated fluorochromes were added, and the cells were fixed with 1% paraformaldehyde. Stained cells were acquired for analysis using a BD FACSCalibur, and results were analyzed using FCS express software. The amount of antibody binding to whole E0771-GFP tumor cells was quantified by relative fluorescence intensity (relative to the average mean fluorescence intensity of control group).

The recombinant SARS-CoV-2 prefusion stabilized S protein and RBD were biotinylated using an EZ-Link Micro Sulfo-NHS-LC Biotinylation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Probes were generated by coupling biotinylated proteins to fluorophore-conjugated streptavidin (SA) molecules for detection by flow cytometry (SA-BV421, SA-APC, and SA-PE, BioLegend). Isolated PBMCs were stained by a panel of antibodies listed in Table S3 as well as S protein and RBD probes (S-APC, S-PE, and RBD-BV421, 100 ng each). The samples were analyzed on a BD Aria III Fusion cell sorter (weeks 6 and 30) or a BD LSRFortessa flow cytometer (all other time points). At weeks 6 and 30, memory B cells (CD3⁻ CD11c⁻ CD14⁻ CD16⁻ CD123⁻ HLA-DR⁺ CD20⁺ IgM⁻ IgG⁺) double-positive for S protein binding were single cell sorted into 96-well plates and frozen immediately on dry ice for subsequent B cell receptor (BCR) amplification. Data were analyzed using FlowJo v.10.7.1 (FlowJo).