Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . CD8+ T cells from 5 donors stimulated 3x with pools of 10 peptides, except pool 9 which had 6 peptides. C . 3D modeling of VP1 pentamer highlighting epitopes (Green: VP1 (100-108), Yellow: VP1 (251-259), (253-262), (274-283), Purple: Published epitopes, Blue: Predicted epitopes from IEDB. D . CD8+ T cells from 3 donors, 10 cell lines each, stimulated 3x with pools of peptides and then individual T2 IFN- γ response.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques:

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . Pooled CD8+ T cell lines from 3 healthy donors were split into three different fractions and stained with low, intermediate, or high tetramer concentration. From samples stained with low and intermediate tetramer concentration, live CD3+ CD8+ T cells were sorted from the 90th percentile tetramer(+) gates. Shown here is the cumulative clone count in the 90th percentile gates (y-axis) plotted against maximum enrichment score out of the 11 calculated for each clone (x-axis). Symbol size corresponds to clone count in tetramer-negative gate. Symbol color indicates clone abundance in the single cell TCR dataset (low tetramer concentration condition). High affinity TCRs have high cumulative clone count, high maximum enrichment score, and small symbol size. SumSort = total number in top 10% tetramer binder gate (i.e. 90th percentile) in the 1:2000 + 1:4000 tetramer staining condition. MaxFold = greatest value for fold enrichment (out of the calculated 11). C . Top 14 T cell receptors were virally transduced into human primary T cells in 2 donors. TCR-transduced cells were stimulated with different amounts of VP1(100-108) peptide and tested for intracellular INF- γ content as ratio of positive control (max). EC50 (M) scores populated in tabular format.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Staining, Concentration Assay, Positive Control

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . TCR 3 and TCR 8 tested against K562 presenting VP1(100-108) on HLA-A2 vs a control TCR (DMF5) with its corresponding peptide (MART1) after 48h incubation at unsorted E:T of 0.5 and 2. Error bars repres nt SEM for 3 technical replicates from one representative experiment. p<0.001 by unpaired T test. C . After 72 hours of co-culture with JCV-infected SVG cells and no T cells (no TCR), sham TCR, TCR 3, and TCR 8 looking at live CD8+ T cell CD137 positivity via flow cytometry. TCR 3 and 8 vs no TCR p value <0.001; TCR 3 vs sham TCR+JCV p=0.0032 and TCR 8 vs sham TCR+JCV p=0.029 D . Percent killing of SVG cells determined by flow cytometry based on gating of loss of live astrocytes. p value <0.001; TCR 3 vs sham TCR+JCV p<0.001 and TCR 8 vs sham TCR+JCV p=0.002 by unpaired T test. Done in duplicate at various unsorted E:Ts, mean results shown.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Control, Incubation, Co-Culture Assay, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: Flow cytometry data of live CD8+ T cells stained with HLA-A2:BK VP1 (100-108) and HLA-A2:JV VP1 (100-108) at 1:50 tetramer dilution with an HLA-A2 positive JCV antibody negative healthy donor on the left and an HLA-A2 confirmed PML survivor on the right.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . CD8+ T cells from 5 donors stimulated 3x with pools of 10 peptides, except pool 9 which had 6 peptides. C . 3D modeling of VP1 pentamer highlighting epitopes (Green: VP1 (100-108), Yellow: VP1 (251-259), (253-262), (274-283), Purple: Published epitopes, Blue: Predicted epitopes from IEDB. D . CD8+ T cells from 3 donors, 10 cell lines each, stimulated 3x with pools of peptides and then individual T2 IFN- γ response.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques:

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . Pooled CD8+ T cell lines from 3 healthy donors were split into three different fractions and stained with low, intermediate, or high tetramer concentration. From samples stained with low and intermediate tetramer concentration, live CD3+ CD8+ T cells were sorted from the 90th percentile tetramer(+) gates. Shown here is the cumulative clone count in the 90th percentile gates (y-axis) plotted against maximum enrichment score out of the 11 calculated for each clone (x-axis). Symbol size corresponds to clone count in tetramer-negative gate. Symbol color indicates clone abundance in the single cell TCR dataset (low tetramer concentration condition). High affinity TCRs have high cumulative clone count, high maximum enrichment score, and small symbol size. SumSort = total number in top 10% tetramer binder gate (i.e. 90th percentile) in the 1:2000 + 1:4000 tetramer staining condition. MaxFold = greatest value for fold enrichment (out of the calculated 11). C . Top 14 T cell receptors were virally transduced into human primary T cells in 2 donors. TCR-transduced cells were stimulated with different amounts of VP1(100-108) peptide and tested for intracellular INF- γ content as ratio of positive control (max). EC50 (M) scores populated in tabular format.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques: Staining, Concentration Assay, Positive Control

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . TCR 3 and TCR 8 tested against K562 presenting VP1(100-108) on HLA-A2 vs a control TCR (DMF5) with its corresponding peptide (MART1) after 48h incubation at unsorted E:T of 0.5 and 2. Error bars repres nt SEM for 3 technical replicates from one representative experiment. p<0.001 by unpaired T test. C . After 72 hours of co-culture with JCV-infected SVG cells and no T cells (no TCR), sham TCR, TCR 3, and TCR 8 looking at live CD8+ T cell CD137 positivity via flow cytometry. TCR 3 and 8 vs no TCR p value <0.001; TCR 3 vs sham TCR+JCV p=0.0032 and TCR 8 vs sham TCR+JCV p=0.029 D . Percent killing of SVG cells determined by flow cytometry based on gating of loss of live astrocytes. p value <0.001; TCR 3 vs sham TCR+JCV p<0.001 and TCR 8 vs sham TCR+JCV p=0.002 by unpaired T test. Done in duplicate at various unsorted E:Ts, mean results shown.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques: Control, Incubation, Co-Culture Assay, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: Flow cytometry data of live CD8+ T cells stained with HLA-A2:BK VP1 (100-108) and HLA-A2:JV VP1 (100-108) at 1:50 tetramer dilution with an HLA-A2 positive JCV antibody negative healthy donor on the left and an HLA-A2 confirmed PML survivor on the right.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques: Flow Cytometry, Staining

Journal: Cell reports

Article Title: Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models

doi: 10.1016/j.celrep.2024.114705

Figure Lengend Snippet: (A) Heatmap showing MetaNeighbor’s AUROC scores between thymocytes split by donor, lineage, and non-effector (c0–11) versus effector (c12–17) clusters. Barplots indicate thymocyte proportions per lineage. (B) Pseudo-bulk differential expression analysis between CD4 + /iNKT and CD8 + /MAIT thymocytes in naive clusters (3, 9, 10, 11). As a negative control, the only three genes that were differentially expressed between CD4 + /MAIT and CD8 + /iNKT thymocytes are displayed in the center of the heatmap. (C) Pseudo-bulk differential expression analysis between CD4 + /CD8 + and iNKT/MAIT thymocytes in naive clusters (3, 9, 10, 11). For both (B) and (C), heatmap displays the expression level of genes (represented with color scale as a Z score of the average normalized expression) that are significantly differentially expressed ( p adj < 0.01). (D) Clustering of hashtag-separated thymic iNKT cells (top), MAIT cells (middle), and γδ T cells (bottom). Right panel shows the score of type I and type III effector gene signature for the corresponding thymic lineage. (E) Kernel density estimates of the normalized expression level of genes of interest. The expression level distribution varies between genes and lineage. The range of kernel density estimate values also varies between each panel (from 0 to 0.04 for the smallest range and 0 to 0.4 for the largest range). A unique color scale was represented to indicate the direction of the values.

Article Snippet: Anti-human CD8ɑ - PE-Cy7 (clone SK1) , Tonbo biosciences , Cat#60-0087; RRID: AB_3106994.

Techniques: Quantitative Proteomics, Negative Control, Expressing

Journal: Cell reports

Article Title: Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models

doi: 10.1016/j.celrep.2024.114705

Figure Lengend Snippet: (A–C) (A) Clustering of hashtag-separated blood iNKT, MAIT, γδ T, CD4, and CD8 T cells, (B) the respective proportion of cells per cluster and donor, and (C) the effector GEP signature scores (as in ) per cell type and cluster. (D) Top GEP usage for each cell type, based on cNMF-derived usage matrix. (E) Pseudo-bulk, pairwise differential gene expression between cell types. (F) Cell-type-specific genes among T inn cells using GEP5. For both (E) and (F), heatmaps depict the expression level of genes (represented with color scale as a Z score of the average normalized expression) that are significantly differentially expressed ( p adj < 0.01). n = 4, 4, 9, 4, and 9 for iNKT, MAIT, γδ, CD4, and CD8 cells, respectively.

Article Snippet: Anti-human CD8ɑ - PE-Cy7 (clone SK1) , Tonbo biosciences , Cat#60-0087; RRID: AB_3106994.

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: Cell reports

Article Title: Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models

doi: 10.1016/j.celrep.2024.114705

Figure Lengend Snippet:

Article Snippet: Anti-human CD8ɑ - PE-Cy7 (clone SK1) , Tonbo biosciences , Cat#60-0087; RRID: AB_3106994.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Multiplexing, Recombinant, Antibody Labeling, Staining, Amplification, Software, Flow Cytometry