Fig. 5: Single nuclei RNA sequencing (snRNAseq) of PBMC from patient NYSCT-03 at day 43.

A Overview of ddPCR VCN kinetics at timepoint selected for analysis, which yielded 4166 nuclei after doublet removal and QC (as shown in Fig. 4B). Of the PBMC population, 1954 nuclei were lentiviral-positive (pLenti + , 46.9%) and 2212 nuclei were lentiviral-negative (pLenti-, 53.1%) (B), while only 10 nuclei were retroviral-positive (pretro + ) and 4156 were retroviral-negative (pretro-) (C). Both pLenti+ and pLenti- cells expressed all immune cell types from Azimuth References at comparable levels in RNA and ATAC UMAPs (D), and all immune cell type states at comparable levels; myeloid cells clustered together, B cells clustered separately, and T cells and NK cells clustered together in RNA and ATAC UMAPs (E). NYESO1 TCRB lentivirus encodes the same amino acids as T cell receptor beta (TRBC1) gene, but is codon optimized. T cells represented 185 nuclei (≈4.5%) of all nuclei, which were equally represented by pLenti+ and pLenti- nuclei (47% and 53%, respectively). Of pLenti+ Tcells, 37% co-expressed genomic TRBC1/2 + codon optimized lentiviral NY-ESO-1 TCR (pRRL-lenti), while 63% exclusively expressed codon optimized pRRL-lenti TCR (F). Of the T cells, 32 nuclei co-expressed the genomic TRBC1/2 + the pLenti TCR, while 98 and 55 nuclei had mutually exclusive expression of either genomic TRBC1/2 or codon optimized pRRL-lenti TCR, respectively (G). T cell CD4:CD8 ratio was 3:1, and the proportion of nuclei belonging to CD4 T cell, CD8 T cell, or other mature T cells was comparable for nuclei expressing exclusively TRBC1/2 (blue) or nuclei expressing codon optimized pRRL-lenti TCR (H). Of all pLenti+ nuclei (1954), 7% co-expressed genomic TRBC1/2 (131), while of all pLenti+ T cells (87 nuclei), 37% co-expressed genomic TRBC1/2 (32 nuclei) (I). Heatmap for expression of pLenti TCR, TRBC2, and TRBC1 per nucleus for pLenti+ T cells revealed allelic exclusion in the majority of nuclei. 45% of the CD4 T cell clusters/state co-expressed the NYESO1 pLenti TCR and genomic TRBC1/2, while 15.4% of the CD8 T-cell cluster/states co-expressed NYESO1 and genomic TRBC2 but not TRBC1, remaining nuclei exclusively expressed the codon optimized NYESO1 pLenti TCR (J).