Journal: Molecular cell

Article Title: Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities

doi: 10.1016/j.molcel.2020.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Fluidigm Access Array Barcodes , Fluidigm , 101–0744.

Techniques: Recombinant, Knock-Out, DNA Extraction, Amplification, Sequencing, Plasmid Preparation, Software

Journal: Frontiers in Immunology

Article Title: MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection

doi: 10.3389/fimmu.2023.1294565

Figure Lengend Snippet: Comparable antigen-specific labeling of TCR-transgenic CD8 + Jurkat and healthy donor-derived viral epitope-specific CD8 + T cell populations with peptide-loaded dsSCD and dtSCT multimers. (A) Schematic representation of dsSCD for the usage as antigen-specific labeling reagent for defined CD8 + T cell populations. Following an overnight peptide pulse, the biotinylated dsSCD are multimerized by the addition of a fluorochrome-labeled streptavidin. (B, C) Cognate dtSCD and dsSCD multimer labeling of CD8 + Jurkat 76 (J76 CD8+ ) stably expressing various published TCR as recombinant chimeric TCR containing murine Cβ and Cα domains. (B) J76 CD8+ cells stably expressing RA14 TCR recognizing HLA-A*02:01/ HCMV pp65 495-503 or DMF5 TCR recognizing HLA-A*02:01/MART-1 26-35 were labeled with HLA-A*02:01 dsSCD previously loaded at varying concentrations with peptide HCMV pp65 495-503 (red symbols) or MART-1 26-35 (blue symbols), respectively, for 18 h followed by multimerization. The upper panel shows representative histograms of RA14 and DMF5 J76 CD8+ labeled with dsSCD multimers loaded with 25 µM peptide (filled line, MFI values in plain font) or dtSCT multimers (dotted line, MFI values in italics ). PE fluorescence-minus-one (FMO) controls of J76 CD8+ are shown in filled gray. The lower panel shows RA14 (circles) and DMF5 (triangles) J76 CD8+ staining efficiencies at varying peptide loading concentrations used for dsSCD loading in triplicates and fitting using non-linear regression. For control, RA14 and DMF5 J76 CD8+ cells were stained with dsSCD loaded with the non-cognate peptide. (C) Antigen-specific staining of published neoepitope or tumor-associated antigen-specific HLA-A*11:01, HLA-C*03:04 and HLA-C*08:02-restricted TCR with multimerized dtSCT and dsSCD harboring the cognate peptide (teal) or a dsSCD/dtSCT-binding control peptide (black). (D, E) Detection of virus-specific human CD8 + T cell populations in healthy donors with dsSCD and dtSCT multimers using combinatorial dual-fluorochrome encoding. (D) Labeling of HLA-A*02:01 + healthy donor 01 (HD-01) CD8 + T cells from PBMC with HLA-A*02:01 multimers generated on the basis of dsSCD, dtSCT or the commercial easYmer. The upper panel shows a representative dot plot of a HCMV pp65 495-503 /HLA-A*02:01-specific CD8 + T cell population. The lower panel shows the identification of four different HLA-A*02:01-restricted multimer + populations found in three independent experiments across all three tested pMHC-I multimer platforms. The data set was statistically analyzed by two-way ANOVA followed by Tukey’s multiple comparison test and no significant (ns) differences between the platforms were found. (E) Labeling of multiple HLA-typed healthy donors with pairs of dtSCT and dsSCD multimers representing 16 known viral and tumor-associated epitopes. Multimer + populations using HLA-A*01:01, A*03:01, B*08:01 or C*06:02 dtSCT and dsSCD in HD-04 and HD-06 are exemplarily shown. (F) Correlation analysis of antigen-specific T cell frequencies detected by HLA-A*01:01, A*02:01, A*03:01 or C*06:02 dsSCD and dtSCT multimers in PBMC-derived CD8 + T cells of HD-01–07. The individual T cell frequencies and specificities are listed in Supplementary Table 2 .

Article Snippet: CD8 + T cells labeled with the DNA-barcoded pMHC-I multimer library were kept in pMHC staining buffer and pMHC-I multimer positive cells were sorted with a FACSAria™ Fusion cell sorter (BD Biosciences) according to the gating strategy shown in into tubes containing 200 μl pMHC staining buffer.

Techniques: Labeling, Transgenic Assay, Derivative Assay, Stable Transfection, Expressing, Recombinant, Fluorescence, Staining, Binding Assay, Virus, Generated, Comparison

Journal: Frontiers in Immunology

Article Title: MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection

doi: 10.3389/fimmu.2023.1294565

Figure Lengend Snippet: Analysis of HLA-A*02:01 dsSCD with cleavable β2m linker. (A) Schematic representation of a disulfide-stabilized β 2 m-HLA-A*02:01 single-chain dimer with an additional HRV 3C cleavage site at the C-terminal end of the glycine-serine linker between β2m and MHC-I ectodomain (dsSCD*). (B) SDS-PAGE analysis of affinity chromatography-purified monomeric HLA-A*02:01 with (dsSCD*) and without (dsSCD) HRV 3C cleavage site incubated overnight in the presence (+) or absence (–) of HRV 3C protease. After dsSCD* cleavage with HRV 3C, free linker-extended β2m is visible. For comparison human β 2 -microglobulin isolated from urine is shown. (C) Antigen-specific labeling of DMF5 and 1G4-TCR transgenic J76 CD8+ cells with multimerized peptide-loaded dsSCD* with non-covalent β 2 m association (+ HRV 3C) and covalent β 2 m association (- HRV 3C). Here, dsSCD* were incubated with HRV 3C overnight followed by a consecutive peptide pulse (25 µM) and multimerization. A corresponding pMHC-I multimer staining using peptide-loaded dsSCD without HRV 3C cleavage site and dtSCT carrying the same peptides is additionally shown. (D) Analysis of the FITC peptide-loading capacity of bead-immobilized dsSCD* and dsSCD treated with HRV 3C. HLA-A*02:01 dsSCD and dsSCD* were incubated overnight in the presence (+) or absence (–) of HRV 3C protease and additional supplementation with a 4.6-fold molar excess of free β2m (+) or no β2m (–) during this incubation step. Treated dsSCD and dsSCD* were immobilized on streptavidin beads and loaded with 1 µM NLVPK FITC VATV peptide overnight followed by flow cytometric analysis. Data represent mean values from 3 independent experiments in triplicates with statistical analysis by one-way ANOVA test followed by Tukey’s multiple comparison test. Error bars show the standard deviation. ns, not significant; *p< 0.05; **p< 0.01; ****p< 0.0001.

Article Snippet: CD8 + T cells labeled with the DNA-barcoded pMHC-I multimer library were kept in pMHC staining buffer and pMHC-I multimer positive cells were sorted with a FACSAria™ Fusion cell sorter (BD Biosciences) according to the gating strategy shown in into tubes containing 200 μl pMHC staining buffer.

Techniques: SDS Page, Affinity Chromatography, Purification, Incubation, Comparison, Isolation, Labeling, Transgenic Assay, Staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection

doi: 10.3389/fimmu.2023.1294565

Figure Lengend Snippet: Detection of neoepitope- and TAA-specific CD8 + T cell populations in the peripheral blood of melanoma patient using multimerized easYmers, dtSCTs and dsSCD (MediMers) in a complementary manner. (A) Identified antigen-specific CD8 + T cell populations in the patient’s peripheral blood by dual color-encoded pMHC-I multimer staining covering all six HLA alleles of a melanoma patient. In total, 148 pMHC-I multimers were generated (107 predicted neoepitopes, 24 non-mutant tumor-associated antigens (TAA) and 17 viral epitopes) on the basis of a dsSCD for HLA-C*08:02 and easYmers covering the remaining five HLA-I allotypes. EasYmer HLA-B*14:01 was used as a surrogate for the patient’s HLA-B*14:02 allotype. For the initial cell staining up to 60 dual color-encoded pMHC-I multimer pairs were used in one single pMHC-I library and detected pMHC-I multimer + populations were verified afterwards by at least two additional independent stainings with up to 10 dual color-encoded pMHC-I multimer pairs. Representative dot plots are shown in Supplementary Figure 3D . Zero values were converted to 0.0001 to allow for plotting on a log scale. (B, C) pMHC-I multimer-guided single-cell TCR repertoire and cell surface protein expression analysis. pMHC-I multimer + CD8 + T cells were cell sorted using a pMHC-I multimer library comprising uniquely DNA-barcoded and population size-dependent dual color-encoded pMHC-I multimer pairs (see Supplementary Figure 2C ) combined with a panel of 30 DNA-barcoded cell phenotyping antibodies ( Supplementary Table 4 ). Multimerized dsSCD, easYmer and dtSCT were used in combination. An experimentally obtained 10x scSeq data set is shown as tSNE plot based on surface marker expression including the pMHC-I multimer labeling of a total of 3236 individual cells represented by dots. Cell clustering based on pMHC-I multimer barcode detection (B) and cell surface expression of selected T cell memory markers (C) is shown. (D, E) Validation of cloned MAGE-A3 and NY-ESO-1-specific TCR from the 10x scSEQ data set. TRBV/TRBJ and TRAV/TRAJ subtypes and respective CDR3 sequences are displayed in the table at the bottom. (D) Antigen-specific staining of a MAGE-A3 168-176 /A*01:01-specific TCR (MM-01) and two NY-ESO-1 139-147 /HLA-C*08:02-specific TCR (MM-02 and MM-03) expressed in J76 CD8+ cells with multimerized dtSCT and dsSCD representing the cognate (teal lines) or a viral control (black lines) epitope. (E) Co-culture of TCR-expressing J76 CD8+ cells with autologous peptide-pulsed B cells. The expression of early T cell activation marker CD69 ± SD in [%] of TCR + J76 CD8+ cells after 18h co-culture in triplicates in presence of cognate (teal) or a control (black) peptide at various concentrations. Two cloned dominant TCRs (MM-04 and MM-05) derived from the OSGEP V91D /HLA-C*08:02 multimer scSEQ cluster lacked expression in J76 CD8+ cells or were not stained by corresponding the pMHC-I multimer, respectively. ND, not determined.

Article Snippet: CD8 + T cells labeled with the DNA-barcoded pMHC-I multimer library were kept in pMHC staining buffer and pMHC-I multimer positive cells were sorted with a FACSAria™ Fusion cell sorter (BD Biosciences) according to the gating strategy shown in into tubes containing 200 μl pMHC staining buffer.

Techniques: Staining, Generated, Mutagenesis, Expressing, Marker, Labeling, Clone Assay, Co-Culture Assay, Activation Assay, Derivative Assay

Journal: bioRxiv

Article Title: Enhancing metabarcoding efficiency and ecological insights through integrated taxonomy and DNA reference barcoding: a case study on beach meiofauna

doi: 10.1101/2024.04.01.587521

Figure Lengend Snippet: A) Map showing the location of the 24 beaches sampled for metabarcoding (blue circles) and locations sampled for reference barcoding (red squares). Note that reference barcoding sites in close proximity to each other are not shown separately due to the map scale. The mini map shows the locations of the sampling area in Europe. B) Schematic view of a beach showing the location of the eight sampling points per transect from dunes to the low tide line.

Article Snippet: Combining morphological identification with molecular methods is increasingly feasible due to the development of rapid DNA barcoding pipelines relying on Oxford Nanopore technology ( , ), potentially allowing the production of molecular references in field locations ( ; ), while taxonomic experts are sorting and identifying specimens.

Techniques: Sampling