Immune system boosting for better substrate delivery

Optimizing the immune system is nowadays a new tool to treat a broad range of diseases such as leukemia, solid-tumors or HIV. The establishment of recent techniques including the generation of Chimeric antigen receptor expressing cells and CRISPR/Cas9 based genome editing give the opportunity to improve or enhance the immune response fairly convenient, thus opening the door for novel immunotherapies.

For this purpose, however, substrate delivery into hematopoietic and blood cells is essential - not only with a focus on high transfection efficiency, but also on cell viability, preservation of functionality as well as patient’s health and safety. Furthermore, delivery methods should be flexible in regard to substrate as researchers editing the cells genome depend on a successful transfection of not only plasmid DNA but also RNA and/or proteins [1].

So far theory, but in practice the transfection of primary immune cells is challenging. Especially the cell types of interest, like T-lymphocytes, Natural Killer cells and B-cells are hard-to-transfect using classical reagent based methods [2]. Major issues resulting from the use of transfection reagents are low transfection efficiencies, cytotoxicity and immunogenicity. The latter is unfavorable in the context of immunotherapies as the cells should maintain their function in the immune system not being activated by the transfection method upfront.

The alternatives to achieve reasonable transfection efficiencies is viral transduction, thus time-consuming and cost-intensive, or improved electroporation techniques, like the Nucleofector^TM Technology, which as a more physical method requires lower biosafety precautions (Table 1).

Overview on advantages and disadvantages of different substrate delivery to primary immune cells

	Reagent-based transfection	Viral transduction	Improved elelectroporation Nucleofector® Technology
Transfection efficiency	Low	Very high stable transfection via insertion	High use of optimized protocols
Viability	Low (cytotoxicity)	High	High use of optimized protocols
Substrate flexibility	Yes	No	Yes
Preservation of functionality	Low	Medium	High
Biosafety precautions	Low	High	Low

Step 1 – Choosing the transfection method

With viral transduction, an integration of the gene of interest into the host genome can be achieved. Most often lentiviral vectors are used, which are shown to have no random integration sites and do not favor proto-oncogene or tumor suppressor gene loci. Furthermore, the use of adenovirus and adenovirus-associated viruses for long-term episomal transgene expression is possible.

Viral transduction is a time and cost-intensive method as for transduction experiments the viral particles need to be produced in packaging cell lines. However, it usually enables high transfection efficiencies with low cytogenicity. One important aspect however is the patients’ safety. Depending on the location of integration into the host genome, which is not predictable, the cell’s functions might be affected.

A convenient technique for substrate delivery into human immune cells is the Nucleofector™ Technology - an improved electroporation technique. The combination of dedicated Nucleofector™ Solutions together with optimized electrical parameters for specific cell types enables researchers to achieve high transfection efficiencies with good viabilities. The substrates are directly delivered into the cytoplasma and nucleus. Immunogenicity is minimized which also preserves functionality of immune cells (Fig. 1).

Figure 1: Cell functionality post Nucleofection. The bar graph displays the relative functionality of mouse dendritic cells, human macrophages and human T cells post Nucleofection (Sample). Functionality is given in percent related to non transfected control (Control). Functionality was analyzed by IL-6 specific ELISA for mouse dendritic cells, TNF-a specific ELISA for human macrophages, IFN-g specific ELISA for stimulated human T cells and by flow cytometry using a CD25 specific antibody for both human T cell states (resting and stimulated). Experiments were performed on a standard Nucleofector^TM Device (mouse dendritic cells) or the 96-well Shuttle^TM System (human macrophages and human T cells). [3]

But not only efficiency and viability are ensured. In view of CAR expressing T-cells [4] and NK-cells [5] as well as genome editing [6] also co-transfection of different substrates is possible – with a large flexibility in size and substrate type like DNA, RNA and proteins.

By using the 4D-Nucleofector™ Technology the transfection application is also closed and scalable. It can easily be transferred from small scale format of 20-100 ul for fundamental research or screening purposes to up to 20 ml for follow-up steps like ex-vivo modifications for cell therapy without any further optimization steps.

Step 2 – Get your immune cells to toe the line

Regardless of the method that is used for substrate delivery, the condition the immune cells are in before transfection pioneers the viability and functionality of the cells thereafter. How robust or sensitive a cell reacts to the transfection process depends on one hand on the isolation procedure, on the other hand it is a question of donor characteristics.

Researchers who are lucky to have access to blood or bone-marrow samples for self-isolation of immune cells should ensure to stick to an at best already published standard operating procedure for isolation, enrichment or stimulation of the cells. Following the instructions precisely will result in comparable and reproducible results.

In case this kind of source is not accessible researchers work with commercially available hematopoietic and immune cells. This facilitates the work in terms of quality testing, already optimized culture systems and a large donor portfolio.

However, no matter what the source of cells is, primary cells are cells from individual organisms. Donor specific differences are inevitable and will result in variations [7].

Step 3 – Ready, steady, substrate delivery

While for viral transduction the preparation of the viral particles is complex and default, the substrate choice for improved electroporation is more diverse.

Promoter choice is the linking piece between transfection efficiencies and expression rates. Only if the cell can express the delivered gene the efficiency of substrate delivery will be optimal. Researchers have to deal with differences in promoter strength depending on the cell types they are working with. What has worked in a specific cell type does not necessarily show the same result in another. Data on different promoters can be found in Lonza’s Bench guide about important vector factors for gene expression.

To ensure optimal conditions for the cells substrate should be of highest quality. This comprises particularly in case of DNA constructs the purity of the substrate preparation (OD A260/A280: 1.6-1.8) as well as the DNA integrity (portion of supercoiled Plasmid-DNA). Furthermore, an endotoxin-free plasmid preparation prevents the immune cells of high intra cellular endotoxin-levels post improved electroporation facilitating optimal cell viabilities.

Substrate size and concentration are additional players in this game. Size alone is usually not a major factor for transfection efficiency, but there is some decrease in efficiency as plasmid size increases above approximately 15 kb. However, larger plasmids are prone to certain issues: particularly the DNA amount needed for the experiment, and plasmid integrity. Increasing DNA amount per reaction generally increases transfection efficiency – but cell viability can be decreased as DNA can be toxic to cells at high concentrations. For Nucleofection experiments the recommended substrate concentrations are usually ranging between 2-5 ug/100 ul for DNA, 2 nM – 2uM for siRNA and 10-20 ug/100 ul for mRNA delivery.

Human cell type	Nucleofection conditions (Kit/pulse)	Transfection efficiency	Viability	Reference
CD34+ cell	P3 / EO-100 (op)	83%	62%	Genovese et al. [8]
B-cell	P3 / EO-117 (op)	28%	70%	Rappocciolo et al. [9]
Natural Killer cell	P3 / FA-100 (ihd) P3 / EK-100 (ihd) >	54% 32%	45% 76%	Rady et al. [10]
T-cell, Unstimulated	P3 / FI-115 (op) P3 / EO-115 (op)	80% 68%	53% 79%	Doherty et al. [11]
T-cell, Stimulated	P3 / EO-115 (op)	70%	59%	Park et al. [6]
Monocyte	P3 / EA-100 (op)	64%	77%	Bharaj et al. [12]
Macrophage	P3 / DP-148 (op)	42%	60%	Daj et al. [13]
Dendritic cell, Immature	P3 / CB-150 (ihd)	69%	84%	Gerdemann et al. [14]

Table 2: Recommended Nucleofection conditions, results and references for primary human immune cells (op: optimized protocol available, ihd: based on in-house data)

Lonza is committed to supporting your research on the immune system. That’s why we offer the Nucleofector® Technology and corresponding Nucleofector® Kits for efficient transfection of immune cells. In addition, we provide hematopoietic and immune cells from a variety of different donors. Please contact Scientific Support for more information.