Matthias De Decker
Senior Scientist, Flow Cytometry
Welcome back to the Flow Matters blog! Last time, we explored the essentials of building a solid clinical flow cytometry assay. This month, we’ll take a closer look at how these principles can be applied for conducting real-world clinical research. Specifically, we’ll be talking about autoimmunity, the role flow cytometry plays in clinical studies of autoimmune diseases, and how the biological context of these studies informs flow assay design.
What is Autoimmunity?
Autoimmune diseases represent some of the most complex and challenging conditions in modern medicine. From psoriasis and rheumatoid arthritis to multiple sclerosis and inflammatory bowel disease, autoimmune disorders affect millions of people worldwide and can impact virtually any organ system. While their symptoms and severity vary widely, they all share a common underlying link: the immune system.
Normally, our immune systems defend the body against pathogens and other foreign invaders; in autoimmunity, these defenses become overactive and mistakenly target the body’s own tissues. This misfiring can be triggered by a variety of factors, including genetics, environmental influences, infections, and dysregulated signaling by immune cells. In the case of the latter, this signaling is often mediated by an important class of molecules called cytokines.
Cytokines are secreted proteins that help regulate and coordinate immune responses. Think of them as short messages cells can exchange with each other: grow, migrate, attack, defend, etc. Based on the types of cytokines present in their local environment, immune cells adjust their activity accordingly to stay in concert with the body’s needs. In autoimmune disorders, the balance of these messages becomes disrupted, causing immune cells to become continuously mobilized to attack (a condition referred to as chronic inflammation). Eventually, they start to attack anything in sight, even other normal cells.
Within autoimmunity, TNF (tumor necrosis factor) is one of the most well-known pro-inflammatory cytokines and plays a central role in diseases like rheumatoid arthritis and Crohn’s disease. Interleukins 1 and 6 (IL-1 and IL-6) also contribute by promoting fever, inflammation, and the activation of immune cells. IL-6 additionally can stimulate T-helper 17 cells (Th17), one of the major T cell subsets involved in the inflammatory response. Th17 cells are some of the most active T cells in the context of autoimmunity and uniquely produce IL-17, another cytokine that further fuels tissue inflammation and is closely tied to psoriasis and multiple sclerosis disease progression. Finally, IL-12 and IL-23 are supporting factors that can drive and sustain the activity of both Th17 cells and more conventional Th1 cells, keeping them going long after the initial inflammation response dies down.
In recent years, new types of treatment for autoimmune diseases have emerged that specifically aim to modulate components of these signaling responses:
- Monoclonal antibodies are immune cell-derived proteins that bind and neutralize their targets. When aimed at disease-causing cytokines or their receptors, they can dampen pro-inflammatory signaling and reduce inflammation as a result.
- Fc fusion proteins combine a receptor fragment with the Fc portion of an antibody to block inflammatory signals, for example, by acting as decoy receptors for cytokines.
- Small molecule inhibitors, such as JAK inhibitors, work intracellularly to disrupt signaling pathways involved in immune activation.
- CAR T cell therapies, which we discussed in a previous post, have recently begun to be developed for the treatment of autoimmune disorders like lupus. These CAR T cells aim to deplete aberrantly active normal immune cells, targeting them in a similar fashion to cancerous immune cells using markers like CD19.
These therapies offer more precise control over the immune system compared to traditional immunosuppressants, improving outcomes and minimizing side effects in many autoimmune conditions.
If all of this sounds complicated, that’s because it is! To make it easier, let’s take rheumatoid arthritis as an example. During rheumatoid arthritis progression, overactive macrophages secrete pro-inflammatory cytokines such as IL-6. IL-6 binds to its receptor (IL-6R) on Th17 cells, promoting their proliferation and activation. Activated Th17 cells then produce large amounts of IL-17, which amplifies joint inflammation and eventually contributes to the cartilage and bone degradation that is a common hallmark of the disease. All these steps in the pathway to disease progression present opportunities for intervention that are being actively explored in clinical studies, as illustrated in Figure 1 below.
Figure 1
Figure 1: Rheumatoid arthritis as a representative model of autoimmune pathogenesis and emerging therapeutic mechanisms. Emerging therapies that target key points in this pathway: IL-6-targeting Fc fusion proteins act as IL-6 decoys to reduce Th17 activation (A); anti-IL-17 monoclonal antibodies (mAb) neutralize IL-17 to dampen inflammation (B); and small molecule JAK inhibitors block JAK-mediated STAT phosphorylation, suppressing IL-17 gene transcription (C).
Flow Cytometry in Autoimmune Disorder Clinical Trials
As our understanding of immune dysregulation deepens, the demand for precise tools to study immune responses increases. In this context, flow cytometry has become an indispensable technology.
In the context of autoimmunity, flow cytometry provides critical insights into both the mechanism of action and pharmacodynamic response. Here are some key applications:
- Baseline immune profiling: Before treatment begins, flow cytometry is used to assess the patients’ immune status. This includes identifying immune cell imbalances that may predict disease severity or therapeutic response.
- Biomarker development: Immune biomarkers, such as increased numbers of T helper 17 (Th17) cells or reduced numbers of regulatory T cells (Tregs), can be monitored over time to evaluate treatment efficacy. In addition, changes in inflammatory or regulatory cytokines involved in disease progression can provide valuable insights into therapeutic efficacy and disease activity.
- Safety monitoring: Some autoimmune therapies, especially biologics or immune modulators, can cause immune suppression or overactivation. Flow cytometry helps detect off-target effects by monitoring changes in immune cell subsets throughout the trial.
- Patient stratification: By identifying unique immune signatures, researchers can stratify patients into subgroups more likely to benefit from a given therapy, thereby supporting a personalized medicine approach.
Developing robust flow cytometry assays for these applications require careful planning to ensure reproducibility, sensitivity, and relevance to the disease mechanism. As highlighted in last month’s blog, a deep understanding of the biology and therapeutic context is essential to inform the assay design. For instance, if the drug being studied targets a cytokine receptor, then a receptor occupancy (RO) assay might be desired to see how effectively the drug binds to its target. A critical aspect of RO assay design is epitope selection. Antibodies must be carefully chosen during assay construction to either compete with the therapeutic agent – allowing measurement of free (unbound) receptors – or bind to distinct non-competing epitopes to quantify total receptor levels regardless of drug binding.
In addition, it is necessary to not only know the priority of your biomarkers, but also to consider where these biomarkers are present in the cell. If detection of intracellular markers such as cytokines (e.g., IL-17, TNF) or transcription factors (e.g., FOXP3 for Tregs, or components of the JAK/STAT signaling pathway) is desired, the cells must be fixed and permeabilized to allow antibodies to penetrate the cell membrane and the nucleus. These steps of the sample processing must be carefully calibrated to preserve epitope integrity while maintaining robust signal quality. Over-fixation can reduce fluorescence intensity or mask epitopes, while under-fixation may lead to cell loss or poor permeability. Fixation can also increase background signal, making it crucial to select sufficiently bright fluorochromes, appropriate compensation controls, and tailor your analysis strategy to enable clear separation of positive and negative populations.
Other general key steps include selecting optimal marker-fluorochrome combinations, titrating antibodies in conditions that closely mimic clinical samples, and validating the full stain panel using real-world conditions (as discussed in blog post #3, Principles of Clinical Flow Assay Design). These measures are critical to generate high-quality, interpretable data suitable for clinical decision-making and trial endpoints.
Figure 2
Figure 2: Flow cytometry panel development for autoimmune clinical trials.
Looking Forward
Autoimmune diseases present a formidable challenge due to their complexity and variability. Flow cytometry offers a window into the immune system, enabling researchers to track disease activity, monitor therapeutic effects, and accelerate clinical trials. By harnessing the power of immune profiling, we move closer to developing safer, more effective, and more personalized treatments for those living with autoimmune conditions.
As new technologies emerge, such as spectral flow cytometry and mass cytometry (CyTOF), researchers can analyze even more parameters simultaneously, yielding deeper insights into immune complexity. Combined with single-cell genomics and machine learning, flow cytometry data is poised to play an even greater role in autoimmune drug development, helping translate cellular insights into clinical breakthroughs.
Thank you for taking the time to read this latest entry of the Flow Matters blog! If you’re involved in autoimmune research or clinical trials, carefully designing your flow cytometry assays can make all the difference in generating meaningful data. Have questions or experiences to share? We’d love to hear from you!
