Journal: Science Advances

Article Title: Simultaneous analysis of pMHC binding and reactivity unveils virus-specific CD8 T cell immunity to a concise epitope set

doi: 10.1126/sciadv.adm8951

Figure Lengend Snippet: ( A ) Biorender experimental flow chart. ( B ) Multimer-library size per virus families selected from IEDB . ( C and D ) Multimer-binding following 24-hour stimulation in three donors. (C) Representative cell sorts of antigen-specific CD8 T cells using barcode-labeled pMHC multimers. Cells were sorted from upper quadrant gates. Stim A1, Stim B8, and Stim All refer to pool-based stimulations with 36 A*01:01-restricted peptides, 50 B*08:01-restricted peptides, and the collective pool of 86 peptides, respectively. All conditions were recorded in technical triplicates. (D) Enrichment scores for select multimer-specific CD8 T cells across three donors and three to four stimulatory settings. Stim A2 refers to the pool-based stimulations with 96 A*02:01-restricted peptides. Samples were pregated for live CD14 − CD19 − CD3 + CD4 − CD8 + lymphocytes. See all remaining populations in fig. S2 (F and G). ( E ) Seventy virus-specific populations were grouped according to whether the respective stimulatory setting included the peptide used for multimer-generation (cognate stim) or not (bystander stim). ( F ) PBMCs from 48 donors were individually stimulated with pools restricted to one to six donor-derived HLA alleles. Multimer + CD8 T cells were sorted and barcodes sequenced . Samples were pregated on live CD14 − CD19 − CD3 + CD4 − CD8 + lymphocytes. Additional flow cytometry is available in fig. S2H. ( G ) Correlation between observed change in multimer frequency [Δmultimer (%)] and change in CD69 + CD137 + frequency [ΔAIM (%)] upon stimulation. The shaded area is the 95% confidence interval. Spearman correlation was used. Unpaired Wilcoxon test between grouped unstimulated/bystander and cognate stimulated samples was performed in (D). P values were calculated using Dunn’s test in (E) and adjusted using Benjamini-Hochberg. Boxplot bounds are 25th and 75th percentiles along with the median. Upper and lower whiskers span the range of data up to 1.5× of the IQR. ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant. All experiments were performed once.

Article Snippet: Assembly of DNA barcode-labeled dextran multimer libraries was performed using biotinylated combinatorial DNA barcodes ( ) (LGC Biosearch, 2.17 μM), fluorescent streptavidin-dextran conjugates (Fina Biosolutions Inc., 160 nM), and custom recombinant, biotinylated, UV-cleavable pMHC monomers (50 μg/ml or approximately 1 μM).

Techniques: Virus, Binding Assay, Labeling, Derivative Assay, Flow Cytometry

Journal: Science Advances

Article Title: Simultaneous analysis of pMHC binding and reactivity unveils virus-specific CD8 T cell immunity to a concise epitope set

doi: 10.1126/sciadv.adm8951

Figure Lengend Snippet: ( A ) Combinatorial encoding of fluorescent pMHC tetramers, experimental setup, and data processing for verification of 62 different epitope-specific CD8 T cell populations across 12 donors. Antigen-specific CD8 T cells for a given fluorophore combination were pregated on CD14 − CD19 − CD3 + CD4 − CD8 + T cells negative for irrelevant tetramer fluorophores. Representative tetramer gating is shown from donor 310. ( B ) Correlation between observed frequency measured through DNA-labeled MHC multimers (see Materials and Methods) and verified frequency using fluorescent-labeled tetramers. ( C ) Markers of memory and effector differentiation CCR7 and CD45RA for bulk and tetramer-positive CD8 T cells of donor 310. ( D ) Summary on tetramer frequency, memory phenotypes, and GzmB expression measured by flow cytometry for antigen-specific T cells split by virus origin. ( E ) Quantification of CD8 MFI, Tn, Tscm, Tcm, Tem, Temra, and GzmB MFI for tet + CD8 T cells grouped by their observed reactivity from initial stimulation and multimer screening. Spearman correlation was performed in (B). P values were calculated using Dunn’s test and adjusted using the Benjamini-Hochberg method. Boxplot bounds show the 25th and 75th percentiles along with the median. Upper and lower whiskers span the range of data up to 1.5× of the IQR. * P < 0.05, ** P < 0.01, *** P < 0.001. Tet, tetramer. Experiment was performed once.

Article Snippet: Assembly of DNA barcode-labeled dextran multimer libraries was performed using biotinylated combinatorial DNA barcodes ( ) (LGC Biosearch, 2.17 μM), fluorescent streptavidin-dextran conjugates (Fina Biosolutions Inc., 160 nM), and custom recombinant, biotinylated, UV-cleavable pMHC monomers (50 μg/ml or approximately 1 μM).

Techniques: Labeling, Expressing, Flow Cytometry, Virus

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sample-multiplexing approaches for single-cell sequencing

doi: 10.1007/s00018-022-04482-0

Figure Lengend Snippet: Characteristics of sample-multiplexing approaches used for single-cell sequencing

Article Snippet: sci-ATAC-seq , 20150522 , Epigenomics , Chromatin accessibility , □ , Combinatorial barcodes are introduced into DNA fragments by Tn5 and PCR , NA , >15000 , > 400 sites , Nuclei , GM12878, HL60, HEK293T , Single-cell combinatorial indexing (x-well plate) , [ 62 ].

Techniques: Membrane, Clinical Proteomics, Transfection, Transgenic Assay, shRNA, Polymerase Chain Reaction, DNA Methylation Assay, CRISPR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sample-multiplexing approaches for single-cell sequencing

doi: 10.1007/s00018-022-04482-0

Figure Lengend Snippet: Schematic overview of five sample-multiplexing strategies used for scRNA-seq. A Natural genetic variation. Without additional labeling, computational demultiplexing is conducted based on SNPs. B Nucleotide-barcode anchoring on cellular or nuclear membranes. The example shown here is Cell Hashing, where oligo-tagged antibodies (hashtags) bind to ubiquitously expressed cell-surface proteins. Oligos with a poly (A) tail are captured along with mRNA. Cells can be assigned to their sample of origin based on different barcodes in the hashtags. C Nucleotide-barcode internalization into the cytoplasm or nucleus. Barcoded DNA traverses the cellular or nuclear membrane by liposomal transfection or directly diffuses into the nuclei. SBO: short barcode oligonucleotide. D Vector-based barcode expression in cells. E Nucleotide-barcode incorporation during library construction

Article Snippet: sci-ATAC-seq , 20150522 , Epigenomics , Chromatin accessibility , □ , Combinatorial barcodes are introduced into DNA fragments by Tn5 and PCR , NA , >15000 , > 400 sites , Nuclei , GM12878, HL60, HEK293T , Single-cell combinatorial indexing (x-well plate) , [ 62 ].

Techniques: Multiplexing, Labeling, Membrane, Transfection, Plasmid Preparation, Expressing