blasticidin  (InvivoGen)

Journal: Nature Communications

Article Title: Whole-protein screening and multi-modal profiling of antigen-specific CD4 + T cells at single-cell resolution

doi: 10.1038/s41467-026-72396-7

Figure Lengend Snippet: a Genetic design encoding the class II SCT protein. The MHC α and β chains are connected to the antigen through flexible linkers (L1, L2, and a partial invariant chain pIi), followed by purification tags (AviTag/HisTag). A secretion signal is placed upstream of the α chain to facilitate protein export. Created in BioRender . b Streamlined workflow for high-throughput SCT plasmid construction and protein expression. High-throughput production of SCT libraries is enabled by automated primer design and an optimized pipeline for plasmid assembly and protein expression/purification. Created in BioRender . c Flow cytometric assay to assess binding of SCT tetramers of various common class II HLA alleles to their cognate TCRs ( n = 3 ). Previously reported TCR-antigen pairs: DRB1*01:01 (DR1) – mutTPI 23-37 :T28I, DRB1*11:01 (DR11) – HIV Gag 299-311 , DRB1*07:01 (DR7) – ADM2 51–65 , DPB1*04:01 (DP4) – mutPly 427–441 (Supplementary Data ). d Comparison of the occurrence of aromatic residues F/W/Y in 2-mers that are positively correlated with SCT expression versus those that are negatively associated. The p value was calculated using Fisher’s exact test on the full 2 × 2 contingency table comparing F/W/Y presence across groups. e A 23-Element DRB1*01:01 CEFT SCT library. SCT expression was evaluated through SDS-PAGE. L, molecular weight ladder; +, protein standard for quantification of expression level. Schematics created in BioRender . f Antigen-specific T cells were detected through dual tetramer staining to enhance the true positive rate. An HIV Gag 41–56 SCT tetramer was used to exclude non-specific binding cells. A representative flow cytometry plot is shown. g Tetramer binding validation of 16 identified CD4 TCRs, each reactive to one of the five CEFT antigens (A–E) ( n = 3 ). Error bars represent standard deviation. h Schematic of peptide-pulsed activation assay. TCR-transduced NFAT-GFP Jurkat cells were co-cultured overnight with peptides and DR1-K562 cells. Created in BioRender . i T cell activation measured through the percentage of GFP+ Jurkats ( n = 3 ). P < 0.01 for each group relative to the negative control peptide. Statistical significance was determined by a one-tailed independent t -test assuming equal variances. All replicates are biological replicates. All bar plots ( n = 3 ) are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: A plasmid encoding for NFAT-GFP reporter (VectorBuilder) was then lentivirally transduced into the TCR- Jurkats and treated with blasticidin (InvivoGen, ant-bl-05) for selection.

Techniques: Purification, High Throughput Screening Assay, Plasmid Preparation, Expressing, Flow Cytometry, Binding Assay, Comparison, SDS Page, Molecular Weight, Staining, Biomarker Discovery, Standard Deviation, Activation Assay, Cell Culture, Negative Control, One-tailed Test

Journal: Nature Communications

Article Title: Whole-protein screening and multi-modal profiling of antigen-specific CD4 + T cells at single-cell resolution

doi: 10.1038/s41467-026-72396-7

Figure Lengend Snippet: a Design of an SCT library with overlapping 15-mer peptides spanning the entire RBD of the SARS-CoV-2 spike protein. b . Schematic overview for the experimental workflow of high-throughput screening. 50 PBMC samples from 22 HLA-DR1+ participants were barcoded by timepoint, pooled, enriched for CD4 + T cells, stained with the 64-element SCT-dextramer pool, FACS-sorted, and sequenced. Single-cell analysis included RNA expression profiling, TCRαβ sequencing, antigen-MHC pairing, and timepoint identification. Comparison of genetic variants between transcriptome and whole-genome sequencing (WGS) was used to demultiplex patient identity (Methods). Created in BioRender . c Correlation between SCT expression levels, quantity of antigen-specific cells captured, and predictions from class II antigen-presentation algorithms. SCT expression quantified prior to purification ( n = 2–4 biological replicates). Data were shown as mean ± SEM. %Rank_EL, percentile rank of eluted ligands. d Clonal and patient diversity in CD4 + T cell responses to each SARS-CoV-2 antigen. Antigens are color-coded based on their parent protein. e Tetramer binding validation of SARS-CoV-2-specific CD4 TCRs. A representative subset of SARS-CoV-2 TCRs ( n = 10) from Fig. 2d were selected and validated through SCT tetramer binding assays with biological triplicates. A DR1-restricted influenza A M1 17–31 SCT was used as the negative control. f Peptide-pulsed activation of SARS-CoV-2 CD4 TCRs in NFAT-GFP Jurkats ( n = 3). e , f P < 0.0001 for each group relative to the negative control peptide, determined by one-tailed independent t -test assuming equal variances. Exact P values are provided in the Source Data. All replicates are biological replicates. All bar plots ( n = 3) are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: A plasmid encoding for NFAT-GFP reporter (VectorBuilder) was then lentivirally transduced into the TCR- Jurkats and treated with blasticidin (InvivoGen, ant-bl-05) for selection.

Techniques: High Throughput Screening Assay, Staining, Single-cell Analysis, RNA Expression, Sequencing, Comparison, Expressing, Immunopeptidomics, Purification, Binding Assay, Biomarker Discovery, Negative Control, Activation Assay, One-tailed Test