Enhanced conversion of T cells into CAR T cells by modulation of the MAPK/ERK pathway

Delivery of chimeric antigen receptors (CARs) to T cells is usually mediated by lentiviral vectors (LVs), which can have broad tropism or be T-cell targeted. To better understand the molecular events during CAR T cell generation, T cell transduction with four different LVs was followed by single-cell multi-omics analysis, distinguishing between transduced T cells and T cells with vector signal but no CAR. We found that only a fraction of the T cells that had encountered vectors converted into CAR T cells. Single-cell transcriptome data revealed that interferon-stimulated genes were upregulated in non-transduced cells, whereas ERK2 phosphatases were upregulated in CAR T cells. This expression pattern was evident in CAR T cells from healthy donors and patients. The role of these genes in T cell transduction was confirmed by chemical inhibitors. These data provide molecular insights into T cell transduction with implications for improving CAR T cell generation. Activated PBMCs from two healthy donors were transduced with lentiviral vectors (LVs). lentiviral vectors pseudotyped with the vesicular stomatitis virus G (VSV-G) protein (VSV-LV), T-cell targeted vectors pseudotyped with paramyxoviral envelope proteins and using CD3, CD4, or CD8 as entry receptors were used for transduction and after 72 hours incubation cells were stained with AbSeqs and sample multiplexing tags and were analyzed with single-cell multi-omics using BD Rhapsody T cell targeted gene panel. (SampleTag11_hs: Donor 1_CD4-LV, SampleTag12_hs: Donor 1_CD8-LV, SampleTag09_hs: Donor 2_CD4-LV, SampleTag10_hs: Donor 2_CD8-LV). (SampleTag12_hs: Donor 1_CD3-LV, SampleTag11_hs: Donor 1_VSV-LV). (SampleTag09_hs: Donor 2_CD3-LV, SampleTag10_hs: Donor 2_VSV-LV)