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: Communications Biology

Article Title: RNA isoform diversity, splicing variants and switching in single cells of the Alzheimer’s disease brain

doi: 10.1038/s42003-026-09759-9

Figure Lengend Snippet: a Schematic representation of experimental workflow for single-nucleus short-read RNA-sequencing and Kinnex long-read RNA-sequencing. Figure 1a. was partially created with BioRender. https://BioRender.com/c44n540 . b UMAP plot colored by cell type assignments. c Bar plot showing the proportions of each cell type in AD and ND samples (ns: not significant; Two-sided t-test). d Differentially expressed genes (DEGs) identified across different cell types in AD and ND samples (absolute log2 fold change > 0.25 and adjusted p -value < 0.05, Wilcoxon Rank Sum test with Bonferroni correction). e–h Circos plots of five selected Gene Ontology (GO) terms for excitatory neurons, microglia, oligodendrocytes, and astrocytes with associated genes (false discovery rate (FDR) of <0.05 with the Benjamini-Hochberg (BH) test). Conserved GO terms are shown in the same color.

Article Snippet: Single-nucleus barcoded cDNA libraries were generated using the 10X Genomics Single Cell 3’ v3.1 kit.

Techniques: RNA Sequencing