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Guest Commentary: A more reproducible approach to creating functional Th17 cells
December 2018
by Kevin Bobofchak of Lonza  |  Email the author
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Closely linked with various inflammatory and autoimmune disorders, Th17 cells are an attractive therapeutic target, and are at present the focus of many cancer immunotherapy research projects. However, current methods of generating Th17 cells are time-consuming, costly and often cause significant intra- and inter-laboratory variability, calling into question the relevance of results.
 
Developing an alternative approach for the creation of functional Th17 cells that improves experimental reproducibility by simplifying the differentiation process has long been a goal of researchers in the field. A novel method of bead-based activation from cryopreserved CD4+ T cells has been developed that offers the potential to generate physiologically relevant Th17 cell populations suitable for a wide range of downstream assays.
 
In this article, we consider the importance of Th17 cells for drug discovery and look at how the use of specialized methods to confirm cellular identity provides a level of validation that has not previously been available.
 
Th17 cells: An attractive therapeutic target
 
Th17 cells are one of a number of different subsets of T helper (Th) cells. Characterized by production of the pro-inflammatory cytokine IL-17, they are derived from CD4+ T cells following interaction with an antigen-MHC Class II complex. Th17 cells play a key role in the immune system’s defense against infection—for example, through recruitment of neutrophils and macrophages to a site of protective inflammation.
 
Excessive Th17 activity is linked to the pathogenesis of inflammatory and autoimmune disorders such as psoriasis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis and asthma. Increased understanding of Th17 cell biology may advance the treatment or management of these conditions, all of which impact negatively upon quality of life and pose a considerable healthcare burden.
 
A further important characteristic of Th17 cells is their potential for longevity, self-renewal and durable memory. It is possible that these stem cell-like properties may be exploited to facilitate long-term responses to cellular immunotherapies. For this reason, Th17 cells have received increasing interest as a target for therapeutic intervention within the field of cancer immunotherapy.
 
Overcoming labor-intensive workflows
 
Production of functional Th17 cells has traditionally relied on the isolation of CD4+ T cells from human whole blood. There are several methods of accomplishing this, with magnetic bead isolation being one of the most common. Once this has been achieved, a cocktail of specific cytokines and antibodies is subsequently used to differentiate the CD4+ T cells into Th17 cells.
 
There are many disadvantages to this method. Working with human whole blood has associated risks, requiring researchers to be vaccinated against conditions such as hepatitis B and necessitating the implementation of health screening. Specialized containment and disposal methods are also needed, which may incur additional costs. Timelines can be extended when supplies of appropriate volumes of fresh blood are unavailable, with further delays often resulting from the need to repeat isolation steps following low CD4+ T cell yields.
 
Several factors can also impact directly on assay performance. Depending on the source, blood stocks can vary substantially depending on the time that has elapsed since donation; this may correlate with cellular changes that manifest themselves as inconsistent downstream readouts. Other sources of variation include the different CD4+ T cell isolation methods used by individual laboratories. This is exacerbated by the fact that a standardized protocol for Th17 cell differentiation has not yet been established, making it difficult to directly compare data from different research groups.
 
A simplified method for Th17 cell creation
 
A novel method for creating Th17 cells from high-quality cryopreserved CD4+ T cells has recently been developed that enhances experimental reproducibility while shortening timelines, cutting costs and eliminating the risks of working with human whole blood.1
 
In this new protocol, highly pure cryopreserved CD4+ T cells are simply thawed and mixed with specialized beads coated with antibodies against CD2, CD3 and CD28 molecules. These mimic antigen-presenting cells (APCs), to which the CD4+ T cells are attracted, and also activate resting T cells. Following the addition of a defined mixture of cytokines and antibodies, and subsequent incubation, the CD4+ T cells differentiate into the Th17 cell lineage (Figure 1).
 
This novel method of Th17 cell creation is far less prone to error than traditional methods because the entire process is greatly simplified. Rather than having to isolate CD4+ T cells from human whole blood, which can result in preparations that may be contaminated with many other cell types, researchers can instead access a highly pure CD4+ T cell population. Bead-based activation of these cells represents a more standardized method of differentiation (Figure 2).
 
An additional advantage of using cryopreserved CD4+ T cells is that each vial contains a defined number of viable cells. This obviates the need to adjust experimental conditions to align with cell populations. Furthermore, with CD4+ T cells available from multiple donors to afford a wide research demographic, and with alternative cell types available from the same donor, a greater breadth of data can be achieved.
 
A fully validated process
 
Validation of Th17 cell creation has traditionally relied on flow cytometric analysis; however, this can be extremely challenging since very few surface molecules are expressed exclusively by Th17 cells and there is a lack of specific antibodies that target these molecules. As a result, cells can often be mistakenly identified.
 
Bead-based activation provides much greater confidence in Th17 cell identification since the process has been validated via multiple methods. During phenotypic monitoring, cell clumping around the beads and increased proliferation are positive indicators of differentiation. Quantitation of IL-17 secretion into the growth medium using a bead-based proximity assay provides further confirmation of Th17 cell identity. This is supported by the use of RNA fluorescence in-situ hybridization (RNA FISH) to detect IL-17 gene expression at the mRNA level. In combination, these techniques provide a more definitive method of confirming Th17 cell creation.
 
Streamlining drug discovery
 
The use of high-quality, commercially available CD4+ T cells in combination with specialized reagents to promote Th17 cell differentiation offers many advantages for drug discovery. Most notably, the quality of data that is produced using Th17 cells is greatly enhanced relative to traditional approaches. Because these cells are more physiologically relevant than preparations that may be contaminated with other cell types, this translates into more effective decision-making that can streamline the drug discovery process.
 
Additionally, this approach is significantly faster, as timelines are shortened by elimination of time-consuming CD4+ T cell isolation steps. This also contributes to safer working practices, while simplification of the differentiation process may also expedite drug development. Moreover, Th17 cells can also provide substantial cost-savings through decreased manual processing steps, a reduced need to perform repeat experiments, and elimination of the expense associated with human whole blood work.
 
Similar combinations of cryopreserved cells, simplified differentiation methods, and user-friendly validation techniques hold enormous potential for the generation of other specialized immune cells to advance research and drug discovery. Bead-based activation represents a major advance in immune cell creation and offers huge possibilities for the future.

Kevin Bobofchak is global product manager for Lonza.
 
FIGURE 1
 
 
FIGURE 2
 
References
1. Holderness K, Crossett T, Larson B and Taubner A. Validation of Th17 cell differentiation from peripheral blood CD4+ T cells through assessment of mRNA expression and cytokine secretion using microplate reading and cellular imaging. Lonza and BioTek; 2016.
 

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