Journal: Nature

Article Title: Latent human herpesvirus 6 is reactivated in CAR T cells

doi: 10.1038/s41586-023-06704-2

Figure Lengend Snippet: a, Quantification of HHV-6B by qPCR at day 19 for four donors normalized to the cell count. Bars are shown in order of increasing abundance. b, Longitudinal qPCR surveillance of HHV-6B U31 gene copies in CAR T cell culture from two donors. c, Schematic of the single-cell sequencing workflow to detect HHV-6+ cells from the CAR T culture. Two models are presented that would explain HHV-6 reactivation: model 1 (top), in which all cells express HHV-6 transcripts; and model 2 (bottom), in which only a subset of cells express HHV-6B. Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d, Summary of HHV-6B expression from an individual donor (98). The top 0.2% of cells contain 99% of the HHV-6B transcript UMIs from this experiment. e, Tabulated summary of scRNA-seq profiling for four CAR T donors, including number of cells profiled, percentage expressing HHV-6, U31 qPCR value and number of shared TCR clones between the HHV-6B+ cells. N/A, not applicable. f, Extended longitudinal sampling of HHV-6B through U31 qPCR. Number at right end of each plot line indicates the fold (×) increase from the first qPCR measurement (day 21) to the final measurement (day 27; black outline) for each donor. g, Schematic and summary of HHV-6B expression in donor 34 after 19 and 25 days, showing evidence of HHV-6B spreading in the culture as depicted in the schematic. h, Correlation analyses of host factor gene expression with HHV-6B RNA abundance in individual cells for donor 34 on day 25. The per-gene correlation statistics are shown in black against a permutation of the HHV-6B expression in grey. Select genes are indicated. i, Pathway enrichment analysis of Molecular Signatures Database Hallmark gene sets for gene set enrichment analysis. A positive normalized enrichment score corresponds to genes that are overexpressed in cells with large amounts of HHV-6B transcript.

Article Snippet: Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d , Summary of HHV-6B expression from an individual donor (98).

Techniques: Cell Counting, Cell Culture, Sequencing, Expressing, Clone Assay, Sampling, Gene Expression

Journal: Nature

Article Title: Latent human herpesvirus 6 is reactivated in CAR T cells

doi: 10.1038/s41586-023-06704-2

Figure Lengend Snippet: (a) Schematic of the experiment where CAR T cells from D98 were profiled using the 10x Genomics Multiome workflow to detect both viral DNA and RNA. (b) Scatter plot of the abundance of viral DNA and RNA at single-cell resolution. Pearson correlation between the log10 abundances is shown. (c) Per-cell viral gene expression signatures. The proportion of viral gene expression belonging to each class (late, early, immediate early) per cell is shown. (d) Same plot as in (c) but colored by the log2 number of viral DNA fragments. The population of cells highly expressing early HHV-6 transcripts show a corresponding high HHV-6 DNA copy number is highlighted by the arrow. (e) Pearson correlation of HHV-6 transcript signatures with their log abundance of DNA fragments per cell. The two-sided p-value for the Pearson correlation test is noted by each bar. (f) Bulk-level RNA and DNA correspondence in the four donors studied in the day 19 allogeneic CAR products.

Article Snippet: Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d , Summary of HHV-6B expression from an individual donor (98).

Techniques: Gene Expression, Expressing

Journal: Nature

Article Title: Latent human herpesvirus 6 is reactivated in CAR T cells

doi: 10.1038/s41586-023-06704-2

Figure Lengend Snippet: (a) Schematic of CAR T product reculture experiment. Donor D97, which at day 19 showed a low but detectable level of HHV-6, was selected for reculture for five days. (b) Summary of RT-qPCR at the control and two treatment levels of Foscarnet. Each dot represents a technical replicate over one biological replicate per condition (validated in panel d). HHV-6 was not detected (n.d.) at the 1 mM concentration. Error bars represent the standard error of the mean. Comparison of foscarnet treated to untreated resulted in significantly lower abundance of HHV-6 RNA (p = 0.00026; two-sided ordinary least squares linear model). (c) Schematic of D34 reculture +/− foscarnet at 1 mM. (d) Difference between untreated and treated in the abundance of HHV-6+ cells. Comparing the two 10x Genomics scRNA-seq data channels, foscarnet-treated cells had a lower incidence of HHV-6 positive cells (OR = 6.25; p = 8.3e-122; Fisher’s exact test, two-sided). (e) Reduced dimensionality analysis of treated and untreated D34 cells profiled with scRNA-seq. Host gene expression was used for the analysis, showing overlapping clustering of populations irrespective of treatment status. (f) Differential gene expression analysis comparing foscarnet treated and control CAR T cells. The three most significant differential genes are noted. 0 genes were differentially expressed with a minimum log2 fold-change exceeding 1 (noted by the red).

Article Snippet: Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d , Summary of HHV-6B expression from an individual donor (98).

Techniques: In Vitro, Quantitative RT-PCR, Control, Concentration Assay, Comparison, Gene Expression

Journal: Experimental dermatology

Article Title: Recommendations on single-cell RNA sequencing of skin xenografts in the study of genetic skin diseases.

doi: 10.1111/exd.15036

Figure Lengend Snippet: FIGURE 1 Workflow for performing single-cell sequencing on xenograft tissue. (A) Human skin xenograft transplanted on the back of nude mice, as described before.1 Grafting area marked. (B) 20× magnification cryosection of the human xenograft 4 weeks after grafting procedure. Stained for human COL7A1 (Green - LH7.2) and mouse keratin 1 (Red). (C) Standard workflow for scRNA-seq including all the QC steps that are performed in our own experiment. After sequencing, numbers of cells in both human and mouse transcriptomes seem sufficient. The human partition of the reads observed only counts a median of 9 genes per cell, with a median of 10 cDNA unique molecules (UMI) found per human cell. Therefore, unlike the mouse partition, human cells sequenced were not adequate for proper analysis. (D) After skin dissociation a trypan blue stain is used to count living and dead cells. As cell type is not identified at this point, we recommend adding an extra cDNA isolation step before continuing with sequencing of the final single-cell barcoded library to test for presence of sequence of interest. (E) During single-cell capture cDNA is generated, this captured cDNA can be evaluated using PCR with primers specific to target and the barcode. With this method, the presence of the target of interest in the sequencing library can be confirmed.

Article Snippet: We then followed the 10X Genomics scRNAseq workflow which contains three quality control (QC) checks that serve as a verification of successful experiments.4 Our samples passed these checks: cell count and viability after dissociation, cDNA quantification and QC through automated electrophoresis, and library QC before the actual sequencing step, also through automated electrophoresis.

Techniques: Sequencing, Staining, Isolation, Generated