Journal: Cancer Discovery

Article Title: Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma

doi: 10.1158/2159-8290.cd-21-1586

Figure Lengend Snippet: Figure 1. CD19 CAR-T cells demonstrate significant transcriptional heterogeneity that changes after infusion into patients. A, Study scheme. Viable CAR-T cells were sorted from the CAR-T cell products or PBMCs of patients with NHL. scRNA-seq and feature barcoding libraries were then prepared and subsequently sequenced. After quality control, dimension reduction was performed and analyzed by cluster or cell subtype with differential gene expression and gene set signature scoring. Validation of memory marker and exhaustion marker expression was performed with flow cytometry. B, tSNE dimension reduction of scRNA-seq data with overlays depicting cluster assignment, cell-cycle analysis, patient number, CD4 or CD8 T-cell group by time point, and T-cell subtype. C, Heat map of differentially expressed genes (adjusted P < 0.05) between all clusters of B by log fold change. The top 3 to 10 genes with highest absolute log fold change are represented. Clusters are annotated with cell subtype proportions.

Article Snippet: The clinical-grade reagents applied in this process were CliniMACS Buffer, TexMACS Media, CliniMACS CD4 reagent, CliniMACS CD8 reagent, TransAct, and the cytokines IL7 and IL15 (Miltenyi Biotec).

Techniques: Control, Gene Expression, Biomarker Discovery, Marker, Expressing, Flow Cytometry, Cell Cycle Assay

Journal: Biomedicines

Article Title: CD5 Deficiency Alters Helper T Cell Metabolic Function and Shifts the Systemic Metabolome.

doi: 10.3390/biomedicines10030704

Figure Lengend Snippet: Figure 6. Removal of CD5 functionally increased glycolysis and mitochondrial respiration in un- stimulated Th cells. (A,E) Using a standard Seahorse protocol, approximately 200,000 CD4+ T cells were seeded into a poly-d-lysine coated 8-well XFp plate and analyzed for metabolic function using stepwise injections of oligomycin, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone plus antimycin A. Three wells were plated for each mouse, and the average was taken for

Article Snippet: Unstimulated CD4+ T cells were selected using positive selection CD4+ (L3T4) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany; kit #130-117-043) according to manufacturer instructions.

Techniques:

Journal: Biomedicines

Article Title: CD5 Deficiency Alters Helper T Cell Metabolic Function and Shifts the Systemic Metabolome.

doi: 10.3390/biomedicines10030704

Figure Lengend Snippet: Figure 7. Unstimulated CD5KO CD4+ T cells were phenotypically different from CD5WT CD4+ T cells. (A) CD4+ T cells were stained with CD44, CD62L, and CD25 to determine activation state and subset polarization. (B) Gating strategy to determine naïve and memory populations between CD5WT

Article Snippet: Unstimulated CD4+ T cells were selected using positive selection CD4+ (L3T4) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany; kit #130-117-043) according to manufacturer instructions.

Techniques: Staining, Activation Assay