Tracking Plasmid Delivery Using a Fluorescent Plasmid in T cells
The relatively low transfection efficiency that was obtained with Lipofectamine in the primary T cells could be due to multiple different steps in the transfection process, including cellular uptake, nuclear translocation, transcription, and translation of the transgene. However, since other groups have achieved high transfection efficiencies (up to 70%) with electroporation of similar plasmids and synthetic mRNAs in primary T cells,24,46–48 we hypothesized that cellular uptake may be the key limiting step for Lipofectamine (instead of nuclear uptake or transcription/translation). To test this hypothesis, we used a fluorescein-labeled plasmid (Mirus Bio™, MIR 7906) to track the delivery of fluorescent plasmid DNA to the cells with flow cytometry and confocal microscopy.
First of all, as shown in Figure 5A, transfection of adherent PC-3 cells (a cell line that is relatively permissive to transfection) with Lipofectamine and pEF-GFP provided a very high percentage of EGFP+ cells (91±1.1% EGFP+). Likewise, a similarly high percentage of the PC-3 cells fluoresced when transfected with the fluorescein-labeled plasmid (96±0.94% fluorescein+), demonstrating that the plasmid was successfully delivered to almost all the PC-3 cells. Interestingly, the transfection efficiency observed with pEF-GFP for the Jurkat cells (51±9.7% EGFP+) was more modest, but the percentage of fluorescein+ cells (74±4.5%) was significantly higher. The mean fluorescence (Figure 5B) of the PC-3 cells was also significantly higher than the mean fluorescence of the Jurkat cells in both types of transfections.
Similar trends were also observed with the primary T cells (Figure 5A/B). The percentage of fluorescein+ primary T cells and the mean fluorescence of the fluorescein+ primary T cells was significantly lower than both Jurkat T cells and the PC-3 cells. There was also a more dramatic disparity between the fraction of fluorescein+ primary T cells (51±11.2% EGFP+) and the percentage of primary T cells expressing EGFP (8.1±0.8% EGFP+). This stark difference suggests that the lipoplexes bind to a large fraction of the primary T cells, but many of those lipoplexes may fail to either enter the cell via endocytosis, escape the endosome into the cytosol, or enter the nucleus.
To determine if the fluorescent lipoplexes were bound to the surface of the cells or taken up into the cytoplasm or nucleus, cells transfected with the fluorescein-labeled plasmid were treated with trypsin-EDTA for up to 30 minutes to disrupt any potential interactions between cell surface proteins on the cell surface and the lipoplex (Figure 5C/D). Hypothetically, if the fluorescent lipoplexes were simply bound to the cell membrane, then this trypsinization would detach them, leading to a significantly lower mean fluorescence and percentage of fluorescein+ cells. Indeed, trypsinization had no significant effect on the percentage of fluorescein+PC-3 cells or their mean fluorescence, suggesting that most of the fluorescent lipoplexes were in the cytosol or nucleus of the PC-3 cells. In contrast, trypsinization significantly decreased the mean fluorescence and the percentage of fluorescein+ Jurkat cells to a level that was comparable to the percentage of EGFP+ cells shown in Figure 5A. A similar, although not statistically significant, decrease in the percentage of fluorescein+ cells was also observed in the primary T cells. However, the mean fluorescence of the primary T cells significantly decreased and was virtually eliminated after 30 minutes of trypsinization (Figure 5D). Altogether, these findings suggest that cellular uptake may be a limiting step for gene delivery in both Jurkat and primary T cells.
To further investigate the localization of the fluorescein-labeled plasmid in the PC-3 and primary T cells, fluorescent microscopy was used to visualize the fluorescent lipoplexes after transfection (Figure 6). In these experiments, cells were also stained with Hoescht 33342 nuclear stain and the Biotium CellBrite red cytoplasmic membrane dye to visualize the nucleus and cytoplasm, respectively. Overall, similar trends in fluorescence were observed across the cell lines. A widespread distribution of bright EGFP fluorescence within the cytoplasm was observed in PC-3 cells transfected with pEF-EGFP (indicating successful transgene expression), while cells transfected with the fluorescein-labeled plasmid exhibited small regions of concentrated fluorescence inside the cytoplasm and nucleus that indicated successful plasmid uptake and nuclear delivery (Figure 6A). In addition, z-stacking with 0.5 µm slices from the top edge to the bottom edge of the PC-3 cells also confirmed that the fluorescent lipoplexes were inside the cell (individual images areshown in Figure 6C, while compiled z-stacks are shown in a video in Figure S5).
In contrast, transfection of CD3+ primary T cells with pEF-EGFP only yielded a small fraction (3.9±0.24%) of EGFP+ cells and the primary T cells that did fluoresce were much dimmer than the PC-3 cells (in agreement with the flow cytometry data shown in Figure 5B). Confocal microscopy z-stacking images also showed that the fluorescent lipoplexes appeared to be localized to the outside of the cell membrane instead of being internalized by the cell (Figures 6C and S6). These observations further support the notion that lipoplexes may successfully bind to primary T cells, but endocytosis of the lipoplexes appears to be limited.
Transcriptome Analysis of Primary T Cells
The experiments with the fluorescein-labeled plasmid (Figures 5-6) seemed to suggest that impaired cellular uptake of the plasmid may be partially responsible for the T cells’ resistance to transfection, but the observation that 30-40% of the primary T cells remained fluorescein+ after trypsinization suggested that other intracellular mechanisms may also impair endosomal escape, nuclear delivery, and/or expression of the transgene. In an effort to detect any additional mechanisms that could inhibit transgene delivery or expression and provide a rationale for the relatively low transfection efficiency of primary T cells, mRNA sequencing was used to investigate the transcriptomes of PC-3, Jurkat, and primary T cells in the absence or presence of lipoplexes. These experiments were motivated by previous studies that showed both viral vectors and Lipofectamine can activate innate immune response pathways which trigger the expression of anti-viral genes (TLRs, MyD88, IRFs) that can hinder transduction in some cell types.54
The complete mRNA-seq data (fastq and bam files, along with a spreadsheet of all FPKM values) from these experiments are available at the NCBI GEO repository (GEO Accession# GSE151759). Overall, one of the most significant differences that was observed in the gene expression profiles of the PC-3 cells and both types of T cells (Table 1) was the absence of several heparan sulfate proteoglycans (HSPGs), which are also known as syndecans (SDCs). As shown in Table 1, HSPG2 and all the sydecans were expressed in PC-3 cells, but mostly absent in both Jurkat and primary T cells (with and without lipoplexes). SDC3 was the only syndecan expressed in Jurkat cells, but its role in endocytosis and gene delivery has not yet been established. SDC4 was expressed in primary T cells (as expected for activated T cells), but SDC2 was not detected. Similar results were reported by one study that showed 100-fold lower levels of HSPG expression in Jurkat T cells compared to HeLa cells.77 Low levels of HSPG expression have also been observed in primary T cells, but T cell activation can upregulate SDC2 and SDC4 expression.58,78
This general lack of syndecan expression may explain the significantly lower transfection efficiencies shown in Figure 5A for the Jurkat and primary T cells relative to the PC-3 cells (which express HSPG2 and all the syndecans). Indeed, while HSPGs are best known for their roles in the attachment of adherent cells to the extracellular matrix or tissue culture plates, they are also directly involved in gene delivery, since they regulate endocytosis and their negatively charged sulfate groups are involved in the initial binding of several viruses and positively charged polyplexes or lipoplexes.79 Indeed, overexpression of SDC1, SDC2, and SDC4 enhances the transfection efficiency of liposomes in K562 cells, although overexpression of SDC2 has also been shown to inhibit PEI-mediated gene delivery.67,80 Alternatively, blocking the sulfation of SDCs has also been shown to inhibit endocytosis and gene delivery.57,67
As previously mentioned, mRNA-sequencing was performed in the different cell lines both in the absence and presence of lipoplexes. The rationale for comparing transfected and untransfected cells was to determine if there were any host cell genes that were upregulated in response to transfection of double-stranded plasmid DNA. Indeed, there are many examples of genes that are induced or upregulated when dsDNA is detected in the cytoplasm and many of these upregulated genes have potent anti-viral functions that are designed to inhibit the replication of viruses.68,69 Unfortunately, many of these genes can also inhibit non-viral transgene delivery or expression.70
Transfection of PC-3 cells led to the induction or upregulation of hundreds of cytokines and cytokine-stimulated genes (CSGs, data not shown), some of which can inhibit transgene delivery (e.g., IFITM 1, 2, and 3) or translation (e.g., IFIT 1 and 2). Nonetheless, the transfection efficiency and mean GFP levels were still high in PC-3 cells, suggesting that many of these upregulated genes may be inconsequential for non-viral pDNA or transgene delivery and expression in PC-3 cells.
In contrast to PC-3 cells, only a few differentially expressed genes were significantly upregulated at least 3-fold in the Jurkat and primary T cells (Table 2). This result is somewhat unanticipated, since the requisite cytosolic DNA sensors (e.g., cGAS and IFI16) and the downstream effectors (e.g., STING, TBK1, and IRFs) that are necessary to detect foreign DNA and induce the expression of cytokines and CSGs were detected in the Jurkat and primary T cells (data not shown). This lack of an innate immune response to dsDNA has been previously reported by other groups, suggesting that T cells may lack an unknown component of the DNA sensing pathways or they somehow repress CSGs.8 Nonetheless, some metallothioneins were significantly upregulated in the Jurkat (MT1F, MT2A) and primary T cell lines (MT1H) after transfection. Metallothioneins are involved mainly in metal binding, often to zinc, but they have also been implicated in immune regulation and the response to bacterial and viral infections.60,61 However, the role of metallothioneins in transgene delivery and expression has not yet been determined.
Although T cells did not significantly upregulate cytokines or CSGs in response to transfection, one interesting trend that emerged when comparing the transcriptomes of the T cells to the PC-3 cells was that multiple CSGs that were upregulated in PC-3 cells after transfection were constitutively expressed at relatively high levels in the T cell lines (Table 2). For example, each member of the pyrin and HIN (PYHIN) domain DNA sensor family (IFI16, AIM2, and PYHIN1/IFIX) was detected in both untransfected and transfected primary T cells. In contrast, AIM2 was only expressed in PC-3 cells after transfection and IFI16 was highly upregulated in transfected PC-3 cells, suggesting that these DNA sensors play an important role in the innate immune response to dsDNA in PC-3 cells. This is a particularly intriguing observation, because after AIM2 and IFI16 bind dsDNA in the cytoplasm, they form an inflammasome complex with PYCARD, Caspase 1/8, and Gasdermin D (all of which were expressed at detectable levels in primary T cells, but not Jurkat cells) that can induce inflammation, apoptosis, and pyroptosis.64Pyroptosis has also been observed in primary T cells during abortive HIV infection, in which double-stranded cDNA is generated in the cytoplasm by the virus.63 Therefore, while the AIM2 and IFI16 inflammasome pathways must be induced (AIM2 & CASP1) or upregulated in PC-3 cells, it appears that primary T cells constitutively express the genes in these pathways, which may lead to higher levels of apoptosis upon transfection with plasmid DNA and the decrease in proliferation shown in Figure 1. Indeed, several other studies have reported that dsDNA is highly toxic in T cells.71,72