Multiomic Study of Cutaneous T-Cell Lymphoma Reveals Single Cell Clonal Evolution in Progression and Therapy Resistance

Description

Methods

Single cell (sc)RNA sequencing were performed on Cutaneous T-cell Lymphoma (CTCL) samples, including skin and lymph node biopsies of CTCL, peripheral blood mononuclear cells, purified malignant and non-malignant T cells and matched normal monocytes or NK cells from peripheral blood, as well as peripheral blood T cells from healthy donors.

Treatment protocol: Peripheral blood mononuclear cells were subjected to CD4+ enrichment (EasySep Human CD4 Positive Selection Kit or CD4+ staining and FACS) and/or monocyte depletion (RosetteSep Human Monocyte (CD36) Depletion Cocktail) and viable cells sorted using a 7-AAD viability stain on a Sony iCyt Synergy SY3200.

Extraction protocol: For each of the PBMC samples, 10,000 to 20,000 viable cells were submitted for processing using the 10x Genomics Chromium Controller and the Chromium Single Cell 5′ Library & Gel Bead Kit v2 (PN-1000006) following the manufacturer’s protocols. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 10-14 cycles before being cleaned up using SPRIselect beads. Samples were then run on a Bioanalyzer to determine the cDNA concentration. TCR target enrichments were performed on the full-length cDNA. GEX and Enriched TCR libraries were prepared as recommended by the 10x Genomics Chromium Single Cell V(D)J Reagent Kits (v1.0 Chemistry) user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Single Cell 5’ Library and Gel Bead Kit v2 (PN-1000006), Chromium Single Cell A Chip Kit (PN-1000152), Chromium Single Cell V(D)J Enrichment Kit, Human, Tcell (96rxns)(PN-1000005), and Chromium Single Index Kit T (PN-1000213) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 151x10x10x151 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each sample.

Data processing: Alignment and gene counting were performed using the Cell Ranger pipeline (10x Genomics, v3.0, Pleasanton, CA) with genome assembly GRCh38/hg38. Genes found in fewer than 15 cells in each sample were removed. For each patient, gene counts and cells were pooled into a single Seurat (v5.1.0)(Butler et al., 2018; Hao et al., 2024; Stuart et al., 2019) object, and cells containing fewer than 200 or more than 3000-3750 expressed genes, more than 8-10% mitochondrial reads, fewer than 300 or more than 10000UMIs, or classification as a doublet by the R package scDblFinder(Germain et al., 2022) with parameters dims = 30, clust.method = “fast_greedy” were removed. In total, 118,608 cells were sequenced, with 92,496 passing initial QC filters after removing dead cells, empty droplets, and suspected multiplets.