title: Single-cell Multi-omics Dissection of CAR T cells Reveals Inhibitors of Gene Delivery Mediated by Lentiviral Vectors
Creators: name: Adabi, Elham
Publisher: name: UNSPECIFIED
Year: 2025

description: CAR T cell therapy has shown promising results in clinical trials and has been approved for use in certain types of cancer, specifically B-cell lymphoblastic leukemia, and large B-cell lymphoma. However, as a relatively new treatment, there are still some challenges to overcome. These include improving efficacy, managing side effects, and making the treatment more widely available and affordable. Lentiviral vectors (LVs) are considered the gold standard tool in the generation of CAR T cells due to their ability to efficiently deliver genetic material into T cells. Several CAR T cell products currently on the market use LVs for CAR T cell generation. During the manufacturing process, T cells are collected from the patient and genetically modified to express the CAR. The modified T cells are then expanded before being infused back into the patient. The amount of CAR T cells in the final product can be influenced by factors such as the purity and quality of the starting T cells, the efficiency of the gene transfer process, and the expansion protocol. Furthermore, the complex and multifactorial manufacturing process of CAR T cells results in a heterogeneous population of which only a fraction are genetically modified T cells.
Among LVs conventional VSV-LV is most frequently used. Besides, T cell targeted vectors using CD3, CD4, and CD8 as entry receptors have been developed. These targeted LVs enable the selective transduction of T cell subtypes, including CD3, CD4, and CD8 T cells. Employing these targeted vectors during in vitro transduction reduces the complexity of manufacturing processes, omits the risk of off target modifications, and holds potential for use in direct in vivo gene transfer applications. Limited information is available about the molecular consequences of vector exposure to T lymphocytes, and the characteristics of CAR T cells generated from the listed types of lentiviral vectors. Therefore, additional evaluation is needed to compare and characterize the phenotype and transcriptome of CAR T cells produced with these targeted vectors compared to those generated with VSV-LV.
T cell transduction is initiated by receptor binding of the LVs followed by endocytosis for VSV-LV and fusion at the cell membrane for the targeted vectors. Whether using conventional lentiviral vectors or targeted vectors, it was unclear whether all cells that bind to LVs would be transduced, or whether there would be cell populations that attach to LVs but do not express the CAR. To address this question, vector binding and CAR expression on T cells were monitored at various time points following the addition of LVs, by using specific antibodies against CAR and LVs. Analysis of vector binding and CAR expression on T cells at different time points after the addition of vector particles revealed that both VSV-LV and targeted vectors attachment rates to T cells were consistently above 60% at 4 hours after vector addition. In contrast, CAR expression 72 hours after adding the vectors was significantly lower than the percentage of vector binding, and it varied between donors and LVs. This thesis aimed at understanding the status and gene expression profiles of cells negative for CAR expression compared to successfully transduced cells that convert into CAR T cells.
For these purposes, single-cell multi-omics analysis was established. CAR T cells generated from two healthy donors were analyzed at early time points after exposure to CD3-LV, CD4-LV, CD8-LV, and VSV-LV. Unlike other single-cell studies that have analyzed CAR T cells cultured for extended periods before infusion, this thesis focused on early time points after vector exposure, analyzing the cells 3 days after encountering lentiviral vectors.
For single-cell analysis, a T cell targeted gene panel, together with primers for the detection of CAR mRNA, was utilized. This method provided sensitive detection of transcripts and allowed for high-resolution differential gene expression analysis, while requiring less sequencing depth. Besides detecting mRNA, this study included barcoded antibodies (AbSeqs) to detect LV binding, CAR, and 30 immune markers in single-cell analysis. AbSeqs were effectively detected and utilized to differentiate various populations of CD4 and CD8 T cells, as well as for the detection of vector bound and CARpos T cells. All AbSeqs showed a bimodal distribution, enabling the separation of cells into positive and negative populations within single-cell data. The percentage of positive cells, as defined by the AbSeqs, was well in line with protein expression data obtained through flow cytometry. This comparison confirmed the validity of the AbSeq data. Based on the AbSeq and transcriptome data, different cell clusters were identified across all samples. Specifically, the CD4-LV sample contained a high proportion of CD4+ cytotoxic cells, and CD8-LV sample predominantly CD8+ naive T cells. The CD3-LV group included the highest percentage of CD4+ central memory T cells, and the VSV-LV sample was notable for the highest frequency of CD4+ effector memory T cells.
When comparing the transcriptome of CAR T cells generated with the targeted LVs to those generated with VSV-LV, distinct gene expression profiles were observed in both CD4, and CD8 CAR T cells derived from each vector type. Specifically, LEF1 and FAM65B, genes that promote a naive phenotype in T cells, were universally upregulated in CD4 and CD8 CAR T cells from targeted vectors. Despite the expression of naive T cell markers both CD4 and CD8 CAR T cells from targeted vectors showed upregulation of cytotoxic markers. Notably, CD4 CAR T cells derived from CD4-LV displayed characteristics of cytotoxic CD4 T cells, marked by high expression of GZMA. These observations indicated that CAR T cells, generated from targeted vectors, had a less differentiated phenotype, while maintaining cytotoxic activity.
Focusing on the heterogeneous population of T cells present during CAR T cell generation, specifically in terms of vector binding and CAR expression, an analysis was conducted on the differentially expressed genes between CARneg T cells with vector binding signal compared to CARpos T cell. In this analysis, a group of antiviral genes stimulated by type I interferon were detected to be upregulated in CARneg T cells. These genes included IFITM2, IFITM3, STAT1, SAMHD1, MX1, IFI6, ADAR, OAS2, TRIM22, TRIM25, and APOEC3G. To discern whether the expression of these genes was induced in T cells after exposure to the LVs, or if it was high in T cells in advance, the expression patterns among different cell subsets were investigated. The expression of these genes, specifically for CD4-LV and CD8-LV treated samples, followed a general pattern: it was low in cells that were negative for both CAR and vector binding (indicating cells that had not encountered the vector particles), while it was high in cells that were CARneg but positive for vector binding, and low in CARpos T cells. This pattern of expression suggested that the expression of interferon stimulated genes was upregulated in T cells after encountering viral vectors.
The examination of the genes commonly upregulated in CARpos T cells, irrespective of the vector used, revealed a consistent upregulation of CSF2, CTLA4, MYB, LAG3, JUNB, and CD2, which are known for their role in the activation, differentiation, and exhaustion of T cells. Additionally, other genes with less understood functions in CAR T cells were also upregulated, including DUSP2, DUSP4, RPDM1, and ZBED2.
To validate the observations made in two healthy donors, single-cell data from anti-CD19 CAR T cell infusion products of 24 patients diagnosed with LBCL were analyzed. The differential expression of genes between CARpos and CARneg T cells confirmed the observations made in healthy donors. Specifically, upregulation of certain interferon-stimulated genes, including IFITM1, IFITM2, and ISG20, in CARneg T cells, as well as an upregulation of DUSP2 and DUSP4 genes in CARpos T cells was detected, despite several days longer cultivation of these cells before analysis.
Considering the upregulation of interferon-stimulated genes in CARneg T cells in both healthy donors and patient’s samples, it was hypothesized that their antiviral activity might be involved in inhibiting LV-mediated gene transfer into T cells. Furthermore, the upregulation of DUSP2 and DUSP4 in CARpos T cells was suggested to play a positive role in T cell transduction.
Based on these observations, various approaches were investigated to either downmodulate interferon signaling or directly inhibit interferon-induced transmembrane proteins (IFITMs).
First, downmodulation of interferon signaling by deucravacitinib (BMS-986165), an inhibitor of TYK2, was investigated. TYK2 is a key kinase at the start of interferon signaling. Its activation leads to the expression of interferon stimulated genes. deucravacitinib (BMS-986165) treatment resulted in increasing CAR expression with up to 3-fold increase in transduction mediated by all targeted vectors and VSV-LV without compromising T cell viability. Although different degrees of enhancement were observed among different LVs and donors.
Next, considering that IFITM genes were commonly upregulated in CARneg T cells of healthy donors and patient’s samples, the direct inhibition of IFITMs with caraphenol A was investigated. Caraphenol A is a resveratrol trimer that has been shown to transiently reduce IFITM protein expression. In this thesis, caraphenol A treatment enhanced gene transfer by targeted LVs up to 2-fold with a less pronounced effect on VSV-LV.
As a final approach to target interferon signaling, ERK inhibitors were utilized to mimic the activity of DUSP2 and DUSP4 proteins in T cells. Upregulation of DUSP2 and DUSP4 were observed in CARpos T cells. This led to the hypothesis that these genes might play a role in the downregulation of interferon-stimulated gene expression by dephosphorylating MAPK1, thereby promoting T cell transduction. Treating T cells with four different ERK inhibitors resulted in increased CAR expression in T cells. The response to various doses of each type of ERK inhibitor varied based on the vector type used for T cell transduction,
but CD4-LV, CD8-LV, and VSV-LV showed an up to a 2-fold increase, while CD3-LV demonstrated an up to a 3-fold increase in response to ERK inhibitor treatment.
In conclusion, by employing single-cell multi-omics, this thesis highlighted the impact of different LVs, on the transcriptomic profiles and the phenotypes of the generated CAR T cells. Furthermore, the heterogeneity observed in CAR T cell products were found to be modulated by interferon-stimulated genes that are induced in T cells by LVs. Some of these genes were identified as inhibitors of CAR T cell generation and could be observed in both healthy donors and patient samples. Moreover, a novel role for the DUSP2 gene in promoting T cell transduction was proposed. The use of different ERK inhibitors, which mimic DUPS2 function, improved transduction with both targeted and VSV-LV vectors, providing the first evidence for this hypothesis and could be used to optimize CAR-T cell production.