In Vitro Immunology Assays

T cells develop in the thymus where positive selection ensures T cells are restricted to MHC (major histocompatibility complex) and lineage selection occurs. Central tolerance removes cells that recognize self through a process called negative selection.

For self-reactive T cells that escape deletion in the thymus, peripheral mechanisms of tolerance operate to keep the activity of these cells in check. This can include regulation and energy mechanisms. When these processes fail, auto-reactive T cells can drive autoimmune disease.

So, controlling or limiting a specific T cell response is an effective strategy to control the autoimmune disease process. Tools like our inflammation and autoimmune T cell assays evaluate your therapy’s efficacy using human T cells in an in vitro system.

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B cells develop and undergo selection in the bone marrow. As for T cells, central and peripheral tolerance mechanisms eliminate or regulate B cells which produce antibodies, which in turn, recognizes self-proteins. When these processes fail, B cells can drive autoimmune disease. B cell activation, maturation, and polarization is intricately regulated via interaction with other cell types such as TFH CD4 T cells.

As a key driver, B cells are an amenable target for those seeking treatments for autoimmune disease and inflammation. Our team can help you determine which B cell assays best support your program, whether you require a screening assay format, tailored in vitro assay design, or disease model.

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Dendritic cells (DC) are professional antigen presenting cells (APCs) that patrol and reside in our tissues where they take up, process, and present antigen on MHCI and MHCII.

Activation and maturation of DCs through danger signals such as DAMPs or PAMPs stimulates them to migrate to the lymph node through which naïve and some memory T cells circulate.

If T cells recognize the MHC/peptide complex presented by DCs, they become activated. DCs not only present MHC/peptides, but also upregulate co-stimulatory ligands and produce cytokines which influence the polarization and type of response a naïve T cell makes.

DCs come in several different types which can be identified by surface markers:

• Dendritic Cell Assays
• Conventional Dendritic Cells Activation Assay
• Plasmacytoid Dendritic Cells Activation Assay

Macrophage and monocytes are components of the innate immune system and they have roles in multiple processes including inflammation and immunity to pathogens, tissue development, and tissue repair.

Macrophages have several origins during ontogeny. Generally, they are either tissue resident and derived from precursors that seed the tissue during development or can develop from circulating monocytes.

The tissue microenvironmental cues influence macrophage polarization and function enabling a quick response to any insult or pathogen. However, if this process is not controlled, this drives inflammatory diseases as well as conditions like fibrosis and atherosclerosis or conversely the cells may be pushed to a more anti-inflammatory phenotype.

Chemotaxis Assay Monocytes: An In Vitro Immunology Assay

The chemotaxis assay assesses the migration of monocytes isolated from the peripheral blood of healthy human donors.

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Platelets are anucleate cell fragments which play a key role in coagulation, preventing bleeding, and starting the process of wound healing.

However, it is now recognized that they play other roles including helping modulate the innate and adaptive immune response through both molecules on their surface and release of soluble factors upon platelet activation.

Neutrophils are a component of the innate immune system and are typically the first responders against pathogens such as bacteria and fungi as well as tissue damage. Neutrophil effector functions include:

• Phagocytosis
• Enzyme release
• ROS and inflammatory mediators
• NETS
• Interaction with various cell types

Chemotaxis Assay Neutrophils: An In Vitro Immunology Assay

The chemotaxis assay assesses the migration of isolated neutrophils from the peripheral blood of healthy human donors.

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Innate lymphoid cells (ILC) are composed of three groups; ILC1, 2, and 3. These cells do not express the diverse lymphoid receptors expressed by B cells and T cells. They are often tissue resident and not only have roles in immune response, but also drive processes such as tissue morphogenesis and repair.

ILC function can become dysregulated during autoimmune or inflammatory diseases and these cells are a potential target for therapies.

The epithelial barrier layers of the gut and skin provide a physical barrier which determines what can and cannot enter the body. These barrier epithelial layers are colonized by a complex cohort of microbes called the microbiome. In addition, the epithelial layers are populated by cells of the immune system. Epithelium/immune system/microbiome exist in a constant state of cross talk to maintain tissue homeostasis and solicit a quick response against any microbes entering the tissue.

These interactions can become dysregulated during autoimmune and autoinflammatory diseases of the gut and skin resulting in chronic inflammation and wounding of the epithelium. Epithelial/immune/microbial cocultures interactions can be modeled in vitro co-culture systems to assess the ability of a therapeutic to drive epithelial repair or limit immune activation.

Haematopoietic stem cells living within the bone marrow give rise to all the haematopoietic lineages and therefore therapies that target these cells are relevant in a variety of therapeutic areas.

Their ability to isolate, expand, and manipulate these cells in vitro provides the opportunity to study how therapeutics targets these populations, and in the case of murine cells, how these cells behave when they are transferred back in vivo.

Microglia are myeloid cells which seed the brain early during fetal development. Here they form a resident self-renewing pool of cells which interact in a complex cross talk with other cell types present within the brain, including neurons.

Microglial/neuron assays can be used to assess microglial targeted therapies in the context of autoimmune disease therapy such as MS, but also for dementia, for example Alzheimer’s disease.

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The tumor microenvironment frequently contains an immune cell component, but this is often switched off by the tumor or subverted into a regulatory phenotype.

Immuno-oncology cell assays allow us to test the ability of therapeutics targeting the immune system to reinvigorate or initiate an immune response.

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Fibroblasts live within the tissue stroma, engage in cross talk with the other cell types within the tissue, and can produce an array of immuno-modulating secretory factors such as cytokines as well as regulate the immune response through cell interactions. They also play a key role in tissue remodeling and wound healing.

Fibroblast assays and co-cultures can be used to determine the effect of a therapeutic on fibroblast function, the impact on immune cell phenotype and function, and vice versa.

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Fibroblast Activation Assay: An In Vitro Immunology Assay

We’ve developed optimized in vitro fibroblast activation assays utilizing human primary fibroblast-like synoviocytes derived from rheumatoid arthritis patients or healthy donors to assess the translational potential of small molecules as novel therapies.

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Flow cytometry is an integral part of many immunology studies providing information on the frequency and number of different cell subsets and how this is regulated by a therapeutic. Diverse aspects of cell behavior can be assessed through a combination of cell surface and intracellular staining including cell proliferation, expression of lineage and activation markers, viability, cytokine, and effector molecules expression. In addition, where high purity cell populations or single cells are required, cell sorting provides a solution for isolating cells.

 

Flow cytometry is not only a key readout for in vivo models, but also for in vitro immunology assays, such as immunophenotyping and functional assays.

 

Cytokines are key mediators of the immune response. They are soluble mediators enabling communication between cells, they regulate diverse aspects of cell behavior in both immune and non-immune cell subsets including altering cells of danger signals, drive immune cell polarization and differentiation, and in the case of chemokines influence cell migration.

Multiplex analysis of cytokines using Luminex or MSD platforms can provide an abundance of information on a panel of analytes from small quantities of biological material, whereas TR-FRET provides a sensitive and quick method of detecting single analytes from small amounts of material. Not all analytes are easily assessed by multiplexing, the more traditional ELISA analysis often provides a solution for single analytes. Where information is required about the frequency or number of cells producing a certain cytokine in response to a specific stimulus, ELIspot can be employed. For example, the number of T cells within a population that produce IFNg in response to stimulation with a specific peptide can be assessed.

Finally, staining allows the analysis of T cell cytokine production in combination with surface markers and effector molecule expression.

Imaging of cells or tissue can provide information not only on whether a cell or tissue is expressing a protein of interest, but also provides information on its localization and distribution. Multicolor immunofluorescence can be used to investigate pharmacodynamic changes and target engagement in clinical samples as well as in samples from pharmacology models.

The use of various dyes can identify various aspects of cell behavior, such as proliferation and apoptosis. Confocal microscopy provides high resolution images in XYZ but is low throughput. Whereas high content imaging (HCS) provides a method of imaging that offers good resolution but is also amenable to screening assays. For assays which include kinetic analysis at low/medium (96 well/364 well) throughput but at a low resolution with few parameters, IncuCyte provides a solution. Imaging can provide answers to questions where the following information is required regarding the MOA of a therapeutic:

• A determination of uptake, delivery, and intracellular compartmentalization
• Study of the target protein expression and localization
• Receptor trafficking, internalization, and recycling
• Co-localization of the target and therapeutic agents

Our experienced immunologists can help you understand what is happening within the cells that are of interest to you as well as help you gain a deeper understanding of the immune responses taking place.

Figure: Uptake of immunoglobulin by moDC and sub-cellular localization. Images taken of moDC exposed to fluorescently labeled IgG. On the left-hand side the cells were stained with Actin (green), IgG (purple), and nuclear stain (blue). On the right-hand side, the cells were stained with IgG (green), LAMP1 (red), and nuclear stain (blue). These images show that IgG is internalized by moDC and subsequently partially co-localizes with lysosomes (LAMP1+).

The effect of a therapeutic on gene expression from isolated tissues of interest or from individual purified cell types can be assessed using several platforms. The choice of platform to assess gene expression will be dictated by the complexity of data required and the question being asked. For example, qPCR can be used to assess gene expression when a small panel of genes have been selected for analysis. A step up in complexity could use NanoString to provide gene expression data on a panel of up to 800 genes.

Finally, RNAseq would provide the most comprehensive analysis of changes in gene expression. Both NanoString and RNAseq may be employed to identify key biomarkers that could be subsequently analyzed by qPCR and verified at the protein level. Other immune specific applications could combine single cell sorting (FACS) for example in the case of T cells, T cell repertoire within a population can be assessed.

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Bioinformatics goes hand in hand with complex data derived from today’s next-generation sequencing technologies. Yet, drug hunters rarely have the computer programming capabilities or expertise to achieve accurate, robust, and in-depth analysis of big data. By partnering with Fios Genomics, we can offer a cost-efficient, rapid turnaround, and high-throughput data analysis service for any of the following modalities with data generated in your own laboratory or that of a 3rd party:

• ASO Design and off-target analysis
• Gene expression profiling
• Proteomics
• Metabolomics
• Epigenetics
• CRISPR analysis

Drive discovery and maximize your ROI using our bioinformatics services.

Most of our assays are ran with either human or murine cells. The conditions for polarization and activation can vary dependent on species, and choice of cells are determined by the MOA of a therapy and the question being asked.

In vitro immunology assays using human cells add value to in vivo efficacy model data as they give you translatable data showing that your compound is on target and drives the expected phenotype in human. This allows you to test the relevant cells using the same material that would progress through the drug development pipeline. If rodent and human immune systems differ, in vitro data using human cells can tease apart any differences in MOA between rodent and human.

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We have worked with a wide range of immune cells and hematopoietic stem cells across species and our experienced immunologists would be happy to discuss custom assays to fit your needs.

Learn more:

• T cells in the Control of Organ-specific Autoimmunity
• B-cell Targeted Therapeutics in Clinical Development

We have standard screening assays that allow you to screen large numbers of molecules with readouts including HCS, proliferation, and cytokine production.

Many of our assays can be run with diseased patient samples. Samples are accessed through a provider. Please note, the number of cells that can be isolated from diseased samples or biopsies need to be taken in to account when setting up your in vitro immunology assay design.

Yes, in early clinical trials which have been traditionally associated only with safety, inclusion of predictive biomarker analysis can provide information on whether your therapy is on target in human. Immune cell assays can be uplifted to GCLP standard and validated for analysis of clinical samples.